Conference Abstracts

60th Annual Fall Conference and Scientific Sessions of

the Council for High Blood Pressure Research in Association with
the Council on the Kidney in Cardiovascular Disease
October 47, 2006 San Antonio Marriott Rivercenter San Antonio, Texas
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ADVANCING RESEARCH.
APPLYING KNOWLEDGE.
IMPROVING THE QUALITY
OF HEALTHCARE.
 e26 Hypertension Vol 48, No 4 October 2006
Oral Presentations
1 dimensions and function, we could examine the role of ACE2 in BP regulation without the
Nuclear Hormone Receptor LXR Induces Murine Mesenchymal Stem Cells
into Renin Expressing Cells: Possible Origin of Juxtaglomerular Cells
Kenichi Matsushita, Fulvio Morello, Richard E Pratt, Victor J Dzau; Duke Univ Med Cntr,
Durham, NC
Mesenchymal stem cell can differentiate into skeletal muscle, smooth muscle, cartilage, fat
and bone. It may play an important role in developmental biology contributing to the genesis
of various tissues. Indeed, it has been reported that juxtaglomerular cells may originate from
the metanephric mesenchyme. In this study we examined for direct evidence that mesenchy-
mal stem cells (MSCs) can be induced to become renin expressing cells. Previously we have
reported that the nuclear hormone receptor liver X receptor (LXR) is a major transcriptional
regulator of renin gene expression. Accordingly, we studied the effect of LXR activation of MSC
renin gene expression. Methods: Murine MSCs were subjected to serum starvation for 16
hours. We then added the LXR synthetic ligand T0901317 (10-6 M) and investigated mRNA
expression of renin, ATP-binding cassette transporter A1 (ABCA1) which is well known
upregulated by LXR activation, LXR-alpha and LXR-beta after 6 hours of pharmacological
treatment by using real-time RT-PCR. We further examined the renin mRNA expression by
adding LXR natural ligand 22-hydroxycholesterol (10-7 M) or cyclic AMP (10-3 M). Results:
Murine MSCs treated acutely (6 hours) with T0901317 exhibited an increase in expression of
renin (1.9 0.4 fold change for MSCs without treatment, P 0.05). There was an increase
in ABCA1 expression (3.7 0.5 fold change for MSCs without treatment, P 0.05) and no
significant change in LXR-alpha or LXR-beta expression, suggesting that T0901317 effectively
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acted only at the post-transcriptional level. Furthermore, 6 hours of 22-hydroxycholesterol or
cyclic AMP treatment also upregulated renin mRNA expression (2.0 0.4 fold and 6.0 1.8
fold changes for MSCs without treatment, respectively. P 0.05). We have also overexpressed
LXR-alpha in MSC using gene transfection. Upon prolonged stimulation with 22-
hydroxycholesterol for 6 weeks or cyclic AMP for 2 weeks, our preliminary results show that
the phenotype of the MSC changed to become cells containing renin granules. Conclusion: Our
results suggest that the activation of LXR induces renin mRNA expression and renin granule
formation in mesenchymal stem cells and suggest that these cells may be the origin of
juxtaglomerular cells during embryonic development.
2 For this purpose, we performed bilateral sino-aortic denervation (SAD) in spontaneous
c-Src Inhibition Attenuates Development of Hypertension and Associated
Cardiovascular and Renal Damage in Ang II-Infused Mice through
Redox-Sensitive Mechanisms
Glaucia E Callera, Ying He, Augusto C Montezano, Univ of Ottawa, Ottawa, Canada; Alvaro
Yogi, Rita C Tostes, Univ of Sao Paulo, Sao Paulo, Brazil; Ernesto L Schiffrin, McGill Univ,
Montreal, Canada; Rhian M Touyz; Univ of Ottawa, Ottawa, Canada
c-Src plays a critical role in Angiotensin II (Ang II)-mediated signaling. Whether this tyrosine
kinase influences development of hypertension and associated target organ damage induced
by Ang II remains unclear. Here we investigated effects of Ang II on blood pressure (BP) and
renal and cardiac fibrosis in mice treated with the c-Src inhibitor, CGP 077675 and in mice
deficient in c-Src (c-Src -/- and c-Src /- ). The systolic blood pressure (SBP) increase induced
by Ang II infusion (400 ng/kg/min, 2-weeks) in c-Src/ mice was significantly reduced by CGP
077675 (25 mg/Kg/day). Ang II-induced hypertension was blunted in c-Src-/- and c-Src/- mice
(1678 vs 1378 and 1057 mmHg). Ang II-induced increase in plasma TBARS (control,
14.40.6 vs Ang II, 19.60.7 nmol/mL) and NAD(P)H oxidase-mediated vascular generation
of .O2- (3,5-fold) were reduced by CGP 077675 (p0.05). c-Src inhibition also inhibited the
increased phosphorylation of ERK1/2 (Ang II, 868 vs Ang II CGP, 613, arbitrary units) and
JNK (Ang II, 12619 vs Ang II CGP, 431, arbitrary units) in aorta and resistance
(mesenteric) arteries. Ang II infusion failed to increase MAPK phosphorylation and .O2-
generation in c-Src/-, c-Src-/- mice (p0.05). The greater expression of proliferating cell
nuclear antigen (PCNA), a marker of cell growth, in mesenteric arteries from Ang II-infused
c-Src/ mice was inhibited with CGP 077675 treatment (Ang II, 1309 vs Ang II CGP, Gene-Targeted Mice
1044, arbitrary units). PCNA protein expression was similar in c-Src/- mice infused with Ang
II and vehicle. Cardiac and renal collagen content induced by Ang II was significantly lower in
in c-Src-/- and c-Src/- mice and in CGP 077675-treated mice. In conclusion, inhibition of c-Src
activity by CGP077675 attenuates Ang II signaling and ameliorates oxidative stress, BP
elevation and cardio-renal fibrosis by Ang II. Studies in c-Src- deficient mice support these
results. Our findings identify c-Src tyrosine kinase as a novel target to blunt Ang II -dependent
hypertension and associated cardiovascular and renal damage.
3 human AGT (hAGT) is expressed only in the kidney, experiments were performed to determine
Enhanced Susceptibility to Ang II-Induced Hypertension and Impaired Ang
II Metabolism in ACE2-Deficient Mice
Susan B Gurley, Thu H Le, Robert Griffiths, Nisha Phillip, Timothy A Haystead, Thomas M
Coffman; Duke Univ, Durham, NC
In order to clarify the physiological roles of ACE2, lines of mice with targeted disruption of the
Ace2 gene have been generated. However, phenotypic features of the different ACE2-deficient
(KO) mouse lines have been variable, especially with regard to blood pressures (BP) and
cardiovascular (CV) functions. One potential source of this variability might be heterogeneity of
genetic background. Thus, we generated inbred ACE2 KO mice by sequential back-crossing of
the Ace2 null mutation onto C57BL/6 (B6) or 129/SvEv (129) background for more than 6
generations. Both lines of inbred ACE2 KO mice are viable, have normal reproduction, and lack
any gross anatomical or structural abnormalities. Because of these mice have normal cardiac
potential confounding effects of impaired heart physiology. Baseline BPs were measured in the
ACE2 KO lines with radiotelemetry. Mean BPs were not significantly different between
B6-Ace2/y and -/y mice, and 129-Ace2/y and -/y mice (1282 vs. 1323 mmHg, n6 all
groups). As in vitro studies suggest that ang II may be a substrate for ACE2, we examined the
effects of ACE2 deficiency on susceptibility to ang II-dependent hypertension, using the inbred
lines. Ace2/y (MWT) and -/y (MKO) mice were infused subcutaneously with angiotensin II (1000
ng/kg/min) with an osmotic pump, while BP was monitored by radiotelemetry. With ang II
infusion, BP increased significantly in both MWT (1332 to 1696 mm Hg; p0.0005) and
MKO mice (1312 to 1956 mm Hg, p0.0002), but the magnitude of this increase was
almost 2-fold greater in the MKO animals (64 mm Hg) compared to MWT (36 mm Hg). After
2 weeks of ang II infusion, BPs were significantly higher in the MKO mice than WT (p0.01).
To examine the mechanism for the more marked BP response to ang II in the ACE2-deficient
mice, we measured kidney ang II levels after ang II infusion using MALDI-TOF mass
spectrometry. Renal ang II content was nearly six-fold higher in MKO mice compared to MWT
(0.280.05 vs. 0.050.002 relative intensity; p0.0008). In summary, we find no effect of
ACE2-deficiency on basal BP regulation or CV function. However, ACE2 protects against ang
II-dependent hypertension by regulating ang II levels in the kidney.
4
Exaggerated Blood Pressure Variability Aggravates Hypertensive Cardiac
Remodeling through the Angiotensin II-Mediated Inflammation
Hiroshi Kudo, Hisashi Kai, Narimasa Takayama, Takahiro Mori, Yusuke Sugi, Daisuke Fukui,
Kiyoko Takemiya, Ayami Ikeda, Masayoshi Futamata, Hideo Yasukawa, Nobuhiro Tahara,
Mitsuhisa Koga, Yumiko Kawai, The Cardiovascular Rsch Institute, Kurume, Japan;
Yoshitaka Hirooka, The Dept of Cardiovascular Medicine, Fukuoka, Japan; Tsutomu
Imaizumi; The Cardiovascular Rsch Institute, Kurume, Japan
It was demonstrated that hypertensive patients with large blood pressure (BP) variability had
greater risk of cardiovascular events and exaggerated end-organ damages. However, the
pathogenesis is currently unknown. The aims of this study were to create a new rat model of
chronic hypertension with exaggerated BP variability and to investigate the effects of
exaggerated BP variability on hypertensive cardiac remodeling and the underlying mechanism.
hypertensive rats (SHRs). Seven weeks after SAD or sham operation (week 7), 24-hour BP was
monitored telemetrically in SADSHRs and shamSHRs. Although mean BP was similar in the
two groups, SADSHRs showed greater BP variability parameters, such as standard deviation
and covariance of mean BP, compared with shamSHRs. At week 7, both groups showed
concentric LV hypertrophy, and SAD enhanced it by 1.3-fold. SADSHRs enhanced myocyte
hypertrophy and myocardial fibrosis by 1.4-fold and 4.7-fold, respectively, relative to
shamSHRs. Perivascular macrophage infiltration was evident in SADSHRs, but not in
shamSHRs. SAD remarkably upregulated expressions of myocardial angiotensinogen and
monocyte chemoattractant protein-1 (MCP-1). To determine the role of angiotensin II in the BP
variability-induced cardiac remodeling, non-depressor dose of angiotensin II receptor blocker,
candesartan, was orally given to SHRs everyday from 1 week after SAD operation
(SADCandSHRs). At week 7, although the small dose of candesartan did not affect BP
variability, the SAD-enhanced LV and myocyte hypertrophy was significantly reduced and the
SAD-induced myocardial fibrosis was almost abolished. Moreover, in SADCandSHRs, the
SAD-induced MCP-1 upregulation and macrophage accumulation were almost reversed to the
levels of shamSHRs. In conclusion, exaggerated BP variability aggravates hypertensive
cardiac hypertrophy and fibrosis through chronic activation of inflammatory process. And,
angiotensin II would play a key role in the activation of the inflammatory process, independently
of the presser effect.
5
Kidney-Specific Enhancement of Angiotensin II Initiates Renal Injury in
Hiroyuki Kobori, Yuri Ozawa, Yuki Suzaki, Tulane Univ Health Sciences Cntr, New Orleans,
LA; Curt D Sigmund, Univ of Iowa, Iowa City, IA; L. G Navar; Tulane Univ Health Sciences
Cntr, New Orleans, LA
We recently reported that concomitant increases in proximal tubular angiotensinogen (AGT)
mRNA and protein participate in increased intrarenal angiotensin (Ang) II leading to progressive
renal injury in AngII-infused rats. However, it has not been established if selective increases in
intrarenal AngII can be responsible for renal injury. Using a transgenic mouse model in which
if selective renal overproduction of AngII elicited by stimulating hAGT present only in the kidney
in the presence of human renin (hR) will cause renal injury. We used 3 groups of mice: 1) single
transgenic (A, N14) expressing hAGT only in the kidney regulated by kidney-specific
androgen regulated protein promoter, 2) double transgenic (D, N13) expressing hR
systemically in addition to hAGT only in the kidney, and 3) wild type mice (W, N12).
Exogenous hAGT protein is inactive in A mice because endogenous mouse renin cannot cleave
hAGT to AngI due to a high species-specificity. All mice were monitored from 12 to 18 wks of
age with free access to a regular diet and water. Systolic BP was progressively increased from
116/-5 mmHg (12 wks) to 140/-7 (18 wks) in D mice during this period. This increase was
not observed in A or W mice. Intrarenal hAGT mRNA and protein were similar in A and D mice;
however, hAGT mRNA or protein was not detectable in kidneys of W mice. While plasma AngII
concentrations were similar among the 3 groups, kidney AngII levels were increased in D
(216/-43 fmol/g) compared with A (117/-16) and W (118/-17) mice. Interstitial
collagen-positive area, stained by PicroSirius Red, was significantly increased in D (0.52/-

0.06%) compared with A (0.36/-0.05) and W (0.34/-0.03) mice. Interstitial macrophage
infiltration, evaluated by CD68-positive cell number, was significantly increased in D (46/-5
cells/mm2) compared with A (20/-3) and W (19/-2) mice. Afferent arteriolar wall thickness,
stained by alpha actin, was significantly increased in D (3.31/-0.41 m) compared with A
(2.21/-0.12) and W (2.16/-0.11) mice. These data indicate that the selective renal
overproduction of AngII initiates renal injury in the gene-targeted mice even before the
development of marked hypertension.
6
A Novel Regulatory Effect of AT1 Receptor-Interacting Molecule on
Vascular Smooth Muscle Cells
Koichi Azuma, Kouichi Tamura, Masashi Sakai, Yuko Tsurumi, Toyoichiro Shigenaga, Yutaka
Tanaka, Motoko Ozawa, Miyuki Matsuda, Tomoaki Ishigami, Yokohama City Univ,
Yokohama, Japan; Marco Lopez-Ilasaca, Harvard Univ, Boston, MA; Masatsugu Horiuchi,
Ehime Univ, Shigenobu, Japan; Satoshi Umemura; Yokohama City Univ, Yokohama, Japan
Activation of tissue AT1 receptor (AT1R) signaling plays an important role in cardiovascular
hypertrophy and remodeling and the C-terminal domain of AT1R is involved in the receptor
internalization and its downstream pathway. We previously cloned a novel molecule interacting
with the C-terminal domain of AT1R, ATRAP (for AT1R-associated protein), using the yeast
two-hybrid strategy. In this study, we tested the hypothesis that vascular smooth muscle cells
(VSMC) express ATRAP and that ATRAP modulates Ang II-induced responses in VSMC. We
identified that the ATRAP mRNA and protein were endogenously expressed in VSMC, and found
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CHBPR ConferenceOral Presentations e27
8
Association between Variants of the Human GSTM Gene Family and
Hypertension
Christian Delles, Univ of Glasgow, Glasgow, United Kingdom; Ana C Braga-Marcano, Patricia
B Munroe, Barts & The London Med & Dental Sch, London, United Kingdom; Sandosh
Padmanabhan, John D McClure, Nick J Brain, Univ of Glasgow, Glasgow, United Kingdom;
Morris J Brown, Univ of Cambridge, Cambridge, United Kingdom; Nilesh J Samani, Univ of
Leicester, Leicester, United Kingdom; David Clayton, Univ of Cambridge, Cambridge, United
Kingdom; Martin Farrall, Univ of Oxford, Oxford, United Kingdom; John Webster, Aberdeen
Royal Infirmary, Aberdeen, United Kingdom; John M Connell, Univ of Glasgow, Glasgow,
United Kingdom; Mark J Caulfield, Barts & The London Med & Dental Sch, London, United
Kingdom; Anna F Dominiczak; Univ of Glasgow, Glasgow, United Kingdom
Background: Glutathione S-transferases (GSTMs) are involved in cellular defences against
oxidative stress. The human GSTM gene cluster consists of 5 genes, GSTM4, 2, 1, 5 and 3. We
have shown that the rat orthologue, Gstm1, is a positional and functional candidate gene for
rodent hypertension. The aim of the current study was to test the hypothesis that this discovery
can be translated to man. Methods: First analysis was performed in 138 white subjects in
whom exons, flanking introns, 3 and 5 regions of GSTM4, 2, 5 and 3 were sequenced to
detect single nucleotide polymorphisms (SNPs) within the GSTM gene family and to examine
linkage disequilibrium (LD) between these SNPs. We have then chosen 10 SNPs across the
GSTM gene cluster and genotyped 1151 families (3453 individuals) from the MRC BRIGHT
Study for these SNPs and for the common GSTM1 deletion genotype. Analysis was performed
by Transmission Disequilibrium Test (TDT) using FBAT Software. Results: We detected 18, 5,
6, and 5 SNPs in GSTM4, 2, 5 and 3, respectively, with minor allele frequencies of 5% or
greater. There were two distinct LD blocks, one between GSTM4 and 2 and one between
GSTM5 and 3. In the BRIGHT TDT Study we detected a highly significant association between
a SNP in the 3 region of GSTM5 (rs11807) and hypertension (z2.698, P0.007) and

a substantial co-localization of ATRAP and AT1R in intracellular compartments in Ang significant associations for two more SNPs within the GSTM5/3 LD block (rs11101992:
II-stimulated VSMC. Overexpression of ATRAP by adenoviral gene transfer significantly inhibited z2.443, P0.015; rs3814309: z2.215, P0.027). The GSTM1 deletion was also over-
Ang II-mediated increases in TGF- mRNA expression (p0.05, n6) and TGF- production transmitted to affected offspring (z2.339, P0.019). Conclusions: The GSTM1 deletion
into the medium (p0.05, n6). Furthermore, this phenomenon was accompanied by genotype and three SNPs in the GSTM5/3 LD block are associated with hypertension as a
inhibition of Ang II-induced activation of BrdU incorporation (p0.05, n6). These results qualitative trait. This is an example for successful translation of findings from a rodent model
indicate that ATRAP significantly promotes internalization of the AT1R and attenuates Ang to human cardiovascular disease. GSTMs, and in particular GSTM1, 5 and 3, are robust
II-mediated proliferative response and synthesis of extracellular matrix in VSMC, and may candidate genes for human essential hypertension. Genetic variants in GSTMs may lead to
suggest a novel strategy to inhibit vascular remodeling. reduced defences against oxidative stress and thereby to endothelial dysfunction and relentless
progression of cardiovascular disease.

7 Tejas V Patel, Gordon H Williams, Naomi D Fisher; Brigham and Womens Hosp, Boston, MA
Human G Protein-Coupled Receptor Kinase Type 4 (hGRK4) Wild-Type
Prevents Salt Sensitivity While its Variant, hGRK4 486V, Promotes Salt
Sensitivity in Transgenic Mice: Role of Genetic Background
Zheng Wang, Laureano D Asico, Crisanto S Escano, Georgetown Univ Sch of Medicine,
Washington, DC; Robin A Felder, Univ of Virginia Health Sciences Cntr, Charlottesville, VA;
Pedro A Jose; Georgetown Univ Sch of Medicine, Washington, DC
The hGRK4 486V variant, either by itself or in conjunction with other hGRK4 variants (65L
and 142V), is associated with salt-sensitive hypertension (Clin Chem.2006;52:352), as
evidenced by its development in hGRK4 486V transgenic mice (but not in hGRK4 wild-type
transgenes) generated in our laboratory. The influence of genetic background on the expression
of salt sensitivity was studied by introducing the transgenes into mice with varying gene ratios
of salt-resistant SJL and salt-sensitive C57BL/6J (B6/J) backgrounds. The mice (6 8 mo old)
were maintained on normal (0.9%) or high (6%) NaCl diet for 3 weeks. With normal NaCl intake,
aortic BPs (measured via the femoral artery under pentobarbital anesthesia) were similar in
transgenic and non-transgenic mice. However, with high NaCl intake, BPs differed among the
groups, and influenced by genetic background (Table). BPs of non-transgenic littermates (NT)
with at least 12% SJL background were not affected by high NaCl. In contrast, high NaCl
increased BPs of 486V mice, despite having 25% SJL background (P0.001). The ability of
high NaCl to elevate BPs of 486V mice decreased as the SJL background increased to 56%.
High NaCl increased BPs of NTs with more than 94% B6/J background. However, hGRK4
wild-type prevented the salt-sensitive hypertension of B6/J mice (P0.001). We conclude that
hGRK4 prevents, while the 486V variant promotes, salt-sensitive hypertension in transgenic
mice. The extent of salt-resistant or salt-sensitive genetic background is crucial in uncovering
the roles of hGRK4 wild-type and 486V variant in salt sensitivity. Inconsistencies in the
association of candidate genes with hypertension may be explained by differences in genetic
background.
% Background Normal NaCl (0.9%) High NaCl (6%)
9
Effect of Age and Angiotensinogen 235 Genotype on Renal Plasma Flow
Responsiveness to Angiotensin II
Objective: In essential hypertensives(HT), blunted renal plasma flow (RPF) response to Ang II
is associated with the Angiotensinogen AGT235TT genotype, suggesting a pathologic increase
in intrarenal Ang II. RPF and its responsiveness to Ang II fall with age. We sought to determine
the interaction of age and genotype on RPF responsiveness in both HT and normotensives (NT).
Method: A total of 315 subjects in high sodium balance had RPF response to Ang II (3ng kg-1
min-1) measured by PAH clearance. Subjects were divided into 45 yrs (N83 HT, 65 NT) and
45 yrs (N145 HT, 22 NT) for age analysis, as 45 was the median age. Result: Age and
AGT235 genotype independently predicted RPF response to Ang II. For both HT and NT, the Ang
II induced fall in RPF was significantly lower in older than in younger subjects (p0.03 and
0.004, respectively). Among HT, this fall with age was seen in subjects carrying both the
AGT235MM and MT genotypes (p0.005, Fig. A). However, AGT235TT homozygotes did not
demonstrate a fall in RPF responses with age (p0.72, young vs. old, Fig. B), as younger
subjects already showed blunted responses. Among NT, all AGT235 genotypes had a significant
fall in RPF response to Ang II. This association between age, RPF response to Ang II and
genotype was not seen with 3 other genes in RAAS: ACE I/D, ATR1 and aldosterone synthase.
Conclusion: In HT but not NT, the AGT235TT variant is associated with a blunted RPF response
to Ang II that does not fall with age. This suggests that young HT with the AGT235TT genotype
warrant a more aggressive management to preserve renal function. This is the first report of
age and genotype interaction, which may have important implications in managing essential
HT. #

SJL B6/J
(salt- (salt- SBP SBP
Mice esistant) ensitive) (mm Hg) N (mm Hg) N P#
10
SJL 100 0 970.6 4 972.2 5 0.87 Disruption of Natriuretic Peptide Receptor A Gene Increases Adrenal
B/6J 0 100 1031.2 4 1231.8* 5 <0.001
Angiotensin II and Aldosterone Levels
NT 25 75 1000.9 4 1003.7 6 0.97
NT 12 88 1052.9 4 1027.7 5 0.76
Di Zhao, Naveen K Somanna, Elangovan Vellaichamy, Kailash N Pandey; Dept of Physiology
NT 6 >94 1021.8 8 1221.2 11 <0.001
and Hypertension and Renal Cntr of Excellence, Tulane Univ Health Sciences Cntr & Sch of
GRK4 6 >94 1012.3 7 1032.3** 7 0.5
Medicine, New Orleans, LA
486V 25 75 1000.5 2 1254.6*** 9 0.03
486V 56 44 963.1 6 1103.6**** 5 0.01
The disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1)
#P 0.9% vs 6% NaCl, ANOVA, Newman-Keuls (6% NaCl): *P0.01, SJL vs B/6J; **P0.001, NT(94% leads to elevated arterial blood pressure, cardiac hypertrophy, congestive heart failure, and
B6/J) vs GRK4; ***P0.001, NT(25% SJL) vs 486V; ****P0.05, 486V (25% SJL) vs 486V (56% SJL). sudden death in mice lacking NPRA. ANP-NPRA signaling is known to counteract the
SBPsystolic blood pressure, Data are MSE. renin-angiotensin-aldosterone system (RAAS). We studied whether Npr1 gene copy number
 e28 Hypertension Vol 48, No 4 October 2006
affects adrenal angiotensin II (Ang II) and aldosterone (ALDO) levels in a gene-dose dependent
manner in Npr1 gene-targeted mice. Adrenal Ang II and ALDO levels increased in 1-copy
(gene-disrupted heterozygous allele, 0.2-fold, p0.05, 0.4-fold, p0.05) mice as compared
with 2-copy (wide type) mice, but decreased in 3-copy (gene-duplicated heterozygous allele,
0.2-fold, p0.05, 0.1-fold, p0.05), and 4-copy (gene-duplicated homozygous allele, 0.3-fold,
p0.01, 0.4-fold, p0.001) mice. Interestingly, renal Ang II levels decreased in 1-copy
(0.3-fold, p0.05), 3-copy (0.4-fold, p0.01), and 4-copy (0.4-fold, p0.01) mice as
compared with 2-copy mice. Low salt diet increased adrenal Ang II and ALDO levels in 1-copy
(0.2-fold, p0.05, 24-fold, p0.001), 2-copy (0.2-fold, p0.05, 23-fold, p0.001), 3-copy
(0.2-fold, p0.05, 4-fold, p0.001), and 4-copy (0.3-fold, p0.05, 5-fold, p0.001) mice.
On the other hand, high salt diet decreased adrenal Ang II levels in 1-copy (0.5-fold, p0.001),
2-copy (0.4-fold, p0.01), 3-copy (0.3-fold, p0.01) mice, and decreased adrenal ALDO
levels in 1-copy (0.3-fold, p0.05), 2-copy (0.2-fold, p0.05) mice. Low salt diet increased
renal Ang II levels in 1-copy (0.5-fold, p0.05), 2-copy (0.4-fold, p0.05), 3-copy (0.6-fold,
p0.05), and 4-copy (0.5-fold, p0.05) mice, whereas high salt diet decreased renal Ang II
levels in 1-copy (0.3-fold, p0.05), and 2-copy (0.3-fold, p0.05) mice. The results suggest
that ANP-NPRA signaling antagonizes adrenal Ang II and ALDO levels in a gene-dose dependent
manner. Our findings implicate that increased adrenal Ang II and ALDO levels play an important
role in elevated blood pressure in Npr1 gene-disrupted mice.
11
The T8590C Polymorphism of CYP4A11 and 20-HETE in Essential
Hypertension
Cheryl L Laffer, New York Med College, Valhalla, NY; James V Gainer, Nancy J Brown,
Michael R Waterman, Jorge H Capdevila, Vanderbilt Univ Med Cntr, Nashville, TN; Michal
Laniado-Schwartzman, Alberto Nasjletti, Fernando Elijovich; New York Med College, Valhalla,
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
NY
CYP4A11 synthesizes 20-HETE in humans. The C allele of the T8590C polymorphism which
codes for an enzyme with a 50% reduction in catalytic activity, is linked to essential
hypertension (EH, population odds ratios of 1.232.31). We have previously shown that salt and
furosemide increase, whereas hyperinsulinemia reduces urine 20-HETE in EH. We now
investigated the effect of C on 20-HETE in 32 hypertensive subjects (18 black, 14 white)
genotyped for T8590C and phenotyped for insulin sensitivity, salt-sensitivity of blood pressure
(BP), and responses of renin, aldosterone, endothelin and catecholamines to salt-loading and
salt-depletion. Genotypes were: 13 TT, 17 CT and 2 CC. Frequency of C was 33% and higher
in blacks (39%) than in whites (25%) as previously reported. C carriers had higher diastolic BP
(943 mmHg vs 862, p0.05) and non-significantly greater waist-to-hip ratios, lower
plasma renin, and blunted aldosterone responses to salt, compared to TT. The slope of the
pressure-natriuresis curves (C1.530.02 radians, TT1.490.02), insulin sensitivity index
(HOMA model, C0.1540.020 ml*L*U-1*mmol-1, TT0.1460.025), glomerular filtration
rate and salt-balance were similar between groups. However, fractional excretions of Na and
K in response to furosemide (1.90.2% vs 2.30.2, p0.06 and 11.30,6% vs 14.01.3,
p0.05) were lower in C than in TT. Urine 20-HETE was similar between C (1.590.29 g/hr)
and TT (1.600.41). However, both insulin and the C-allele significantly and independently
blunted the 20-HETE response to salt-loading (20-HETEsalt-load2.36 - 0.054xInsulin -
0.49xC-allele, F11.2, p0.001). From this multivariate model it can be calculated that the
C allele and a 9 U/ml increase in serum insulin are equipotent in determining a 0.5 g/hr
lesser response of urine 20-HETE to salt. Our data suggest that C influences 20-HETE
responses to salt and 20-HETE-mediated, furosemide-induced natriuresis and kaliuresis.
However, allele-based analysis does not reveal relationships with salt-sensitivity of BP. This will
likely require enough homozygous CCs for genotype-based analysis, as suggested by
population studies in which odds ratios for hypertension are higher in CC homozygous subjects
than in C allele carriers.
12
Characterization of Blood Pressure and Protein Excretion in Chromosome
18 Congenic Strains of Dahl S Rats
Carol P Moreno Quinn, Roberta Rogge, Mary L Kaldunski, Bernardo Lopez, Jozef Lazar,
Allen W Cowley, Jr, Howard J Jacob, Howard J Jacob; Med College of Wisconsin,
Milwaukee, WI
The aim of the present study was to identify the region within Chr 18 involved in the
development of salt-induced hypertension in the Dahl salt-sensitive (SS) rat. Previous linkage
and chromosomal substitution studies suggest the presence of a locus for salt-sensitive
hypertension in chromosome 18. Consomic SS-18BN rats were backcrossed to the SS rat, and
the offspring intercrossed to generate a series of overlapping congenic strains covering the
entire length of chromosome 18. Blood pressure and protein excretion was measured by
telemetry in male rats during low salt and three weeks of high salt diet (8% NaCl) in parental
(SS and SS-18BN) and congenic strains. Mean arterial pressure (MAP) did not differ significantly
between any of the strains studied during low salt diet (ranging from 110 to 115 mmHg). After
three weeks of high salt, SS rats developed hypertension (MAP 16710 mmHg), while in the
SS-18BN consomics the development of hypertension was significantly attenuated (MAP 1405
mmHg). Protein excretion was also significantly higher in the SS than in the SS-18BN consomic
rats. Initial characterization of the overlapping congenic strains suggests that there is a 17 Mbp
region in chromosome 18 that provides significant protection from the effects of high salt diet
in the SS rats. We also phenotyped consomic BN-18SS rats, in which choromosome 18 from the
hypertensive SS rat was introgressed onto the genetic background of the normotensive Brown
Norway (BN) rat, and found that this reverse consomic strain had no increased blood pressure
sensitivity to a high salt diet. These results suggest that there is a region in chromosome 18
that confers protection to the development of salt-sensitive hypertension but is not enough by
itself to increase susceptibility to hypertension, probably needing the additional contribution of
other genes.
13
Sexual Dimorphism in the Regulation of NOS 3 and Oxidative Stress in the
Renal Cortex of Spontaneously Hypertensive Rats (SHR)
Jennifer C Sullivan, David M Pollock, Jennifer S Pollock; Med College of Georgia, Augusta,
GA
Female SHR have a slower progression of renal injury compared to male SHR, although the
mechanisms protecting females are unknown. NO is an important regulator of kidney function,
and NO levels have been reported to be greater in females. The aim of this study was to test
the hypothesis that (1) NOS3 is differentially regulated in the renal cortex of male and female
SHR, and (2) oxidative stress is less in female SHR such that there is greater NO bioavailability
in females. Experiments used 14 16 week-old male and female SHR (n6 7). Microalbumin
(ualb) excretion and macrophage-specific ED-1 staining in the renal cortex were used to assess
injury. Blood pressure was measured by telemetry. At 14 weeks of age, blood pressures were
comparable in male and female SHR (1293 and 1311 mmHg respectively). Male SHR had
greater ualb excretion (4.40.3 mg/day/gram) compared to females (1.00.2 mg/day/gram,
p0.05). In addition, male SHR had increased ED-1 staining (ED-1 cells/glomerulus: male,
2.00.3, female, 1.30.3, n3) verifying a gender difference in renal injury. Total NOS 3
protein expression was comparable in the renal cortex from male and female SHR (male:
1.40.11 RDU, female: 1.50.12 RDU). Phosphorylation of serine 1177, serine 633 and
dephosphorylation of threonine 495 have been associated with increased NO production in
endothelial cells. Renal cortical expression of phosphoSer1177 NOS3 in female SHR was
increased compared to male SHR (male: 0.050.02 RDU, female: 0.110.04 RDU, p0.04).
However, phosphoSer633 and phosphoThr495 were comparable from male and female SHR
(phosphoSer633NOS3: male: 0.210.01 RDU, female: 0.220.01 RDU; phosphoThr495NOS3:
male: 0.160.01 RDU, female: 0.160.01 RDU). Basal superoxide production (lucigenin
chemiluminescence in renal cortical homogenates) was higher in male SHR (male: 3.30.7
cpm/g; female: 1.60.2, p0.04). Glomerular nitrotyrosine staining was also greater in male
SHR compared to female SHR. Therefore, these data indicate that female SHR have a greater
capacity to maintain NO bioavailability in the renal cortex than male SHR through enhanced
phosphorylation of NOS3 as well as maintaining reduced levels of superoxide; this effect
appears to be unrelated to blood pressure.
14
17b-Estradiol (e2)-Mediated Protection from Renal Injury is Associated
with Angiotensin Converting Enzyme 2 (ace2) Up Regulation
Hong Ji, Georgetown Univ Schl Of Med, Washington, DC; Carlo Pesce, Stefano Menini, Univ
of Genova, Genova, Italy; Xie Wu, Wei Zheng, Kathryn Sandberg; Georgetown Univ Schl Of
Med, Washington, DC
The newly discovered member of the renin angiotensin system, angiotensin converting enzyme
2 (ACE2), exhibits cardioprotective effects through its conversion of angiotensin II (Ang II) to
Ang-(17). Objective: To investigate whether ACE2 contributes to the clinical observation that
chronic renal disease from several etiologies progresses at a slower rate in women compared
to men, we determined if E2 regulates the activity of renal ACE2 in the renal wrap model of
hypertension (RW). Methods: Sprague Dawley rats (n6 8/group) were divided into 2
sham-operated control groups: male (Sham-M) and female (Sham-F) and 4 RW groups: RW-M,
RW-F, RW-OVX and RW-OVX-E2 (0.24 mg E2/60 day pellet). After 6 wk on a high sodium (4%)
diet, the RW kidney was removed for determination of renal pathology by morphometry. In
addition, renal cortical expression of ACE2 mRNA and protein were determined by real-time
PCR, expressed as fold increase over RW-F (Fold), and Western blot, expressed in arbitrary
units normalized to -actin (AU); significant differences were defined as *p0.05. Results: In
the RW kidney, ovariectomy reduced the expression of ACE2 mRNA [Fold: RW-OVX, 0.26
0.03* vs RW-F, 1.00 0.17 or RW-OVXE2, 1.07 0.3] and protein [AU: RW-OVX, 35.9
4.2* vs RW-OVXE2, 82.8 17], while E2 replacement prevented these effects. Compared to
ovariectomized females (RW-OVX) and male (RW-M) rats, estrogen replete females (RW-F &
RW-OVXE2) also exhibited less renal injury including tubulointerstitial fibrosis, glomeruloscle-
rosis, and mean glomerular volume (MGV) [MGV (m3 x 106): RW-M, 2.25 0.16* vs RW-F,
1.25 0.06; RW-OVX, 2.02 0.11* vs RW-OVXE2, 1.33 0.03 or RW-F]. Conclusions:
These results demonstrate that E2-mediated protection from renal damage induced by RW
hypertension is associated with E2-mediated up regulation of renal ACE2, suggesting that
females are protected from progressive renal disease by increased metabolism of Ang II to
Ang-(17).
15
Ovariectomy is Protective Against Salt-Induced Renal Injury in the Older
Female mRen2.Lewis Strain
Liliya M Yamaleyeva, Brian M Westwood, Leanne Groban, Mark C Chappell; Wake Forest
Univ Health Sciences, Winston-Salem, NC
Estrogen depletion (ovariectomy) at an early age increases blood pressure and exacerbates
salt-dependent hypertension and renal injury in the congenic mRen2.Lewis strain. The loss of
estrogen in mRen2.Lewis rats is associated with impaired endothelial nitric oxide synthase
expression in the kidney, as well as enhanced expression of Ang II, aldosterone and ACE, which
is reversed by estrogen replacement. Although these and other studies clearly support the
protective effects of estrogen, recent clinical trials in older females do not substantiate this role.
Therefore, the current study assessed the outcome of estrogen depletion in older mRen2.Lewis
rats subjected to a high salt diet. Heterozygous intact or ovariectomized (OVX, 15 weeks of age)
mRen2.Lewis were aged to 60 weeks and then placed on a high salt (HS, 4% sodium) diet for
4 weeks. The systolic blood pressures were similar between groups [intact: 182 7 versus
OVX: 169 6 mmHg, p0.22] following the 4 week diet; however, the HS diet significantly
increased proteinuria in the intact versus OVX rats [intact: 31.3 7; OVX: 1.8 0.3 mg/mg
creatinine/kg body weight, p0.01, n6]. Creatinine clearance (GFRc) tended to decrease in
intact rats maintained on the HS diet relative to the high salt OVX mRen2.Lewis rats. Both the

mean kidney-to-body weight (MK/BW) and left ventricle to BW (LV/BW) ratios were significantly
greater in the intact HS group [intact MK/BW: 3.7 0.2 versus OVX: 2.5 0.04 mg/gm,
p0.01, n6 and intact LV/BW: 2.8 0.1 versus OVX: 2.1 0.1 mg/gm, p0.01, n6]. In
summary, ovariectomy of adult mRen2.Lewis rats prevents proteinuria, renal and cardiac
hypertrophy as well as maintains GFRc during the course of a HS diet. We conclude that in older
female hypertensive mRen2.Lewis rats with increased sodium intake, ovarian hormones may
have deleterious actions within the kidney.
16
Gender Differences in Cortical and Medullary Function in Adults Rats with
a Reduction in Ang II during Nephrogenic Period
Analia S Loria, Virginia Reverte, Francisco Salazar, Fara Saez, M T Llinas, F. J Salazar; Dept
of Physiology, Sch of Medicine, Univ of Murcia, Murcia, Spain
A recent study of our group has demonstrated that there are important gender differences in
renal structural response to the reduction of angiotensin II (Ang II) during the nephrogenic
period. It was found that renal cortex was more affected in males than in females treated with
an AT1 Ang II receptor antagonist (ARAII) during the first two weeks of life and that only treated
males had an important papillary atrophy. This study was designed to test the hypothesis that
there are gender differences in the cortical and medullary function in adult rats in which Ang
II effects were reduced during the nephrogenic period. Newborn SD rats were treated with
vehicle or an ARAII (L-158.809, 7 mg/kg/day) during the first 14 postnatal days. Systolic arterial
pressure, renal clearances, proteinuria and the urinary concentrating ability in response to
dehydration was examined in rats at 12 months of age (n8 in each group). Arterial pressure
was greater (P0.05) in treated males (134 0.9 mmHg) and treated females (131 0.5
mmHg) than in control rats (cr) (115 0.3 mmHg in both sexes). Urinary protein excretion
(mg/ml/24hrs) increased (P0.05) in treated males and females, but the elevation was greater
(P0.05) in treated males (264 24 vs. 101 10 in cr) than in treated females (95 19
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CHBPR ConferenceOral Presentations e29
(MA), 15 month old middle-aged rats that were ovariectomized at 4 months of age (ovx), and
15 month old middle-aged rats that were ovariectomized at 4 months of age and supplemented
with estrogen (E; n6 each group). LV levels of extracellular matrix (ECM) proteins, matrix
metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) were evaluated
by immunoblotting, with densitometry values normalized to actin. Results. LV mass increased
from 6976 mg in Y to 125731 mg in MA and 119925 mg in ovx (p0.05 Y vs MA and
ovx). LV mass in the E group was not significantly different from Y. Fibronectin (Fn) levels
increased from 1.390.03 units in Y to 1.670.04 units in MA (p0.05). Collagen III and
collagen IV levels were also elevated in MA, indicating increased fibrosis. Multiple MMPs/TIMPs
increased in MA, including MMP-3, MMP-7, MMP-9, MMP-13, MMP-14, and each TIMP (-1, -2,
-3, and -4). Ovx showed similar increases in fibrosis and MMP/TIMP profiles. The increase in
collagen levels was attenuated in the E group. MMP-12 increased from 1.190.03 units in MA
to 1.360.1 units in ovx (p0.05) and the 150 kD Fn fragment increased from 1.200.02
units in MA to 1.270.03 units in ovx (p0.05). Compared to MA levels, MMP-2, MMP-8, and
TIMP-4 increased in E, as did the 75 kD fragment of collagen III. Compared to ovx, MMP-8
increased 18025% and the collagen III fragment increased 1135% in the E group.
Conclusion. Hypertension superimposed on aging stimulated ECM turnover through increased
MMP/TIMP production and ECM degradation. Estrogen replacement attenuated the hypertro-
phic response and increased collagenolysis, which may serve as one mechanism for the
protective effects of estrogen.
19
MicroRNAs in the Kidney: Regional Distribution and Physiological
Implications

vs. 40 3 in cr). As compared to the values found in the control group, basal urine osmolality
was lower in male than in female treated rats. In response to a prolonged dehydration period,
the increment in urinary osmolality was significantly greater (P0.05) in female (706 92
mOsm/kg) than in male treated rats (386 54 mOsm/kg). Changes in tubular solute free water
reabsorption (TcH2O) (ml/24 hr/gr kidney w.) in response to dehydration were not affected in
female treated rats with respect to female control rats. However, the decrease of TcH2O in
response to dehydration was lower (P0.05) in male treated rats (8.0 0.8 to 3.9 0.5) than
in male control rats (12.8 1.8 to 7.0 0.9). In summary, the results of this study present
new evidences showing that the reduction of Ang II during the nephrogenic period elicits the
development of hypertension in males and females. This reduction alters more significantly
renal cortical and medullary function in adult males than in adult females.
17
Phytoestrogens Inhibit Human Aortic Smooth Muscle Cell Growth and
ERK1/2 Expression via PPAR-, but not Estrogen, Receptors
Raghvendra K Dubey, Univ Hosp Zurich, Zurich, Switzerland; Edwin K Jackson, Delbert G
Gillespie, Univ of Pittsburgh Med Cntr, Pittsburgh, PA; Bruno Imthurn, Marinella Rosselli;
Univ Hosp Zurich, Zurich, Switzerland
Plant-dervied estrogens (phytoestrogens), such as genistein (abundant in soybeans), may
induce cardioprotective effects by inhibiting smooth muscle cell (SMC) growth and neointima
formation. The prevailing view is that the biological effects of phytoestrogens are mediated by
estrogen receptors (ERs). Our recent findings that the effects of estradiol are ER independent
and mediated via its metabolite (methoxyestradiol) suggest that the actions of phytoestrogens
may also be due to alternative mechanisms. Because PPAR- is a nuclear receptor that can
interact with steroids, we investigated the role of PPAR- in mediating the antimitogenic
actions of phytoestrogens in human aortic SMCs. In cultured human aortic SMCs, treatment
with 0.1100M genistein or estradiol inhibited fetal calf serum (2.5%)-induced DNA synthesis
(3H-thymidine incorporation), cell proliferation (change in cell number), collagen synthesis
(3H-proline incorporation), cell migration (modified Boydens chamber) and ERK 1/2 expession.
All of the inhibitory effects of genistein, but not estradiol, were completely blocked by GW9662
(2M, PPAR- antagonist). For example, genistein (50M) inhibited cell proliferation by
724%, and GW9662 reduced this to 71%. Treatment of SMCs with rosiglitazone (PPAR-
agonist) inhibited SMC growth, and this effect also was blocked by GW9662. Importantly,
genistein was more potent than rosiglitazone with regard to inhibiting SMC growth (50M
inhibited SMC growth by 72% and 30%, respectively). The ER-antagonist ICI182780 (10M)
did not attenuate the inhibitor effects of either estradiol or genistein. Moreover, inhibitors of
CYP450 and COMT (inhibit metabolism of estradiol to methoxyestradiol) blocked the antimi-
togenic actions of estradiol, but not genistein. Similar to genistein, the inhibitory effects of other
phytoestrogens equol and daidzein on SMC growth were abrogated by GW9662. In conclusion,
our findings suggest that some phytoestrogens exert inhibitory effects on SMCs via activation
of PPAR-. Regardless of whether this is a direct or indirect effect, activation of PPAR- by
phytoestrogens may confer cardiovascular protection.
18
Estrogen Effects on Left Ventricular Hypertrophy and Matrix
Metalloproteinase Profiles in Dahl Salt-Induced Hypertension
Qiuxia Dai, Teresa Craig, Carmen Hinojosa-Laborde, Merry L Lindsey; Univ of Texas Health
Science Cntr, San Antonio, TX
Background. Female Dahl salt-sensitive (DS) rats fed a low salt diet develop hypertension with
age. DS rats ovariectomized at 4 months of age show accelerated hypertension, which is
attenuated with estrogen replacement. While acute pressure overload is known to induce
structural and functional changes in the left ventricle (LV), the effects of chronic hypertension
on LV size and structure have not been examined in the DS rat model. Methods. Four groups
of DS rats were examined: young intact control rats (Y), 15 month old middle-aged intact rats
Mingyu Liang; Med College of Wisconsin, Milwaukee, WI
MicroRNAs are a class of small regulatory RNA, the discovery of which has been hailed as one
of the most significant breakthroughs in biology in recent years. Mature microRNAs are
approximately 20 nucleotides long, and can bind to the 3 untranslated region of their target
mRNAs and suppress protein translation. A mammalian genome has at least a few hundred
genes that specifically encode microRNAs. The known mammalian microRNAs, which are
highly conserved among mammalian species, have been predicted to regulate the protein
expression of thousands of genes since microRNAs only need to match their targets partially.
The relevance of specific microRNAs to complex mammalian physiology, however, is largely
unknown. To begin analyzing the physiological role of microRNAs in mammalian species, we
examined the expression profile of microRNAs in the renal cortex and the renal medulla of male
Sprague-Dawley rats (n6). We constructed a microRNA microarray containing probes for
nearly 400 microRNAs that have been identified in human, mouse, or rat. MicroRNAs were
isolated from the renal cortex and the renal medulla using size-based fractionation. A 3
extension method was used to label microRNAs, which were then hybridized to microRNA
microarrays. Approximately 100 microRNAs were detectable in each region of the kidney.
Statistical analysis identified 11 and 9 microRNAs as preferentially expressed in the renal cortex
and the renal medulla, respectively, four of which were further validated using a modified
real-time PCR method. Computationally predicted target genes for the differentially expressed
microRNAs include transporters, transcriptional regulators, and cell cycle regulators, several of
which appeared to be consistent with known physiological characteristics of the renal cortex
and the renal medulla. The present study was the first to have systematically analyzed
microRNAs in mammalian kidneys. The results suggest that microRNAs might play an important
role in controlling renal function through regulating the expression of their target genes.
20
Collecting Duct-Specific Knockout of PPAR Impairs Sodium Retaining
Ability and Reduces Blood Pressure during Sodium Depletion
Tianxin Yang, Aihua Zhang, Zhanjun Jia, Hui Zhang; Univ of Utah, Salt Lake City, UT
We have recently created mice with collecting duct (CD)-specific disruption of PPAR (termed
CD PPAR KO) by crossing PPARf/f mice with AQP2-Cre mice and the KO mice were resistant
to thiazolidinedione-induced fluid retention, revealing a novel role of PPAR in regulation of
distal tubular fluid reabsorption (Zhang et al. PNAS 104:9406 11, 2005). The present study
examined a potential physiological role of CD PPAR in the setting salt depletion. We used
telemetry to monitor daily mean arterial blood pressure (MAP). During one-week low salt (LS)
diet PPARf/f mice were able to maintain constant MAP while CD PPAR KO mice exhibited a
significant reduction of MAP (105.0 3.1 vs. 94.6 2.4 mmHg, on day 7, n7, p0.05).
After switching from low to normal salt diet, MAP in CD PPAR KO mice returned to normal.
Sodium balance studies showed that CD PPAR KO mice had impaired ability to effectively
reduce urinary sodium excretion during LS intake. This was associated with exaggerated
plasma renin and plasma aldosterone responses to LS. In response to LS, PPARf/f mice
exhibited a 2.5-fold increase in the abundance of the subunit of the epithelial sodium channel
(-ENaC) in the kidney as assessed by immunoblotting and a nearly complete trafficking of
-ENaC to the apical membrane of the CD by immunohistochemistry, that were both abolished
in CD PPAR KO mice. LS treatment induced more than 10 fold increases in urinary excretion
of the endogenous ligand of PPAR 15d-PGJ2 in both mouse strains. In primary cultures of wild
type CD cells, 1 M rosiglitazone treatment for 24 hours induced a 2-fold increase in Sgk1
mRNA as assessed by real time RT-PCR that was significantly suppressed in the PPAR KO
cells. In summary, this study has characterized 15d-PGJ2/PPAR/Sgk1/-ENaC pathway in the
distal nephron that appears to be important for maintenance of sodium balance and blood
pressure during sodium depletion.
 e30 Hypertension Vol 48, No 4 October 2006

21
Enhanced Renal Interstitial Fluid ATP and the Tubuloglomerular Feedback
Mechanism in Dahl Salt-Sensitive Hypertensive Rats
Akira Nishiyama, Matlubur Rahman, Youichi Abe, Masakazu Kohno, Kagawa Univ Med Sch,
Kagawa, Japan; L G Navar; Tulane Univ Health Sciences Cntr, New Orleans, LA
Participation of renal interstitial fluid (RIF) ATP as a mediator of the tubuloglomerular feedback
(TGF) mechanism has been indicated in recent studies. Here, we investigated the possible
contributions of RIF ATP and TGF mechanism to the development of salt-dependent
hypertension. Dahl salt-sensitive (DS) rats were maintained on a low (L: 0.3% NaCl) or high salt
(H: 8% NaCl) diet for 4 weeks. Using an intravital tapered-tip lens-probe videomicroscopy
system, the superficial afferent arteriolar diameter (AAD) was measured before and during
enhanced TGF activity with acetazolamide (2 mg/kg, bolus 4 mg/kg/h, infusion, i.v.) in
anesthetized rats. RIF ATP levels were measured by a microdialysis method. P0.05 was
considered statistically significant. Systolic blood pressure in DS/L (n28) and DS/H (n36)
rats was 1342 and 2178 mmHg, respectively. Compared to DS/L rats (9.90.8 m), DS/H
rats showed smaller basal AAD (7.70.9 m). In DS/L rats, acetazolamide significantly
decreased AAD (8.70.7 m) without changing the efferent arteriolar diameter. Inhibition of
TGF activity with furosemide (1 mg/kg, bolus 4 mg/kg/h, infusion, i.v.) reversed the
acetazolamide-induced afferent arteriolar vasoconstriction (9.60.8 m). In DS/H rats,
acetazolamide did not alter basal AAD (7.6 0.9 m), whereas the addition of furosemide
significantly increased AAD (8.51.0 m). Basal RIF ATP levels were much higher in DS/H rats
(4.30.5 nmol/L) than DS/L rats (1.20.4 nmol/L). Acetazolamide increased RIF ATP levels in
DS/L rats (2.20.7 nmol/L), whereas ATP levels were not altered by acetazolamide in DS/H
rats (3.80.8 nmol/L). Treatment with suramin (20 mg/kg/day, i.p.), a non-selective P2
receptor antagonist, markedly attenuated the development of hypertension in DS/H rats
(1383 mmHg, n25). Suramin also significantly increased basal AAD (10.60.9 m) in
DS/H rats. However, AAD did not change during the administration of acetazolamide and
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and feature the metabolic syndrome. We studied these transgenic and non-transgenic control
rats (SD) under standard diet for 22 weeks. Body weight (BW) gain was similar in both groups
until the age of 7 weeks, although energy expenditure (EE) and lipid oxidation (LOX) were
greater in TGR(hREN) vs. SD. Thereafter, BW gain was significantly greater in TGR(hREN) vs.
SD. At age 22 weeks, BW was 5709 g in TGR(hREN) and 49720 g in SD. Food intake was
significantly higher in TGR(hREN) than SD, but EE and LOX were similar in both groups.
MRI-determined body composition analysis showed a significantly higher body fat mass in the
TGR(hREN) (886 g) vs. SD (553 g) rats. Additionally, TGR(hREN) had increased serum
triglyceride levels (1088 vs. 665 mg/dl) and impaired glucose tolerance tests, compared
with SD. Telemetric mean arterial blood pressure was not different in TGR(hREN) and SD
(1042 and 1032 mm Hg). Chronic ACE inhibitor treatment did not influence BW,
triglycerides, or glucose tolerance impairment. When pair-fed, TGR(hREN) rats were restricted
to the caloric intake of SD rats, the developed a lower BW than SD. In vitro, we found that
neither renin nor renin-inhibitor treatment had any influence on adipogenesis of human
preadipocytes. We conclude that renin initiates a process leading to increased appetite, obesity
and metabolic alterations independent of blood pressure. Human renin does not seem to have
a direct effect on adipogenesis.
24
Impaired Renal Vasodilation in SHR Fed a High Fat Diet Precedes Changes
in Insulin Sensitivity
Sarah Knight, Ahmed El-Marakby, John D Imig; Med College of Georgia, Augusta, GA

furosemide in suramin-treated DS/H rats. These results suggest that augmented TGF activity
associated with increases in RIF ATP levels contributes to the increased afferent arteriolar tone
and the development of salt-dependent hypertension.
22
Localization of the Renin Receptor in Renal Tubules and its Upregulated in
Diabetes
Chun Xue, Jiqian Huang, John Gildea, Univ of Virginia, Charlottesville, VA; Genevieve
Nyugen, INSERM Tenon Hopital, Paris, France; Helmy M Siragy; Univ of Virginia,
Charlottesville, VA
Recent studies demonstrated presence of the renin receptor (RR) in the glomerular mesangium
and the subendothelial layer of the renal arteries. In this study we hypothesized that in addition
to the presence of RR in glomerular mesangium, it is also present in renal tubules and is
upregulated in diabetes via angiotensin AT1 receptor (AT1R) and oxidative stress. Using real
time PCR, western blot analysis and immunostaining, we studied renal RR expression at 6
weeks after induction of diabetes in rats injected with streptozotocin (65 mg/kg ip) and in
response to 1 week treatment with the AT1R blocker valsartan (10mg/kg/d) or the NADPH
oxidase inhibitor DPI (0.5mg/kg/d). Both RR mRNA and protein expressions were present in
renal glomeruli and tubules in normal rats. Diabetes significantly upregulated RR expression in
the kidney (Figure A& B). Valsartan or DPI treatments reduced the RR expression in diabetic rats
(Figure A& B). To confirm the presence of the RR expression in renal glomerular mesangium
and tubules, we used Western blot analysis to detect RR protein in isolated renal mesangial and
proximal tubular cells (Figure C). Similarly, immunostaining localized the RR in glomeruli and
tubules. These results demonstrate that RR is present in renal glomeruli and tubules and
upregulated in diabetes. AT1R and oxidative stress upregulate RR expression.
Obesity, hypertension and insulin resistance have been identified as cardiovascular risk factors
which are believed to contribute to the progression of end-stage renal disease. To examine the
mechanism by which a HF diet and hypertension contribute to endothelial dysfunction, 8 week
old male spontaneously hypertensive rats (SHR) were fed either a high fat (HF, 36% fat) or a
normal fat (NF, 7% fat) diet for 6 weeks (n6). Systolic blood pressure increased in a similar
manner in NF (from 163 4 mmHg to 219 5 mmHg) and HF diet fed rats (from 166 3
mmHg to 217 4 mmHg). In the NF group body weight increased from 218 7 g to 288
6 g, whereas the HF groups displayed a 15% greater increase, from 217 5 g to 319 9 g
after 6 weeks. Non-fasting blood glucose levels were 13% higher in the HF group than the NF
group at 109 3 mg/dL and 95 2 mg/dL respectively (P0.05). Using the euglycemic
clamp method, glucose infusion rates of 40 2 mg/kg/min in the NF group and 39 3
mg/kg/min in the HF group were required to maintain blood glucose levels of 125 mg/dL,
indicating comparable insulin sensitivity (n3 4). We measured renal afferent arteriole
vascular reactivity to acetylcholine (ACh) (n6). Mean baseline diameter averaged 23 2 m
and 26 5 m in the NF and HF groups respectively. The relaxation of pre-constricted
arterioles in response to doses of 1.0x10-8 to 1.0x10-5 ACh was reduced in the HF compared
with the NF group. Maximum relaxation reached 84% of baseline diameter in the NF but only
57% of baseline in the HF group (P0.05). In order to elucidate a possible mechanism for the
reduced renal vascular relaxation to ACh we measured the cortical expression of CYP2C23, an
enzyme responsible for the production of vasodilatory EETs, by western blot in rats fed a HF
or NF diet. We found that cortical CYP2C23 protein expression was reduced in the HF group
with levels of 20 4 densitometric units (DU) compared to 29 1 DU in the NF group (n4).
Our results indicate that a 6 week HF diet with hypertension impairs the renal vascular
response to ACh, in the absence of insulin resistance or a change in blood pressure and the
decrease in CYP2C23 metabolites could contribute to the impaired renal vasorelaxation.

25
Glucose Reduces Renal Medullary Circulation by Induction of Oxidative
Stress in Renal Medulla

Chun-hua Jin, Takefumi Mori, Susumu Ogawa, Satoshi Endo, Yoshimi Yoneki, Kazuhiro
Nako, Sadayoshi Ito; Div of Nephrology, Endocrinology and Vascular Medicine, Tohoku Univ
Graduate Sch of Medicine, Sendai, Japan

23
Obesity in Human Renin Transgenic Rats - A Novel Role for Renin?
Petra Gratze, Ralf Dechend, Michael Boschmann, Jeanette Malchow, Stefan Engeli, Jurgen
Janke, HELIOS, Franz-Volhard Clinic, Charite, Berlin, Germany; Jochen Springer, Dept of
Cardiology, Charite, Berlin, Germany; Ralph Plehm, Max-Delbruck Cntr, Berlin, Germany;
Susanne Klaus, German Institute of Human Nutrition, Potsdam, Germany; Friedrich C Luft,
HELIOS, Franz-Volhard Clinic, Charite, Berlin, Germany; Dominik N Muller; Max-Delbruck
Cntr, Berlin, Germany
Renin is known to function as a protease initiating angiotensin (Ang) II formation. However, we
found that transgenic rats over-expressing the human renin gene (TGR(hREN)) develop obesity
Although high blood pressure is often observed in diabetes and metabolic syndrome, little is
known whether renal medullary circulation is involved. Previous studies have shown that
glucose increase superoxide production in medullary thick ascending limb. Studies were
designed to determine the role of glucose and insulin in the regulation of renal medullary
circulation. 50% glucose solution was infused from femoral vein at a rate of 0.5ml/h in
anesthetized male Sprague-Dawley rats and blood pressure was monitored with the catheter
implanted from the femoral artery. Local renal blood flow was measured with implanted optical
fiber attached to the laser-Doppler flowmetry. Drugs were infused from catheter implanted to
the border of inner and outer medulla to determine the local renal medullary effect. Intravenous
infusion of glucose resulted in 215 mg/dl increase of blood glucose and 124% (n7)
reduction of medullary blood flow (MBF) without significant reduction on mean arterial
pressure. Interstitial infusion of 50% glucose solution at a rate of 0.5 ml/h also reduced MBF
by 165% (n6) without reduction of blood glucose. However, the level of MBF was remained
low when 20mU/kg/min. of insulin were additionally infused to medullary interstitial space even
with 358 mg/dl reduction of blood glucose. Renal medullary infusion of superoxide scavenger
Tiron (90g/kg/min., n7) and sodium-glucose transporter (SGLT) inhibitor phloridzin
(6.9g/kg/min., n5) abolished the reduction of MBF by intravenous infusion of glucose. We
conclude that glucose but not insulin reduces medullary blood flow with induction of oxidative
stress, and SGLT is involved in the glucose transport for reduction of MBF. We conclude that
glucose induced reduction of MBF could involve in the mechanism of high blood pressure in
diabetes and metabolic syndrome.

26
Dynamic Renal Autoregulation: Abnormal in Diabetes
Gerald DiBona, Univ. Ia. College of Medicine, Iowa City, IA; Tracy D Bell, Michael W Brands;
Med College of Georgia, Augusta, GA
Within 24 hours of induction of diabetes mellitus (DM, stretptozotocin) in conscious rats, arterial
pressure (AP) and renal blood flow (RBF) are increased, achieving levels of 10% and 22%
respectively above control at DM day 7 (submitted). In addition, dynamic RBF autoregulation (AP
to RBF transfer function) was impaired on DM day 1 with positive gain values at both
tubuloglomerular feedback (TGF, 0.02 0.05 Hz) and myogenic (myo, 0.1 0.3 Hz) frequencies.
To identify whether there was a change in the RBF frequencies and amplitudes used by TGF
and myo during evolution from Control to DM, the dominant frequency in both the TGF (Ftgf)
and myo (Fmyo) ranges was identified and the amplitude at that dominant frequency (Atgf, Atgf)
was extracted over 1000 seconds during both Control and DM periods using a time-frequency
representation approach (Wang et al, Ann Biomed Eng, 2006). *P0.01 DM vs Control,
P0.01 Atgf vs Amyo. The dominant frequencies chosen for the TGF (Ftgf) and myo (Fmyo)
components of dynamic RBF autoregulation were not different between Control and DM.
However, the RBF amplitude at both the dominant frequencies for TGF (Atgf) and myo (Amyo)
was significantly increased in DM compared to Control. Atgf was greater than Amyo during both
Control and DM. As increased RBF amplitude at the TGF and myo frequencies indicates less
attenuation by the renal vasculature of the corresponding AP amplitude at the same
frequencies, this is consistent with impairment of both the TGF and myo mechanisms for
dynamic RBF autoregulation in DM. That these changes are apparent on DM day 1, prior to any
renal structural damage, suggests that this is a functional alteration related to the altered
metabolic state.
Control DM
Ftgf, Hz 0.0330.001 0.0340.001
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
Fmyo, Hz 0.1740.001 0.1680.001
Atgf, amplitude 0.1010.006 0.3960.007*
Amyo, amplitude 0.0320.001 0.0900.001*
27
The Splanchnic Circulation as a Critical Target for Chronic Low Dose
Angiotensin II Hypertension
Andrew J King, Melissa W Li, Gregory D Fink; Michigan State Univ, East Lansing, MI
We have shown that chronic low dose angiotensin II (AngII), only when administered in
combination with a high salt diet, engages the sympathetic nervous system (SNS) to increase
venomotor tone. We propose this contributes to AngII hypertension by increasing the driving
force for venous return to the heart, resulting in a translocation of blood from the highly
compliant venous system to the less compliant arterial circulation. In particular the splanchnic
venous bed, which is richly innervated by the SNS mostly via the celiac ganglion, and is the
most important active capacitance bed in the body, may be an important target for AngII
mediated sympathoactivation. Therefore we hypothesized that celiac ganglionectomy (CGx) will
attenuate the hypertensive response to AngII only in rats fed a high salt diet. To test this, rats
were acclimatized to a 2% or 0.4% NaCl diet for 7 days and then instrumented with a
radiotelemetry blood pressure transmitter at the same time as surgical CGx or sham operation
(SHAM). Following 10 days of recovery and 4 days of control, an AngII or saline filled minipump
was implanted subcutaneously to deliver AngII (150ng/kg/min) or vehicle for 14 days. During
control CGx reduced MAP in rats on 2% (SHAM 102 1.4, CGx 95 1.7 mmHg, p0.05) and
0.4% NaCl (SHAM 100 1.3, CGx 93 2.3 mmHg, p0.05). CGx markedly attenuated the
hypertensive response to AngII in rats on 2% NaCl (SHAM 145 6.3, CGx 114 7.9 mmHg,
p0.05), but had little effect on rats fed 0.4% NaCl (SHAM 114 4.2, CGx 104 4.5 mmHg).
CGx was confirmed by measuring norepinephrine levels in splanchnic organs by HPLC. To
investigate the role of the renal nerves and the possibility that CGx was exerting its effects via
renal denervation (RDx), a separate group of rats fed 2 % NaCl were subjected to the same
protocol but received selective bilateral RDx. RDx decreased MAP (SHAM 103 1.7, RDx 97
1.9 mmHg, p0.05) during control but had no protective effect (SHAM 128 9.9, RDx 127
4.9 mmHg) on the hypertensive response to AngII and appeared to exacerbate the pressor
response over the first 5 days. We conclude that CGx attenuates the hypertensive response to
chronic low dose AngII, only in rats fed a high salt diet, and this suggests the splanchnic
circulation is a critical target for AngII induced sympathoactivation.
28
Probing Functional Differences in Sympathetic Input to Mesenteric Arteries
and Veins in Normotensive and DOCA-Salt Hypertensive Rats Using in vitro
Continuous Amperometry and Video Imaging
Greg M Swain, Jinwoo Park, James J Galligan, Gregory D Fink; Michigan State Univ, East
Lansing, MI
Arteries and veins make different contributions to overall hemodynamics and there are likely
to be differences in sympathetic transmission to arterial and venous smooth muscle cells in
normal and hypertensive subjects. Therefore, we used amperometric methods with diamond or
carbon fiber microelectrodes and video imaging to study neurogenic constrictions and the
kinetics of norepinephrine (NE) release and clearance in perivascular sympathetic neuroeffector
junctions in mesenteric arteries (MA) and veins (MV) in vitro. We used tissues from control and
DOCA-salt rats. Electrically-evoked constrictions and NE release in MA and MV were blocked
by tetrodotoxin (TTX, 0.3 M) indicating that these responses were neurogenic. NE release and
contractile responses were frequency dependent (130 Hz) with maximal responses at 20 Hz.
The frequency-response curves for constriction and NE release in MV were shifted to the left
of those for MA. The half maximum stimulation frequency was 1.9 0.2 Hz in veins and this
value was lower (P 0.05) than in MA (4.8 0.3 Hz). Peak responses were similar in MA and
MV. The 2-adrenergic receptor antagonist, yohimbine (1.0 M), increased NE release and
CHBPR ConferenceOral Presentations e31
constriction in MA but not MV; the effects of yohimbine were impaired in tissues from
DOCA-salt rats. Cocaine (10 M) which blocks NE uptake increased the amplitude and duration
of NE oxidation currents and constrictions in MA but not MV; the effect of cocaine was larger
in MA from DOCA-salt rats. The latency to onset and duration of constriction in MA were shorter
than MV but the rate of constriction and rise time of NE release were faster in MV from control
rats; there were no differences in the rise time of constriction or NE oxidation currents in MA
and MV from DOCA-salt rats. These data indicate that there are fundamental differences in the
dynamics of sympathetic neurotransmission to MA and MV. NE uptake and prejunctional 2
autoreceptors play a more prominent role in modulating NE disposition in MA compared to MV.
Furthermore, DOCA-salt hypertension is associated with an impairment of autoreceptor
function and upregulation of NE uptake in the neuroeffector junction of MA. These changes
contribute to the hemodynamic disturbances occurring in DOCA-salt hypertension. #
29
Renal Denervation Does not Abolish Sustained Baroreflex-Mediated
Reductions in Arterial Pressure
Thomas E Lohmeier, Drew A Hildebrandt, Terry M Dwyer, Austin M Barrett, Univ. of
Mississippi Med Cntr, Jackson, MS; Eric D Irwin, North Memorial Med Cntr, Robbinsdale,
MN; Martin A Rossing, Robert S Kieval; CVRx, Inc., Maple Grove, MN
We previously demonstrated that prolonged bilateral electrical stimulation of the carotid sinus
activates the baroreflex producing sustained reductions in plasma norepinephrine concentra-
tion (NE), mean arterial pressure (MAP), and heart rate (HR) in normotensive and hypertensive
dogs. Prolonged baroreflex activation (PBA) also decreases renal sympathetic nerve activity and
increases renal excretory function, responses expected to lead to long-term reductions in MAP.
The goal of this study was to determine whether PBA mediated reductions in MAP are
dependent upon renal innervation. Six dogs were chronically instrumented to allow PBA and
continuous recordings (24h/day) of MAP and HR. Sodium intake was maintained at 60
mmol/day. After control measurements, the animals underwent 7 days of PBA. On day 1,
sodium excretion was reduced by 20 mmol while MAP (control 953 mmHg) and HR
(control653 bpm) decreased to 813 mmHg and 552 bpm, respectively. On subsequent
days of PBA, sodium balance was achieved despite a sustained fall in MAP and HR (day 7
794 mmHg and 552 bpm, respectively). Plasma NE concentration decreased 50%
during PBA (control 9613 pg/ml). After termination of PBA, there was a natriuresis on day
8, followed by sodium balance as MAP and HR returned to control levels during 7 days of
recovery. Two weeks after bilateral denervation, the above experiment was repeated. MAP, HR,
and NE were 914 mmHg, 704 bpm, and 1008 pg/ml before PBA. During 7 days of PBA,
reductions in MAP, HR, and NE were equivalent to those observed when the renal nerves were
intact. Tissue levels of NE were reduced 100-fold in denervated kidneys (control 98780
ng/g), confirming denervation. Thus, the renal nerves are not essential for achieving long-term
reductions in MAP during PBA. As denervated kidneys may be supersensitive to NE, decreased
NE during PBA may account for increased renal excretory function and attendant reductions in
MAP in the absence of the renal nerves.
30
ASIC2-/- Mice: A Genetic Model of Impaired Baroreceptor Activation and
Oxidative Stress-Dependent Neurogenic Hypertension
Rasna Sabharwal, Harald M Stauss, Eric Lazartigues, Carol A Whiteis, Margaret P Price,
Michael J Welsh, Robin L Davisson, Francois M Abboud, Mark W Chapleau; Univ. of Iowa,
Iowa City, IA
Acid Sensing Ion Channel 2 (ASIC2) is highly expressed in sensory neurons and brain including
medulla. Our previous work suggests a role of ASIC2 in baroreceptor activation. Most recently
we reported that ASIC2-/- mice exhibit decreased baroreflex sensitivity (BRS), sympathoexci-
tation, and mild hypertension (FASEB J. 20(5):2006). Neuronal oxidative stress contributes to
sustained sympathoexcitation in a variety of pathological states. In this study, we tested the
hypothesis that oxidative stress mediates the baroreflex/autonomic dysfunction in ASIC2-/-
mice. Blood pressure (BP) and heart rate (HR) were measured by telemetry in conscious control
(n7) and ASIC2/ (n8) male mice before and after administration of the antioxidant tempol
(drinking water, 1 mM) for one week. Results are shown in the table. BRS calculated from
spontaneous fluctuations in BP and HR using the sequence method was markedly decreased
in ASIC2-/- mice. Cardiac parasympathetic and sympathetic activities as determined from HR
responses to methylatropine and propranolol were abolished and enhanced, respectively in
ASIC2-/- mice. HR and BP (24 hr. avgs.) were significantly elevated. Tempol reversed the
baroreflex/autonomic impairment and normalized HR and BP in ASIC2-/- mice, but had no
effect in control mice (see Table, *ASIC2-/- vs. control, P0.05; Tempol vs. baseline). We
conclude that oxidative stress causes baroreflex and autonomic dysfunction and mild
hypertension in ASIC2-/- mice. The role of baroreceptor sensory vs. central nervous system
defects in mediating the functional changes as well as the mechanism linking loss of ASIC2 and
oxidative stress remain to be determined. (NIH HL14388 and AHA#0525745Z)
Control Control Tempol
24 hr. avg. BP (mmHg) 1093 1083
24 hr. avg. HR (bpm) 54713 53316
BRS (ms/mmHg) 2.400.17 2.270.21
Atropine ( bpm) 15029 9220
Propranolol ( bpm) -8821 -7020
ASIC2-/- ASIC2-/- Tempol
24 hr. avg. BP (mmHg) 1173* 1083
24 hr. avg. HR (bpm) 5788* 5337
BRS (ms/mmHg) 0.940.07* 2.070.52
Atropine ( bpm) 1512* 1117
Propranolol ( bpm) -20233* -6411
 e32 Hypertension Vol 48, No 4 October 2006
31
Alpha-Glucosidase Inhibition, a Novel Treatment for Postprandial
Hypotension in Autonomic Failure
Cyndya Shibao, Alfredo Gamboa, Andre Diedrich, Cynthia Dossett, Ginnie Farley, Italo
Biaggioni; Vanderbilt Univ, Nashville, TN
Postprandial hypotension (PPH), is an important clinical condition associated with syncope,
falls, angina and cerebrovascular events. Those at most risk are elderly and patients with
autonomic dysfunction. Because the enteric glucose availability has been proposed to
contribute to the pathophysiology of postprandial hypotension (PPH), we hypothesized that
acarbose, an alpha-glucosidases inhibitor, that decrease the glucose absorption, and the
postprandial peak of insulin, would improve PPH. The effect of 100 mg of acarbose was studied
in 7 patients with pure autonomic failure (5 females, 2 males, age 657 years, BMI 24.2
4.9 Kg/m2) in a double blind, non-randomized, crossover study. All participants had severe PPH,
defined as a decrease in systolic blood pressure of at least 20 mm Hg within 2 hours of meal
ingestion. Baseline measurements were taken for 30 minutes every 5 minutes on the supine
position. Acarbose (or placebo) was administered 20 minutes before ingestion of a meal
consisting of 423 Kcal. Blood pressure and heart rate were monitored for 90 minutes.
Neurohumoral parameters were measured at baseline, 15, 30, 45, 60 and 90 minutes of meal
ingestion. During placebo, the fall in systolic blood pressure (SBP) was 429 mm Hg as
compared to 206 mm Hg with acarbose, P0.05 (Figure). Acarbose significantly reduced
plasma levels of insulin (193 at 60 min compare to 285 pg/ml with placebo, P0.028). We
conclude that acarbose 100 mg successfully improved postprandial hypotension in patients
with severe autonomic failure.
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32
Antenatal Betamethasone Causes Angiotensin II-Mediated Impairment of
Cardiovascular Function
Hossam A Shaltout, Wake Forest Univ, Sch of Medicine, Hypertension and Vascular Disease
Cntr, Winston Salem, NC; Jorge P Figueroa, James C Rose, Wake Forest Univ, Sch of
Medicine, Dept of Obstetrics and Gynecology, Winston Salem, NC; Mark Chappell, Debra I
Diz, David B Averill; Wake Forest Univ, Sch of Medicine, Hypertension and Vascular Disease
Cntr, Winston Salem, NC
Exposure of fetuses that are candidates for premature delivery in the early part of the third
trimester to synthetic steroids is an accepted strategy to accelerate lung development and to
protect neonates from other early developmental problems. However, recent studies show that
exposure of the fetus at this point of gestation to synthetic steroids perturbs normal
nephrogenesis and may lead to elevated blood pressure as these individuals reach adoles-
cence. We hypothesize that exposure of the fetus to synthetic steroids leads to activation of the
renin-angiotensin system as the individual enters early adulthood. To investigate this
hypothesis, fetal sheep were exposed to betamethasone (Beta) or vehicle (Veh) at the 80th day
of gestation and then delivered at full term. Sheep (average age 1.8 years) were instrumented
for conscious recording of arterial pressure and heart rate and infusion of drugs. Beta sheep
(n6) had significantly (p 0.01) higher baseline mean arterial pressure (MAP) (93 2 mm
Hg, n6) than Veh sheep (84 2 mm Hg, n5). Blockade of AT1 angiotensin (Ang) II
receptors with candesartan (CV; 0.3 mg/kg, i.v.) reduced MAP in Beta sheep (82 3 mm Hg)
whereas MAP was not changed in Veh sheep (82 3 mm Hg). Baroreceptor reflex control of
cardiac interval was evaluated by sequential infusion of phenylephrine and sodium nitroprus-
side. Prior to AT1 blockade baroreflex sensitivity was significantly less in Beta sheep compared
to Veh sheep (11 13 vs. 26 3 msec2 mmHg-1, respectively; p0.0.01). Moreover, 45
minutes after CV injection baroreflex sensitivity was increased in Beta (21 5 msec2 mmHg-1)
and Veh (35 4 msec2 mmHg-1) sheep. Finally, the left ventricle to body weight ratio (LV/BW)
of Beta sheep was significantly greater than Veh sheep (p0.01). These results demonstrate
that in utero exposure of the fetus to synthetic steroid can have long-lasting programming
effects to alter the circulatory control.
33
Enhanced Depressor Response by Activating TRPV1 During High Salt
Intake: Role of a Novel Endovanilloid N-Arachidonoyldopamine
Youping Wang, M, Donna H Wang; Michigan State Univ, East Lansing, MI
Ttransient receptor potential vanilloid type 1 (TRPV1) channels play a role in preventing high salt
(HS) induced increases in blood pressure (BP), but mechanisms for TRPV1 activation by HS is
unclear. We hypothesized that increased sensitivity of BP to N-arachidonoyldopamine (NADA),
an endovanilloid metabolized from dopamine, occurs during HS intake. NADA (1, 4, 10 mg/kg,
iv) dose-dependently decreased mean arterial pressure (MAP) in conscious Wistar male rats fed
a HS diet for 10 days, and its depressor effect was greater in HS vs. normal salt (NS)-treated
rats (e.g. at 4mg/kg, 16 3 vs. 8 2 mmHg, p0.05, n6 7). NADA (4 mg/kg) induced
depressor effect was abolished by capsazepine (CAPZ, 3 mg/kg), a selective TRPV1 antagonist,
or CGRP8 37 (1 mg/kg/min), a selective CGRP receptor antagonist (p0.05). Capsaicin (CAP, 10
or 30 g/kg, iv), a selective TRPV1 receptor agonist, or CGRP (1 or 5 g/kg, iv),
dose-dependently decreased MAP in HS and NS rats, with a greater effect in the former (CAP,
11 2 vs. 6 1; 21 3 vs. 12 2; CGRP, 22 3 vs. 12 2; 41 4 vs. 25 3, p0.05).
CGRP levels in plasma were higher in HS compared to NS rats (58.7 5.7 vs. 40.3 4.6
pg/ml, p0.05), and that NADA (4 mg/kg) caused a greater increase in plasma CGRP levels in
HS compared to NS rats (84.2 7.2 vs. 55.3 4.6 pg/ml, p0.05). TRPV1 receptor protein
expression in the mesenteric arteries was increased in HS compared to NS-treated rats
(0.81 0.06 vs. 0.59 0.04, p0.05). Our data show that 1) HS upregulates mesenteric
TRPV1 expression; 2) HS increases sensitivity of BP to NADA; 3) HS increases basal and
NADA-induced release of CGRP; and 4) the enhanced depressor effect induced by NADA during
HS intake is blocked by antagonists of TRPV1 or CGRP receptors. These results indicate that
NADA may serve as a novel endogenous TRPV1 agonist to prevent salt induced increases in BP
via enhancing CGRP release.
34
Cardiovascular and Sympathetic Responses to Direct Injection of Leptin
into the Arcuate Nucleus of the Hypothalamus
Kamal Rahmouni, Donald A Morgan; Univ of Iowa, Iowa City, IA
Leptin is an adipocyte-derived hormone that plays an important role in the regulation of energy
homeostasis through its action in the central nervous system. The arcuate nucleus (ARC) is
considered as a major hypothalamic nucleus for leptin action on energy homeostasis. Here, we
tested whether leptin action in the ARC plays a dual role in the regulation of sympathetic nerve
activity (SNA) deserving thermogenic and cardiovascular tissues. The sympathetic and
cardiovascular responses to intra-ARC leptin were compared to those evoked by intracerebro-
ventricular (ICV) administration of leptin. Sprague-Dawley rats were equipped with an ICV or
intra-ARC cannula one week before the study. Arterial pressure and multifiber SNA to
thermogenic brown adipose tissue (BAT) and kidney were recorded simultaneously under
anesthesia (-chloralose), at baseline and for 6 h after treatment. As expected, ICV
administration of leptin (10 g in 2 L, n6) caused a significant increase in BAT (250
16%, P0.00001 vs. vehicle) and renal (73 20%, P0.003 vs. vehicle) SNA. ICV leptin also
increased mean arterial pressure (from 83 4 to 100 8 mmHg, P0.04). Intra-ARC
administration of vehicle (200 nL) caused no significant change in regional SNA or arterial
pressure. However, intra-ARC leptin mimicked the effect of ICV administration of this hormone.
Indeed, direct injection of leptin (500 ng, n7) into the ARC increased both BAT (244 51%,
P0.0036 vs. vehicle) and renal (95 29%, P0.0035 vs. vehicle) SNA. Intra-ARC
administration of leptin also increased mean arterial pressure (from 82 3 to 100 7 mmHg,
P0.02). These data demonstrate that leptin action in the ARC is important for the control of
thermogenic sympathetic nerve activity. These results also suggest that the arterial pressure
effects of leptin might be evoked by the action of this hormone in the arcuate nucleus of the
hypothalamus.
35
Cardiovascular and Renal Function in Dahl Salt-Sensitive Rats Vaccinated
against Endogenous Inhibitors of Na/K ATPase
Christine G Schnackenberg, Melissa H Costell, Roberta E Bernard, Mark A Pullen, Robert W
Coatney, Karen F Kozarsky, Theodore M Danoff; GlaxoSmithKline, King of Prussia, PA
Endogenous inhibitors of Na/K ATPase, which are thought to be structurally related to ouabain,
are reportedly increased in hypertension. We tested the hypothesis that antibodies directed
against these postulated inhibitors of Na/K ATPase attenuate the development and maintenance
of salt-sensitive hypertension and cardiac hypertrophy. Dahl salt-sensitive (DahlS) rats with
radiotelemetry were maintained on low salt diet and divided into three groups: control (n8),
vehicle vaccinated (n9), and ouabain vaccinated (n8). When the ouabain binding capacity
of the plasma antibodies from ouabain vaccinated rats was high (ELISA), baseline measure-
ments were determined and high salt (8% NaCl) feeding began in all groups. There were no
differences in basal MAP (125 3 mmHg), renal function, or cardiac function (ECHO) in DahlS
rats on low salt diet. Four weeks of high salt intake significantly increased mean arterial
pressure (182 5, 167 6, 171 6 mmHg), proteinuria (186 4, 141 8, 169 27
mg/d/100g) and left ventricular mass/body weight ratio in all groups. Ouabain vaccinated DahlS
rats produced ouabain specific, nM affinity IgG that neutralized ouabain inhibitory activity of
Na/K ATPase in vitro. Despite the blockade of ouabain activity, ouabain vaccinated rats had
similar blood pressure, renal function, and cardiac function as vehicle vaccinated rats. Similar
results were found in digoxin vaccinated DahlS rats. Finally, in a separate group of naive
hypertensive DahlS rats (n5), intravenous administration of antibodies to digoxin that also
bind ouabain (Digibind, 60 mg/kg) had no effect on mean arterial pressure. These studies
suggest that neutralizing antibodies to endogenous inhibitors of Na/K ATPase do not prevent the
development of salt-sensitive hypertension, renal dysfunction, and cardiac hypertrophy or
attenuate the maintenance of high blood pressure in DahlS rats.

36
High Plasma Norepinephrine Levels Determined By 2-Adrenoceptor
Polymorphisms Predict the Future Renal Injury in Nonobese, Normotensive
Subjects
Kazuko Masuo, Baker Heart Rsch Institute, Melbourne Vic, Australia; Tomohiro Katsuya,
Hiromi Rakugi, Hideki Kawaguchi, Toshio Ogihara, Osaka Univ Graduate Sch of Medicine,
Suita City, Osaka, Japan; Michael L Tuck; Sepulveda VA Med Cntr and the UCLA Sch of
Medicine, Sepulveda, CA
Background: Renal injury is one of the common organ damage caused by hypertension. The
2-adrenoceptor polymorphisms and heightened sympathetic nerve activity are associated
with hypertension. In the present study, we examined the relationships between alterations of
renal function (blood urea nitrogen (BUN) and creatinine), sympathetic nerve activity (SNA) and
the 2-adrenoceptor polymorphisms in a longitudinal design over 5 years. Methods: In 219
nonobese, normotensive, normal renal function men at entry, we measured serum BUN,
creatinine, plasma norepinephrine (NE) levels, the homeostasis model assessment of insulin-
resistance (HOMA), BMI, total body fat-mass, blood pressure (BP), and the 2 (Arg16Gly,
Gln27Glu)-adrenoceptor polymorphisms annually over 5 years. The subjects were stable in
body weight over 5 years and non-diabetic. Results: Hyper-SNA was defined as a plasma NE
level of mean1SD of 1.37 pmol/mL in the participants at entry. There were 37 subjects who
had entry hyper-SNA and 182 subjects who had entry normal-SNA (plasma NEmean1SD).
At entry, plasma NE was significantly higher in subjects with hyper-SNA compared to those
with normal-SNA, but BUN, creatinine, HOMA, BMI, total body fat-mass and BP levels were
similar. Changes in serum BUN, creatine, uric acid, plasma NE, HOMA, total body fat-mass, BMI
and BP levels over a 5-year period were significantly greater in subjects with hyper-SNA.
Subjects with hyper-SNA had significantly higher frequencies of the Gly16 and Glu27 alleles of
2-adrenoceptor polymorphisms. Subjects carrying the Gly16 or Glu27 alleles had higher levels
of plasma NE, HOMA and total body fat-mass at entry, and greater increases in BUN, creatinine,
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
plasma NE levels and total body fat-mass over a 5-year period. Conclusions: The Gly16 and
Glu27 alleles are associated with high plasma NE at entry and elevations in BUN and creatinine
over a 5-years period accompanied by greater increases in plasma NE. Although renal injury
is known of hypertension-related end-organ damage, high plasma NE (heightened sympathetic
nerve activity) and high HOMA (insulin-resistance) in part determined by 2-adrenoceptor
polymorphisms might be related with the onset of renal injury.
37
Blood Pressure Lowering Effect Of The Mitochondrial Inhibitor Rotenone In
Mice With DOCA-salt Hypertension
Tianxin Yang, Aihua Zhang; Univ of Utah, Salt Lake City, UT
Oxidative stress plays a pivotal role in the pathogenesis of various forms of hypertension but
its source is debatable. Despite emphasis on NADPH oxidase, attention has been paid to the
mitochondria as a potential source of oxidative stress in the development of hypertension. The
present study was undertaken to examine the effect of the mitochondrial complex I inhibitor
rotenone on blood pressure (Bp) in mice treated with deoxycorticosterone acetate and 1% salt
(DOCA-salt). Wild type 129-C57/BL6 mice received DOCA-salt treatment for 4 days followed by
an one-week treatment with vehicle or rotenone at 600 ppm in a gelled diet, and daily mean
arterial pressure (MAP) was monitored by telemetry. The second day of DOCA-salt, MAP in the
vehicle treated mice increased from 110 1.33 to 138 2.04 mmHg that remained constant
throughout the entire experimental period. Rotenone treatment induced an immediate and
significant reduction of MAP (136.0 2.9 vs. 114 3.5 mmHg, n6, p0.01). DOCA-salt
increased urinary excretion of 8-isoprostane from 275.47 44.65 to 1654.97 316.15
pg/24h that was reduced to 826.04 141.24 pg/24h after rotenone treatment. Conversely,
DOCA-salt decreased urinary excretion of nitrate/nitrite from 466.73 84.14 to 68.30 15.17
that was restored to 238.11 44.53 pmol/24h by rotenone treatment. Reactive oxygen
species (ROS) production in isolated kidney mitochondria as measured by enhanced luminal or
isoluminol chemiluminescence was 1.0 0.1, 2.48 0.4 (p0.01), and 1.26 0.28
(p0.01) (fold changes over control) in vehicle, DOCA-salt, and DOCA-salt plus rotenone
groups, respectively; levels of protein carbonyls in the three groups in the same order were
4.38 1.35, 11.10 5.56 (p0.01), and 5.91 2.28 (p0.05) nmol/mg protein.
Histological analysis of heart and kidneys did not reveal significant abnormalities after rotenone
treatment. In contrast to the significant Bp lowering effect of rotenone, DOCA-salt hypertension
in mice with mutation of the p47phox gene did not appear to be affected. In conclusion, the
present study provides new evidence favoring mitochondria as an important source of oxidative
stress in DOCA-salt hypertension.
38
Salt-Sensitivity in African Americans: Is it Initiated by an Abnormal
Vascular Response?
Olga Schmidlin, Alex Forman, Anthony Sebastian, R. C Morris, Jr; Univ. of California San
Francisco, San Francisco, CA
It is widely formulated that salt-sensitivity is: 1) initiated by an increased renal reclamation of
salt that induces a transient increase in cardiac output (CO) by expanding plasma volume (PV):
and later, 2) sustained by a persisting increase in systemic vascular resistance (SVR). Although
many observations accord with this nephrogenic hypothesis, recent observations in animal
models suggest that an abnormal vascular response to salt may initiate salt-sensitivity. Neither
of these hypotheses has been rigorously tested in humans. In 20 mostly normotensive African
Americans we determined a) whether dietary NaCl loading induces greater increases in Na
balance (NaB), PV and CO in those who are salt-sensitive (SS) compared to those who are
salt-resistant (SR); and b) whether initial NaCl-induced hemodynamic events can predict
salt-sensitivity. Employing impedance cardiography, we measured CO daily at 4-hr intervals
during the last 3 of 7 days of low NaCl intake, 35 mmol/d, and on all 7 days of NaCl loading,
250 mmol/d. We estimated changes in PV from changes in serum protein concentration.
CHBPR ConferenceOral Presentations e33
Results: Within 2 days, NaCl loading induced similar increases in PV and CO in SS (MAP
10mmHg, n9) and SR (MAP 0mmHg, n11). In both SS and SR, CO reached its
maximum on day 3, 8% above its low NaCl value, and returned to baseline by day 7 of the NaCl
load. In SR but not in SS the NaCl-induced increase in CO was accompanied by significant initial
decreases in BP (-6%) and SVR (-14%). In SR, SVR returned to low NaCl levels by day 7 of the
NaCl load, whereas in SS, SVR rose above low NaCl levels by day 4 and continued to rise to
a final value of 11% above low NaCl. NaCl load induced an increase in bodyweight (BW) of 2.1
kg in SS and 2.5 kg in SR, respectively, Pns. This increase in BW was accompanied by a
positive NaB of 390 mmol in SS and of 520 mmol in SR, P0.05. Changes in BP as early as
9 hours after initiating NaCl loading were highly predictive of salt-sensitivity, i.e. changes in
MAP on days 5 and 6, r0.64, P0.003 for DBP. Changes in NaB, BW, PV and CO are not
predictive of salt-sensitivity at any time point. Conclusions: We propose a vasogenic
hypothesis of salt-sensitivity: In African Americans, the pressor effect of dietary NaCl depends
from its outset on an abnormal vascular response to NaCl loading.
39
The Na-K-2Cl Cotransporter as a Mediator of the Na Retention in African
Americans
Tae-Yon Chun, Indiana Univ Sch of Medicine, Indianapolis, IN; Lise Bankir, INSERM Unite
652, Paris, France; George J Eckert, Chandan Saha, Mary Anne Wagner, Beth Deem,
Indiana Univ Sch of Medicine, Indianapolis, IN; Daniel G Bichet, Hopital du Sacre-Coeur de
Montreal, Montreal, Canada; J. Howard Pratt; Indiana Univ Sch of Medicine and the VA Med
Cntr, Indianapolis, IN
Identifying differences between ethnic groups in the renal handling of Na could provide insight
into mechanisms for hypertension. In the current study, we made measurements in African
Americans (AAs) and European Americans (EAs) that had potential relevance to the renal
Na-K-2Cl cotransporter (NKCC2). Urine osmolality (Uosm) served as an indirect index of NKCC2
function since it is highly dependent on the medullary Na concentration achieved by NKCC2.
Healthy subjects (99 AAs, 78 EAs; 18 35 yr) were admitted to the GCRC where they had free
access to water overnight (12 hr) and during an early morning meal. This was followed by a
continuous infusion of 0.9% saline (60 ml/hr x 8 hr) (no additional food or water). After
equilibration and 2 hr basal period, 40 mg furosemide (F) was administered i.v. Analysis of
covariance adjusted for sex was used to compare the 2 groups. During the overnight period,
Uosm (mosm/kg H2O) was higher in AAs than EAs, 552 25 (SE) vs. 413 17 (p0.0001),
whereas during saline infusion baseline values were not significantly different: 537 19 and
492 21 in AAs and EAs, respectively (p0.12). Thirty min after F administration, Uosm fell
in both groups close to plasma osmolality (300), indicating complete inhibition of NKCC2. In
the subsequent recovery period, Na excretion was less in AAs (p0.0013 in initial 2 hr)
consistent with greater Na reabsorption. Following the post-F nadir, Uosm increased but more
slowly in AAs than EAs (p0.0001). This appeared explainable in part by reduced levels of
renin activity (PRA) (p0.0041) and aldosterone (PA) (p0.0008) in AAs than EAs. Recovery of
Uosm correlated with both PRA (r0.17; p0.057) and PA (r0.24; p0.0027) leading us to
speculate that in AAs the suppressed levels resulted in less vascular tone in vasa recta thereby
enhancing washout of the medullary gradient. Plasma vasopressin measured in a subset of 60
subjects did not explain the ethnic differences. In summary, AAs had higher Uosm under free
living conditions, less Na excretion in post-F recovery period, and slower Uosm recovery that
correlated with PRA/PA. Although the indirect assessments limit making specific conclusions,
the findings suggest that increased Na retention in AAs could be representative of greater
NKCC2 activity.
40
Non-Modulating Hypertension is Associated with Insulin Resistance and
the Lys528Arg Variant of Human Adipocyte-Derived Leucine
Aminopeptidase
Jonathan S Williams, Annaswamy Raji, Gordon H Williams, Paul R Conlin; Brigham and
Womens Hosp/Harvard Med Sch, Boston, MA
Intermediate phenotyping provides a powerful means to test susceptibility loci for human
hypertension. Two intermediate phenotypes, insulin resistance (IR) and adrenal responsiveness
to angiotensin II (Ang II) have demonstrated familial aggregation in hypertensive sib-pair
studies. Adipocyte-derived leucine aminopeptidase (ALAP) degrades Ang II in the kidney,
suggesting its possible role in BP regulation. This is supported by reports that the Lys528Arg
polymorphism, which reduces ALAP activity, is associated with human hypertension. Non-
modulating hypertension (NM) is characterized by blunted renal vascular and adrenal responses
to Ang II, which are corrected by ACE-inhibition. NM patients are also more likely to have IR.
We investigated the heritability of NM and its association with IR and the Lys528Arg variant of
ALAP. We analyzed 436 hypertensive individuals categorized by their adrenal response to Ang
II (NM aldosterone response to Ang II 15 ng/dl, N98). Within this cohort, results from 138
sib-pairs were examined to test familial aggregation of the NM phenotype. IR was estimated
using homeostasis model assessment (HOMA). Sib-pair analysis demonstrated familial
aggregation of NM (OR 2.6, 95%CI, 1.1, 6.78). NM was also associated with IR (OR 1.8, 95%CI
1.1, 2.9). Allele frequencies for Lys528Arg were in Hardy-Weinberg equilibrium. NM was
associated with a higher odds of carrying the 528Arg variant of ALAP (OR 2.2, 95% 1.0, 4.9,
P 0.047). There was a significant genotype-HOMA dose-response relationship for Lys528Arg
with higher HOMA values associated with the 528Arg genotype (P-trend 0.03). Individuals
with both IR and NM were more likely to have the 528Arg polymorphism (OR 4.55, 95%CI, 1.4,
15.0, P 0.008). These results show that NM is associated with IR, aggregates in families,
and that both NM and IR are associated with the 528Arg variant of ALAP. Our results support
the involvement of ALAP in both blood pressure regulation and IR in NM. This suggests a
common underlying mechanism for both IR and hypertension in NM, which may be linked
through abnormal ALAP activity.
 e34 Hypertension Vol 48, No 4 October 2006
41
Reactive Oxygen Species (ROS)-Dependent Hypertension in D2 Receptor
Deficient Mice
Ines Armando, Xiaoyan Wang, Van A Villar, John E Jones, Laureano Asico, Pedro A Jose;
Georgetown Univ Med Cntr, Washington, DC
Alterations in dopamine D2-like receptor function have been reported in essential hypertension.
A polymorphism in exon 6 of the D2 receptor (D2R) gene is associated with elevated blood
pressure and a Taq1 polymorphism is associated with human essential hypertension. We have
previously shown that disruption of D2R in mice (D2-/-) results in high blood pressure that is
associated with increased aldosterone production and defective aldosterone suppression by
high salt diet. Aldosterone has been shown to increase the expression of NADPH oxidase
subunits as well as oxidative stress. We hypothesized that the increased blood pressure in D2-/-
is related to increased NADPH oxidase activity and ROS. We determined the mRNA expression
of the NADPH oxidase subunits p22phox, p40phox, p47phox, p67phox, Rac1, Rac2, Nox1, Nox2 and
Nox4 by RT-PCR in kidneys of D2-/- and their wild type littermates, D2/.The mRNA
expression of Nox1, Nox2 and Nox4 was increased 303, 141 and 292%, respectively in
D2-/- (p0.05 vs D2/, n5 per group).The mRNA expression of the other subunits tested
was similar in both groups. The protein expression of Nox1, Nox2 and Nox4, determined by
western blot, was increased in kidneys of D2-/- mice by 458, 8910 and 555%,
respectively (p0.05 vs D2/). The urinary excretion of 8-isoprostane (7715 vs 358
ng/mg creatinine [D2/]; p0.04, n9), a marker of oxidative stress, and renal NADPH
oxidase activity (121,30015,310 vs 81,2009,870 [D2/] LU/mg prot; p0.05; lucigenin
assay) were also increased in D2-/- mice. Treatment with hemin, an inducer of heme
oxigenase-1 (50 mol, IP, for 24 h), normalized blood pressure in anesthetized (Avertin) D2-/-
mice (systolic: vehicle: 1235; hemin: 896 mmHg; p0.05; n5) but had no effect in
D2/ mice (systolic: vehicle: 1032; hemin: 1001 mmHg; n5). Treatment with
apocynin, a NADPH oxidase inhibitor (3 mg/k/day, via osmotic mini-pumps, 10 days), also
normalized blood pressure in D2-/- mice (systolic: D2-/-, 962; D2/, 961 mmHg; n5
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
per group). Our results show that the D2R is involved in the regulation of ROS production and
suggest that by direct and indirect mechanisms altered D2R function may result in
ROS-dependent hypertension.
42
Endothelin-1 Induced Oxidative Stress Reduces Endothelial Progenitor Cells
via an ETA-NADPH Oxidase Pathway in Salt-Sensitive Hypertension
Dandan Chen, Michigan State Univ, East Lansing, MI; YuGang Dong, The First Affiliated
Hosp of Sun Yat-Sen Univ, Guangzhou, China; Eric J Marrotte, Jame J Galligan, Gregory D
Fink, Alex F Chen; Michigan State Univ, East Lansing, MI
Background: The circulating endothelial progenitor cells (EPCs) derived from the bone marrow
home to the ischemic site to form mature EC and new blood vessels. Circulating EPCs are
reduced in patients of cardiovascular diseases including hypertension, and correlates inversely
with their mortality. However, the mechanisms underlying reduced availability of EPCs in
hypertension are poorly understood. DOCA-salt hypertension exhibits increased levels of ET-1
and oxidative stress. We tested the hypothesis that ET-1-induced oxidative stress contributes
to reduced EPCs in DOCA-salt rats. Methods: Rat EPCs were isolated from peripheral blood and
cultured in EC growth medium-2 for 5 days. EPCs that were DiI-acLDL and lectin
double-positively stained were counted. The level of intracellular reactive oxygen species (ROS)
in EPCs was assessed by dichlorofluorescein (DCF) fluorescence microscopy. Results: The
number of EPCs was significantly reduced in DOCA-salt compared to Sham rats (3.940.30
vs. 10.320.25cells/hpf, n3 6, p0.01). Treatment with ROS generator LY83583 (10 M,
18 hrs) profoundly elevated the ROS level in EPCs of DOCA-salt rats (212.48.3% vs. Sham,
n3, p0.05), but only slightly in EPCs of normal rats (110.01.0%, n3, p0.05). ET-1
treatment (10 nM) significantly reduced the number of EPCs in a time-dependent manner
(24 96 hrs) in normal rats. ET-1 (48 hrs) induced decrease of EPCs (6.910.11 vs.
10.320.25 cells/hpf, n3 6, p0.01) was rescued by the selective ETA receptor antagonist
ABT-627, NADPH oxidase inhibitor apocynin, or adenoviral-mediated gene transfer of
dominant-negative Rac1 that inhibits endogenous NADPH oxidase subunit Rac1 (9.850.28,
10.480.38, and 9.130.38 cells/hpf, respectively, n3 6, p0.01), but not by ETB receptor
antagonist BQ788 (7.620.41 cells/hpf, n3, p0.05). ET-1 treatment also significantly
augmented the ROS level in EPCs of normotensive rats (212.48.3%, n3, p0.05), which
was blunted by ABT-627 and apocynin (103.32.0% and 114.52.5%, respectively, n3,
p0.05). Conclusion: These results demonstrated, for the first time, that EPCs of DOCA-salt
hypertensive rats exhibit both reduced number and antioxidant capacity, mediated at least in
part, via an ETA-NADPH oxidase pathway.
43
Acute Oxidative Stress Inhibits HO-Dependent Generation of Vascular CO in
Renal Arteries
Brian D Lamon, Frank F Zhang, Huan Deng, Michael S Wolin, Alberto Nasjletti; New York
Med College, Valhalla, NY
Carbon monoxide (CO) is produced endogenously via HO-dependent and HO-independent
pathways. CO is liberated during the metabolism of heme by HO-1 and -2 along with biliverdin
(BV) and iron. HO-independent CO is generated by peroxidizing lipids under conditions of
elevated oxidative stress. The source of endogenous CO may have functional implications, as
CO alone causes vasoconstriction while the combination of CO and BV causes vasodilation.
These studies were designed to determine the effects of oxidative stress on CO generation by
renal interlobar arteries (RIA). We studied rats treated with vehicle, with the superoxide
dismutase (SOD) inhibitor diethyldithio-carbamate trihydrate (DETC; 1g/kg, i.p.) or with DETC
tempol (an SOD mimetic). RIA were obtained 3 hours after treatment and used to measure
superoxide generation, HO protein levels, and endogenous CO release. An inhibitor of HO,
chromium mesoporphyrin (CrMP; 30uM), was used to discriminate between HO-independent
and HO-dependent CO generation and to assess the significance of HO products on the internal
diameter of renal interlobular arteries from treated and untreated rats. DETC increased vascular
superoxide production (475 vs. 746 cpm/ug protein; p0.05) and HO-1 protein, without
altering HO-2 protein levels. Yet, total CO generation was reduced (112696 vs. 682147
pmol/mg/hr), reflecting a near abolition of HO-dependent CO generation (52963 vs. 2920
pmol/mg/hr) while HO-independent generation was marginally increased. Pretreatment with
tempol significantly reduced HO-independent CO and minimized DETC-induced inhibition of
HO-dependent CO production (2920 vs. 28075 pmol/mg/hr; p0.05). Assessment of the
functional response to HO inhibition in pressurized renal interlobular arteries revealed that CrMP
reduced internal diameter in vehicle-treated rats while having no effect in DETC-treated
animals (-28.30.8 vs. -1.00.6 um; p0.05). These data suggest an inhibitory role of
O2- or one of its derivatives on HO activity that prevents the vascular HO system from supporting
vasodilatory mechanisms. Hence, vascular dysfunction during acute oxidative stress may be
related, in part, to deficient generation of HO-derived products.
44
Chronotropic Actions of Angiotensin II Are Mediated by a PKC-Dependent
Activation of NADPH Oxidase in Brain Neurons
Chengwen Sun, Colin Sumners, Mohan K Raizada; Dept of Physiology and Functional
Genomics, College of Medicine, Univ of Florida, Gainesville, FL
We have previously established that via stimulation of AT1 receptor (AT1R), Angiotensin II (Ang
II) increases neuronal firing, which induces cascades of signaling events leading to cardiovas-
cular effects of this hormone in the brain. Although we have identified NADPH oxidase-derived
ROS as a key signaling intermediate in the chronotropic actions of Ang II, the precise
mechanism underlying this effect remains elusive. We hypothesized that NADPH oxidase
activation involves protein kinase C (PKC) since inhibition of this enzyme prevents Ang
II-induced neuronal activity. Thus, our objective in the present study was to examine the role
of PKC-mediated NADPH oxidase activation in the chronotropic actions of Ang II in the neurons
co-cultured from hypothalamus and brainstem. Incubation of neurons with Ang II (100 nM)
causes 2 fold increases in NADPH oxdase activity. This action of Ang II, mediated by
neuronal AT1R, is abolished by a PKC inhibitor GF109203X (GF, 1 M) and mimicked by a PKC
activator, Phorbol 12-myristate 13-acetate (PMA, 1 M). In addition, Ang II-induced increase
in neuronal firing rate (from 0.99 0.18 Hz to 1.81 0.29 Hz) is blocked by GF (1 M).
Treatment of neurons with PMA (1 M) causes a 2-fold increase in neuronal firing rate that is
attenuated by a ROS scavenger, TEMPOL (100 M) or a NADPH oxidase inhibitor, gp91ds (5
M). Finally, treatment of neurons with Ang II (100 nM, 5 min) increases membranous p47phox
and decreased cytoplasmic p47phox protein as measured by Western blotting. This Ang
II-induced translocation of p47phox is abolished by pretreatment of neurons with GF (1 M).
These observations indicate that Ang II-induced stimulation of neuronal firing is mediated by
activation of NADPH oxidase in a PKC-dependent manner and that PKC is proximal to NADPH
oxidase activation in ROS-mediated actions of Ang II in the regulation of neuronal activity.
45
Reduction of Oxidative Stress-Induced Vasoconstriction by Tempol is
Mediated by Hydrogen Peroxide
Adam Pearlman, Christopher Wilcox, Yifan Chen; Georgetown Univ, Washington, DC
Reduction of oxidative stress in hypertension by the superoxide dismutase (SOD)-mimetic
tempol improves vascular dysfunction. Tempol reduces the concentration of superoxide anion,
(O2-), but increases the concentration of hydrogen peroxide (H2O2). We tested the hypothesis
that the vasodilatory effect of acute exposure to tempol is primarily due to generation of H2O2.
We applied tempol to mouse cremaster arterioles in vivo (n7) and to pressurized rat
mesenteric arteries in vitro (n9). Vessels were exposed to angiotensin II (cremaster) or
U46619, a thromboxane receptor agonist, (mesenteric) for 1530 minutes. Acute application of
tempol caused significant vasodilation in vivo (18935%) and in vitro (212%) with preserved
vasodilation in endothelium denuded vessels. The vasodilatory effect of tempol was signifi-
cantly reduced in vivo (397%) or in vitro (102%) in the presence of catalase. Similarly,
addition of 30mM KCl to the mesenteric vessel bath abrogated the effect of tempol. Tempol
was not effective following prolonged treatment with alternative preconstrictors not associated
with substantial production of O2-: phenylephrine in vivo (154%) or norepinephrine in vitro
(0%). In contrast to tempol, nitro blue tetrazonium, (NBT), a O2- scavenger of equal potency to
tempol that does not result in increased H2O2, was an ineffective vasodilator when applied in
vivo (105%) or in vitro (03%). Direct applications of H2O2 to the vessels caused a
dose-dependent dilation in vitro (0.01100M) that was independent of intact endothelium. We
conclude that prolonged stimulation of angiotensin II or thromboxane receptors produce
vascular oxidative stress. Subsequent vasodilation by tempol is largely due to generation of
H2O2 which likely produces a hyperpolarizing effect. In a variety of pathophysiological
conditions associated with oxidative stress, an adequate amount of H2O2 may be an important
counterbalance to the vasoconstrictive effect of O2-.
46
Angiotensin II Causes Elevated Superoxide Levels in the Paraventricular
Nucleus during Hypertension
Carrie A Northcott, Andrew J King, Gregory D Fink, Joseph R Haywood; Michigan State
Univ, East Lansing, MI
Circulating angiotensin II (Ang II) acts on the brain to stimulate neural pathways to the
paraventricular nucleus (PVN) that use Ang II as a neurotransmitter. In renal wrap hypertension,
an Ang II dependent model, superoxide (O2-) levels in the PVN are elevated. Thus, we
hypothesized that Ang II was the stimulus for increased O2- in the PVN during hypertension. We
examined this hypothesis in several ways; ex vivo addition of Ang II (100 nmol Ang II for 15,
30 and 60 minutes) to PVN brain punches to determine if Ang II could directly stimulate O2-
production in the PVN, measurements of O2- levels in PVN punches from rats treated acutely

with Ang II (30 ng/kg/min, 15ul/min intravenously for 1 hour) and rats treated chronically with
Ang II (150 ng/kg/min subcutaneously for 14 days). Because high blood pressure itself has
been shown to increase O2- levels in some tissues, we examined O2- levels in the PVN of chronic
DOCA-salt hypertensive rats, a model lacking elevated circulating Ang II. Blood pressures were
measured in the conscious state. The brains were removed, quick frozen on dry ice and
immediately cryostat sectioned (600 um) and PVN punched for chemiluminescent O2-
measurements. After a 15 minute ex vivo incubation of the PVN with Ang II there was a
significant increase in O2- (p0.05), demonstrating direct Ang II-induced O2- production. Acute
Ang II infusion resulted in a significant increase in blood pressure (p0.05), and O2- in the PVN
(Saline: 0.22 0.02 nmol/min*mg, Ang II: 0.36 0.02 nmol/min*mg, p0.05). Chronic Ang
II infusion also resulted in a significant increase in blood pressure (Mean Arterial Pressure: 104
3 mm Hg vs. 134 8 mm Hg, p0.05) and increased O2- in the PVN (Vehicle: 0.23 0.02
nmol/min*mg, Ang II: 0.44 0.09 nmol/min*mg, p0.05). In contrast, in DOCA-salt rats
[Systolic Blood Pressure (SBP) 178 3 mm Hg] there was no significant increase in O2- in
the PVN compared to sham normotensive rats (SBP136 5 mm Hg). These data
demonstrate that circulating Ang II acts on neural pathways in the brain to increase PVN O2-.
Further, the O2- changes in the PVN are not due to increased blood pressure per se. In
conclusion, Ang II-mediated changes in O2- may contribute to increased excitatory neurotrans-
mitter activity in the PVN and ultimately result in hypertension.
47
Heterozygous eNOS-Deficiency Produces Oxidative Stress and Endothelial
Dysfunction in a Model of Diet-Induced Obesity and Type II Diabetes
Sean P Didion; Univ of Iowa Carver College of Medicine, Iowa City, IA
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
Although the incidence of obesity and type II diabetes is increasing, very little is known
regarding mechanisms that contribute to endothelial dysfunction in these conditions. The goal
of this study was to test the hypothesis that loss of a single gene for endothelial nitric oxide
synthase (eNOS) produces endothelial dysfunction in a mouse model of diet-induced obesity
and type II diabetes. Responses of carotid arteries from wild-type and heterozygous
eNOS-deficient (eNOS) mice were examined in vitro following 32 weeks on either a control
(10% kcal from fat) or a high fat (45% kcal from fat) diet. Body weight, fat deposition (ie,
perirenal and reproductive fat mass) and blood glucose levels were greater (P0.05) in high-fat
fed mice compared to mice fed a control diet and were not affected by genotype. Responses
to acetylcholine (ACh, an endothelium-dependent agonist) and nitroprusside (NP, an
endothelium-independent agonist) were similar in wild-type and eNOS mice fed a control diet
(eg, 10 mol/L ACh produced 926 and 853% relaxation in wild-type and eNOS mice,
respectively). In contrast, responses to ACh were markedly reduced (P0.05) in eNOS, but
not wild-type, mice fed a high-fat diet (eg, 10 mol/L ACh produced 853 and 496%
relaxation in control and high-fat fed eNOS mice, respectively). This effect was selective
since NP produced similar (P0.05) relaxation in vessels from high-fat fed wild-type and
eNOS mice. Responses to ACh in high-fat fed eNOS mice were restored toward normal by
tempol (a superoxide scavenger, 1 mmol/L) suggesting a role for superoxide in this response.
These findings provide the first genetic evidence that loss of a single eNOS gene produces
oxidative stress and endothelial dysfunction in a model of diet-induced obesity and type II
diabetes. These findings have important implications in terms of disease states and/or genetic
polymorphisms associated with reductions in eNOS expression and/or activity.
48
Sympathetic Neurons Isolated from Apolipoprotein E Deficient Mice Exhibit
Increased Excitability: Role of Oxidative Stress
Xiuying Ma, Zhi-Yong Tan, Carol A Whiteis, Francois M Abboud, Mark W Chapleau; Depts of
Internal Medicine and Physiology & Biophysics, Univ of Iowa, and Veterans Affairs Med Cntr,
Iowa City, IA
We recently demonstrated that O2- is increased in sympathetic ganglia of apolipoprotein E
deficient (apoE-/-) mice with atherosclerosis. Previous studies have shown that oxdative stress
in the central nervous system increases sympathetic nerve activity. In the present study, we
tested the hypothesis that oxidative stress in apoE-/- sympathetic ganglia increases membrane
excitability of postganglionic neurons. Neurons were isolated from sympathetic ganglia of
wild-type (WT, n6), apoE-/- (n6), and apoE-/- mice treated with the SOD mimetic tempol
(n4) for 2 weeks in the drinking water (30 mg/kg/day). Resting membrane potential (RMP)
and action potentials (APs) were recorded by patch-clamp at rest and in response to
depolarizing current injection. RMP was more negative in apoE-/- (n24) than in WT (n20)
neurons (-43.60.6 vs. -41.10.9 mV, P0.05) but membrane resistance did not differ. The
majority neurons from WT mice (17 of 20, 85 %) were electrically quiescent at RMP with only
3 of 20 neurons (15 %) firing APs spontaneously. In contrast, a large percentage of apoE-/-
neurons fired APs spontaneously at RMP (10 of 24, 42 %, P0.05 vs. WT). In quiescent
neurons, the current threshold for triggering a single AP was significantly lower in apoE-/- than
in WT mice (7.51.9 vs. 14.52.4 pA), and the frequency of AP firing with equivalent current
injection (100 pA for 1 s) was higher (15.01.7 vs. 9.52.1 spikes) (apoE-/- vs. WT, P0.05).
Chronic treatment of apoE-/- mice with tempol reduced the proportion of neurons firing APs
spontaneously (22 vs. 42 %, tempol treated vs. untreated apoE-/-, P0.05). Interestingly,
tempol-treatment did not affect either current threshold or AP firing during current injection in
quiescent apoE-/- neurons. We conclude that membrane excitability is increased in sympa-
thetic neurons isolated from apoE-/- mice. The reduction in number of spontaneously firing
neurons isolated from tempol-treated apoE-/- mice suggest oxidative stress plays a role in the
increased neuronal excitability of apoE-/-sympathetic neurons. We speculate that increased
excitability of sympathetic neurons may contribute to excessive sympathetic nerve activity and
baroreflex impairment in atherosclerotic states. (VA and NIH HL14388)
CHBPR ConferenceOral Presentations e35
49
Agonistic Autoantibodies to the AT1 Receptor in Rat Models of
Preeclampsia: Induced by Chronic Reductions in Uterine Perfusion Pressure
(RUPP) and Low Dose TNFalpha Infusion
Ralf Dechend, Helios-Klinikum, Franz-Volhard-Klinik, Charite, Berlin, Germany; Mytae Llinas,
Univ of Mississippi Med Cntr, Jackson, MS; Silvia Caluwaerts, Universitair Ziekenhuis
Gasthuisberg, Leuven, Belgium; Florian Herse, Helios-Klinikum, Franz-Volhard-Klinik, Charite,
Berlin, Germany; Babbette Lamarca, Univ of Mississippi Med Cntr, Jackson, MS; Dominik N
Muller, Max-Delbrueck Cntr, Berlin, Germany; Robert Pijnenborg, Universitair Ziekenhuis
Gasthuisberg, Leuven, Belgium; Gerd Wallukat, Max-Delbrueck Cntr, Berlin, Germany; Joey
P Granger; Univ of Mississippi Med Cntr, Jackson, MS
The IgG fraction from preeclamptic women contains an angiotensin II receptor autoantibody
(AT1R-AA) that stimulates the angiotensin II type I (AT1) receptor. AT1R-AA could contribute to
the pathogenesis of preeclampsia. We investigated the uterine perfusion pressure (RUPP) and
low-dose TNF infusion models to see if these models develop AT1R-AA. We detected
AT1R-AA by the chronotropic responses to AT1 receptor-mediated stimulation of cultured
neonatal rat cardiomyocytes coupled with receptor-specific antagonists. Mean arterial pressure
(MAP) was significantly higher in RUPP rats (1371 mm Hg) than in normal pregnant (NP) rats
(1071 mm Hg). The hypertension in the RUPP rats was associated with significant elevations
in agonistic antibodies capable of activating the angiotensin AT1 receptor (RUPP 15.31.6 vs.
NP 0.60.3 beats/min). Pregnant rats with low dose TNF infusion (5 days at a rate of 50
ng/d during days 14 to 19 of gestation in pregnant rats) had higher blood pressures (125
1 mm Hg) than NP rats (1012 mm Hg). They also developed AT1R-AA (9.22.3 vs 1.0 0.8
beats/min). Low dose TNF in virgin rats did not increase MAP nor lead to AT1R-AA. TNF
expression in the placentas of RUPP rats was upregulated (p 0.05) Serum TNF levels were
nearly 3-fold higher in the RUPP rats compared with NP (p 0.05). These findings suggest that
the generation of AT1R-AA is a secondary event to reduction in placental perfusion leading to
chronic inflammation.
50
RGS2 Ameliorates Angiotensin II-Induced Hypertension in Mice
Michael Obst, MDC, Berlin, Germany; Jens Tank, Med Faculty of the Charite, Franz Volhard
Clinic, HELIOS Klinikum-Berlin, Berlin, Germany; Ralph Plehm, MDC, Berlin, Germany; Jens
Jordan, Med Faculty of the Charite, Franz Volhard Clinic, HELIOS Klinikum-Berlin, Berlin,
Germany; Friedrich C Luft, MDC and Med Faculty of the Charite, Franz Volhard Clinic,
HELIOS Klinikum-Berlin, Berlin, Germany; Volkmar Gross; MDC, Berlin, Germany
The regulator of Gq G protein signaling (RGS) 2 accelerates the rate of G protein deactivation
by stimulating GTP hydrolysis. Angiotensin II (Ang II) activates signaling pathways predomi-
nantly through the G-protein-coupled Ang II type 1 receptor (AT1AR). We hypothesized that RGS2
deletion increases the response to Ang II and changes blood pressure regulation because
signaling would be abnormally prolonged. To address this issue, we infused Ang II chronically
in conscious RGS2 deleted (RGS2 -/-) and wild-type (RGS2 /) mice. We combined
telemetric arterial blood pressure recordings with fast Fourier analysis (FFT) of mean arterial
blood pressure (MAP) and heart rate (HR) and pharmacological autonomic testing to elucidate
Ang II effects in these mice. During Ang II infusion, BP increased to a greater degree in RGS2
-/- animals ( MAP day (113 vs. 33 mmHg and ( MAP night 123 vs. 73 mmHg) while
HR was not affected and ranged between 56315 and 59515 beats/min. Baroreflex
sensitivity was not different between the groups. Therefore, baroreflex resetting was more
pronounced in RGS2 -/- compared with wild type animals. Together, these observations
suggest, that RGS2 attenuates Ang II mediated resetting of baroreflex heart rate regulation. The
profoundly decreased urinary norepinephrine excretion rates in Ang II-treated RGS2 -/- and
RGS2 / mice (17.25.0 vs. 24.16.9 ng/day), compared to untreated RGS2 -/- and RGS2
/ mice (332.864.6 vs. 172.216.2 ng/day), suggest a reduced sympathetic activity.
Furthermor, the depressor response to prazosin was similar before and after Ang II infusion in
RGS2 -/- animals. Thus, the increased pressor response in RGS2 -/- mice cannot be explained
by an excessive increase in alpha-adrenoreceptor mediated vasoconstriction. The results
suggest that the interaction between RGS2 and Ang II-dependent vasoconstriction via the AT1A
receptor is important for blood pressure homeostasis. Deletion of RGS2 sensitizes Ang
II-dependent signaling in vivo. 1712
51
AAV Delivery of NADPH Oxidase gp91-shRNA Attenuates Cold-Induced
Hypertension and Vascular Hypertrophy
Xiuqing Wang, Lucille H Skelley, Zhongjie Sun; Univ Fl Coll Med, Gainesville, FL
Cold exposure increases NADPH oxidase gp91 protein expression. The aim of the present study
was to determine the effect of RNAi inhibition of gp91 expression on cold-induced
cardiovascular dysfunction. We hypothesized that RNAi inhibition of gp91 expression attenuates
cold-induced hypertension (CIH) and vascular hypertrophy. Recombinant AAV carrying short-
hairpin small interference (si)RNA for gp91 (AAV.gp91-shRNA) was constructed and tested for
the ability to inhibit gp91 to control CIH. Three groups of rats received AAV.gp91-shRNA
(1.25X1010 particles/rat, IV), AAV carrying scrambled shRNA (AAV.Control-shRNA., 1.25X1010
particles/rat, IV), and phosphate buffer solution (PBS), respectively. Following gene delivery, all
rats were exposed to moderate cold (44F) continuously. Blood pressure was monitored using
a telemetry system. AAV delivery of gp91-shRNA significantly attenuated cold-induced
elevation of blood pressure (BP). BP was 1215 mmHg, 1486 mmHg, and 1545 mmHg
for the AAV.gp91-shRNA-treated group, AAV.Control-shRNA-treated group, and PBS-treated
group, respectively. One single injection of AAV.gp91-shRNA can attenuate hypertension for up
to 10 weeks (length of the study). Western blot analysis indicated that gp91 protein expression
was inhibited by AAV.gp91-shRNA. DHE staining showed that the in situ superoxide production
was significantly decreased in aorta by AAV delivery of gp91-shRNA. AAV.gp91-shRNA
attenuated vascular hypertrophy. AAV delivery of gp91-shRNA significantly decreased TGF-
 e36 Hypertension Vol 48, No 4 October 2006
expression. Conclusions: (1) AAV delivery of gp91-shRNA effectively silenced gp91 expression
in vivo. (2) RNAi inhibition of gp91 may serve as a new approach for long-term control of
oxidative stress and related cardiovascular dysfunction. (Supported by NIH R01 HL077490).
52
Total Body Na and Osmotically Inactive Na Content Are Dependent on
the Agt Genotype in Mice
Jens Titze, Nada Cordasic, Claudia Handtrack, Bernd Klanke, Friedrich-Alexander-Univ
Erlangen-Nurnberg, Erlangen, Germany; Peter Dietsch, Charite, Berlin, Germany; Hubertus
Wagner, Federal Cntr for Nutrition and Food, Kulmbach, Germany; Karl F Hilgers;
Friedrich-Alexander-Univ Erlangen-Nurnberg, Erlangen, Germany
Water-free Na retention, either by osmotically inactive Na storage, or by osmotically neutral
Na/K-exchange, may play an important role for volume and blood pressure homeostasis.
However, the molecular mechanisms of water-free Na retention are unclear. We hypothesized
that increasing the gene dose of angiotensinogen (Agt) gene would increase the total body Na
content and alter the Na-to-water ratio in mice. From mice carrying either a deletion or a
duplication of the Agt gene, we bred fifty-eight animals with one, two, three or four copies
(Agt-1: n20; Agt-2: n 7; Agt-3: n13; Agt-4: n18) which were fed a normal (0.6%) salt increases NO dependency of BP and RVR in FHH. Despite lower BP, RVR tended to increase,
diet. At the end of the experiment, the carcasses were ashed and the total body Na, K, and
water content was measured. Increasing the Agt gene dose increased the total body Na
(0.1340.028 versus 0.1440.029, versus 0.1660.017, versus 0.1740.021 mmol/g dry
weight in Agt-1, Agt-2, Agt-3 and Agt-4 mice, respectively; p0.05), while the total body water
content was only moderately increased (0.6060.046 versus 0.6310.059, versus
0.6450.013, versus 0.6520.033 ml/g wet weight; p0.05). As increases in total body Na Effect of Cardiomyocyte Overexpression of Angiotensin II Type 2 Receptor
were not balanced by a corresponding K loss in the mice, the presence of increasing AGT
gene copies increased the total body (NaK)-to-water ratio (0.1940.015 versus
0.2000.010, versus 0.2080.013, versus 0.2110.007 mmol/ml; p0.05), indicating
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
osmotically inactive Na storage. In separate animals (n218), we confirmed that there was
a small but significant gene-dose effect on systolic blood pressure which increased by 3.51.1
mmHg for each Agt gene copy (p0.05). We conclude that increasing the Agt gene dose leads
to total body Na surplus and pronounced osmotically inactive Na storage in the mice.
Whether pronounced osmotically inactive Na storage in Agt-4 mice is secondary to total body
Na surplus, or directly regulated by the renin-angiotensin-system, remains to be investigated.
We speculate that osmotically inactive Na storage may contribute to buffer the effect of Na
accumulation on volume retention and blood pressure in this model.
53
P38 Map Kinase Inhibition Ameliorates Cardiac Damage without Affecting
Albuminuria in Angiotensin (Ang) II-Induced End-Organ Damage
Joon-Keun Park, Dept of Nephrology, Hannover Med Sch, Hannover, Germany; Ralf
Dechend, Robert Fischer, Erdenechimeg Shagdarsuren, Andrej Gapeljuk, Maren Wellner,
HELIOS, Franz-Volhard Clinic, Charite, Berlin, Germany; Silke Meiners, Dept of Cardiology,
Charite, Berlin, Germany; Petra Gratze, Nidal Al-Saadi, Anette Fiebeler, HELIOS,
Franz-Volhard Clinic, Charite, Berlin, Germany; Jeffery B Madwed, Boehringer Ingelheim
Pharmaceuticals, Ridgefield, CT; Alexander Schirdewan, HELIOS, Franz-Volhard Clinic,
Charite, Berlin, Germany; Hermann Haller, Dept of Nephrology, Hannover Med Sch,
Hannover, Germany; Friedrich C Luft, HELIOS, Franz-Volhard Clinic, Charite, Berlin,
Germany; Dominik N Muller; Max-Delbruck Cntr, Berlin, Germany
Ang II induces cardiac and renal damage. We investigated whether or not p38 inhibition
ameliorates end-organ damage in a rat model with high Ang II levels. We studied cardiac and
renal damage in double transgenic rats harboring both human renin and angiotensinogen genes
(dTGR) in protocol I from week 4 to 7 and protocol II from week 4 to 8, with or without p38
inhibitor (p38i; 30 mg/kg/d in the diet) treatment. Non-transgenic Sprague-Dawley (SD) rats
were controls. Systolic blood pressure (BP) was moderately increased at week 5 (1613 mm
Hg) and reached 2044 mm Hg at week 7 in untreated dTGR. P38i treatment partially reduced
BP (1667 mm Hg), while SD were normotensive. Albuminuria was not different between
untreated and p38i treated dTGR (335 vs. 305 mg/d), but increased compared to SD
(0.30.09 mg/d). While cardiac hypertrophy index (heart weight/tibia) was unchanged in
untreated and p38i-treated dTGR (3106 vs. 3076 mg/cm), the beta-MHC expression of
p38i-treated hearts was significantly lower compared to dTGR, indicating a delayed isoform
switch to the fetal phenotype. Fibrosis, macrophage infiltration, TNF- and IL-6 immunostain-
ing were significantly reduced in the heart by p38i, but only slightly reduced (IL-6, TNF- and
macrophages) or not (fibrosis) in the kidney. Vascular damage seemed to be improved in the
heart and the kidney. In protocol II, we focused on mortality and arrhythmogenic electrical
remodeling. Mortality of untreated dTGR was 100% (10/10) at week 8, whereas only one of 10
died in the p38i group (10%). Cardiac Magnetic Field Mapping showed prolongation of
depolarization and repolarization in untreated dTGR compared to SD (QRS 24.30.4 ms vs.
18.70.8 ms, QT 118.614.3 ms vs. 821.7 ms), which was partially reduced by treatment
with p38i (QRS 22.60.3 ms, QT 99.33 ms). Our findings indicate that p38i improved
survival, vascular, and cardiac damage/arrhythmogenic electrical remodeling, with a slight
effect on BP. In contrast, renal damage was not ameliorated to the same extent. Therefore, p38
inhibition appears to benefit the heart more than the kidney in this model.
54
Long-Term Effects of Perinatal Nitric Oxide Supplementation on Blood
Pressure and Glomerulosclerosis in Fawn-Hooded Hypertensive Rats
Maarten P Koeners, Branko Braam, Hein A Koomans, Roel Goldschmeding, Jaap A Joles;
UMC, Utrecht, The Netherlands
Perinatal treatments, that supposedly increase NO bioavailability, persistently reduced adult
blood pressure (BP) in SHR (Racasan ea 2004). Whether such perinatal treatment can also
prevent hypertension and associated renal injury in rats with a different genetic background is
unknown. We studied effects of brief perinatal molsidomine (NO donor) treatment in
Fawn-Hooded Hypertensive (FHH) rats, a genetic model of hypertension-associated renal injury
with defective regulation of preglomerular resistance. FHH dams received oral molsidomine
(120 mg/l) during the last 2 wk of pregnancy and all 4 wk of lactation. Systolic BP (SBP),
proteinuria and urinary excretion of NO metabolites (NOx) were measured in male and female
offspring. Mean arterial pressure (MAP) and renal vascular resistance (RVR) was measured
under pentobarbital anesthesia at 36 wk in males and 42 wk in females and during NO
synthase inhibition with L-NNA in males. Perinatal molsidomine persistently reduced SBP from
4 to 42 wk in females: 1231 vs. 1373 and from 4 to 36 wk in males: 1434 vs. 1513
mmHg (all P0.05). Urinary NOx excretion was significantly increased at 8 wk, i.e. 4 wk after
stopping molsidomine treatment in males and females (P0.05). At the end of the experiment
MAP was lower, significantly in females (P0.05). Despite lower MAP, renal vascular
resistance (RVR) increased, significantly in females (P0.05). During acute NOS inhibition at 36
wk in male FHH the increase in RVR was significantly higher (P0.05) in perinatally treated
FHH. There were no significant effects on proteinuria or glomerular counts, but perinatal
molsidomine caused about 50% decrease in glomerulosclerosis (GS) in males and females
(p0.01). Perinatal NO supplementation had clear programming effects on BP, NOx excretion
and development of GS in female and male FHH. In contrast to perinatally treated SHR, delta
MAP and delta RVR during NOS inhibition in FHH were higher in perinatally treated males
compared to controls, supporting that perinatal administration of a NO donor permanently
possibly indicating restoration of preglomerular resistance, either by structural adaptation or
increased activity of a vasoconstrictor.
55
on Cardiac Remodeling and Dysfunction in Mice with Myocardial Infarction
Ying Sun, Oscar A . Carretero, Jiang Xu, Chunxia Lin, Edward G. Shesely, Xiao-Ping Yang;
Henry Ford Hosp, Detroit, MI
Angiotensin II (Ang II) acts mainly on two receptors: AT1 and AT2. AT1 is known to mediate most
of the biological actions of Ang II, whereas the role of AT2 remains controversial. We previously
found that lack of AT2 diminished the beneficial cardiac effect of AT1 receptor blockers (ARB),
indicating a cardioprotective role of the AT2 receptor. Using transgenic mice overexpressing AT2
in ventricular myocytes (9 copies of the AT2 transgene, Tg-AT2), we tested the hypothesis that
cardiac remodeling and dysfunction post-myocardial infarction (MI) would be less severe and
the response to ARB would be greater in Tg-AT2 mice. Both Tg-AT2 and their wild-type
littermates (WT) were divided into 1) sham MI; 2) MI vehicle; and 3) MI ARB (losartan, 30
mg/kg/day, via drinking water). Treatment were started 4 weeks after MI and continued for 8
weeks. Systolic blood pressure (SBP) was measured weekly by tail cuff. Left ventricular ejection
fraction (EF) and systolic (LVDs) and diastolic dimension (LVDd) were evaluated by echocar-
diography. In sham-MI, these parameters did not differ between strains. MI increased LVW and
chamber dimensions and decreased EF to a similar extent in both strains. Losartan decreased
LV weight (LVW), LVDd and LVDs and increased EF in WT and these effects were diminished
in Tg-AT2 mice (Table). Our data suggest that overexpression of the AT2 receptor only in
cardiomyocytes does not provide cardiac protection, nor aggravate cardiac dysfunction and
remodeling post-MI; however, it diminishes the cardioprotective effect of AT1 blockade. The
precise mechanism needs to be studied further.
Sham MI MIvehicle MI losartan
WT TG WT TG WT TG
LVW (mg/10g) 321 331 431* 442* 391# 412
EF (%) 782 781 373* 353* 484# 362
LVDd (mm) 2.70.1 2.50.1 4.10.2* 4.20.2* 3.50.2# 4.30.1
LVDs (mm) 1.20.1 1.20.1 3.00.2* 3.20.2* 2.40.3# 3.40.1
*p0.01 vs sham MI within strains; #p0.05 vs MIvehicle within strains;
p0.05 vs WT-MI losartan
56
Genetic Variation at the CYP11B Locus Accounts for Heritabilities of
Aldosterone Metabolite (THAldo) Excretion and 11Beta-Hydroxylase Activity
Marie Freel, Univ of Glasgow, Glasgow, United Kingdom; Bernard Keavney, Peter Avery,
Univ of Newcastle, Newcastle, United Kingdom; Bongani Mayosi, Univ of Cape Town, Cape
Town, South Africa; Nicole Gaukrodger, Helen Imrie, Michelle Baker, Univ of Newcastle,
Newcastle, United Kingdom; Robert Fraser, Mary Ingram, Univ of Glasgow, Glasgow, United
Kingdom; Hugh Watkins, Martin Farrall, Univ of Oxford, Oxford, United Kingdom; Eleanor
Davies, John Connell; Univ of Glasgow, Glasgow, United Kingdom
Aldosterone is a key cardiovascular hormone: 15 % of hypertensives have altered aldosterone
regulation, defined by a raised ratio of aldosterone to renin. However, the causes of aldosterone
excess are not understood. Polymorphic variation in the gene encoding aldosterone synthase
(CYP11B2) is associated with hypertension, but the best characterised intermediate phenotype
is a relative reduction in efficiency of 11-hydroxylation (conversion of deoxycortisol to
cortisol), which reflects function of the enzyme,11-hydroxylase, encoded by the adjacent
gene (CYP11B1). This is expressed in zona fasciculata, and is not involved in aldosterone
synthesis. To characterise better the genetic regulation of aldosterone synthesis we have used
a family study to define heritability of steroid phenotypes and identify key genetic determinants.
We genotyped 6 polymorphisms in CYP11B2 and 3 in CYP11B1 in 248 nuclear families and
measured urinary excretion rates of the major metabolites of aldosterone (THAldo), deoxycor-
tisol (THS) total cortisol (F) and androgens in 573 subjects from 105 families. The efficiency of
11-hydroxylation, previously noted to be associated with CYP11B2, was assessed by the
THS/F ratio. THAldo and THS/F were highly heritable (p0.0001). THAldo excretion and THS/F
associated most strongly with polymorphisms in CYP11B1 (exon 1 and intron 3) (0.001).
THAldo excretion was closely correlated with F, androgens and THS/F (all p0.001). We have
 CHBPR ConferenceOral Presentations e37
shown, for the first time, that aldosterone production is heritable, reflecting genetic regulation,
and confirmed the heritability of THS/F, the index of 11-hydroxylation. The same polymor-
phisms in CYP11B1 account for variability in THAldo excretion and reduced efficiency of
11-hydroxylation, consistent with the hypothesis that a genetically determined change in
11hydroxylase efficiency leads to an increase in adrenal ACTH drive that, over years, amplifies
aldosterone production. This proposal is strongly supported by the correlations demonstrated
between THAldo and ACTH-dependent steroids (cortisol and androgens) and between THAldo
and the index of 11hydroxylase efficiency. These data provide novel insights into the possible
origins of aldosterone-associated hypertension.

57
Adrenal Steroids and the Metabolic Syndrome in African Americans

Srividya Kidambi, Jane M Kotchen, Clarence E Grim, Shanthi Krishanswami, Hershel Raff,
Theodore A Kotchen; Med College of Wisconsin, Milwaukee, WI

The Metabolic Syndrome describes the cluster of hypertension with metabolic CVD risk factors.
African Americans have a high prevalence of hypertension-related CVD and adrenal cortical
adenomas/hyperplasia. We evaluated the hypothesis that adrenal steroids are associated with
metabolic syndrome risk factors in African Americans. Ambulatory blood pressures, anthropo- 59
metric measurements, and standardized measurements of PRA, aldosterone, fasting lipids,
glucose, and insulin were obtained in 261 African Americans (48% hypertensive, 47% female),
Rapid Aldosterone-Mediated Effects in Vascular Smooth Muscle Cells
studied on an inpatient CRC after discontinuing antihypertensive and lipid lowering medications
Robert Gros, Qingming Ding, Souzan Armstrong, Caroline ONeil, J. Geoffrey Pickering, Ross
for 1 and 4 weeks, respectively. Hypertension was defined as an average ambulatory blood
D Feldman; Robarts Rsch Institute, London, Canada
pressure 130/85 mmHg. Insulin resistance was estimated with the Homeostasis Model
Assessment (HOMA). Late night and early morning salivary cortisol, and 24-hour urine free
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It has been increasingly appreciated that aldosterone (ALDO) elicits acute vascular effects
cortisol excretion were measured in a sub sample of 68 subjects (41% hypertensive). Results
through non-genomic signalling pathways. Our previous studies demonstrated that ALDO
are described in the table below.
attenuated phenylephrine-mediated constriction in intact vessels (via PI3 kinase-dependent
METABOLIC/HORMONAL TRAITS IN STUDY SUBJECTS NOS activation) but enhanced vasoconstrictor responses in endothelium-denuded arteries. To
determine the mechanism of this contractile response, we assessed the effect of ALDO on
Normotensives Hypertensives p-value
myosin light chain (MLC) phosphorylation and contraction in clonal adult human vascular
Age (years) 42 1 SE 45 1 0.01 smooth muscle cells (HITC6). Acute incubations of HITC6 cells with increasing doses of ALDO
BP (mmHg) 115 1/69 1 147 1/89 1 (0.1100 nmol/L) mediated a dose-dependent increase in MLC phosphorylation. ALDO (100
Waist circumference (cm) 87 1 94 1 0.0001 nmol/L)-mediated MLC phosphorylation was inhibited by pretreatment with either spironolac-
Plasma glucose (mg/dl) 88 1 90 1 ns
tone (10 mol/L) or the PI3 kinase inhibitor, LY294002 (50 mol/L). These rapid effects of
Plasma insulin (mIu/ml) 11.2 0.5 12.7 0.5 0.01
Total cholesterol (mg/dl) 175 3 185 4 0.05 ALDO on MLC phosphorylation were mimicked by 17-estradiol (100 nmol/L) and hydrocor-
LDL (mg/dl) 108 3 117 3 0.04 tisone (100 nmol/L). Paralleling the effects on MLC phosphorylation, ALDO-mediated a
HDL (mg./dl) 50 1 47 1 0.01 dose-dependent contraction response in HITC6 cells (EC50 of 19910 pmol/L, Emax 596%
Triglycerides (mg/dl) 93 4 105 5 0.09 of ionomycin-mediated contraction), that was inhibited by pretreatment with spironolactone as
HOMA index 1.4 0.1 1.6 0.1 0.01 well as by LY294002. In addition, comparable contractile responses were seen with both
Plasma Aldosterone (ng/dl) 6.3 0.4 8.4 0.4 0.0001 17-estradiol (EC50 of 9.20.1 nmol/L, Emax 778% of ionomycin-mediated contraction) and
PRA (ng/ml/hr) 1.7 0.2 1.1 0.1 0.01
hydrocortisone (EC50 of 1.70.1 nmol/L, Emax 599% % of ionomycin-mediated contrac-
Late night salivary cortisol 1.5 0.2 2.3 0.3 0.02
(nmol/L) tion). In total, these data are consistent with a mechanism of acute ALDO-mediated contraction
acting via PI3 kinase-dependent activation. Further, this effect is common to both glucocor-
ticoids and estrogen. Steroid-mediated vasoconstriction may represent an important patho-
Hypertensives had greater waist circumference, less favorable lipid profiles, were more insulin
biological mechanism of vascular disease- especially in the setting of pre-existing endothelial
resistant, had lower PRA and higher plasma aldosterone and late night salivary cortisol
dysfunction.
concentrations. Early morning salivary cortisol and 24-hour urine free cortisol did not differ.
Based on Adult Treatment Panel III criteria, 45% of hypertensive and 4% of normotensive
subjects had the Metabolic Syndrome (p 0.0001). Overall, ambulatory systolic blood pressure
was correlated with plasma aldosterone (r 0.31, p 0.0001) and late night salivary cortisol
(r0.31, p 0.01). The relationship of blood pressure with both aldosterone and cortisol raises
the possibility that adrenal mineralocorticoids and glucocorticoids contribute to hypertension
and associated metabolic CVD risk factors in African Americans. 60
Aldosterone and Salt-Induced Cardiac Fibrosis is Exaggerated in
Angiotensin II Type 1a Receptor Knockout Mice

58
Aldosterone Deficiency Attenuates Angiotensin II-Stimulated Plasminogen
Activator Inhibitor-1 Gene Expression
James M Luther, Zuofei Wang, Ji Ma, Vanderbilt Univ, Nashville, TN; Natalia Makhanova,
Hyung-Suk Kim, Univ of North Carolina, Chapel Hill, NC; Nancy J Brown; Vanderbilt Univ,
Nashville, TN
To test the hypothesis that Ang II increases plasminogen activator inhibitor-1 (PAI-1) expression
mediated by aldosterone, we administered Ang II (600ng/kg/min IV x 4 hrs) or vehicle to male
wild type (WT) and aldosterone synthase deficient (KO) mice, both on a C57BL/6 background
(n5/group). Two additional KO groups were given aldosterone (500 ng/d via minipump) for 7
days prior to study. Carotid arterial and jugular venous catheters were implanted 35 days prior
to study. Baseline systolic blood pressure in KO mice was lower than in WT (1253 vs 1113
mmHg, P0.006) and K (5.50.2 vs. 4.70.2 mmol/L, P0.02) was higher. Urinary K
(38040 mol/24hr) and Na (300130 mol/24hr) excretion was similar between groups.
Plasma BUN (263 vs. 171 mg/dL, P0.01) was higher, while Na (142.10.6 vs.
146.81.8 mmol/L, P0.004) and Cl- (1120.7 vs. 115.71.6 mmol/L, P0.03) were lower
in KO mice. Aldosterone replacement normalized SBP (1206 mmHg), K (4.30.2, P0.001
vs. KO), Na, and BUN. Ang II infusion increased SBP in WT (19.67 mmHg) and KO (16.49),
and increased SBP to a greater extent in KO mice after aldosterone replacement (34.84.4
mmHg, P0.05 for time x group). Body weight and relative heart weight (5.40.4 vs. 5.70.2
mg/g) were similar between groups while relative kidney weight was lower in KO (6.10.3 vs.
7.70.4 mg/g, P0.003). Ang II increased cardiac PAI-1 gene expression in WT but not KO
(Figure 1). Aldosterone replacement normalized Ang II-stimulated cardiac PAI-1 gene expres-
sion. Aldosterone enhances Ang II-stimulated PAI-1 gene expression in vivo. These data have
implication for the development of pharmacological inhibitors of aldosterone synthesis.
Shuntarou Kagiyama, Kiyoshi Matsumura, Masayo Fukuhara, Kanae Higuchi, Koji Fujii,
Mitsuo Iida; Kyushu Univ, Fukuoka, Japan
Background: Aldosterone infusion with high salt treatment induces interstitial and perivascular
fibrosis in the heart of rats. Several studies reported that aldosterone enhanced angiotensin II
(AngII) induced proliferation and increased the expression of AngII receptor mRNA and AngII
binding in vascular smooth muscle cells. To investigate the role of AngII type 1a receptor
(AT1aR) in aldosterone and salt (Ald-NaCl)-induced cardiac fibrosis, we used AT1aR knockout
mice (AT1aR-KO). Methods and Results: Aldosterone (0.15 g/hr) was infused subcutane-
ously with 1% NaCl (Ald-NaCl) in drinking water on AT1aR-KO or wild type mice (Wt). To
examine the role of NaCl on cardiac fibrosis, we fed some of the AT1aR-KO receiving
aldosterone with tap water (Ald-H2O). After Ald-NaCl treatment, systolic BP in both strains was
significantly higher than that in mice not receiving aldosterone, but systolic BP in Ald-NaCl-
treated AT1aR-KO was still significantly (p0.05) lower than that in Wt. Left ventricular
weights/body weights in Ald-NaCl-treated AT1aR-KO and Wt were significantly higher than in
the mice receiving only NaCl but did not differ between the strains.Severe cardiac fibrosis was
seen only in Ald-NaCl-treated AT1aR-KO and not in Ald-NaCl-treated Wt. Ald-H2O-treated
AT1aR-KO did not show any cardiac fibrosis. We performed RT-PCR to investigate the effect of
Ald-NaCl treatment on the expressions of mRNA of sodium-handling related genes in cardiac
tissues. Epithelial Na channel and subunit were not detected, and subunit was detected
but did not differ in strain or treatment. Aldosterone increased the expression of Na-K
ATPase 1 subunit in Wt but not in AT1aR-KO. The expression of 2 subunit was decreased
in AT1aR-KO, but 1 subunit was unchanged. Na/Ca2 exchanger was decreased in
AT1aR-KO and was further decreased by aldosterone treatment.Conclusions: These results
suggest that the Ald-NaCl-induced cardiac fibrosis required both aldosterone and NaCl
administration. As cardiac fibrosis was exaggerated in AT1aR-KO mice, AT1aR may not be
essential for this phenomenon. Although precise mechanisms remain unresolved, alterations in
sodium handling genes transcription were present and might be implicated in the present
findings.
 e38 Hypertension Vol 48, No 4 October 2006

Comparative Effects of Mineralocorticoid Receptor Antagonism and
Aldosterone Synthase Inhibition on Angiotensin II-Induced End Organ
Damage
William B Lea, Eun Soo Kwak, Agnes B Fogo, Nancy J Brown; Vanderbilt Univ, Nashville, TN
Mineralocorticoid receptor antagonism or the aldosterone synthase inhibition reduces Ang
II-induced end organ damage. However, the effect of the two pharmacologic strategies has
never been compared. We compared effects of spironolactone (5.8mg/kg/d) and FAD286
(4mg/kg/d) in uninephrectomized Sprague-Dawley rats treated with high sodium intake (1%
saline) and either saline or Ang II (1/h) via osmotic minipump for 8 weeks. Spironolactone or
FAD286 prevented the hypertensive response to uninephrectomy alone but did not attenuate
the SBP response to Ang IIuninephrectomy. FAD286, but not spironolactone, significantly
decreased plasma aldosterone during Ang II infusion. Serum potassium, sodium and urine
potassium and sodium excretion were comparable in spironolactone and FAD286 treatment
groups. Ang II induced cardiac hypertrophy and either spironolactone or FAD286 prevented this
effect. Unilateral nephrectomy caused hypertrophy of the remaining kidney, which was
increased by Ang II. Spironolactone and FAD286 prevented Ang II induced renal hypertrophy,
albuminuria and azotemia. Mineralocorticoid receptor antagonism and aldosterone synthase
antagonism similarly protect against Ang II induced cardiac hypertrophy and renal injury
through pressure-independent mechanisms. Aldosterone synthase inhibition also attenuated
Ang II-induced aortic medial thickening.
Uninephrectomy Uninephrectomy
Sham Uninephrectomy Spironolactone FAD286
SBP 157.72.7 168.02.4* 159.12.2 162.92.5 172.22.5*
(mmHg)
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61 experiments were performed in the presence of catalase (100 U/ml). We found that O2-
treatment selectively increased PKC alpha activity by 177% (from 4.7 1.2 to 13.2 1.7 A.U;
p0.02; n5) and PKC delta activity by 37% (from 12.6 1.0 to 17.3 1.4 A.U.; p0.05;
n5). O2- did not activate the other isoforms tested (beta, gamma, epsilon, theta, iota, lambda
and zeta). In isolated perfused tubules, O2- treatment increased JCl by 42 13% (p0.05;
n5). In the presence of the PKC alpha/beta-selective inhibitor Go6976, O2- did not significantly
increase JCl (from 132.2 24.5 to 136.7 28.2 pmol/min/mm; n5). Pre-treatment of thick
ascending limb tubules with rottlerin induced cell detachment from the basal membrane, cell
swelling and necrosis at all concentrations tested. We conclude that: 1) O2- stimulates specific
isoforms of PKC in the thick ascending limb; 2) activation of PKC alpha is required for O2- to
stimulate NaCl absorption in the thick ascending limb; and 3) the role of PKC delta in O2-
-stimulated NaCl absorption in the thick ascending limb could not be tested because it appears
to be necessary for cell survival.
64
cGMP Decreases Surface NKCC2 Levels in Thick Ascending Limbs
Gustavo R Ares, Pablo A Ortiz; Henry Ford Hosp, Detroit, MI
NaCl absorption in the TAL is mediated by the apical Na/K/2Cl cotransporter, NKCC2. We
showed that NKCC2 levels in the apical membrane determine NaCl absorption by the TAL. Nitric
oxide and atrial natriuretic peptide decrease NaCl absorption in the thick ascending limb (TAL)
via cGMP. However, the mechanism by which cGMP decreases NaCl absorption in TALs is not
Uninephrectomy Uninephrectomy known. We hypothesized that cGMP decreases NKCC2 levels in the apical membrane of TALs.
Uninephrectomy Ang II Ang II We used surface biotinylation and Western blot to measure NKCC2 levels in the apical
Ang II Spironolactone FAD286 membrane of rat TAL suspensions. Under basal conditions, surface NKCC2 levels were 3.4
171.72.5* 167.42.4*
0.7% (n9) of total NKCC2, similar to what we previously reported. We first tested whether the
membrane-permeant cGMP analog, dibutyryl cGMP (db-cGMP) could decrease surface NKCC2
levels. Adding db-cGMP (100 M) to the bath for 20 min decreased surface NKCC2 levels by

Aldosterone (pg/ml) 20875 16840 14218 13136 472159* 374160 13910

HW/BW (mg/g) 3.50.2 3.80.3 3.80.3 3.80.1 4.90.2*
Aortic media (m) 139.010.1 149.26.8 171.215.8 141.55.4 181.315.2*
KW/BW (mg/g) 3.80.3 5.60.2* 5.50.3* 5.80.4* 6.80.6*||
BUN (mg/dL) 22.52.1 24.92.0 26.61.1 26.21.9 46.78.0*
Albumin/Cr (g/mg) 460226 1027407 28272514 1062488 35831606*
*P0.01 versus sham, P0.05 versus uninephrectomy, uninephrectomyspironolactone, or uninephrectomyFAD286, P0.05 versus
uninephrectomyAng IIFAD286, P0.05 versus uninephrectomyAng IIspironolactone, || P0.05 versus uninephrectomyAng IIspironolactone,
P0.05 versus uninephrectomyFAD286
62
Synergetic Interaction between Aldosterone and Angiotensin II Induces
Activation of c-Src and NADPH Oxidase but not of MLC in Vascular Smooth
Muscle Cells
Augusto C Montezano, Glaucia E Callera, Ottawa Health Rsch Institute, Ottawa, Canada;
Ernesto L Schiffrin, Lady Davis Institute for Med Rsch, Montreal, Canada; Rhian M Touyz;
Ottawa Health Rsch Institute, Ottawa, Canada
Aldosterone (Aldo) exerts a synergistic mitogenic effect with angiotensin II (Ang II) through
ERK1/2 activation in vascular smooth muscle cells (VSMC). Whether similar interactions
influence contractile and redox signaling pathways remain unclear. Here we investigated the
cross-talk of c-Src-sensitive NADPH oxidase-generated O2- signaling between Aldo and Ang II
and assessed whether this interaction influences pro-contractile events. Cultured rat mesen-
teric vascular smooth muscle cells were studied. Activation of c-Src was determined by
immunoblotting using a phospho-specific antibody. NADPH-dependent generation of O2- was
measured by enhanced lucigenin (5mol) chemiluminescence. Phosphorylation of myosin light
chain (MLC) was assessed as a molecular signaling marker of contraction. Whereas high
concentrations (10-8 mol/L) of Aldo and Ang II significantly increased activation of c-Src
(2-fold) and NADPH oxidase (2-fold), low concentration (10-10 mol/L) of Aldo and Ang II
were without significant effect. High dose Ang II and Aldo induced phosphorylation of MLC.
Co-stimulation of VSMCs with combined low dose Aldo and Ang II significantly increased c-Src
phosphorylation (0.5-fold above basal, p0.05) and NAD(P)H-dependent generation of O2-
(1-fold, p0.05). MLC phosphorylation was not affected by low dose Aldo/Ang stimulation.
c-Src phosphorylation was inhibited by a selective AT1 receptor antagonist, Ibersartan (10-6
mol/L), or a mineralocorticoid receptor antagonist, eplerenone (10-6 mol/L). Our results suggest
that Aldo exerts a synergistic c-Src/redox signaling effect with Ang II. However, signaling events
associated with activation of MLC, typically involved in VSMC contraction, are independent of
synergistic actions between Aldo and Ang II. Whereas synergism may be important in mitogenic
responses in VSMCs, pathways involved in contraction may not be. These findings indicate
differential regulation of Ang II-mediated signaling events by Aldo.
PKC Alpha Activation is Required In O2- -Stimulated NaCl Absorption in
Thick Ascending Limb
Guillermo B Silva, Pablo A Ortiz, Jeffrey L Garvin; Henry Ford Hosp, Detroit, MI
Superoxide (O2-) is an important regulator of kidney function. The thick ascending limb
reabsorbs 20 30% of the filtered load of NaCl. Abnormal sodium retention in this segment has
been shown to be involved in hypertension. O2- stimulates NaCl absorption by the thick
ascending limb by enhancing Na/K/2Cl- co-transport activity. Protein kinase C (PKC)
stimulates thick ascending limb transport. Thus we hypothesized that O2- stimulates NaCl
absorption by selective activation of specific PKC isoforms. To test this, we measured the effect
of O2- on activation of classical, novel and atypical PKC isoforms by membrane fractionation and
Western blot. In isolated perfused tubules we measured the effect of the PKC alpha/beta-
selective inhibitor Go6976 and the PKC delta-selective inhibitor rottlerin on O2--induced Cl-
absorption (JCl). Medullary thick ascending limbs were exposed to O2- by adding xanthine
oxidase (1 mU/mL) and hypoxanthine (0.5 mmol/L) to the bath. To avoid formation of H2O2,
3.80.1 4.10.3 26 7% (n 4, p0.02) whereas db-cGMP at 500 M decreased surface NKCC2 by 46
181.812.6* 151.816.6 10% (n 5, p0.01). A higher concentration of db-cGMP (1 mM) did not decrease surface
5.30.2* 5.80.3*
NKCC2 levels further (n5). Parallel control experiments showed no biotinylation of intracel-
23.80.9 30.80.6
26211074 25371694
lular proteins, indicating that only surface proteins were being biotinylated. To test whether
cGMP regulates NKCC2 trafficking rather than total NKCC2 expression, we measured the effect
of db-cGMP (500 M) on total NKCC2. We found no change in total NKCC2 expression
compared to basal (basal: 100%, db-cGMP: 114 14 %, n 4, n.s.). We concluded that
cGMP decreases NKCC2 levels in the apical membrane of TALs by regulating NKCC2 trafficking.
This may be the primary mechanism by which NO and other natriuretic hormones that act via
cGMP inhibit NaCl absorption by the TAL.
65
Extracellular Renal Cyclic Gmp is Required for Nitric Oxide-Induced
Natriuresis in the Rat
Farah Ahmed, Nancy L Howell, Brandon A Kemp, Helmy M Siragy, Robert M Carey; Univ
Virginia Sch Med, Charlottesville, VA
Previous studies from our laboratory have demonstrated that extracellular guanosine cyclic 3,
5-monophosphate (cGMP) inhibits Na transport directly in proximal tubule cells and that renal
interstitial (RI) cGMP induces natriuresis and modulates pressure-natriuresis in vivo in the rat.
The present study addresses the hypothesis that nitric oxide (NO)-induced natriuresis is
abolished when cGMP is prevented from being transported outside its synthesizing cells in vivo.
Sprague-Dawley rats (N13) were anesthetized and uninephrectomized and the remaining
kidney was implanted with a renal cortical interstitial micro-infusion catheter and a separate
microdialysis probe for measuring RI cGMP. Rats were studied during a 1 h control period
(vehicle infusion) following which they received a RI infusion of NO donor S-nitroso-N-
acetylpenicillamine (SNAP; 0.12 nmol/kg/min; N7) or SNAP combined with organic anion
transporter inhibitor probenecid (PB; 10 g/kg/min; N6) for two consecutive 1 h experimental
periods, followed by a 1 h post-control period when vehicle was infused. All RI infusions were
at 2.5 l/min and blood pressure (BP), urinary Na excretion (UNaV) and RI cGMP were
quantified for each control and experimental period. In response to intrarenal infusion of SNAP
alone, UNaV increased from a control of 0.05 0.02 to 0.12 0.06 mol/min (PNS) at 1 h
and to 0.14 0.03 mol/min (P0.01) at 2 h, followed by a reduction to 0.07 0.03
mol/min (PNS) during the post-control period. In contrast, SNAP failed to increase UNaV
when co-infused with PB during either experimental period. Indeed, PB decreased UNaV from
0.07 0.01 to 0.03 0.01 mol/min during the second SNAP infusion period (P0.01 from
SNAP alone). SNAP alone increased RI cGMP from 2.2 0.6 to 3.6 1.1 pmol/ml (PNS) in
the first experimental period and to 4.5 1.3 pmol/ml (P0.01) during the second
experimental period. In contrast, PB abolished the increase in RI cGMP in response to SNAP
alone in both experimental periods (P0.01 from SNAP alone in the second period). The data
demonstrate that export of cGMP from its synthesizing cells into the RI compartment is required
63 for the natriuretic action of NO donor SNAP. Extracellular RI cGMP mediates NO-induced
natriuretic responses.
66
Increased Renal Na Transporters and Altered AT1R and Dopamine
Receptors Are Associated with Hypertension in D3 Dopamine Receptor
Deficient Mice
Xiaoyan Wang, Ines Armando, Laureano Asico, John E Jones, Zheng Wang, Van Anthony M
Villar, Crisanto S Escano, Pedro A Jose; Georgetown Univ Med Cntr, Washington, DC
D3 dopamine receptor (D3R) deficient mice (D3-/-) have impaired ability to excrete a sodium
load and renin-dependent hypertension. Lack of one dopamine receptor gene may alter the
expression of other G protein-coupled receptors (GPCR) and renal Na transporters which may
contribute to hypertension. We studied the expression of dopamine receptors and renal Na
transporters in D3-/- and their wild type (D3/) littermates. On a normal NaCl (0.8%) diet,

systolic blood pressure (SBP) (telemetry), was higher in D3-/- than in D3/ (night time:
1323 vs. 1223 mm Hg; n 7; p 0.001). Renal D1R and D2R proteins were the same;
D4R was decreased (599%) while D5R was increased in D3-/- (16923%) relative to
D3/ (10010%; p0.05). Infusion of SCH 23390, a D1/D5R antagonist (120 ng/k/min x
1h) into anesthetized mice increased BP in D3-/- (from 1131 to 1283, n3; p0.05) but
not in D3/, suggesting that D5Rs partially compensated for the lack of D3Rs. AT1R
expression (autoradiography) in renal proximal tubules was greater in D3-/- (955 fmol/mg
prot) than in D3/ (76 5; p 0.05); AT1 receptor expression in other areas was similar.
On a normal salt diet, NHE3 (major proximal tubule Na transporter) and NCC (distal convoluted
tubule Na transporter) proteins were increased in D3-/- (28427% and 59641%, respec-
tively). On a low NaCl diet (0.04%), D3-/- had increased expression of alpha ENaC (39455%)
and gamma ENaC (14418%, 75 kDa, active form). On high NaCl diet (1.6%), NCC
(20924%) and alpha ENaC (25839%) proteins were increased in D3-/- (all data relative to
D3/). Our results suggest that by direct or indirect mechanisms the D3R regulates the renal
expression of other GPCR (e.g., D4R, D5R, and AT1R) and Na transporters. The high BP in
D3-/- is mitigated by upregulation of D5R. Eventual BP is the result of the interaction of several
genes (e.g., dopamine receptors and AT1R), consistent with the polygenic regulation of BP.
67
Cholesterol Facilitates Hypertension by Stimulating the Renal Epithelial
Sodium Channel
He-Ping Ma, Jing Wang, You-You Liang, David G Warnock; Univ of Alabama at Birmingham,
Birmingham, AL
CHBPR ConferenceOral Presentations e39
vasculature may lead to vascular dysfunction and hypertension. To test this hypothesis, we
generated transgenic mouse models targeting dominant negative mutants of hPPAR (either
P467L or V290M) to the vascular E and SM using the vascular E-specific Ve-cadherin promoter
or the SM-specific SM myosin heavy chain promoter, respectively. The dominant negatives
should produce cell-specific knock-downs of PPAR. Expression of each transgene was
confirmed and differentiated from the endogenous PPAR via RNase protection assay. No
expression of the transgene was observed in non-transgenic (NT) littermates. Physiologic
characterization of the E-V290M model revealed significant impairment of E-dependent
relaxation of aorta to acetylcholine (Ach, 454% vs 544% in NT at 10M, n6), whereas
the response to nitroprusside was normal. In the basilar artery, there was also a trend toward
impaired relaxation to Ach (656% vs 767% in NT at 100 M, n7) and the calcium
ionophore A23187 (585% vs 676% in NT at 0.1 M, n7). Interestingly, preliminary
studies suggest that the impairment in endothelial function was greater in females than males.
We also examined contraction of the aorta in response to several vasoconstrictors. Contraction
to PGF2 was greater in E-V290M (1.70.1g, n7) than NT littermates (1.40.1g, n6).
Contraction of the aorta in females in response to endothelin-1, a PPAR target gene, was
greater in E-V290M (585 mg, n5) than in NT (328 mg, n3). Our data suggest that
PPAR plays and important role in the regulation of vascular tone. Studies identifying the
specific molecular and signaling pathways regulating vascular function, and physiological
analysis of SM-P467L and SM-V290M mice are currently in progress.
70
Ac-SDKP Attenuates Angiotensin II-Induced Collagen Deposition and
Macrophage Infiltration in the Aortic Media

Hypertension can be eventually developed due to the well-known effect of cholesterol on
vascular walls. However, a significant portion of the patients with hypercholesterolemia
appears to have hypertension before any morphological changes in vascular walls. This clinical
observation indicates that cholesterol may also cause hypertension by affecting an organ rather
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
Chunxia Lin, Xiaoping Yang, Tangdong Liao, Martin D Ambrosio, Oscar A Carretero; Henry
Ford Hosp, Detroit, MI
We previously reported that N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibits interstitial

than cardiovascular system. Since excessive sodium reabsorption by the kidney due to and perivascular fibrosis in the heart and kidney and this effect may be partly due to inhibition
enhanced activity of the renal epithelial sodium channel (ENaC) can result in hypertension, as of macrophage infiltration and collagen deposition. In the present study, we hypothesized that
seen in Liddles syndrome, we hypothesized that cholesterol might facilitate hypertension by in angiotensin (Ang II) - induced hypertension, Ac-SDKP prevents vascular inflammation and
stimulating the renal ENaC. To test this hypothesis, patch-clamp experiments and transepi- fibrosis by decreasing macrophage infiltration and collagen deposition. SD rats were divided
thelial current measurement were performed by using A6 distal nephron cells cultured on into 4 groups and treated for 1 week with: 1) vehicle; 2) Ac-SDKP (800 g/kg/D, via osmotic
permeable support. The data demonstrated that application of cholesterol (final concentra- minipump); 3) Ang II (750 g/kg/D, via osmotic minipump); or 4) Ang IIAc-SDKP. We found:
tion 50 g/ml) to the basolateral bath significantly increased ENaC open probability from 1) Ang II significantly increased systolic blood pressure (SBP), aortic medial area (hematoxylin
0.28 0.12 to 0.63 0.17 in five cell-attached patches (p 0.01) and also elevated and eosin staining), collagen I and III deposition (picrosirius red staining) and macrophage
amiloride-sensitive current across A6 cell monolayer (n 24; P 0.001). However, number (immunohistochemical staining) in the aortic media; 2) Ac-SDKP had no effect on Ang
application of cholesterol to the apical bath did not affect ENaC open probability (0.31 0.09 II-induced hypertension and medial hypertrophy; however, 3) Ac-SDKP greatly reduced Ang
versus 0.29 0.10) in four cell-attached patches and did not affect amiloride-sensitive current II-induced collagen I and III deposition and macrophage infiltration in the aortic media (see
across A6 cell monolayer (n 18). To monitor cholesterol transport across the cell membrane, table). These data suggest that in Ang II-induced hypertension, Ac-SDKP inhibits macrophage
confocal microscopy experiments were performed by using a fluorescence-labeled cholesterol infiltration and collagen deposition in the aortic media via a hypertension-independent
(NBD-cholesterol). The data demonstrated that there was an acute cholesterol uptake across mechanism.
the basolateral membrane, but not the apical membrane. These data together suggest that
basolateral cholesterol uptake, somehow, significantly stimulates ENaC in A6 distal nephron vehicle Ac-SDKP Ang II Ang IIAc-SDKP
cells. This finding may uncover a novel pathway for cholesterol to induce hypertension. SBP (6th day,mm Hg) 1191 1214 1897** 19113
Aortic medial area 59.166.00 66.302.11 102.301.66** 87.876.62
(10000, m2)
68 Collagen I (% of medial area) 10.751.66 11.640.40 19.082.03* 9.291.94#
Collagen III (% of medial area) 2.590.20 2.620.36 4.850.62* 1.550.37##
NOS Uncoupling in the Kidney of Dahl S Rats - Role of Dihydrobiopterin Macrophage number 2.370.85 1.870.94 19.380.35** 9.761.72##
(per mm2 of medial area)
Norman E Taylor, Med College of Wisconsin, Milwaukee, WI; Kristopher G Maier, SUNY Values are mean SE, *p0.05, p0.01, Ang II vs vehicle; # p0.05, p0.01, Ang II vs Ang
Upstate Med Univ, Syracuse, NY; Richard J Roman, Allen W Cowley, Jr.; Med College of IIAc-SDKP
Wisconsin, Milwaukee, WI

Nitric oxide synthase (NOS) can paradoxically contribute to the production of reactive oxygen 71
species (ROS) when L-arginine or the cofactor R-tetrahydrobiopterin (BH4) become limited. The Decreased Physiological Levels of Ac-sdkp Increase Perivascular Fibrosis
present study examined whether NOS contributes to superoxide (O2-) production in kidneys of
hypertensive Dahl salt-sensitive rats (SS) compared to an inbred consomic control strain Maria A Cavasin, Tang-Dong Liao, Xiao-Ping Yang, Oscar A Carretero; Henry Ford Health
(SS-13BN) and tested the hypothesis that elevated dihydrobiopterin (BH2) levels are importantly System, Detroit, MI
involved in this process. This was assessed by determining the effects of L-nitroarginine methyl
ester (L-NAME) inhibition of NOS on O2- production and by comparing tissue concentrations of We previously showed that prolyl oligopeptidase (POP) is the enzyme responsible for release of
BH4 and BH2. A RP-HPLC method was applied for direct measurements of BH4 and BH2 using N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) from its precursor thymosin-4, and oral
(S)-tetrahydrobiopterin as an internal standard. O2- concentrations were measured in-vivo treatment with a specific POP inhibitor (POPi) significantly decreased plasma and tissue
from medullary microdialysis fluid using dihydroethidine and in-vitro using lucigenin. The concentrations of Ac-SDKP. Chronic infusion with Ac-SDKP prevents and/or regresses fibrosis
results indicate: 1) O2- levels were doubled in the outer medulla of SS rats fed a 4% salt diet in hypertension and post-myocardial infarction; however, it is not known whether normal levels
and could be inhibited by L-NAME, from 17.72.9 to 11.421.9 RLU/min/mg dry tissue. In of this peptide have a physiological role in preventing collagen deposition. Therefore, we
contrast, L-NAME raised O2- levels in consomic SS-13BN rats from 7.32.1 to 13.12.2 hypothesized that decreasing circulating levels of Ac-SDKP using an oral POPi induces heart
RLU/min/mg dry tissue. 2) SS rats showed a reduced ratio of BH4/BH2 (1.40.2 vs. 5.90.6) and kidney fibrosis. SD rats were divided into two groups: a) control, and b) POPi (S17092) and
in the outer medulla that was driven by increased concentrations of BH2 (14315 vs. 558 killed after 60 days of treatment. Myocardial and renal hydroxyproline were measured, and
pg/mg tissue). 3) A 28% decrease in superoxide dismutase activity together with a 42% tissue slices were stained with picrosirius red to determine collagen deposition. We found that
decrease in catalase activity contributed to elevated ROS in SS samples. Based on the shift of rats treated with POPi had significantly lower cardiac and renal levels of Ac-SDKP compared
BH4 to BH2 and the observation of L-NAME inhibitable O2- production, we conclude that NOS to the controls. POPi significantly increased total collagen content in the heart and kidney as
uncoupling occurs in the renal medulla of hypertensive SS rats fed a high salt diet and well as perivascular fibrosis (PVF). To confirm that the increase in collagen was due to the
contributes to the observed medullary renal injury. decrease in Ac-SDKP and not to an increase in any other POP substrates with potential fibrotic
effects, we measured the neuropeptides arg-vasopressin and substance P in tissue. We found
no changes in concentration in the heart and kidney, although both neuropeptides were
69 significantly increased in the brain. We conclude that Ac-SDKP may participate in the regulation
Vascular Dysfunction in Transgenic Mice Expressing a Dominant Negative of collagen deposition, since decreasing its normal concentration in tissue promotes fibrosis.
Mutant of PPAR
Groups Control POPi (40 mg/kg/day).
Andreas M Beyer, Carmen M Halabi, Henry L Keen, Mary L Modrick, Frank M Faraci, Curt D HEART Hydroxyproline (g/mg tissue) 3.270.3 4.270.3*
Sigmund; Univ of Iowa, Iowa City, IA Cardiac PVF (collagen / vessel area) 0.570.06 0.700.04*
KIDNEY hydroxyproline (g/mg dry tissue) 3.400.15 4.430.24*
Patients carrying dominant negative mutations in the PPAR gene exhibit an attenuation of Renal PVF (coll / vessel area) 0.630.02 0.790.06*
PPAR transcriptional activity and present with insulin resistance and hypertension. Gene HEART Ac-SDKP (pg / mg tissue) 34.33.3 5.71*
targeted mice carrying equivalent dominant negative mutations also exhibit hypertension with KIDNEY Ac-SDKP (pg / mg tissue) 11324 37.25.8*
HEART Arg-vasopresin (fg/mg tissue 173 205
varying degrees of metabolic abnormalities. As PPAR is expressed in both endothelial (E) and KIDNEY Arg-vasopresin (fg/mg tissue 8722 7316
smooth muscle (SM) cells, we hypothesized that diminished activity of PPAR in the
 e40 Hypertension Vol 48, No 4 October 2006
Groups Control POPi (40 mg/kg/day).
HEART Substance P (pg / mg tissue) 0.330.12 0.250.03
KIDNEY Substance P (pg / mg tissue) 0.100.01 0.100.01
* p 0.05 vs control.
72
Inhibition of Progression and Stabilization of Plaques by Therapeutic
Interferon- Function Blocking in Apoe-/- Mice
Koga Mitsuhisa, Cardiovascular Rsch Institute, Kurume, Japan; Hisashi Kai, Kurume Univ,
Kurume, Japan; Mamiko Kai, Fukuoka Univ, Fukuoka, Japan; Nobuhiro Tahara, Kiyoko
Takemiya, Hiroshi Kudo, Yusuke Sugi, Takahiro Mori, Narimasa Takayama, Daisuke Fukui,
Ayami Ikeda, Masayoshi Futamata, Kurume Univ, Kurume, Japan; Yumiko Kawai, Fukuoka
Univ, Fukuoka, Japan; Hideo Yasukawa, Yasufumi Kataoka, Tsutomu Imaizumi; Kurume
Univ, Kurume, Japan
A role of interferon- (IFN) is suggested in early development of atherosclerosis. However, it
remains unknown whether inhibition of IFN function will prevent progression of advanced
atherosclerotic plaques and stabilize them. Atherosclerotic plaques were induced in ApoE-/-
mice by feeding with high-fat diet from 8-week old. IFN function was postnatally inhibited by
repeated gene transfers of a soluble mutant of IFN receptors (sIFN-R), an IFN inhibiting
protein, into the thigh muscle every 2 weeks. Prophylactic protocol: sIFN-R or the mock
plasmid treatment was initiated at 8-week old (pre-atherosclerotic stage). Mock-treated
ApoE-/- mice showed atherosclerotic plaques in the ascending aorta, extending to the aortic
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
arch, at 12 week old. sIFN-R prevented the early plaque formation, resulting in 60% reduction
of the en-face plaque area relative to the mock treatment. Therapeutic protocol: sIFN-R or the
mock treatment was started at 12-week old (atherosclerotic stage). Mock-treated ApoE-/- mice
showed marked progression of atherosclerotic plaques, extending to the abdominal aorta, at 16
week old. The plaque of the mock-treated mice consisted of massive lipid core and bulky
macrophage accumulation, but had very thin fibrous cap with little smooth muscle cells,
suggesting low plaque stability. sIFN-R prevented further plaque progression, and the en-face
plaque area at 16-week old was similar to the pre-treatment level at 12 week old. Also,
sIFN-R stabilized advanced plaques: sIFN-R decreased the lipid and macrophage accumu-
lations and increased fibrotic area with more smooth muscle cells. Moreover, sIFN-R
downregulated expressions of pro-inflammatory cytokines (IL-1 and IL-6), chemokines
(MCP-1 and macrophage inflammatory protein-1), vascular cell adhesion molecule-1, and
matrix metalloproteinases-9 and -13, but upregulated type I pro-collagen. sIFN-R did not
affect serum cholesterol levels. Accordingly, the role of IFN was suggested in not only the
early plaque development but also further progression and destabilization of advanced plaques.
Blocking of IFN function would be a new strategy to inhibition plaque progression and to
stabilize advanced plaques through the anti-inflammatory effects.
73
Differential Effects of Hypertension and Hypercholesterolemia on Carotid
Vasa Vasorum Density and Endothelial Function
Daniele Versari, Mario Gossl, Dallit Mannheim, Offer Galili, Lilach O Lerman, Amir Lerman;
Mayo Clinic Rochester, Rochester, MN
Objective: Hypertension and hypercholesterolemia are two of the most important risk factors
for carotid atherosclerosis and cerebrovascular ischemic events. However, little data are
available on the different effects of these risk factors on carotid function and structure. The aim
of the present study was to compare the effects of hypertension and hypercholesterolemia on
carotid endothelial function, structure and vasa vasorum (VV). Methods: Sixteen pigs were
randomized to 12-week normal diet without (N; n5) or with renovascular hypertension (HT;
n4), or high-cholesterol diet (HC; n6). For each pig one carotid artery was injected with a
lead chromate-doped silicon polymer and scanned by microCT for evaluation of VV structure.
Tissue from the other carotid was obtained for staining (trichrome and Sirius red for fibrosis),
immunoblotting (VEGF, bFGF), and assessment of endothelial function by organ chambers
(vasodilation (VD) to acetylcholine, ACH, and sodium nitroprusside, SNP). Results: HT and HC
showed increased carotid artery fibrosis as compared to N, but in HT Sirius red revealed a
higher content of organized collagen fibers than in HC; also intima-media thickness was
significantly (p0.05) increased in HT (0.780.06 mm) compared to N (0.540.04 mm) and
HC (0.590.02 mm). Immunoblotting densitometry, microCT and endothelial function results
are shown in the Table. Conclusions: Both HT and HC induce endothelial dysfunction in pig
carotid artery. However, HT is also associated with greater fibrosis and vascular wall
thickening, which might impair endothelium-independent VD, as well as VV growth. On the
other hand, HC is associated with increased VEGF expression and VV vascularization of the
arterial wall, suggesting that carotid atherosclerosis can evolve in several different pathways.
These findings might have significant implications for our understanding of the pathophysiology
and complications of carotid atherosclerosis.
Mean VV diameter (um) 48.013.65 0.340.02* 18.3722.21*
N HC HT
VV density (n/mm2) 0.080.01 0.340.02* 0.090.01
Mean VV diameter (um) 64.51.3 63.02.1 66.91.1
VEGF/actin 0.10.1 1.920.17* 0.490.63
FGF/actin 0.460.33 0.440.25 0.470.21
Max %VD ACH 48.013.65 23.821.6* 18.3722.21*
Max %VD SNP 85.09.7 82.68. 61.617.61*
* p0.05 vs N; p0.05 vs HC
74
Interferon-Gamma-Mediated Pathway -New Target for Prevention of
Vascular Remodeling after Vascular Injury-
Ken Kusaba, Hisashi Kai, Mitsuhisa Koga, Narimasa Takayama, Takahiro Mori, Yusuke Sugi,
Kiyoko Takemiya, Hiroshi Kudo, Daisuke Fukui, Ayami Ikeda, Masayoshi Futamata, Yumiko
Kawai, Nobuhiro Tahara, Hideo Yasukawa, Tsutomu Imaizumi; Kurume Univ, Kurume, Japan
Interferon-gamma is a multi-functional cytokine with both pro- and anti-atherogenic properties.
Thus, it is still controversial whether the net effect of interferon-gamma promotes or attenuates
vascular remodeling after vascular injury. In vascular smooth muscle cells (SMCs), interferon-
regulating factor-1 (IRF-1) is induced by interferon-gamma, and in turn upregulates expression
of inducible nitric oxide synthase (iNOS), which results in SMC proliferation. Recently, we have
shown that blocking of interferon-gamma by overexpressing a soluble mutant of the
interferon-gamma receptor (sIFNgR), an interferon-gamma inhibiting protein, regresses and
stabilizes advanced atherosclerotic plaque in apoE-deficient mice. Thus, we investigated the
role of interferon-gamma on neointima formation after vascular injury by inhibiting the
interferon-gamma-mediated pathway in vivo. For this purpose, naked DNA plasmid encoding
the sIFNgR or the empty plasmid (mock) was injected into the thigh muscle of male Wistar rats
2 days before balloon injury (day -2)(n6/group). sIFNgR gene transfer significantly elevated
serum levels of sIFNgR protein at days 2 to 14. In the mock-treated rats, balloon injury induced
a transient proliferation of neointimal SMCs with a peak at day 7, assessed by PCNA labeling,
and the maximum neointimal thickening was observed at day 14. At day 7, balloon injury
markedly upregulated IRF-1 and iNOS mRNAs in the mock-treated rats. sIFNgR gene transfer
almost inhibited the balloon injury-induced inductions of IRF-1 and iNOS at day 7. And, the
number of proliferating neointimal SMCs was reduced by 50% in the sIFNgR-treated rats
relative to the mock-treated rats. Moreover, at day 14, sIFNgR gene transfer prevented
neointima formation, resulting in 45% reduction of the intima/media area ratio compared with
mock-treated rats. In conclusion, the role of interferon-gamma was suggested in the neointima
formation through IRF-1 regulation of iNOS expression and subsequently SMC proliferation in
balloon-injured artery. The present study provided an insight into a new strategy for prevention
of vascular remodeling after vascular injury by targeting interferon-gamma.
75
Sleep Disordered Breathing in Children with Hypertension
Alisa A Acosta, Kathy Franco, Monesha Gupta-Malhotra, Richard J Castriotta, Ronald J
Portman; Univ of Texas - Houston, Med Sch, Houston, TX
Obstructive sleep apnea (OSA) is known to be a risk factor for hypertension (HTN) in adults, and
few studies have shown a similar association in children. Recently, we have included in our
Pediatric Hypertension Clinic protocol a routine screen of risk factors for sleep disordered
breathing (SDB). In the past year, 26 out of 350 (7.4%) of the hypertensive patients were
identified for nocturnal polysomnography based on the following risk factors: snoring, enlarged
tonsils, BMI 85th percentile, or nocturnal HTN by ambulatory blood pressure monitoring.
Twenty patients completed nocturnal polysomnography. The mean age at the time of
polysomnography was 12.6 3.97 years (range 4 18 years), 15 were male (75%), all 20 had
a diagnosis of HTN, 11 (55%) also had nocturnal HTN, 17 (85%) had adenotonsillar
hypertrophy, and all 20 patients had a history of snoring. At the time of the initial visit to
Pediatric Hypertension Clinic, 17 (85%) had a BMI the 95th percentile, and one was at the
90th percentile. Of the 20 patients who completed the nocturnal polysomnography, 12 (60%)
had SDB: 7 (35%) had obstructive sleep apnea ( 1 obstructive apnea/hour), 4 (20%) had
obstructive hypoventilation (maximum pCO2 53 torr during sleep and/or 50 torr for 10%
of sleep), and 1 (5%) had mild SDB (1.5 apneas hypopneas/ hour). Of the remaining 8
subjects studied, 6 (30%) had primary snoring disorder (PSD), and 2 had normal polysom-
nography without snoring. Furthermore, SDB was found in 10 (59%) pts with adenotonsillar
hypertrophy and HTN [OR2.16, CI 1.18 - 3.95, p0.007 vs adenotonsillar hypertrophy alone]
and 11 (65%) pts with obesity and HTN [OR2.18, CI 1.19 4.01, p0.007 vs obesity alone].
This compares to an overall prevalence of 2% in all children, 40% in children with
adenotonsillar hypertrophy, and 46% in obese children reported in the literature. These data
suggests HTN in children constitutes an additional risk factor for SDB, and further studies are
needed to explore the relationship between SDB and HTN.
76
Difference in Aldosterone Levels in Male and Female Subjects with
Resistant Hypertension
Krishna K Gaddam, Monique N Pratt-Ubunama, Mari K Nishizaka, David A Calhoun; Univ of
Birmingham at Alabama, Birmingham, AL
Background: Several centers worldwide including ours have recently reported a high incidence
of primary aldosteronism (PA) among subjects with resistant hypertension. The reason for the
high prevalence of hyperaldosteronism in this high risk group is unknown. The current study
was designed to identify potential stimuli of aldosterone secretion in subjects with resistant
hypertension. Methods: Consecutive subjects referred for resistant hypertension (blood
pressure 140/90 on 3 antihypertensive agents) were prospectively evaluated with plasma
aldosterone concentration (PAC), plasma rennin activity, a 24-hr urine collection for aldoste-
rone, sodium and potassium during a normal diet. All subjects were on a stable antihyper-
tensive regimen without use of potassium sparing diuretics. Results: A total of 271 subjects
were evaluated, including 132 males and 139 females. Age (5411 vs. 5511), BMI (335.3
vs. 338.2), clinic blood pressure (14522/8717 vs. 14421/8316) were similar in men
and women. PAC was higher in men compared to women (14.89.54 vs. 11.89.28ng/dl,
p0.0091). In a subset of subjects (n96) 24-hr urinary cortisol, aldosterone, sodium,
potassium and protein were measured. In this subset, PAC (11.437.6 vs. 10.46.2ng/dl) and
urinary aldosterone (13.27.2 vs. 9.55.3g/24-hr, p0.0053) were higher in men
compared to women. Urinary cortisol (l0842.1 vs. 65.630g/24-hr, p0.0001), sodium
(233.8111.8 vs. 155.984.2meq/24-hr, p0.0002) and potassium (79.633.6 vs.

50.523.9meq/24-hr, p0.0001) were also higher in male subjects. Urinary aldosterone
excretion was significantly correlated with cortisol excretion in males (p0.0003, r 0.51)
with a similar trend in females (p0.062, r0.27). Conclusion: This data is the first to
demonstrate a gender difference in plasma aldosterone and urinary aldosterone excretion
levels in subjects with resistant hypertension. Based on our findings, we propose that potential
stimuli of aldosterone underlying this gender difference include greater dietary ingestion of
potassium by the male subjects; and/or ACTH stimulation as suggested by the same gender
difference in aldosterone and cortisol excretion.
77
Plasma Aldosterone is Related to Severity of Obstructive Sleep Apnea in
Subjects with Resistant Hypertension but not Subjects with Normal Blood
Pressure
Monique N Pratt-Ubunama, Mari K Nishizaka, UAB Hypertension Program, Birmingham, AL;
Krisha K Gaddam, UAB Hypertension Prgm, Birmingham, AL; Robyn L Boedefeld, UAB Sleep
Disorders Cntr, Birmingham, AL; Stacey S Cofield, UAB Dept of Biostatistics, Birmingham,
AL; Susan M Harding, UAB Sleep Disorders Cntr, Birmingham, AL; David A Calhoun; UAB
Hypertension Program and Sleep Disorders Cntr, Birmingham, AL
Objective: Untreated obstructive sleep apnea (OSA) may contribute to the development of
resistant hypertension by stimulating aldosterone release. This study compares plasma
aldosterone, renin levels and OSA severity in subjects with resistant hypertension and in control
subjects without resistant hypertension but with equally severe obstructive sleep apnea.
Methods: Seventy-one consecutive subjects (41 males, 31 females) referred to University of
Alabama at Birmingham for resistant hypertension (blood pressure uncontrolled with 3
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medications) and 29 consecutive subjects (15 males, 14 females) referred to the UAB Sleep
Disorders Center for suspicion of OSA and without resistant hypertension (normotensive or
blood pressure controlled on 2 or less antihypertensive medications) were evaluated. All
subjects were prospectively evaluated by an early morning, paired plasma aldosterone
concentration (PAC) and renin level and overnight, attended polysomnography. The number of
apnea and hypopnea events/hr (apnea-hypopnea index or AHI) and the percent of sleep time
spent with oxygen saturation 90% (hypoxic index or HI) was determined in all subjects.
Results: The prevalence of OSA (AHI 5 events/hr) was 85% in subjects with resistant
hypertension (median AHI 15.3 events/hr) and 83% in control subjects (median AHI 14.3
events/hr). Median PAC was significantly higher (11.0 vs. 5.5 ng/dL, p0.002) and renin levels
were significantly lower (8.0 vs. 19.0 Units/mL, p0.0005) in subjects with resistant
hypertension compared to controls subjects. PAC correlated with AHI (rho0.44, p0.0002)
and HI (rho0.38, p0.001); there was no correlation between PDR and AHI or HI in subjects
with resistant hypertension. Median plasma aldosterone levels were not related to AHI
(rho0.12, p0.52) or HI (rho0.002, p0.99) in control subjects. Conclusions: We
demonstrate that PAC correlates with OSA severity in subjects with resistant hypertension. No
relationship between PAC and OSA severity was observed in control subjects with equally
severe OSA but without resistant hypertension. These data suggests that OSA contributes to the
development of resistant hypertension by stimulating aldosterone release.
78
Sleep Apnea and Hypertension Prevalence in Chronic Kidney Disease
John J Sim, Stephen F Derose, Scott A Rasgon; Kaiser Permanente Los Angeles Med Cntr,
Los Angeles, CA
Introduction: Hypertension (HTN) is a known complication and risk factor for chronic kidney
disease (CKD). The rate of HTN in CKD correlates with declining renal function exceeding 80%
in those with end stage renal disease. Sleep apnea (SA) also has a strong association with
systemic HTN. Both CKD and SA have been associated with cardiovascular and cerebrovascular
disease where HTN may be an intermediary variable. We evaluated the prevalence of HTN in
patients who have CKD and SA compared to the CKD population overall. CKD was defined by
depressed glomerular filtration rate (GFR) and/or the presence of proteinuria and classified into
National Kidney Foundation Kidney/Disease Outcomes Quality Initiative (K/DOQI) stages. We
compared the prevalence of HTN in patients in each stage of CKD against those with CKD and
SA. Methods: The population of subjects age 18 years and enrolled in an integrated health
plan during 2002 - 2004 was studied. CKD was determined by GFR and proteinuria. GFR was
estimated by the modified MDRD equation. Two GFRs taken 90 days apart were required to
establish stable GFR according to predefined algorithms. HTN was determined using a
combination of ICD-9 codes, HTN medications, and comorbid conditions. SA was defined by
ICD-9 codes for obstructive or central SA, or dispensation of CPAP or BIPAP. Rates of HTN and
SA were compared by chi-squared tests. Results: See table. HTN is significantly more
prevalent in patients (1) with SA than without SA among CKD patients with GFR 1590
mL/min/1.73m2 (p 0.001 for CKD Stage 2 & 3, p 0.05 for CKD Stage 4); (2) with SA than
without SA among patients with GFR 90 (p 0.001 for no CKD and CKD Stage 1); and (3)
with proteinuria than without proteinuria among patients with GFR 90 (p 0.001 for no SA
and SA). Conclusion: HTN is very common in SA with or without CKD. Rates of HTN increase
when SA coexists with CKD and in patients with normal GFR who have proteinuria. Rates of
HTN increase as GFR declines.
HTN AND SLEEP APNEA BY CKD STAGE
CKD
Stage Renal Function Total Patients HTN (%) SA (%) HTN & SA (%)
0 GFR 90 No Proteinuria 395,634 109,144 (28%) 8,151 (2%) 4,182 (51%)
1 GFR90 and Proteinuria 4,917 3,626 (74%) 261 (5%) 228 (87%)
2 GFR 6089 258,238 141,947 (55%) 8,486 (3%) 5,573 (66%)
3 GFR 3059 59,300 48,217 (81%) 1,901 (3%) 1,642 (86%)
4 GFR 1529 2,789 2,614 (94%) 91 (3%) 91 (100%)
CHBPR ConferenceOral Presentations e41
79
Higher Ambulatory Blood Pressure and Less White-Coat Effect in Subjects
with Hyperaldosteronism and Resistant Hypertension
Eduardo Pimenta, Sr., Monique N Pratt-Ubunama, Krishna K Gaddam, Mari K Nishizaka,
David A Calhoun; Univ of Alabama at Birmingham, Birmingham, AL, USA, Birmingham, AL
Background: Ambulatory blood pressure (BP) better predicts cardiovascular outcomes than
office BP. The effects of hyperaldosteronism on ambulatory BP levels and circadian BP variation
are not well established. Objective: The purpose of this study was to compare the 24-hr
ambulatory blood pressure monitoring (ABPM) profile and the degree of white-coat effect (WCE
- daytime ambulatory BP at least 10 mmHg office BP) in resistant hypertensive subjects with
or without hyperaldosteronism. Methods: One hundred thirty-eight subjects with resistant
hypertension were prospectively evaluated with an early-morning plasma aldosterone and
plasma renin activity (PRA), 24-hour urinary aldosterone and sodium, and 24-hr ABPM.
Daytime, nighttime and 24-hr blood pressure as well as nocturnal BP decline and the morning
BP surge were determined. Hyperaldosteronism (H-Aldo) was defined as suppressed PRA
(1.0 ng/mL/hr) and elevated 24-hr urinary aldosterone excretion (12 g/24h) in sodium
replete subjects (200 mEq/24-hr). Results: Overall, the mean office BP was 160.325.9/
89.816.3 mmHg on an average of 4 medications. There was no difference in mean office BP
values between the 45 subjects with H-Aldo and the 93 subjects with normal aldosterone levels
(N-Aldo). However, systolic daytime (149.215.2 vs 138.617.4mmHg, p0.0007), diastolic
daytime (89.48.8 vs 80.513.7mmHg, p0.0001), systolic nighttime (143.116.4 vs
130.917.8mmHg, p0.0002), diastolic nighttime (82.810.1 vs 73.514.2mmHg,
p0.0001), 24-hr systolic (146.814.6 vs 135.717.0mmHg, p0,0003) and 24-hr diastolic
(86.618.8 vs 77.913.8mmHg, p0.0002) BP were all higher in H-Aldo than N-Aldo
subjects. A WCE was present in 46% of the H-Aldo subjects but only 13% of N-Aldo. Nocturnal
dipping was not different in the 2 aldosterone groups. The systolic morning surge was
17.411.3 mm Hg in the H-Aldo subjects and 19.213.7 mm Hg in the N-Aldo subjects
(pNS). Conclusions: In spite of similar office BP, daytime and night ambulatory BP levels are
higher and there is less WCE in resistant hypertensive subjects with high aldosterone levels.
These results suggest that high aldosterone levels impart increased cardiovascular risk not
reflected by office blood pressure measurements.
80
Blood Pressure and Mortality in Patients with Cardiovascular Disease
Jing Chen, Tulane Univ, New Orleans, LA; Donfeng Gu, Chinese Acadewmy of Med
Sciences and Peking Union Med College, Beijing, China; P. Whelton, Chung-Shiuan Chen,
Tulane Univ, New Orleans, LA; Xigui Wu, Chinese Academy of Med Sciences and Peking
Union Med College, Beijing, China; Xingfeng Duan, Chinese Academy Med Sciences and
Peking Union Med College, Beijing, China; Kristi Reynolds, L Lee Hamm, Paul K Whelton,
Jiang He; Tulane Univ, New Orleans, LA
Background: Blood pressure (BP) has been associated with an increased risk of cardiovascular
disease (CVD) mortality in the general population. However, direct and J-shaped associations
between BP and mortality have been reported among patients with CVD. Methods: We
examined the association between BP and CVD mortality among 2,253 male and 1,942 female
patients with CVD. Data on BP and co-variables were obtained at a baseline examination in
1991 using a standard protocol. Follow-up evaluation was conducted in 1999 2000 with a
response rate of 93.4%. Results: After adjustment for age, gender, cigarette smoking, alcohol
consumption, body mass index, physical inactivity, education, diabetes, geographic region
(north vs. south) and urbanization (urban vs. rural), a direct association between BP and CVD
mortality was observed (p0.001). Compared to those with a systolic BP 120 mm Hg,
relative risks (95% CI) for patients with a systolic BP of 120 129, 130 139, 140 159,
160 179 and 180 mm Hg were 1.21 (0.79,1.84), 1.69 (1.15, 2.48), 2.09 (1.47, 2.96), 2.83
(1.98, 4.03), 3.83 (2.66, 5.52) for stroke, 1.29 (0.94, 1.77), 1.63 (1.22, 2.19), 1.96 (1.50, 2.56),
2.37 (1.80, 3.12), 2.99 (2.25, 3.99) for CVD, and 1.05 (0.82, 1.33), 1.23 (0.98, 1.53), 1.34
(1.09, 1.64), 1.62 (1.32, 2.00), 2.00 (1.61, 2.50) for all-cause mortality, respectively. The
associations between BP and risk of CVD and all-cause mortality were similar in males and
females. Conclusion: These data indicate that there is no J-shaped association between BP
and mortality among patients with CVD. Furthermore, our findings support a lower BP treatment
goal among patients with CVD in order to reduce mortality.
81
Effect of Cardiac Overexpression of Bradykinin B2 Receptors in a Rat
Model of Pressure Overload
Marcos E Barbosa, Max-Delbruck-Cntr, Berlin, Germany; Heloisa A Baptista, Escola Paulista
de Medicina, Sao Paulo, Brazil; Robert Fischer, Max-Delbruck-Cntr, Berlin, Germany; Joao B
Pesquero, Escola Paulista de Medicina, Sao Paulo, Brazil; Michael Bader;
Max-Delbruck-Cntr, Berlin, Germany
Hypertension resulting in left ventricular hypertrophy and fibrosis can lead to cardiac
dysfunction. Using a myosin light chain 2 promoter, a transgenic rat model overexpressing
bradykinin B2 receptors (BKB2) specifically in the heart (TGR(MLC2B2)) was developed to
investigate the cardiac function of the kallikrein-kinin system (KKS). Overexpression of BKB2
receptors was verified by a ribonuclease protection assay, by radioligand binding studies as
well as by the response to bradykinin in isolated hearts perfused in a modified Langendorff
preparation. Isolated hearts from TGR(MLC2B2) rats presented higher left ventricular pressure
(68.66.6mmHg) when compared with Sprague Dawley (SD) rats (49.51.3 mmHg,
p0.001). Accordingly, blood pressure measured by tail-cuff plethysmography was increased
in TGR(MLC2B2) (122.71.9 mmHg) compared to SD rats (107.81.9 mmHg, p0.001).
Telemetric measurements of systolic blood pressure confirmed the difference between SD
(120.92.8 mmHg) and TGR(MLCB2) rats (130.62.7 mmHg, p0.05). Treatment with the
BKB2 antagonist, icatibant, annulled this difference (121.52.5 mmHg). To study the function
of the KKS in the pathogenesis of cardiac hypertrophy, SD and TGR(MLC2B2) rats were
 e42 Hypertension Vol 48, No 4 October 2006

submitted to abdominal aortic constriction. After 8 weeks, hemodynamic parameters were
measured by a Millar tip catheter and the pressure overload after aortic banding in TGR(MLCB2)
(sham 113.23.9 to banded 166.39.1 mmHg) was 76% higher than the observed in SD rats
(sham 106.3 2.7 to banded 136.37.4 mmHg, p0.05). Cardiac Magnetic Field Mapping
showed a slight prolongation of the early part of repolarization at baseline in TGR(MLCB2)
compared to SD rats (QTPeak 302.5 and 25.70.6, p0.053). Alterations in repolarization
were more pronounced in TGR(MLCB2) submitted to aortic constriction compared to SD rats.
Rotation in magnetic field orientation during early repolarization was higher in TGR(MLCB2)
(137.08.0 to 101.06.7 degrees) compared to SD rats (134.06.0 to 122.09.0 degrees,
p0.05). In conclusion, overexpression of the BKB2 receptor in cardiomyocytes of transgenic
rats results in increased cardiac performance at baseline and after pressure overload.
The increase in intracellular Ang II (in cell lysates) was even greater (64 25 and 70 12
%, in CMs and FBs, respectively) in the presence of candesartan, suggesting the increase was
due to intracellular synthesis, not to uptake. Similarly, high glucose treatment resulted in a
significant increase in total Ang II synthesis. However, in CMs the increase in the concentration
of Ang II was mainly observed in the cell lysates (704 46 %), with a minimal increase in the
culture medium (14 1 %), compared to control. In the presence of candesartan, the Ang II
concentration increased 556 78 % intracellularly and 34 4 % in the medium. This latter
observation suggested that under hyperglycemic conditions, intracellularly synthesized Ang II
in CMs is retained inside the cell. In contrast, Ang II synthesized by FBs is primarily secreted
extracellularly. This study demonstrates intracellular synthesis of Ang II by isolated CMs and
FBs and suggests a possible role for intracrine (intracellular) Ang II in CMs, under high glucose
conditions.

82
The Nonpeptide Angiotensin-(17) Receptor Mas Agonist AVE 0991
Attenuates Heart Failure Induced by Myocardial Infarction
Anderson J Ferreira, Bruno J Jacoby, Ccero A Araujo, Filipe A Macedo, Gerluza A Silva,
Alvair P Almeida, Robson A Santos; Federal Univ of Minas Gerais, Belo Horizonte, Brazil
The nonpeptide AVE 0991 has been reported as a selective ligand of the Ang-(17) receptor
Mas, which actions are similar to those attributed to the cardioprotective fragment of the
renin-angiotensin system, Ang-(17). In this study we evaluated the cardiac effects of AVE
0991 (0.58 mol/kg, 7 days) in normal and infarted male Wistar rats. Myocardial infarction was
induced by left coronary artery ligation. At the end of the treatment, the Langendorff technique
was used to analyze cardiac function. Left ventricles serial sections were stained with Gomori
trichrome stain to quantify the infarted area. AVE 0991 produced a significantly decrease in
perfusion pressure and an increase in systolic tension, dT/dt, and heart rate. These effects
were completely blocked by the perfusion of the hearts with a solution containing the selective
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85
Myocardial Performance Index in Childhood Onset Essential Hypertension
Monesha Gupta-Malhotra, Rabih K Hamzeh, Tim Poffenbarger, Karen McNiece, Ronald J
Portman; Univ of Texas, Houston, TX
Objectives. As a measure of global ventricular systolic and diastolic function, the myocardial
performance index (MPI) may be an early indicator of left ventricular dysfunction in
hypertensive children. Thus, we performed the following study to determine the MPI in children
with essential hypertension (HTN). Methods. Study subjects were untreated newly diagnosed
children with HTN and controls. HTN was defined as BP 95th percentile based on the Fourth
Working Group criteria and confirmed by 24-hour ambulatory blood pressure monitoring
(ABPM). BP is reported as the patients BP factored by their age, gender, height-specific 95th
%ile (BP index). Left ventricular mass index (LVMI) was calculated as LVM (g)/height (m) 2.7, and
left ventricular hypertrophy (LVH) was defined as LVMI 95th percentile. Control subjects had

Ang-(17) antagonist, A-779 (2 nM). L-NAME treatment (10 mg/kg) abolished the AVE
0991-induced vasodilation in isolated hearts. AVE 0991 significantly attenuated the decrease
in systolic tension (sham-operated: 13.00 1.02 g; infarction: 7.18 0.66 g; AVE-treated:
9.23 1.05 g, n5), dT/dt (sham operated: 209.9 17.9 g/s; infarction: 104.5 12.5
g/s; AVE treated: 136.2 19.8 g/s, n5), - dT/dt and heart rate induced by myocardial
infarction. Infartion-induced vasoconstriction was completely abolished by AVE 0991 treatment.
Furthermore, AVE 0991 significantly decreased the infarted area. These data indicate that the
compound AVE 0991 produces beneficial effects in isolated perfused rat hearts involving the
Ang-(17) receptor Mas and the release of nitric oxide. In addition, our results indicate that AVE
0991 attenuates post-ischemia heart failure.
normal casual BPs and 24-hour ABPM. 2-D and M-mode echocardiography with pulse Doppler
were performed on all pts prior to therapy. Endocardial shortening fraction (SF), midwall left
ventricular shortening (MWS) and circumferential end systolic stress (cESS) were calculated.
MPI was calculated from simultaneous mitral valve inflow and left ventricular outflow velocities
using pulse Doppler. Results. The left ventricular diastolic parameters (peak E and A velocities,
E/A ratio, isovolemic relaxation time, acceleration and deceleration times) were similar between
the two groups. MPI was significantly higher among HTN children compared to non-
hypertensive controls. Conclusions. MPI is significantly higher in children with essential HTN
compared to controls, albeit with preserved function as this early stage. These findings suggest
that MPI may be a sensitive parameter for detecting early cardiac dysfunction in hypertensive
children.

83 HTN Control P value

Genetic Ablation of Bach1, a Transcriptional Repressor of Heme Number 24 36
Oxygenase-1 Gene, Leads to Myocardial Protection against Pressure Age, years 13.5 1.8 13.5 1.7 NS
Overload in Mice BMI 27 9 26 3 NS
Casual systolic BP index 1.11 .06 1.05 .06 0.002
Casual diastolic BP index .95 .14 .93.12 NS
Shinji Mito, Ryoji Ozono, Yuichiro Watari, Yoshiyuki Yamamoto, Yoko Yano, Masao
Ambulatory systolic BP index 1.04 0.05 0.93 0.04 0.0001
Yoshizumi; Hiroshima Univ Graduate Sch of BioMed Sciences, Hiroshima, Japan Ambulatory diastolic BP index 0.93 0.1 0.87 0.07 0.006
Systolic BP load, % 65 17 21 13 0.0001
Bach1 is a stress-responsive transcriptional factor that is thought to control the expression Diastolic BP load, % 28 24 14 11 0.003
levels of cytoprotective factors including heme-oxygenase(HO)-1. In the present study, we LVMI, g/m2.7 36.7 9 35.8 8 NS
investigated the roles of Bach1 in the development of left ventricular hypertrophy (LVH) induced Relative wall thickness 0.37 0.05 0.35 0.04 NS
by transverse aortic constriction (TAC) in vivo using mice lacking the Bach1 gene (Bach1-/- SF, % 42 10 42 13 NS
MWS, % 20 0.4 15 0.4 NS
mice). Three weeks after TAC, the heart weight to body weight (HW/BW) ratio, myocardial
cESS, g/cm2 82 34 75 29 NS
cross-sectional area, and an index of interstitial fibrosis were significantly increased both in MPI 0.36 0.4 0.2 0.1 0.04
Bach1-/-mice and wild-type control (Bach1/) mice. However, the increases in all of these
parameters were significantly less in Bach1-/- than in Bach1/ mice (HW/BW: 5.741.17 vs.
7.250.53 mg/g, p0.05). Myocardial HO-1 protein expression was significantly greater in
sham-operated Bach1-/- than in Bach1/ mice. TAC increased myocardial expression levels of 86
HO-1 protein to a larger extent in Bach1-/- than in Bach1/ mice. Treatment of Bach1-/- mice Stimulation of Cytokines Gene Expression in Hypertrophied Hearts of Mice
with tin-protoporphyrin, an inhibitor of HO, completely abolished the hypertrophy-reducing Lacking Guanylyl Cylase/Natriuretic Peptide Receptor-A
effect of Bach1-knockout, indicating that the reduction of hypertrophy was dependent on the
HO activity.The results of present study suggest that Bach1 plays a significant role in repressing Elangovan Vellaichamy, Di Zhao, Naveen K Somanna, Kailash N Pandey; Dept of Physiology
the myocardial HO-1 expression both in basal and stressed conditions, thereby setting a and Hypertension and Renal Cntr of Excellence, Tulane Univ Health Sciences Cntr & Sch of
threshold of deployment of anti-hypertrophic factors. Inversely, an inhibition of Bach1 could be Medicine, New Orleans, LA
a novel approach to protect myocardium from pressure overload.
Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP), are released in the
circulation and elicit natriuretic, diuretic, vasorelaxant, and anti-proliferative responses, all
84 directed to the reduction of blood pressure and blood volume. Natriuretic peptide receptor-A/
Evidence of Intracellular Synthesis of Angiotensin II in Cardiac Myocytes guanylyl cyclase-A (NPRA/GC-A) is considered the principal receptor for both ANP and BNP, and
and Fibroblasts binding of these hormones to NPRA leads to the generation of the intracellular second
messenger cGMP. Mice carrying targeted-disruption of Npr1 gene (encoding for NPRA) exhibit
Vivek P Singh, Kenneth M Baker, Rajesh Kumar; Div of Molecular Cardiology, Cardiovascular hypertension, marked cardiac hypertrophy, and congestive heart failure with sudden death
Rsch Institute, The Texas A&M Univ System Health Science Cntr, College of Medicine; Scott after six months of age. In the present studies, we analyzed the cardiovascular phenotype in
&White; Central Texas Veterans Health Care System, Temple, TX Npr1 (genetic determinant of NPRA) gene-disrupted mutant mouse model. Homozygous null
mutant Npr1 (0-copy; 0/0) mice showed almost 35 40 mmHg higher systolic blood pressure
The current paradigm is that cardiac angiotensin II (Ang II) is synthesized in the interstitial space and 60 65% greater left ventricular weight as compared with Npr1 wild-type (2-copy; 1/1)
from components of the circulating or local renin angiotensin system. Local production of Ang mice. The expressions of cytokines and growth factors gene were significantly increased in
II has a significant role in cardiac homeostasis, through autocrine/paracrine actions. The recent ventricles of 0-copy Npr1 mice hearts as compared with 2-copy wild-type mice hearts. The
discovery of intracrine effects of Ang II in the heart, prompted us to determine if there was electrophoretic mobility shift assay (EMSA) showed a 4-to-5-fold increase in NF-B binding
intracellular Ang II synthesis. Cardiac myocytes (CMs) and fibroblasts (FBs) were isolated from activity in nuclear extracts of 0-copy mice hearts as compared with 2-copy mice hearts.
neonatal rat hearts and separated by double-differential plating. Before treatment, the cells Captopril or hydralazine treatments attenuated systolic blood pressure; however, only captopril
were incubated in serum-free medium for 24 h to allow for degradation of any exogenous treatment significantly decreased the left ventricular weight and fibrosis in 0-copy Npr1 mice
source of Ang II. Cells were treated with either isoproterenol (10 M) or high glucose (25 mM) hearts. Nevertheless, the captopril treatment also decreased cytokine expression in 0-copy
in the absence or presence of the AT1 antagonist, candesartan (1 M). The latter was included Npr1 mice hearts. cGMP levels were significantly reduced 5-fold in plasma and 10-fold in
to prevent receptor-mediated internalization of Ang II. After 24 h of treatment, Ang II was ventricular tissues of mutant mice hearts relative to wild-type controls. The present findings
measured in cell lysates and the culture medium using a competitive ELISA. Isoproterenol provide direct evidence that ablation of NPRA/cGMP signaling activates inflammatory process
treatment increased the Ang II concentration in cell lysates (38 18 and 24 12 %) and the leading to an enhanced cardiac hypertrophy and fibrosis in homozygous null mutant mice
medium (52 24 and 144 22 %), of both CMs and FBs, respectively, compared to control. lacking NPRA.

87
A Novel Mechanism of Afferent Arteriole (Af-Art) Regulation: A Cross-Talk
between the Connecting Tubule (CNT) and Af-Art
Yilin Ren, Jeffrey L Garvin, Ruisheng Liu, Oscar A Carretero; Henry Ford Hosp, Detroit, MI
In most nephrons the superficial connecting tubule (CNT) comes into close contact with the
afferent arteriole (Af-Art). However, the physiological significance of this contact is not
clear. The NaCl concentration in the CNT varies over a wide range. We hypothesized that
there is cross-talk between the CNT and Af-Art that is initiated by changes in NaCl
concentration in the CNT lumen. Rabbit Af-Arts were perfused at 60 mm Hg while the NaCl
concentration in the lumen of the adherent CNT was changed from 10 to 80 mM. We first
found that in nonconstricted Af-Art, increasing luminal NaCl in the CNT caused slight
dilatation but not constriction. Then we tested whether increasing luminal NaCl in the CNT
induced dilatation of Af-Arts preconstricted with norepinephrine (NE). With 10 mM NaCl in
the CNT lumen, adding NE to the bath decreased Af-Art diameter from 18.4 0.7 to
10.1 1.3 m. When the solution was changed to 80 mM NaCl, Af-Art diameter increased
to 17.3 1.6 m (n 6; p 0.05, high vs. low NaCl). We then tested whether entry
of Na, Cl- or some combination of these ions is required to induce Af-Art dilatation. During
the control period, high NaCl induced preconstricted Af-Art dilatation from 13.0 1.9 to
17.8 1.3 m (n 4; p 0.05, high vs. low). We then replaced Na in the CNT lumen
with choline chloride. Unlike NaCl, increasing choline chloride from 10 to 80 mM did not
dilate the preconstricted Af-Art (from 11.9 1.6 to 12.4 1.8 m; n 3). We next
studied which Na transporter mediates these effects. When we blocked amiloride-sensitive
Na channels by adding 10-6 M amiloride to the CNT lumen, the dilatation induced by 80
mM NaCl was eliminated (from 12.0 1.2 to 11.9 1.6; n 4). Finally, we added 10-3
M thiazide to the CNT lumen to block NaCl cotransport. Thiazide did not block
NaCl-induced dilatation of the preconstricted Af-Art. We concluded that: 1) there is
cross-talk between the CNT and the Af-Art; 2) increases in CNT Na cause dilatation of
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
preconstricted Af-Arts; and 3) this phenomenon is mediated by amiloride-sensitive
epithelial Na channels. Cross-talk between the CNT and Af-Art may be a novel
mechanism of regulation of the renal microcirculation and may explain the dilatation
observed during high salt intake.
88
Angiotensin Type 2 Receptor Contributes to Vascular Responses in
Resistance Arteries of Type 2 Diabetic Hypertensive Patients Treated with
Angiotensin Type 1 Receptor Blockers
Carmine Savoia, Lady Davis Institute for Med Rsch, SMBD Jewish General Hosp, McGill
Univ, Montreal, Canada; Rhian M Touyz, Kidney Rsch Cntr, Ontario Health Rsch Institute,
Univ of Ottawa, Ottawa, Canada; Ernesto L Schiffrin; Lady Davis Institute for Med Rsch,
SMBD Jewish General Hosp, McGill Univ, Montreal, Canada
The role of angiotensin (Ang) AT2 receptors on blood pressure and vascular responses to
Ang II in humans is unclear. We questioned whether AT2 is expressed and functionally
active in peripheral resistance arteries of hypertensive diabetic patients treated for one
year with either the Ang receptor blocker (ARB) valsartan or the beta blocker (BB) atenolol.
Twenty-six hypertensive type 2 diabetic patients (30 to 70 years of age) treated with oral
hypoglycemic and antihypertensive agents (not receiving ARBs or BBs) were randomized
to one-year treatment with valsartan (80 to 160 mg) or atenolol (50 to 100 mg), added to
their previous therapy. Ten normal subjects were studied as a control group. Resistance
arteries dissected from gluteal subcutaneous tissue were assessed on a pressurized
myograph. Vasodilatory response curves to Ang II (10-9-10-6 mol/L) were performed in
norepinephrine pre-contracted vessels in presence of valsartan (10-5 mol/L) the AT2
inhibitor PD123319 (PD, 10-6 mol/L). AT2 expression in small resistance arteries was
evaluated by confocal microscopy. After one year of treatment, systolic and diastolic BP
were well controlled in both the valsartan and atenolol groups (1433/832mmHg vs
1222/732mmHg, p0.005; 1442/832mmHg vs 1283/752mmHg, p0.005,
respectively). Endothelium-dependent and -independent relaxation did not change in the
treated groups between the beginning and end of the study. Ang II evoked a significant
vasodilatory response only in norepinephrine-pre-contracted resistance arteries from
patients treated with valsartan. PD significantly blocked this effect. AT2 expression was
increased (four-fold, p0.005) only in valsartan-treated patients. In conclusion, AT2
receptors are upregulated and contribute to Ang II-induced vasodilation in human
resistance arteries of hypertensive diabetic patients treated with an ARB, and may mediate
in part the beneficial actions of these drugs in the treatment of hypertension in high risk
patients.
89
High Fat Diet Increases Oxidative Stress and Inflammatory Markers
Expression without Worsening of Lipid Profile in Forko Mice
Danesh Javeshghani, Lady Davis Institute, Montreal, Canada; Malur R Sairam, IRCM,
Montreal, Canada; Ernesto L Schiffrin, Lay Davis Institute, Montreal, Canada; Rhian M
Touyz; Kidney Rsch Cntr, Ottawa, Canada
The potential effect of ovarian hormone replacement therapy (HERS) on the development of
atherosclerosis is unclear. To probe this question we determined the effect of 3 4 month high
fat diet (HFD) feeding on vascular function and changes in the expression of inflammatory
markers in 3 groups of adult mice : wild-type (WT), heterozygote (HT, lower estrogen) , and
homozgygous (FORKO, Follitropin Receptor KnockOut, estrogen deficient). Bradykinin relaxation
response of resistance arteries was less in FORKO (33.7%13.7, p0.003) and HT
(44.4%5.4, p0.04) than WT (60.3%10.5). ACh relaxation response was also less in
FORKO than the WT (75.112.2 vs 96.6%3, p0.04). These responses were not reduced
further by HFD in HT or FORKO groups. In L-NAME-incubated vessels HFD-fed groups
ACh-relaxation response was less in WT (28.2%12.4, p0.004) compared to WT without
CHBPR ConferenceOral Presentations e43
HFD (96.6%3), but not in HT or FORKO. Angiotensin II-constrictor responses were not altered
by HFD in all groups. Incubation of the vessels with antioxidant cocktail (SOD, catalase,
TEMPOL) significantly increased the ACh response only in HFD-fed FORKO (59.6%5.9,
p0.005). Reactive oxygen species (ROS) generation was increased in FORKO (0.29.11
arbitrary unit) and further increased by HFD (10-fold). These findings suggest that endothelial
dysfunction in FORKO mice is mediated by ROS which is aggravated by HFD. Oil-red-O staining
revealed deposition of fat in aortic and coronary sinuses that was associated with increased
expression of VCAM-1 in all HFD-fed groups. The percent change in lipid profile (total
cholesterol, HDL, LDL and TRIG by FD) was not significantly different in FORKO compared to
the same change by HFD in WT and HT. Our findings demonstrate that HFD aggravates vascular
function by increasing oxidant radicals in FORKO mice without worsening lipid profile.
90
Differential Function and Trafficking of ETA and ETB Receptors in
Mesenteric Arterial and Venous Smooth Muscle and HEK 293 Cells
Xiaoling Dai, James J Galligan; Michigan State Univ, East lansing, MI
ET-1 is more potent in contracting veins than arteries and this might be due to differential
localization of ETA (ETAR) and ETB receptors (ETBR) on vascular smooth muscle cells
(VSMCs). We investigated ETAR and ETBR trafficking in HEK 293 cells transfected with ETA
and/or ETB receptors and in rat mesenteric arteries (MA) and veins (MV). Using [Ca2]i
imaging in HEK 293 cells transfected with ETAR and ETBR, we found that ETBR desensitized
faster (t1/2211s) than ETAR (t1/2481s) upon ET-1 (0.1 M) stimulation. The t1/2 for
desensitization in cells co-transfected with ETAR and ETBR was 400s. In HEK 293 cells
transfected with ETAR or ETBR alone, 83% of ETAR and 54% of ETBR, colocalized with the
plasma membrane marker pan cadherin. After ET-1 treatment for 1 hr 76% of ETAR, but
only 11% of ETBR, colocalized with pan cadherin indicating a preferential internalization of
ETBR. When ETAR and ETBR were co-transfected, 93% of ETAR and 84% of ETBR
co-localized with pan-cadherin, and 79% of ETAR and 71% of ETBR colocalized with pan
cadherin after ET-1 treatment. We next studied ET-1-induced constriction of MA and MV
in vitro. The maximal ET-1 constriction was 50% for MV and 20% for MA. MA desensitized
by 50% during a 20-min ET-1 application, while MV maintained maximal constriction. In
dissociated MA VSMCs, 26% of ETBR colocalized with ETAR in the membrane in contrast
to 43% in MV VSMCs. In MA VSMCs, 50% of ETAR and 15% of ETBR colocalized with pan
cadherin. In MV VSMCs, 55% ETAR and 40% of ETBR colocalized with pan cadherin. After
1 hr of ET-1 treatment, colocalization of ETAR and ETBR with pan cadherin increased to
62% and 33% respectively; in MA VSMCs this increased to 60% and 53% for ETAR and
ETBR, respectively. We conclude that co-expression of ETAR with ETBR in HEK 293 cells
increases ETBR membrane expression/stability and this is associated with decreased ETR
desensitization. There is increased ETBR membrane expression in MV compared to MA and
MV are resistant to desensitization. ET-1 stimulation promotes increased membrane
localization of ETAR and ETBR and this effect is more prominent for ETBR in MV. These data
indicate ETBR receptors traffic to the plasma membrane more efficiently in MV than in MA
and this difference may contribute to the enhanced contractile sensitivity of MV to ET-1.
91
Glutathione-s-Transferase -1 is a Candidate Modifier Gene for Renal
Vascular Pathology
Kelly K Parsons, Mitchel D Harris, Thomas M Coffman, Thu H Le; Duke Univ Med Cntr,
Durham, NC
On susceptible genetic backgrounds such as C57BL/6 (B6), mice with targeted disruption of the
AT1A receptor gene locus (Agtr1a) develop renal vascular pathology characterized by medial
thickening of interlobular arteries and arterioles, resembling lesions seen in patients with
hypertensive nephrosclerosis. We identified a locus, msrvh1, from B6 conferring susceptibility
for renal vascular pathology. Msrvh1 spans 5 cM on chromosome 3 and contains at least
40 known genes. As one approach to identify candidate genes, we performed microarray
analysis with RNA from kidneys of Agtr1a-/- mice with susceptible (B6) and resistant (129/SvEv)
genetic backgrounds. We identified 2 differentially expressed genes located within msrvh1. Of
interest, both were members of the glutathione-s-transferase (Gst) super-family of enzymes
involved in the detoxification of reactive oxygen species (ROS). Expression of one of these, Gst
-1 (Gstm1), was previously reported to be reduced in stroke-prone spontaneously hyperten-
sive rats, perhaps contributing to the increased oxidative stress seen in SHRSPs. In the arrays,
Gstm1 expression was reduced by 50% in kidneys from B6 compared to 129 Agtr1a-/- mice
(6.70.5 vs. 13.50.4; p0.004). To verify the differences in Gstm1 expression between the
strains, we performed Northern analysis and found a similar, significant reduction in Gstm1
mRNA levels in kidneys from B6 compared to 129 Agtr1a-/- mice (270.2 vs. 392.7
%GAPDH; p 0.01). A corresponding difference in Gst1 protein levels was also seen by
Western blotting. We next assessed Gst activity in kidney tissues of B6 and 129 Agtr1a-/- mice
using a spectrophotometric assay (Sigma). Total Gst activity tended to be lower in kidneys from
B6 compared to 129 Agtr1a-/- mice, p0.051. After treatment with Cibacron which inactivates
the Gst and classes, leaving Gst intact, we found a significant 30% reduction in Gst
activity in B6 kidney tissues compared to 129 (9.2 0.9, n6 vs 13.5 0.5 nmol/ml/min,
n6 p 0.002). In summary, we have identified strain-specific differences in expression of
Gstm1 in the kidney. Genetic variants associated with reduced Gstm1 expression have a
reduced capacity for ROS inactivation in tissue and this may confer susceptibility to renal
vascular damage.
 e44 Hypertension Vol 48, No 4 October 2006

92
Chymase-Dependent Processing of Big ET-1 in Arteries but not Veins
Keshari Thakali, Catherine Rondelli, Gregory D Fink, Stephanie W Watts; Michigan State
Univ, East Lansing, MI
The endothelin (ET) family of peptides are vasoconstrictors more potent in contracting veins
than arteries. The precursor big endothelin-1 (big ET-1) can be processed by multiple vascular
enzymes to form contractile substances. We focused on metabolism of big ET-1 by endothelin
converting enzyme (ECE; to ET-1[121]), matrix metalloproteases (MMPs; to ET-1 [132]) and
chymase (to ET-1[131]). The overall hypothesis was that arteries and veins would be
differently dependent on these three systems. Immunohistochemical, western, zymographic
and isometric contractile assays in rat aorta and vena cava were used. Big ET-1 contracted
aorta [6017% maximal phenylephrine (PE) contraction] but was more efficacious in vena
cava [47861% maximal norepinephrine (NE) contraction]. Dependence on ECE in both artery
and vein was supported by immunohistochemical localization of ECE and its product ET-1
[121] to endothelial cells, and the reduction of big-ET-1 induced contraction by ECE inhibitors
phosphoramidon (10 uM; aorta: 33% control; vena cava: 37% control) and CGS-26393 (10 uM;
aorta: 26% control; vena cava: 80% control). By contrast, inhibition of MMPs with minocycline
(50 uM) and GM6001 (5 uM) did not reduce big ET-1 -induced contraction in aorta or vena cava,
though zymographic analyses confirmed active MMPs in all tissues. The product of MMP2,
purified ET-1[132] (100 nM), contracted aorta (269% max PE) and vena cava (25288%
max NE). Chymase was detected in both aorta and vena cava via Westerns and immunohis-
tochemistry; inhibition of chymase (chymostatin, 100 uM) reduced arterial (19% control) but not
venous constriction to big ET-1. Both vessel types contracted to the product of chymase,
ET-1[131] (100 nM; aorta 628% max PE; vena cava 16351 max NE). These were randomly divided into four groups: 1) CCR2 / controls, 2) CCR2 / infused with Ang
results suggest at least one significant difference, the role of chymase, in the impact made by
enzymatic processing of big ET-1 in arteries and veins. These findings are relevant to
hypertension because chymase processes not only big ET-1, but also angiotensin peptides.
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90mM KCl were also similar across groups (18758 and 13330 nM in AngHS and control
cells; respectively). Therefore, Ang II hypertension plus high salt markedly attenuates
preglomerular calcium signaling responses to P2 receptor activation, with particular impair-
ment of P2X1 receptor-mediated responses. These data are consistent with the hypothesis that
P2X1 activation of calcium signaling pathways plays a central role in pressure-mediated
autoregulatory responses. Impairment of P2X1 receptor dependent responses may explain the
hypertension-induced impairment of renal microvascular autoregulatory capability.
95
Role of CCR2 in the Development of Renal Damage in Angiotensin
II-Induced Hypertension
Tang-Dong Liao, Xiao-Ping Yang, Edward G. Shesely, Yun-He Liu, Jiang Xu, Mingzhu Tian,
Maria A. Cavasin, Oscar A. Carretero; Henry Ford Hosp, Detroit, MI
Angiotensin II-induced hypertension is associated with an inflammatory response that may
contribute to the development of target organ damage. Monocyte chemoattractant protein-1
(MCP-1) plays a role in the recruitment and activation of macrophages/ monocytes via CC
chemokine receptor-2 (CCR2). We tested the hypothesis that in Ang II-induced hypertension,
reduction of the inflammatory response will decrease target organ damage in CCR2 -deficient
mice (CCR2 -/-). CCR2 -/- mice and age-matched C57BL/6J wild-type controls (CCR2 /)
II (5.2 ng/10g/min), 3) CCR2 -/- controls and 4) CCR2 -/- infused with Ang II. After 4 weeks of
Ang II infusion via osmotic mini-pump, systolic BP increased significantly compared to controls;
however, there was no difference between the two strains. In CCR2 -/-, macrophage infiltration

Because ET-1 and angiotensin II are mediators in the pathogenesis of hypertension, this work and cell proliferation in the kidney decreased significantly (table). 24-hour urinary albumin was
underscores the potentially different roles played by the arterial and venous circulations in lower in CCR2 -/-, while GFR was higher (table). Glomerular matrix and Renal collagen content
elevation of blood pressure. was significantly Lower in CCR2-/- than in CCR2/ with Ang II infusion. We concluded that
CCR2 is required in the development of renal damage in Ang II-induced hypertension.

93 CCR2 CCR2 CCR2 CCR2

Parameters /(controls) /(Ang II) -/-(controls) -/-(Ang II)
Reduction of NOX by siRNA Ameliorates Tubuloglomerular Feedback (TGF)
during Angiotensin II Infusion SBP (mm Hg) 1062.9 1563.7*** 952.4 1594.9***
Urinary Albumin (g/24 hr) 364.4 26287.6* 394.1 5910.8#
Renal Macrophages 333 7010** 395 352##
Pouneh Nouri, Pritmohinder Gill, Christopher S Wilcox, William J Welch; Georgetown Univ,
(No./mm2)
Washington, DC Renal proliferating Cells 375 9410** 665 597##
(No./mm2)
Angiotensin II infusion increases oxidative stress, renal vasoconstriction and tubuloglomerular GFR (ml/min/100gBW) 1.040.08 0.590.08* 1.080.10 0.980.08##
feedback (TGF), but the specific role and source of superoxide (O2-) in these responses in Glomerular Matrix 8.950.76 14.480.99** 8.820.60 8.210.76##
unclear. We showed that reduction of the p22phox subunit of NADPH oxidase (NOX), the major (% glomerular area)
source of superoxide (O2-) in the kidney, by small interfering RNA (siRNA) treatment reduced Renal collagen content 22.81.6 28.00.8* 20.71.6 22.01.1##
(g/mg dry weight)
the blood pressure response to a slow pressor infusion of Ang II (200 ng/kg/min). To determine
whether NOX mediates the effects of Ang II on TGF, we measured single nephron GFR from Values are Mean SE ***p 0.001, **p 0.01, *p 0.05 Ang II vs controls; ##p 0.01, #p 0.05,
samples collected in the proximal tubule (PT) and distal tubule (DT) in rats undergoing a slow CCR2 -/- Ang II vs CCR2 / Ang II; p 0.01 CCR2 -/- vs CCR2 /
pressor infusion of Ang II, 2 days after IV injection of siRNA directed to p22phox (Ang II
siRNA) or vehicle (Ang II). Proximal SNGFR (which does not reflect macula densa-TGF control)
was not different between groups (Ang II: 423 vs Ang II siRNA: 473 nl/min, ns). However,
distal SNGFR (which does reflect macula-densa TGF control) was higher in Ang II siRNA rats
(293 vs 403 nl/min, n79, p0.05). Distal SNGFR in turn reflected the differences in
whole kidney GFR (0.580.07 vs 0.790.07 ml/min/100 g BW, n79, p0.05). PT flow was 96
not different, but DT flow was higher in Ang II siRNA rats (DT: 5.60.7 vs 7.30.4 nl/min, Aquaporin-1-Dependent NO Transport is Essential for Full Expression of
p0.05), suggesting NOX-dependent O2- enhances reabsorption in the loop of Henle. The
regulation of SNGFR by TGF, determined by the difference between Prox-Dist SNGFR was
Endothelium-Dependent Relaxation
reduced by in Ang II siRNA (Ang II: 12.4 2.1 vs Ang II siRNA: 6.9 1.1 nl/min, n79,
Marcela Herrera, Jeffrey L Garvin; Henry Ford Hosp, Detroit, MI
p0.05). We conclude that O2- generated by NOX in the kidney contributes to the renal
vasoconstriction and fall in GFR during Ang II infusion via activation of the macula densa
Nitric oxide (NO) released by vascular endothelial cells relaxes adjacent smooth muscle cells.
mechanism that enhances the TGF response.
It has been generally assumed that NO freely diffuses out of the endothelial cells without need
of a specific transporter. However, we have recently shown that the water channel aquaporin-1
(AQP-1) transports NO across cell membranes. Vascular endothelial cells express AQP-1, but
94 the role these water channels play in the release of NO and endothelium-dependent relaxation
P2 Receptor Mediated Calcium Signaling Responses are Impaired in is unknown. We hypothesized that AQP-1 mediates the release of NO out of endothelial cells,
Preglomerular Smooth Muscle Cells (PVSMC) of Ang II Hypertensive Rats and thus endothelium-dependent relaxation is impaired in AQP-1 knockout (KO) mice. We
Fed a High Salt Diet measured a) NO release by endothelial cells from wild-type (WT) and AQP-1 KO mice, in
response to 1 M acetylcholine (Ach) using a NO-selective sensor and b) the ability of Ach to
Edward W Inscho, Andrea Clarke, Shali Zhang, Crista Royal, David Osmond; Med College of relax aortic rings from WT and AQP-1 KO mice, using a wire myograph. In confluent aortic
Georgia, Augusta, GA endothelial cells from WT mice, Ach increased NO release by 111.5 24.3 pmol NO/mg
protein after 1 min. In contrast, Ach increased NO release only by 32.9 12.2 pmol NO/mg
P2X1 receptors play a critical role in pressure-mediated afferent arteriolar autoregulatory protein in endothelial cells from AQP-1 KO mice, 76 % less (n 3, p 0.044). In intact aortic
responses. These autoregulatory responses are significantly impaired in kidneys from Ang II rings, the ability of Ach to cause vasorelaxation was significantly reduced in AQP-1 KO
hypertensive rats fed a high salt diet for 14 days (AngHS), and this impairment is associated compared to wild-type mice at almost all concentrations tested (table). We found no differences
with impaired afferent arteriolar responsiveness to P2X1 receptor activation. We tested the in the relaxation induced by the NO donor sodium nitroprusside in endothelium-denuded aortic
hypothesis that PVSMC from AngHS exhibit compromised calcium signaling responses to P2 rings among strains. We conclude that: 1) AQP-1 transports endogenously produced NO out of
receptor activation. Rats were infused with angiotensin II (65ng/min) for 14 days by osmotic endothelial cells; 2) this is essential for full expression of endothelium-dependent relaxation;
minipump while being fed an 8% salt diet. PVSMC were isolated and their calcium signaling and 3) the impaired relaxation observed in AQP-1 KO mice is not due to changes in the
responses were determined using a microscope-based fura 2 technique. Calcium signaling signaling cascade beyond NO. AQP-1-dependent NO transport could be an important regulatory
responses were determined for ATP, the P2X1 receptor agonist, , -methylene ATP, or the mechanism of vascular resistance. Inadequate transport of NO by AQPs may be an alternate
P2Y2 receptor agonist, UTP. Systolic blood pressure averaged 1165 mmHg in control rats explanation for diseases believed to be caused by reduced NO production or availability, such
(n8) compared to 2058 mmHg in AngHS rats after 14 days (P0.05; n9). The increase as hypertension.
in calcium induced by ATP (10M) in cells from AngHS rats was significantly reduced and
% Relaxation
averaged 19836 nM compared to 35780 in control cells (P0.05; n28 35). Consistent
with impaired P2X1-receptor mediated vasoconstriction in hypertensive rats, calcium signaling Ach [M] 0.003 0.01 0.03 0.1 0.3 1 3
responses were markedly attenuated in AngHS rats. , -methylene ATP (10M) increased WT 134 316 469 668 776 834 834
calcium by 699 nM in cells from AngHS rats compared to 33392 nM (P0.05; AQP-1 KO 00.5 51 204 477 577 576 577
n10 15). Responses to UTP (10M) were similar across the two groups and averaged p value 0.01 0.001 0.02 n.s 0.05 0.01 0.02
529125 and 737136 nM for the AngHS and control groups; respectively. Responses to

97
Resetting of Pressure Natriuresis by Decoy of Transcription Factor
HIF-1alpha in the Renal Medulla
Ningjun Li, Li Chen, Pin-Lan Li; Med College of Virginia, Virginia Commonwealth Univ,
Richmond, VA
Hypoxia inducible factor-1 (HIF-1), a transcription factor, is abundantly expressed in the
renal medulla and regulates many oxygen-sensitive genes such as nitric oxide synthase (NOS),
cyclooxygenase-2 (COX-2) and heme oxygenase-1 (HO-1). Given that the expression of these
genes are importantly involved in the long term control of arterial blood pressure, the present
study is designed to test the hypothesis that HIF-1 serves as an antihypertensive factor to
regulate renal medullary function and sodium excretion by activating regional expression of
these oxygen-sensitive genes. A new gene transfection strategy with ultrasound-
microbubble technique was used for local delivery of HIF-1 decoy oligodeoxynucleotide
(ODN) into the renal medulla in Sprague Dawley rats. This HIF-1 decoy ODN would block
the binding of HIF-1 and thereby inhibit the transcription of its target genes. Ten days
after ODN transfection, the HIF-1 binding activities were significantly inhibited by 45%
and the mRNA levels of HO-1, one of the HIF-1 target genes, were decreased by 51% in
renal medullary tissues in decoy ODN transfected rats compared with the scrambled ODN
transfected rats. The diuretic and natriuretic responses to the elevation of renal perfusion
pressure (RPP) (from 80 - 160 mmHg) were significantly blunted by 50% in HIF-1 decoy
OND infused rats, accompanied by a 37% inhibition in the increases of renal medullary
blood flow (MBF) in response to the elevations of RPP. Intravenous infusion of angiotensin
II (Ang II) at 0.2 nmol/Kg/min produced a much larger reduction of renal MBF in HIF-1
decoy ODN-treated rats than in scrambled ODN-treated rats (66 6.1% vs. 84 3.9 %
of the control MBF), while there was no difference in Ang II-induced decreases in cortical
blood flow in both rat groups. In summary, local delivery of transcription decoy ODN with
ultrasound-microbubble technique effectively blocked the activity of HIF-1 in the renal
medulla, and that decoy of HIF-1 blunted pressure-natriuresis and enhanced the
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
CHBPR ConferenceOral Presentations e45
hemodynamic response to Ang II in the renal medulla. It is suggested that HIF-1-
mediated gene activation importantly participates in the regulation of renal medullary
function and pressure natriuresis. (Supported by NIH grants DK 054927 and HL 70726).
98
Preventing an Increase in RIHP Reduces Renal 20-Hete Levels and Inhibits
Pressure Natriuresis
Jan M Williams, Robert P Ryan, Averia K Flasch, Richard J Roman; Med College of
Wisconsin, Milwaukee, WI
Previous studies from our laboratory have demonstrated that inhibitors of the formation of
20-hydroxyeicosatetraenoic acid (20-HETE) attenuate the pressure natriuretic response.
However, the signal that increases 20-HETE release following elevations in renal perfusion
pressure (RPP) is unknown. This study examined the effects of changes in renal interstitial
hydrostatic pressure (RIHP) on renal 20-HETE levels and the pressure natriuretic response in
male Sprague Dawley rats. In control animals, 20-HETE levels in renal cortical tissue rose from
0.760.26 to 1.420.33 ng/g of tissue (n6) following elevations in RPP from 103 to 166
mmHg. After opening the renal capsule to prevent the rise in RIHP, 20-HETE levels were not
significantly altered and averaged 0.490.14 and 0.600.14 n/g of tissue (n6) at low and
high pressure, respectively. Preventing a rise in RIHP by opening the renal capsule blunted the
pressure diuretic and natriuretic responses by 41% and 36%, respectively without altering
glomerular filtration rate (GFR) or renal blood flow (RBF). Pre-treating the animals with
N-hydroxy-N-(4-butyl-2methylphenyl) formamidine (HET0016, 1 mg/kg/hr, i.v.), a selective
inhibitor of the synthesis of 20-HETE, completely prevented the rise in 20-HETE levels in the
renal cortex following an elevation in RPP and blunted the diuretic and natriuretic responses by
47% and 39%, respectively. HET0016 had no significant effect on GFR or RBF. These results
suggest that elevations in RPP increase 20-HETE levels in the kidney secondary to increasing
RIHP and that 20-HETE contributes to the pressure natriuretic response.
 e46 Hypertension Vol 48, No 4 October 2006

Poster Presentations
P1
Gender Differences in Baroreflex Sensitivity and Lifespan in
Renin-Angiotensinogen Transgenic Hypertensive Mice
Veronica A Peotta, Rasna Sabharwal, Harald M Stauss, Eric Lazartigues, Carol A Whiteis,
Francois M Abboud, Mark W Chapleau; Univ. of Iowa, Iowa City, IA
Men and women differ in baroreflex sensitivity (BRS), susceptibility to cardiovascular disease,
and lifespan. Decreased BRS predicts increased mortality post-myocardial infarction. The goal
of this study was to characterize BRS and age-related mortality in male vs. female mice with
and without hypertension (HT). We hypothesized that BRS is impaired at a young age in HT,
most prominently in male mice; and that age-related mortality is greater in male vs. female HT
mice. Blood pressure (BP) was measured in conscious control and HT (renin-angiotensinogen
transgenic, RA) mice by radiotelemetry. Spontaneous BRS was calculated using the
sequence technique. BRS was markedly impaired in male RA mice compared with male
control mice at a young age (20 wks) (see Table, *P0.05). BRS tended to be reduced in
female control mice, yet was slightly but significantly higher in female RA compared with
male RA mice (**P0.05). Consequently, the HT-induced BR impairment was not
statistically significant in females. Age-related mortality defined by Kaplan-Meier plots
(Controls, n90; RA, n36) was markedly increased at one year of age in male RA
mice (43%), an effect not seen in female RA mice (8%). Mortality at one year was low in
male (7%) and female (3%) control mice. The differences in BRS and mortality could not be
attributed to BP which was comparably increased in male and female RA mice (1653 vs.
1564 mmHg, respectively). We conclude that male mice with angiotensin-dependent HT
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months of age. Female sheep were synchronized in their estrous cycles by withdrawal of a
progesterone-releasing implant. ACE and ACE2 activities were determined in serum by
assessing the metabolism of radiolabeled Ang I and Ang II to Ang II and Ang-(17), respectively.
Ang I was readily converted to Ang II, Ang-(17) and Ang-(15) in serum and Ang II production
was abolished with the ACE inhibitor lisinopril (LIS); however, Ang-(19) was not detected in
control or LIS-treated samples. Both male and female sheep exhibited similar ACE activities
based on the rate of Ang I to Ang II conversion [male: 49 5 fmol/ml/min, n5; female: 45
7 fmol/ml/min, n3]. Ang II was primarily converted to Ang-(17) in serum and its formation
was abolished with the ACE2 inhibitor MLN4760. Serum ACE2 activity based on Ang II to
Ang-(17) formation was higher in male versus female [male: 38 4 fmol/ml/min, n5;
female: 22 2 fmol/ml/min, n4; p0.05]. ACE2 inhibition increased the half-life of Ang II
6 fold in male serum [101 24 min vs. 602 196 min, n3] and 2.9 fold [150 29 min
vs. 439 150 min, n3] in female serum. Immunoblots using an N-terminally directed
antibody revealed a 120 kD band in both male and female serum samples consistent with the
activity measurements. ACE2 activity was readily detectable in sheep serum and constituted
the primary pathway for Ang II metabolism, although males exhibited higher serum activity than
females. Serum ACE2 did not directly convert Ang I to Ang-(19), however, Ang I to Ang II
conversion lead to increased levels of Ang-(17) via ACE2. We conclude that a soluble form of
ACE2 is evident in serum at activity levels comparable to ACE and that both enzymes may
contribute to the circulating levels of Ang II and Ang-(17).
P4
Role of Angiotensin II in Female Mouse Model of Systemic Lupus

exhibit a profound decrease in BRS at a young age accompanied by very high premature
mortality while female RA mice are protected from HT-induced baroreflex dysfunction and
do not die prematurely. Decreased BRS along with other factors may contribute to increased
mortality in males with angiotensin-dependent hypertension. (VA and HL14388)
Males-BRS (ms/mmHg) Females-BRS (ms/mm Hg)
Control Mice 2.330.35 (n5) 1.610.40 (n8)
RA Mice 0.720.10*(n8) 1.000.19**(n9) treat its cardiovascular complications, the contribution of RAS to the pathophysiology of
P2
Gender Differences in the Renal Structural Changes in Response to the
Reduction of Ang II during Nephrogenic Period
Fara Saez, Dept of Physiology, Sch of Medicine, Univ of Murcia, Murcia, Spain; M T Castell,
Adelina Zuasti, Dept of Hystology, Sch of Medicine, Univ of Murcia, Murcia, Spain; Analia
Loria, Francisco Salazar, Virginia Reverte, F. J Salazar; Dept of Physiology, Sch of Medicine,
Univ of Murcia, Murcia, Spain
It is well documented that angiotensin II (Ang II) is involved in nephrogenic development, and
that Ang II inhibition during perinatal period induces an elevation in arterial pressure in adults.
This study was designed to test the hypothesis that normal renal development is more Ang
II-dependent in males than in females and consequently, there are gender differences in the
renal response to Ang II inhibition during perinatal period. Newborn SD rats were treated with
an AT1 Ang II receptor antagonist (ARAII) (L-158.809, 7 mg/kg/day) during the first 14 postnatal
days. Renal structural changes were examined at 3 and 9 10 months of age using appropriate
techniques in male and female rats treated with vehicle or ARAII. At three months of age,
systolic arterial pressure increased (P0.05) to the same extent in treated males and females
(127 0.5 vs. 115 0.7 mmHg in control rats, cr). Nephron number/gram b.w. decreased
similarly (P0.05) in treated males (54 5 vs. 98 3 in cr) and treated females (81 5
vs. 133 10 in cr) with the consequent elevation in mean glomerular volume that was 21%
greater (P0.05) in males than in females. Glomerulosclerosis was greater (P0.05) in treated
males (56 2%) than in treated females (31 3%). Tubulointerstitial damage was also
greater (P0.05) in treated males than in treated females in renal cortex (22 3% vs 10
2%), outer medulla (17 3 vs. 9 2), and inner medulla (36 13 vs. 8 2). The ratio
arteriolar wall thickness/luminal diameter was elevated in adult rats treated with ARAII during
the nephrogenic period but the increment was greater (P0.05) in males (3.5 0.1) than in
females (2.3 0.1). Another important gender difference in the response to the Ang II inhibition
during the nephrogenic period was that only treated males had a significant decrease in papilla
volume (50%, P0.05). At nine months of age, there was a further elevation of arterial
pressure in treated males (134 0.9 mmHg) and treated females (131 0.5 mmHg), and the
previously mentioned gender differences in renal structure were significantly accentuated. In
summary, the results of this study provide new evidences showing important gender
differences in the response of renal structure to the reduction of Ang II effects during
nephrogenic period.
P3
Gender Differences in ACE2-Dependent Metabolism of Angiotensin II in the
Serum of Sheep
Brian M Westwood, Jorge Figueroa, James C Rose, Mark C Chappell; Wake Forest Univ Sch
of Medicine, Winston-Salem, NC
Increasing evidence suggests that angiotensin converting enzyme 2 (ACE2) is an enzymatic
component of the renin-angiotensin system (RAS) that may modulate the conversion of Ang II
to Ang-(17). We have shown in heart and kidney that tissue-bound or membrane-associated
ACE2 plays a key role in the metabolism of Ang II, but not Ang I. The issue of circulating ACE2
and its contribution to angiotensin metabolism has not been investigated; therefore the current
studies evaluated ACE2 activity in the serum from both male and female adult sheep at 18
Erythematosus
Michael J Ryan, Gerald R McLemore, Jr.; Univ of Mississippi Med Cntr, Jackson, MS
Systemic Lupus Erythematosus (SLE) is an autoimmune inflammatory disorder that predomi-
nantly affects women. Individuals with SLE have a high incidence of hypertension, renal, and
vascular disease. While blockade of the renin angiotensin system (RAS) is commonly used to
hypertension during SLE is unclear. We tested the hypothesis that activation of the RAS
contributes to hypertension and altered angiotensin (AngII) dependent renal blood flow (RBF)
responses in a mouse model of SLE (NZBWF1). Anesthetized female SLE mice or controls
(NZW/LacJ) were instrumented with a RBF probe and carotid artery and jugular vein catheters.
Mean arterial pressure (1083 vs. 873 mmHg, n8, p0.01) and renal vascular resistance
(212 vs. 152 RU, p0.03) were significantly increased in SLE mice. Acute infusion of the
AngII receptor blocker losartan (10g, i.v. bolus) caused a similar increase in RBF in control and
SLE mice (457% vs. 456%, n10); however, losartan caused a greater decrease in
mean pressure in SLE mice compared to controls (-142 vs. -82 mmHg, n10, p0.04).
Compared to controls, the RBF response to infusion of exogenous AngII (1ng, i.v. bolus) in SLE
mice was blunted (284% vs. 123%, p0.01). After placing mice on a high salt diet (4%,
7 days) to suppress endogenous RAS, the pressor response to AngII infusion was unchanged
(4.71.1 vs. 4.41.0 mmHg) in control mice and increased in SLE mice (3.20.9 vs. 5.51.2
mmHg). Similarly, the RBF response to AngII was restored after high salt treatment in SLE mice
(-224%, n8, p0.05) but unchanged in controls (-325%, n7). These data suggest an
increased activity of RAS and support a potential role for RAS in SLE induced hypertension.
P5
Up-Regulation of Renal Ace2 is Associated with Gender Differences in a
Model of Fetal Programming of Hypertension Induced by Placental
Insufficiency
Daniela Grigore, Norma Ojeda, Elliott B Robertson, Barbara T Alexander; Univ of Mississippi
Med Cntr, Jackson, MS
Hypertension and cardiovascular disease are programmed by exposure to adverse conditions
in utero. Reduced uterine perfusion initiated at day 14 of gestation in the pregnant rat results
in intrauterine growth restriction (IUGR) and hypertension by 4 weeks of age. Gender
differences are present since only male IUGR offspring remain hypertensive after puberty. A role
for RAS involvement in IUGR hypertension is suggested as hypertension in adult male IUGR is
abolished by RAS blockade. Thus, the purpose of this study was to determine if age and gender
specific RAS alterations are associated with IUGR-induced hypertension. At 12 weeks of age
MAP by telemetry was significantly elevated (P0.05) in male IUGR as compared to male
control, female control, and female IUGR offspring (1351, 1211, 1171, 1262 mmHg,
respectively). Real-time PCR was used to measure renal cortical mRNA expression of RAS
components in male and female IUGR and control offspring. At 6 weeks of age, no significant
alterations in renal RAS components were present in male or female IUGR offspring as
compared to their sex-specific control counterpart. However, by 12 weeks of age, or after
establishment of hypertension, renal cortical mRNA expression of angiotensinogen (Aogen) was
significantly increased 11-fold in male IUGR (n 6) compared to male control (n 6); renin
24-fold and ACE 13-fold. Renal cortical mRNA expression of Aogen was also significantly
increased 1.6-fold in female IUGR (n 6) compared to female control (n 6) offspring as was
renin, 4.4-fold, and ACE, 5.5-fold. However, the difference in expression for each RAS
component, comparison of IUGR to control, was significantly less in female compared to male
offspring. At 12 weeks of age ACE2 which is known to counterbalance ACE activity was
significantly decreased 6-fold in male IUGR relative to male control offspring. ACE2 expression,
however, was significantly increased 6-fold in female IUGR relative to female control offspring.
Thus, these results suggest inappropriate activation of ACE pathway components may
participate in the maintenance of hypertension in adult male IUGR offspring. However, an
increase in ACE2 may be a compensatory mechanism that protects female IUGR offspring from
sustained hypertension into adulthood.

P6
Differential Vascular Tissue Distribution of Angiotensin II Receptor
Subtypes during Pregnancy
Vera V Koledova, Yaroslavl State Med Academy, Yaroslavl, Russian Federation; Raouf A
Khalil; Brigham and Womens Hosp, Boston, MA
Several forms of hypertension are associated with upregulation of the renin-angiotensin
system. Although the renin-angiotensin system is upregulated during normal pregnancy,
reduction in blood pressure and decreased vasopressor response to AngII are often observed;
however, the vascular mechanisms involved are unclear. AngII activates not only angiotensin
type 1 receptor (AT1R) to induce vasoconstriction, but also AT2R to release vasodilator
substances. We tested whether the pregnancy-associated reduction in the pressor response to
AngII reflects differences in the vascular tissue distribution of AT1R and AT2R subtypes.
Systolic blood pressure was measured in pregnant and virgin Sprague-Dawley rats, the
thoracic aorta was isolated and cryosections (6 micron) were prepared for histological
morphometric analysis using hematoxylin and eosin staining, and immunohistochemical
analysis using antibodies to AT1R and AT2R and VectaStain Ellite ABC Kit. Blood pressure was
lower in pregnant (1002 mmHg) than virgin rats (1043 mmHg). The vessel wall thickness
was thinner in pregnant (158) compared with virgin rats (206 microns). The tunica intima
thickness as % of total was smaller in pregnant (2) than virgin rats (4), while the % tunica
media thickness was greater in pregnant (68) than virgin rats (63), possibly related to increased
hemodynamic forces during pregnancy. Significant AT1R and AT2R staining could be identified
in tissue sections from pregnant and virgin rats. In virgin rats, AT1R staining was abundant and
evenly distributed in the tunica media and smooth muscle (49.5%) and smaller AT2R staining
was detected in the tunica intima and endothelium (26.6%). In pregnant rats, AT1R staining in
the smooth muscle layer was significantly reduced (32.3%), while AT2R staining in the
endothelium was enhanced (59.2%). AT1R and AT2R staining in the adventitia was not different
between virgin (18.3 and 22.9%) and pregnant rats (13.1 and 26.6%, respectively). These data
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suggest differential recruitment of the vasodilator AT2R and vasoconstrictive AT1R subtypes
between the vascular endothelium and smooth muscle during pregnancy, and may in part
explain the reduction of blood pressure despite upregulation of the renin-angiotensin system
during pregnancy.
P7
Presence of Activating Autoantibodies against the AT1-Receptor in Women
with a History of Preeclampsia
Ralf Dechend, Helios Klinikum, Franz-Volhard-Klinik, Charite, Berlin, Germany; Myles Wolf,
Renal Unit Massachusetts General Hosp, Boston, MA; Nina Markovic, Magee-Womens Rsch
Institute and Dept of Obstetrics and Gynecology, Pittsburgh, PA; Florian Herse, Helios
Klinikum, Franz-Volhard-Klinik, Charite, Berlin, Germany; Gerd Wallukat, Max-Delbrueck
Cntr, Berlin, Germany; Carl A Hubel; Magee-Womens Rsch Institute and Dept of Obstetrics
and Gynecology, Pittsburgh, PA
Activating autoantibodies against G protein-coupled AT1-receptor (AT1R-AA) and the alpha-
adrenergic receptor have been detected in the serum of preeclamptic patients. Whether or not
these activating autoantibodies persist after delivery is not known. If they persist, they might
be implicated in the increased cardiovascular risk after preeclampsia. We included 29
normotensive women with a history of preeclampsia and 32 women with normal pregnancies
during their first completed (nulliparous) pregnancy at 18.0 9.7 months postpartum in the
study. The activating antibodies were detected by the chronotropic responses to AT1
receptor-mediated stimulation of cultured neonatal rat cardiomyocytes coupled with receptor-
specific antagonists (losartan and prazosin). 17% of former preeclamptic patients and 3%
former normal pregnant patients showed a positive bioassay for antibodies directed at the
AT1-receptor (p0.05). Activating autoantibodies against the alpha-adrenergic receptor were
found in 10% of formerly normal pregnant and 14% of formerly preeclamptic patients. In an
earlier study, we found that sFLT1, indicating altered angiogenesis, is upregulated in the
formerly preeclamptic group (41.6 6.7 vs. 30.4 10.2; P 0.01). There was no correlation
between patients with AT1R-AA and sFLT-1. We conclude that AT1R-AA did not invariably
disappear after delivery but persist in nearly 20% of formerly preeclamptic patients. Further
follow-up is necessary to determine if AT1R-AA represent a subsequent risk in these women.
Lack of correlation between AT1R-AA and sFLT may indicate heterogeneous mechanisms
leading to preeclampsia.
P8
Low Dose Angiotensin II Paradoxically Lowers Blood Pressure in Female
Rats
Amanda Sampson, Rebecca L Flower, Robert E Widdop, Kate M Denton; Monash Univ,
Melbourne, Australia
There are striking sex-related differences in the regulation of arterial pressure and the
incidence of cardiovascular disease. The mechanisms are incompletely understood, but it is
suggested that sex-hormones contribute by differentially modulating the renin-angiotensin
system, one of the major regulators of blood pressure. The two main angiotensin receptors,
angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptor (AT2R), exert opposing
effects on the cardiovascular system. All the classical excitatory effects evoked by AngII
(vasoconstriction, sodium reabsorption) result from AT1R stimulation, while AT2R stimulation
causes vasodilatation and natriuresis, although the latter effects may be masked by the
overriding effects of AT1R stimulation. Indeed, it is now appreciated that there is a low level of
AT2R expression in the vasculature, although these data are almost universally obtained from
male animals. Thus there is a dearth of knowledge on the influence of gender on both AT1R
and AT2R expression and functional responsiveness in the cardiovascular system. We made the
remarkable observation that 2 weeks low dose AngII decreases blood pressure in female rats
(-81 mmHg, P0.05) at levels (50 ng/kg/min s.c) that increase blood pressure in males
(51 mmHg, P0.05). This startling finding is consistent with opposing vascular effects of
CHBPR ConferencePoster Presentations e47
AT1R and AT2R activation. Few studies have examined the effects of chronic AngII infusion in
females, and even fewer measure blood pressure via the sensitive method of radiotelemetry.
This finding is potentially of great significance as it suggests that females have in place
mechanisms to counter the classic pressor effects of AngII to increase blood pressure, that are
not present or less effective in males.
P9
Angiotensin Ii Type I Receptor Blockade Prevents Interleukin (IL-6)-Induced
Hypertension in the Pregnant Rat
Babbette LaMarca, Josh Speed, Lillian Fournier, Kathy Cockrell, Joey P Granger; UMMC,
Jackson, MS
Increases in inflammatory cytokines such as tumor necrosis factor (TNF) alpha and
interleukin-6 have been proposed to be an important link between placental ischemia,
endothelial dysfunction, and hypertension in women with preeclampsia. We have recently
reported that blood pressure and IL-6 are significantly elevated in response to reductions in
uterine perfusion in pregnant rats. We have also reported that a 5 day infusion of IL-6, at a rate
to mimic serum levels in the rat placental ischemic model, significantly increases blood
pressure in normal pregnant rats. We also found an increase in plasma renin activity in
pregnant rats with IL-6-induced hypertension, suggesting a role for activation of the
renin-agiotension system. Therefore, the purpose of this study was to determine the role of
angiotensin II in mediating IL-6-induced hypertension in pregnant rats. To achieve this goal, we
compared the effects of increasing serum IL-6 ( 5ng/day for 5 days, IV) in normal pregnant rats
and in pregnant rats pretreated with an angiotensin II type I receptor antagonist (Losartan, 40
mg/kg/day). Arterial pressure increased in normal pregnant rats (n6) from 102 2 to 118
4 mm Hg when serum IL-6 levels were increased from 63 5 pg/ml to 230 54 pg/ml.
In sharp contrast, blood pressure in losartan-pretreated pregnant rats infused with IL-6 was not
significantly different (854 vs 854 mmHg) than control pregnant rats pretreated with
losartan (n8). The results of this study indicate that activation of the angiotensin type 1
receptors play an important role in mediating IL-6-induced hypertension in pregnant rats.
P10
Ovariectomy Induces Hypertension in Adult Growth Restricted Female
Offspring in a Model of Fetal Programming due to Placental Insufficiency
Norma Ojeda, Daniela Grigore, Elliott B Robertson, Barbara T Alexander; Univ of Mississippi
Med Cntr, Jackson, MS
In a rat model of fetal programming induced by placental insufficiency, male intrauterine
growth restricted offspring (IUGR) are hypertensive in adulthood whereas female IUGR offspring
are normotensive. The renin angiotensin system (RAS) appears to play a role in IUGR-induced
hypertension as ACE inhibition abolishes hypertension in adult male IUGR offspring. The
purpose of this study was to determine whether removal of estrogen would reveal an increase
in arterial pressure in female IUGR offspring through unopposed activation of the RAS. Female
control and IUGR offspring underwent either ovariectomy (OVX) or sham surgery (intact) at 10
weeks of age followed by insertion of a telemetry probe for 24 hour measure of mean arterial
pressure (MAP) from 12 to 16 weeks of age. ACE inhibition (Enalapril, 40 mg/kg/day) or vehicle
was initiated in a subset of all groups at 14 weeks of age. At 16 weeks of age MAP was similar
upon comparison of intact female control (1178 mmHg) and intact female IUGR offspring
(1182 mmHg). Ovariectomy led to a significant increase in MAP in female IUGR offspring
(1422 mmHg, P0.05 vs. intact female IUGR), while it remained without effect in female
control offspring (1242 mmHg). ACE inhibition abolished hypertension induced by ovariec-
tomy in adult female IUGR offspring (961 mmHg, P0.05 vs. untreated OVX female IUGR).
However the decrease in blood pressure in response to Enalapril, in IUGR offspring was greater
in OVX group, treated vs. untreated, as compared to the intact group, treated vs. untreated
(difference of 462 mmHg vs. 245 mmHg, respectively, P0.001) indicating enhanced RAS
activation in the absence of estrogen. However, circulating RAS components were not
significantly altered following ovariectomy (plasma renin activity and plasma renin substrate)
suggesting activation of tissue RAS or increased sensitivity to angiotensin II may occur in the
absence of estrogen in female IUGR. Thus, these results suggest that development of
hypertension in ovariectomized female IUGR offspring may occur through unopposed activation
of the RAS.
P11
Differential Expression of Sry and Tyrosine Hydroxylase in Adrenal Gland of
WKY and SHR/y Rats
Daniel L Ely, Amy Milsted, Gail Dunphy, Monte Turner; Univ of Akron, Akron, OH
Our laboratory has shown that a locus on the SHR Y chromosome (Yc) increases blood pressure
(BP) in the SHR rat and in WKY rats that had the SHR Yc locus crossed into their genome (SHR/y
rat). We have shown a novel function for the locus of interest, Sry, a transcription factor on the
Yc which elevates tyrosine hydroxylase (TH) promoter activity and norepinephrine (NE)
production. SHR/y rats have increased NE content and turnover in kidney and heart. Also we
have identified at least 6 different copies of the gene on the Yc. When Sry was transfected to
a normotensive WKY, BP increased. To determine the time course of both Sry and TH adrenal
expression,the following study examined endogenous Sry and TH expression using real time
PCR in the adrenal glands in WKY and SHR/y strains, before and during the time when BP was
increasing (215 weeks). The adrenal epinephrine content increased 60% between 215
weeks in both strains. Adrenal NE content showed a 70% increase between 2 6 weeks in the
SHR/y strain, whereas the WKY strain showed only a 26% increase between 215 weeks. The
adrenal NE content may be a trigger for the sharp rise in BP around 6 weeks in the SHR/y strain.
In order to determine if exogenous Sry would elevate adrenal TH, NE and BP, we delivered
10g of either the expression construct, Sry1/pcDNA 3.1 or control vector, into the adrenal
medulla by electroporation. One week after exogenous Sry delivery there was a 98% increase
in TH compared to vector controls. After 3 weeks there were significant increases in resting
 e48 Hypertension Vol 48, No 4 October 2006
plasma NE and adrenal TH content compared to vector controls. BP was 30mmHg higher in Sry
injected animals (160mmHg) compared to vector controls (130mmHg) after 3 weeks. In
conclusion, Sry, a transcription factor on the Y chromosome, modulates adrenal medullary
function and blood pressure.
P12
Decreased Autonomic Contribution to Obesity-Associated Hypertension in
African Americans Women
Cyndya Shibao, Alfredo Gamboa, Andre Diedrich, Ginnie Farley, Italo Biaggioni; Vanderbilt
Univ, Nashville, TN
Obesity affects nearly 30% of the US population and is a risk factor for hypertension. Those
most affected, with the higher prevalence of obesity and hypertension, are African-Americans,
particularly women. Increased sympathetic activation has been associated with obesity and it
may contribute to obese-related hypertension, however blood pressure is poorly controlled with
sympatholytics agents in hypertensive African-Americans. We tested the hypothesis that the
autonomic nervous system contributes less to blood pressure regulation in obese African-
Americans as compared to obese Caucasians. A total of 17 obese healthy females were studied
(9 Caucasians, Age 374 years, BMI 351.2 Kg/m2, BP 1255/744 mm Hg, and 8 African
Americans, Age 352 years, BMI 340.9 Kg/m2 ,BP 1267/744 mm Hg, P0.05).
Complete autonomic blockade with trimethaphan decreased mean arterial blood pressure
(MAP) more in Caucasians (-194 mm Hg as compared to African Americans (-82 mm Hg,
Figure). Intrinsic MAP, in the absence of autonomic influences, was higher in African Americans
(836 vs 723 mm Hg in Caucasians, P0.124). The decreased in MAP was negatively
associated with the androgen to gynecoid ratio (A/G), an index of abdominal fat distribution
(R-0.6, P0.03). We conclude that the autonomic nervous system contributes less to blood
pressure regulation in African Americans as compared to Caucasians obese women.
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P13
Molecular Mechanisms of Sexual Dimorphism in Renal Injury in
Spontaneously Hypertensive Rats (SHR): Involvement of Angiotensin II and
the JAK/STAT Pathways
Amy Banes-Berceli, Mario B Marrero, Jennifer S Pollock, Jennifer C Sullivan; Med College
of Georgia, Augusta, GA
Male SHR have a more rapid progression of renal injury compared to female SHR. The
molecular mechanism(s) responsible for the sexual dimorphism in renal injury, however, is
unknown. We hypothesized that altered angiotensin II receptor 1 (AT1) signaling to the Janus
Kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in the renal
cortex from male and female SHR promotes renal injury and reno-protection in male and female
SHR respectively. While no studies have investigated differential regulation of the JAK/STAT
pathways by gender, previous studies have demonstrated in male diabetic rats that angiotensin
II-induced activation of the JAK2/STAT1 pathway increases proteinuria and renal injury.
Whereas, STAT5 activation has been shown to be anti-apoptotic in cultured endothelial cells.
Male and female SHR (14 16 week-old) were used in all studies, n5 8. A terminal plasma
sample was collected and kidneys were isolated for Western blot analysis. Plasma angiotensin
II levels were higher in female SHR compared to males (male: 10.21.2 pg/mL; female:
20.11.5, p0.001). Despite females having higher levels of plasma angiotensin II, male SHR
had greater AT1 protein expression in the renal cortex compared to female SHR (male:
0.0450.003 RDU; female: 0.0190.002 RDU, p0.001). We then assessed the level of
activation of a known AT1 pathway target, JAK/STAT. Activated JAK1 and JAK2 levels in the
renal cortex from female SHR were significantly higher compared to male SHR (expressed as
% phosphorylated/total protein, JAK1: male, 11.11.4 %; female, 44.78.3 %,p0.01; JAK
2: male, 56.74.4 %; female, 72.59.9 %, p0.05). Male SHR had significantly higher levels
of STAT6 (male: 65.517.2 %; female: 32.44.3 %, p0.05) and STAT1 (male: 45.13.8 %;
female 31.26.2%, p0.05) activation in the renal cortex, while female SHR had significantly
higher levels of STAT5 (male: 4.03.2 %; female 25.65.5%, p0.05) activation. These data
suggest that differential activation of the AT1/JAK/STAT pathways may be involved in promoting
renal injury in male SHR and the reno-protection afforded to female SHR.
P14
Prolonged Enhancement of Vascular Inflammatory Process and Oxidative
Stress Aggravates Hypertensive Myocardial Fibrosis and Diastolic
Dysfunction in Ovariectomized Rats
Takahiro Mori, Hisashi Kai, Hiroshi Kudo, Yusuke Sugi, Narimasa Takayama, Kiyoko
Takemiya, Mitsuhisa Koga, Yumiko Kawai, Daisuke Fukui, Ayami Ikeda, Masayoshi
Futamata, Hideo Yasukawa, Tsutomu Imaizumi; Kurume Univ, Kurume, Japan
The prevalence of diastolic dysfunction is high in hypertensive women, particularly in the
post-menopausal state. However, the underlying mechanism remains unknown. Recently, we
have shown that in male Wistar rats with a supra-renal aortic constriction (AC), pressure
overload induces a transient perivascular inflammation, which leads to reactive myocardial
fibrosis and the resultant diastolic dysfunction. The aims of this study were to determine
whether the estrogen withdrawal would aggravate pressure overload-induced myocardial
fibrosis and the resultant diastolic dysfunction and if so to examine whether these changes
would be associated with inflammatory process. For these purposes, female Wistar rats were
randomized to the following 2 groups: ovariectomy (OVX)AC rats received bilateral OVX and
ShamAC rats received the sham-operation at 6 week-old. In both groups, rats underwent AC
at 10 week-old (Day 0). OVX had no effects on blood pressure elevation during the observation
period up to 28 days after AC (Day 28). There were no significant differences in LV weight/body
weight ratio and myocyte diameter between the two groups at Day 28. However, OVX enhanced
the AC-induced myocardial fibrosis and aggravated diastolic dysfunction assessed by Doppler
echocardiography. In ShamAC rats, myocardial MCP-1 induction and perivascular macro-
phage infiltration were transiently induced with a peak at Day 3, and they returned to the
baseline levels by Day 28. In OVXAC rats, the AC-induced MCP-1 induction and macrophage
infiltration at Day 3 were enhanced and remained high levels at Day 28. In ShamAC rats,
dihydroxyethidium (DHE) staining revealed a transient superoxide generation in the intramyo-
cardial arterioles, peaking at Day 3 and decreasing to the insignificant level by Day 7. In
contrast, the vascular DHE signals remained higher levels at Day 28 in OVXAC rats. In
conclusion, the estrogen withdrawal aggravated the pressure overload-induced myocardial
fibrosis and diastolic dysfunction through prolonged enhancement of vascular inflammatory
process and oxidative stress.
P15
Dysregulation of the Circulating and Local Renin-Angiotensin System in
Preeclampsia
Florian Herse, Ralf Dechend, Helios Klinikum, Franz-Volhard-Klinik, Charite, Berlin, Germany;
Nina Harsem, Dept of Obstetrics and Gynecology, Ulleval Univ Hosp, Oslo, Norway; Gerd
Wallukat, Max-Delbrueck Cntr, Berlin, Germany; Jurgen Janke, Henning Damm, Helios
Klinikum, Franz-Volhard-Klinik, Charite, Berlin, Germany; Dominik N Muller, Max-Delbrueck
Cntr, Berlin, Germany; Anne C Staff; Dept of Obstetrics and Gynecology, Ulleval Univ Hosp,
Oslo, Norway
The renin-angiotensin system (RAS) has been implicated in the pathogenesis of preeclampsia,
although conflicting results have been demonstrated, especially regarding locally tissue-related
RAS. Plasma renin activity (PRA) and local RAS gene expression was investigated in
preeclamptic (n11) and uneventful pregnancies (n10) at cesarean delivery. Maternal
serum, subcutaneous adipose tissue, placental biopsies and decidual vacuum suction material
was collected. Autoantibodies against the AT1 Receptor (AT1R-AA) were measured by cardiac
contraction bioassay. We also analyzed mRNA expression of renin, renin receptor (ReRe),
angiotensinogen (Aogen), angiotensin-converting enzyme (ACE), AT1 Receptor and AT2
Receptor in decidua, placenta and adipose tissue by quantitative real time RT-PCR. PRA was
significantly reduced in the maternal circulation of preeclamptic patients. We detected AT1R-AA
in 78% of preeclamptic patients compared to 0% of control patients. AT1 receptor expression
was 4-fold increased in the decidua of preeclamptic patients compared to controls. There was
a 1.5 fold increased AT1-expression in adipose tissue of preeclamptic patients. AT2 receptor
expression was nearly absent in preeclamptic placenta, whereas weak expression was
detectable in most of the controls (10% vs 60%). No difference between the two groups was
observed for renin, ReRe, ACE or Aogen expression in either tissues investigated. However,
renin expression was nearly 114-fold increased in decidua compared to placenta for both
patient groups. The substrate Aogen and ACE were also much higher expressed in decidua as
compared to placenta biopsies. In conclusion,Tissue RAS genes and PRA are dysregulated in
preeclampsia. The activating AT1 receptor autoantibody is present in most preeclamptic
women. The AT1 receptor expression is upregulated in decidua and adipose tissue of
preeclamptic women. These findings suggest a role for the RAS in preeclampsia.

P16
Estrogen Blunts Angiotensin II- Induced Activation of NAD(P)H Oxidase in
Female Mice: Role of Thioredoxin
Talin Ebrahimian, Ernesto L Schiffrin, Lady Davis Institute for Med Rsch, SMBD Jewish
General Hosp,McGill Univ, Montreal, Canada; Rhian M Touyz; Kidney Rsch Cntr, Ottawa
Health Rsch Institute, Ottawa, Canada
Thioredoxin (Trx) is a small ubiquitous protein, which could act directly as an antioxidant by
modulating Reactive Oxygen Species (ROS). Estrogen has been shown to have protective
effects against oxidative stress. In addition there is gender dimorphism with respect to the Trx
system and differential regulation of the cardiac Trx system and NAD(P)H oxidase activity by
Angiotensin II (Ang II) in male and female mice. We questioned whether estrogen has a
protective effect in Ang II-induced hypertension and if the Trx system contributes to this
protective effect in female mice. C57bl/6 female intact (Cont) or ovariectomized (Ovx) mice
were infused with Ang II (400 ng/kg/min) for 14 days. Systolic blood pressure (SBP) was
measured by tail cuff. Oxidative stress was evaluated by dihydroethidine staining and
chemiluminescence. The expression of Trx was measured by western- blot. The SBP, similar
in Cont and Ovx groups (P0.05, n6), increased by Ang II to similar levels in both groups
(1253 vs. 897 mmHg in Cont and 1362 vs. 1126 mmHg in Ovx, P0.001 and
P0.0002 respectively). Aortic Trx basal expression similar in both groups, was decreased (0.4
fold) by Ang II in Cont mice (P0.01, n6), and was unchanged in Ovx mice compared to the
control counterpart (P0.05, n4). Aortic *O2- level as an index of oxidative stress, was higher
(1.5 fold) in Ovx group compared to Cont group, Ang II increased *O2- level (1.5 fold) only in Ovx
mice compared to Cont mice (P0.01, n4). Moreover NAD(P)H oxidase activity was similar
under basal conditions between both groups, was decreased by Ang II in Cont group (P0.01,
n4 5), whereas it was significantly increased (1.3 folds) in Ovx group (P0.05, n4 5).
These results demonstrate that estrogen negatively modulates the Trx and AngII-induced
NAD(P)H oxidase activity in Ang II infused female mice independently of the increase in blood
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pressure.
P17
Differential Regulation by ER Alpha and ER Beta for ACE and ACE2
K. Bridget Brosnihan, Liomar A Neves, Patricia Gallagher, Wake Forest Univ Sch of
Medicine, Winston-Salem, NC; Jeffrey B Hodgin, Oliver Smithies, Nobuyo Maeda; Univ of
North Carolina, Chapel Hill, NC
ACE and ACE2 are key enzymes in the regulation of Ang II and Ang-(17) formation. ACE is
known to be down-regulated by estradiol, but it is not known if estrogen (E2) regulates ACE2.
To investigate the role of estrogen receptor-alpha (ER) in estrogens regulation of ACE and
ACE2 mRNA in lung, we ovariectomized female mice lacking apolipoprotein E (AAee) or
apolipoprotein E and ER (ee) and treated them with estradiol (6 g/day) or placebo for 3
months. ACE and ACE2 mRNA were measured using RT-PCR. E2 treatment of ovariectomized
AAee mice downregulated ACE mRNA (1.00.05 vs 0.76 0.6 units, p0.005); a similar
reduction (1.10.1 vs 0.760.1 units) was observed in ee. E2 treatment had no effect on
ACE2 mRNA in AAee mice, but resulted in significant reduction in ACE2 mRNA in ee (0.96
0.1 vs 0.640.6 units, p 0.01). These results suggest that an ER-independent receptor,
most likely the ER, plays a role in ACE mRNA regulation. On the other hand, E2
down-regulation of ACE2 mRNA is revealed only in the absence of ER. Under conditions of
estrogen treatment where E2 downregulates ACE mRNA and has no effect on ACE2 mRNA, the
formation of Ang-(17) would be favored.
P18
Maternal Plasma and Placental Cytokine Profile in Hypertensive
Preeclamptic Women
Babbette LaMarca, UMMC, Jackson, MS; Bartira E Pinheiro da Costa, Pos-graduacao em
Medicina e Ciencias da Saude, FAMED/IPB/HSL, Pontifcia Universidade Catolica do RGS,,
Porto Alegre, Brazil; Lillian Fournier, UMMC, Jackson, MS; Kiele Hoffmann, Pos-graduacao
em Medicina e Ciencias da Saude, FAMED/IPB/HSL, Pontifcia Universidade Catolica do
RGS,, Porto Alegre, Brazil; Giovani Gadonski, Pos-graduacao em Medicina e Ciencias da
Saude, FAMED/IPB/HSL, Pontifcia Universidade Catolica do RGS, Porto Alegre, Brazil; Carlos
E Poli de Figueiredo, Pos-graduacao em Medicina e Ciencias da Saude, FAMED/IPB/HSL,
Pontifcia Universidade Catolica do RGS,, Porto Alegre, Brazil; Joey P Granger; UMMC,
Jackson, MS
Preeclampsia is a hypertensive disorder of human pregnancy that causes high morbidity and
mortality. Women with preeclampsia present with increased blood pressure, systemic
endothelial dysfunction, placental ischemia, and enhanced production of inflammatory
cytokines. Cytokines alter many immune response pathways and other biological responses
like endothelial function and angiogenesis. The aim of the present study was to examine the
cytokine profile in maternal plasma and placenta in normal pregnant and preeclamptic women.
Recruitment of preeclamptic and healthy pregnant women was performed at So Lucas Hospital,
Brazil. Patients with severe preeclampsia were diagnosed with blood pressure above 160/110
mmHg after 20 weeks of gestation accompanied by proteinuria of at least 2 g/24 hour. Samples
of blood and placental bed biopsy were collected immediately before and after caesarian,
respectively. Protein array analysis was used to characterize 120 cytokines in both plasma and
placenta tissue. Relevant cytokines were classified as pro-angiogenic (n37), inflammatory
(n60), anti-angiogenic (n4). The Student t and Chi-square tests were used to determine
statistical significance, p0.05. Approximately 50% of all cytokines examined were signifi-
cantly up-regulated in preeclamptic patients with greater differences seen in protein isolated
from placenta. In plasma samples 2 pro-angiogenic, 14 inflammatory and 2 anti-angiogenic
were significantly altered in preeclamptic patients. In protein isolated from placenta, 16
pro-angiogenic, 19 inflammatory and 1 anti-angiogenic was significantly altered in preeclamp-
sia. In summary, we found that placental and plasma cytokine profile was significantly altered
in preeclamptic women. Proinflammatory cytokines, such as TNF alpha and various interleu-
CHBPR ConferencePoster Presentations e49
kins, may be important mediators of endothelial dysfunction and hypertension in preeclamptic
women. Furthermore, the increased expression of the angiogenic cytokines from the ischemic
preeclamptic placenta, such as CNTF and angiogenin, may be evidence of an innate attempt
to improve placental blood flow via vascular remodeling.
P19
Human Renal Mesangial Cells Can Produce Aldosterone in Response to
Low-Density Lipoprotein(LDL)
Tetsuo Nishikawa, Jun Saito, Sachiko Suematsu, Yuzuru Ito, Yoko Matsuzawa, Hiroko Ito,
Yokohama Rosai Hosp, Yokohama, Japan; Tomoshige Kino, George Chrousos; National
Institute of Child Health and Human Development, Bethesda, MD
Aldosterone is classically produced by the zona glomerulosa cells of the adrenal cortices.
Systemic aldosterone plays an important role in the development of the microvascular disease
and glomerular damage of the kidney. Here, we investigated the possibility of local production
of aldosterone in the kidney, using human primary glomerular mesangial cells. These cells
produced both pregnenolone and aldosterone measured by specific radioimmunoassay and/or
GC/MS methods. The production of both steroids was significantly stimulated by treatment with
LDL, while angiotensin II had a synergistic effect. ACTH and (Bu)2cAMP, on the other hand,
failed to stimulate aldosterone production by these cells, suggesting that the local production
of this steroid by mesangial cells is regulated differently from that of adrenal zona glomerulosa
cells. LDL-induced aldosterone production by mesangial cells was further enhanced when
incubated with high concentration of glucose in the culture media. Mesangial cells expressed
the mRNA of the LDL receptor and steroidogenic enzymes, such as P450scc, 3b-HSD,
21-hydroxylase and CYP11B2. Expression of CYP11B2 mRNA was remarkably enhanced by
LDL. Mesangial cells also expressed mRNA of the mineralocorticoid receptor (MR), and LDL
stimulated its abundance by 3 fold, while spironolactone completely abolished this LDL effect.
Since MR is a known mineralocorticoid-response gene as well as an intracellular receptor
molecule for this steroid, these results suggest that locally produced aldosterone is biologically
active, stimulating the transcription rates of the mineralocorticoid-responsive genes by
activating the MR in mesangial cells. Our data indicate that human mesangial cells are an
aldosterone-producing tissue in which LDL plays a major regulatory role. Therefore, human
renal mesangial endocrine system may contribute to local aldosterone concentrations and
effects in the renal glomerulus independently of the systemic RAA system and may participate
in the development and progression of glomerular damage in several pathologic conditions
such as diabetic nephropathy.
P20
Non-Genomic c-Src-Dependent Signaling by Aldosterone Occurs through
Caveolin-Enriched Membrane Microdomains
Glaucia E Callera, Augusto C Montezano, Univ of Ottawa, Ottawa, Canada; Alvaro Yogi, Rita
C Tostes, Univ of Sao Paulo, Sao Paulo, Brazil; Rhian M Touyz; Univ of Ottawa, Ottawa,
Canada
We previously demonstrated that aldosterone (Aldo) rapidly increases activation of NAD(P)H
oxidase through c-Src-dependent pathways in vascular smooth muscle cell (VSMC). Aldo
induces c-Src trafficking into membrane cholesterol rich domains. Whether non-genomic
c-Src-dependent signaling by Aldo involves the specialized caveolin-enriched membrane
microdomains remains unclear. In the present study we hypothesized that Aldo activates c-Src
through Caveolin-enriched/cholesterol rich domains. Detergent resistant fractions from rat
VSMC were obtained by sucrose gradient centrifugation and identified by flotillin-2 expression.
Methyl--cyclodextrin (10-2) was used to disrupt membrane cholesterol rich domains.
Knockdown of Cav 1 protein by small interfering RNA was performed in VSMC. Aldo (10-7 mol/L)
effects on c-Src expression and phosphorylation were assessed in total protein and in
membrane fractions. NAD(P)H-dependent generation of .O2- were evaluated by lucigenin-
derived luminescence assay. The translocation of p47phox subunit was assessed in cholesterol
rich domains. Cav 1 knockdown abolished Aldo-induced c-Src phosphorylation (p 0.05). The
phosphorylation of cortactin, a c-Src downstream target, was inhibited by Cav 1 knockdown
(p 0.05). Aldo induced NAD(P)H-dependent generation of .O2- in VSMC (1-fold, p0.01) and
this effect was inhibited by cholesterol rich domains disruption (p 0.05). Aldo stimulated
p47phox translocation into cholesterol rich domains (2-fold, p0.05). Aldo-mediated c-Src
translocation into cholesterol rich domains (1.5-fold) was decreased by PP2 (0.2-fold, p0.05),
a c-Src activity inhibitor. Aldo actions were inhibited by eplerenone, a mineralocorticoid
receptor antagonist. Our results suggest that 1) c-Src activation by Aldo in VSMCs mediates
NAD(P)H oxidase activation through cholesterol rich domains, 2) c-Src phosphorylation
precedes its translocation into cholesterol rich domains, 3) caveolin-enriched membrane
microdomains contribute to non-genomic c-Src-dependent signaling by Aldo. Findings from
these studies further identify molecular mechanisms underlying Aldo-induced non-genomic
actions, which are mediated through classical mineralocorticoid receptors.
P21
Aldosterone Suppresses Insulin Signaling via the Downregulation of Insulin
Receptor Substrate-1 in Rat Vascular Smooth Muscle Cells
Hirofumi Hitomi, Hideyasu Kiyomoto, Dept of Cardiorenal and Cerebrovascular Medicine,
Kagawa Univ, Kagawa, Japan; Akira Nishiyama, Dept of Pharmacology, Kagawa Univ,
Kagawa, Japan; Taiga Hara, Kumiko Moriwaki, Naoki Kondo, Kumiko Kaifu, Genei Ihara,
Yoshiko Fujita, Masakazu Kohno; Dept of Cardiorenal and Cerebrovascular Medicine,
Kagawa Univ, Kagawa, Japan
Reports indicate impaired glucose tolerance in the patients with primary aldosteronism;
however, the relationship between aldosterone and insulin signaling pathway has not been
clarified. In this study, we examined the effects of aldosterone treatment on insulin receptor
substrate-1 (IRS-1) expression and Akt phosphorylayion in rat vascular smooth muscle cells
(VSMCs). IRS-1 protein expression and Akt phosphorylation were determined by Western blot
 e50 Hypertension Vol 48, No 4 October 2006
analysis with anti-IRS-1 and phosphorylated-Akt antibodies, respectively. P0.05 was
considered significant. Aldosterone (1100 nmol/L) dose-dependently decreased IRS-1 protein
expression with a peak at 18 h (n4 7 for each). Aldosterone-induced degradation of IRS-1
was markedly attenuated by treatment with a selective mineralocorticoid receptor antagonist,
eplerenone (10 mol/L, n4). Treatment with antioxidants, N-acetylcysteine (10 mmol/L) or
ebselen (40 mol/L), also abrogated aldosterone-induced IRS-1 degradation (n4 7 for each).
Additionally, proteasome inhibitor, MG132 (1 mol/L) prevented IRS-1 degradation (n4).
Aldosterone treatment abolished Akt phosphorylation induced by insulin (100 nmol/L, 5 min,
n4). These data indicated that aldosterone decreases IRS-1 expression via reactive oxygen
species stimulation in VSMCs; thus aldosterone may be involved in the pathogenesis of
vascular insulin resistance.
P22
Spironolactone Reduces Ischemic Infarct Size in Male, but not Female
Hypertensive Rats
Christine S Rigsby, Anne M Dorrance; Med College of Georgia, Augusta, GA
Data from the Framingham Heart Study suggests that women may be more sensitive to the
deleterious cardiovascular remodeling effects of aldosterone. Our laboratory has previously
shown that male spontaneously hypertensive stroke-prone rats (SHRSP) treated chronically
with spironolactone, a mineralocorticoid receptor antagonist, have 50% smaller ischemic
infarcts after the induction of middle cerebral artery occlusion (MCAO), without a change in
blood pressure. Therefore, we hypothesized that female SHRSP treated with spironolactone
would also have less damage after MCAO. Female SHRSP were treated for six weeks from six
weeks of age with spironolactone (25 or 50 mg/kg/day) and compared to non-treated controls
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and Wistar Kyoto (WKY) rats. At 12 weeks of age, cerebral ischemia was induced using the
permanent MCAO technique. After 18 hours, the brain was removed, sliced (2 mm), and stained
with 2% TTC to visualize the infarcted region; infarct volume was analyzed and the percent of
the hemisphere infarcted (%HI) was determined using the Swanson equation. Surprisingly,
spironolactone-treated female SHRSP did not have decreased infarct sizes compared to control
SHRSP (table). Mean arterial pressure was not altered by spironolactone treatment (table), as
evidenced by the use of telemetry. We also examined the effect of acute administration of
spironolactone on ischemic infarct size in female SHRSP. Spironolactone (25 mg/kg, IP) was
administered 24 hours before and again at the time of MCAO; infarct sizes were analyzed 24
hours post-MCAO, as described above. Acute administration of spironolactone also had no
effect ischemic infarct size (65.0 1.2%HI control vs. 63.8 1.5%HI spironolactone-treated,
p0.05). These interesting studies indicate an apparent sexual dimorphism in the actions of
spironolactone.
TABLE.
Experimental Group (n) Mean Arterial Pressure (mmHg) %HI
SHRSP - Control (9) 145.04.9 66.31.3
SHRSP - Spir 25 mg/kg (8) 129.76.3 63.21.3
SHRSP - Spir 50 mg/kg (7) - 61.81.3
WKY - Control (4) 98.21.6* 22.97.0*
All values are mean SEM. *p0.05 vs. SHRSP groups. Spir spironolactone
P23
Pentoxifylline Decreases Cardiac Hypertrophy and Renal Damage in Rats
with Mineralocorticoid Dependent Hypertension
Anita D Smith, Anne M Dorrance; Med College of Georgia, Augusta, GA
Rats with deoxycorticosterone acetate (DOCA)-salt hypertension have elevated levels of
proinflammatory cytokines. Pentoxifylline (PTX) is a phosphodiesterase inhibitor that inhibits the
release of tumor necrosis factor-, we have previously shown that treatment of DOCA-salt rats
with PTX reduces their blood pressure and increases the levels of the anti-inflammatory
cytokine interleukin-10. We hypothesized the PTX treatment would also reduce oxidative stress
and inflammation in DOCA-salt treated rats and that this would result in a reduction in cardiac
hypertrophy and urinary protein excretion. Male Sprague-Dawley rats were uninephrectomized
and treated with DOCA (200mg/kg) PTX (9.6mg/day), both groups of rats were given salt
water to drink (1% NaCl and 0.2%KCl), control rats were uninephrectomized and were given
regular tap water. After 28 days of treatment 24-hour urine samples and terminal blood
samples were obtained. PTX treatment significantly reduced the blood pressure in the
DOCA-salt treated rats and reduced cardiac hypertrophy as evidenced by a reduction in the
heart / body weight ratio. Plasma thiobarbitutic acid reactive substances (TBARS) were used as
a measure of oxidative stress; these were increased in the DOCA-salt rats and reduced by PTX
treatment. Plasma monocyte chemoattractant protein-1 (MCP-1) was used as a marker of
inflammation; this was also elevated in the DOCA-salt rats and reduced by PTX treatment. 24
hour urinary albumin excretion was lower in the DOCA-salt rats treated with PTX compared to
the rats treated with DOCA-salt alone, suggesting that PTX treatment may reduce renal
damage, interestingly, this occurred without a reduction in kidney hypertrophy (Table1). These
results suggest that PTX may be a valuable therapeutic agent for the treatment of hypertension
related end-organ damage.
TABLE 1
TBARS Urinary Heart/ Kidney/
Blood Pressure MCP-1 (nmol protein body body
(mmHg) (pg/ml) MDA/ml) (mg/dl) weight weight
DOCA-salt 2202.91 71521185 2.490.28 13219 0.500.02 0.670.02 prevented the increased mRNA levels of ENaC subunits but not SGK1. Na-rich aCSF increased
DOCAPTX 1887.54* 2354526* 1.240.21* 4111* 0.440.01* 0.650.03 MR binding densities in the SON (p0.05), whereas the mRNA levels did not change. In the
SHAM 1533.41* 1741339* 1.450.11* 206* 0.340.01* 0.470.01
Significantly different from DOCA-salt (p0.05), n6 10 in each group.
P24
Angiotensin II-Induced Protein Kinase D Activation is Mediated by Protein
Kinase C in H295R Human Adrenocortical Cells
Damian G Romero, Bronwyn L Welsh, Silvia Rilli, Licy L Yanes, Elise P Gomez-Sanchez,
Celso E Gomez-Sanchez; Univ of Mississippi Med Cntr, Jackson, MS
Angiotensin II (Ang II) is one of the most important physiological modulators of adrenal cell
physiology where Ang II regulates aldosterone secretion and proliferation. Abnormalities in
aldosterone secretion have been associated with nearly 10 % of the cases of primary
hypertension. However, Ang II signaling in adrenal cell remains poorly understood. H295R
human adrenal cells are a widely used experimental model to study adrenal cell physiology.
Protein kinases are crucial mediators in intracellular signaling. We screened for protein kinase
expression levels using the KinetworksTM system in H295R cells after 3 h Ang II treatment.
Protein kinase D (PKD) was the protein kinase that exhibits the most dramatic changes. PKD
is a member of a new class of serine/threonine protein kinases that is activated by
phosphorylation. PKD has been implicated in the regulation of a variety of cellular functions,
including signal transduction, membrane trafficking, protein transport, and cell survival,
migration, differentiation, and proliferation. We show that Ang II time- and dose-dependently
increase PKD phosphorylation in H295R cells. PKD phosphorylation occurs within 2 min of Ang
II treatment and at concentrations as low as 1 nM. PKD phosphorylation was dose-dependently
increased by the protein kinase C (PKC) activator PMA. Ang II-mediated PKD phosphorylation
was blocked by several PKC inhibitors as Bisindolylmaleimide I, Go 6983, Ro-31 8220 and
Ro-32 0432. PKC inhibitor profiles and PKC isozyme expression profile in H295R cells suggest
that PKC could mediate PKD phosphorylation. We used different experimental approaches to
interfere with PKC function and study its role on PKD phosphorylation. PKC translocation
inhibitor peptide decreased Ang II-mediated PKD phosphorylation (-47 %, p 0.05). PKC
expression downregulation by plasmid delivered RNA interference also decreased PKD
phosphorylation (-25 %, p 0.05) mediated by Ang II. This study reveals for the first time that
PKD is an intracellular signaling mediator of Ang II in human adrenal cells. These findings show
a novel intracellular signaling system in adrenal cells that could be involved in pathological
conditions related to alterations in Ang II or aldosterone secretion.
P25
The Effect of Aldosterone in Angiotensin Ii/High Salt Induced Heart
Damage and the Relationship with Oxidative Stress
Hong Wang, Tatsuo Shimosawa, Hiromitsu Matsui, Tomoyo Kaneko, Sayoko Ogura, Toshiro
Fujita; the Univ of Tokyo, Tokyo, Japan
Angiotensin II can cause organ damage by increasing oxidative stress directly. Recently,
aldosterone, which is released by angiotensin II, has also been shown to induce cardiac
damage partly by increasing oxidative stress. Most basic research revealed that aldosterone-
induced organ damage are closely related to salt intake. In order to clarify the relation between
salt status and aldosterone in cardiac damage and the role of oxidative stress, we examined
the effect of circulating aldosterone by salt restriction, local aldosterone by angiotensin II and
high salt loading and the aldosterone antagonist, eplerenone, in Sprague Dawly rat. Rats were
allocated to five groups as follows: 1. angiotensin II high salt diet; 2. angiotensin II low
salt diet; 3. high salt diet; 4. low salt diet; and 5. angiotensin II high salt diet eplerenone.
The blood pressure was highest in the angiotensin II high salt group and eplerenone did not
decrease the blood pressure. Diastolic heart function was monitored by both deceleration time
(DcT; measured with Doppler echocardiography) and time constant (T; measured with Left
ventricular pressure curve). Diastolic function was deteriorated by angiotensin II high salt
(DcT; 69.29 6.17 ms and T; 18.63 3.59 ms) which was reversed by aldosterone blockade
of eplerenone (DcT; 59.58 0.98 ms and T; 12.64 2.19 ms). Although aldosterone was
stimulated by angiotensin II, angiotensin II low salt group did not show diastolic dysfunction.
The oxidative stress, which is known to induce heart damage, was higher in high salt group
and it was decreased by eplerenone. These results suggest salt status is essential in oxidative
stress and angiotensin-aldosterone axis to induce heart damage. Eplerenone can attenuate
heart dysfunction independent of blood pressure, but by blocking the aldosterone effect and
decreasing NADPH oxidase-derived oxidative stress. Salt restriction is also effective in
preventing cardiac dysfunction by the inhibition of oxidative stress.
P26
Changes in Mineralocorticoid Receptors (MR) and Epithelial Sodium
Channels (ENaC) in Rat Brain by Central Sodium
Hong-Wei Wang, M.Shahrier Amin, Junhui Tan, Frans H Leenen; Univ of Ottawa Heart
Institute, Ottawa, Canada
Both MR and ENaC subunits (, , ) are expressed in rat brain on epithelia and neurons.
Central blockade of MR or ENaC prevents sympathetic hyperactivity and hypertension induced
by icv infusion of Na-rich aCSF. Mechanisms regulating ENaC in the brain are still unknown.
To assess transcriptional and post-transcriptional regulation, we evaluated MR and ENaC mRNA
and protein levels in the brain in response to Na-rich aCSF by real-time qRT-PCR,
immunohistochemistry and aldosterone receptor binding assay. mRNA levels of serum/
glucocorticoid regulated kinase 1 (SGK1) , an important regulator of ENaC, were also examined.
Male Wistar rats were infused icv for 2 weeks with regular or Na-rich aCSF with or without
spironolactone. Brain areas studied included ependyma of the anteroventral 3rd ventricle, SON,
PVN and SFO. In the ependyma, Na-rich aCSF increased protein levels (p0.05) for both
and ENaC, which were prevented by spironolactone. In the SON, Na-rich aCSF did not affect
the protein levels of the ENaC subunits but tended to increase (p0.05) and ENaC (p0.08)
mRNA levels. Na-rich aCSF also increased SGK1 transcripts (p0.005). Spironolactone
PVN, Na-rich aCSF increased ENaC protein levels (p0.05) and MR binding densities
(p0.06) but ENaC mRNA levels appeared to decrease (p0.08). Na-rich aCSF did not

affect the transcription of , ENaC, MR and SGK1 in the PVN. In the SFO, Na -rich aCSF only
significantly increased ENaC mRNA abundance and MR binding densities. Spironolactone did
not prevent the changes of ENaC expression in the PVN and SFO. These results suggest that
regulation of ENaC expression in response to Na-rich aCSF is quite region specific and may
reflect primary (ependyma, SON) and downstream (PVN, SFO) responses to CSF [Na]. The
upregulation of the SGK1 and ENaC transcription by Na-rich aCSF in the SON suggests that
SGK1 is involved in ENaC mRNA regulation. Spironolactone prevented the increases in ENaC
subunits expression only in the ependyma and SON, implying that, in addition to aldosterone,
other regulators (e.g. vasopressin) play an important role.
P27
Aldosterone Augments the Vasoconstriction and Reduced Vascular
Relaxation in AngII/L-NAME Treated Rats on High Salt Diet
Jeffrey C Lin, Harvard Med Sch, Boston, MA; Amanda K Stennett, Gordon H Williams,
Brigham and Womens Hosp, Boston, MA; Raouf A Khalil; Harvard Med Sch, Boston, MA
Aldosterone (Aldo) is a major component of RAAS and renal control mechanisms of blood
pressure particularly during sodium overload; however, the role of Aldo in the vascular control
mechanisms is less clear. We tested whether Aldo enhances the vasocostrictive effects of AngII
particularly during high salt diet. Male Sprague-Dawley rats were placed on normal (NS, 0.4%
NaCl) or high sodium diet (HS, 1% NaCl) for 14 days. To minimize endogenous Aldo, rats were
adrenalectomized (ADX) and supplemented with dexamethazone. Rats were treated with
L-NAME in drinking water (40 mg/kg/day for 14 days). During days 8 14, rats were treated
with AngII (225 g/kg/day) and Aldo (80 g/kg/day) via subcutaneous osmotic minipumps
Aldo antagonist eplerenone (Epl, 100 mg/kg/day by oral gavage). Rats were sacrificed and
thoracic aortic segments were prepared for measurement of isometric contraction/relaxation
and NO production using DAF-FM fluorescence. Phenylephrine (10-5 M)-induced vascular
contraction was enhanced in HS (0.66) compared with NS rats (0.47), but not in ADX HS rats
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(0.61g/mg). AngIIL-NAME treatment did not enhance contraction in ADX rats, suggesting that
endogenous Aldo is needed to sensitize blood vessels to AngII. Aldo administration enhanced
vascular contraction in AngIIL-NAME treated rats. Acetylcholine (Ach, 10-5 M)-induced %
vascular relaxation was smaller in HS (41) than NS rats (71) and further reduced in
AngIIL-NAME treated HS rats (11%), suggesting significant NO production during HS diet. Epl
partially restored Ach relaxation in AngIIL-NAME treated rats (25%). Aldo administration
abolished Ach relaxation in AngIIL-NAME treated rats, which was slightly restored by Epl
(5%). Ach-induced vascular NO production was reduced in HS (0.103/mg) compared with NS
(0.122/mg), but not in ADX HS rats (0.117/mg). The reduction in NO production in
AngIIL-NAME treated rats (0.106/mg) was reversed by Epl (0.127/mg). Vascular relaxation to
exogenous NO donor SNP was also reduced in Aldo-treated rats. Thus Aldo enhances the
vasoconstrictive effects of AngII in part by reducing vascular NO production and by decreasing
vascular sensitivity to NO, which may compromise the vascular control mechanisms of blood
pressure particularly during high salt diet.
P28
Role of Rho-Kinase in Aldosterone-Induced Vascular Remodeling
Kayoko Miyata, Yukiko Nagai, Shoji Kimura, Hideyasu Kiyomoto, Masanao Hosomi,
Masakazu Kohno, Akira Nishiyama; Kagawa Univ Med Sch, Kagawa, Japan
Aldosterone plays a critical role in the pathogenesis of cardiovascular injury. We recently
demonstrated that MR is abundantly expressed in cultured rat vascular smooth muscle cells
(VSMCs). In this study, we investigated whether Rho-kinase, an effector of the small G protein
Rho, is involved in aldosterone-induced vascular remodeling. Phosphorylation of myosin
phosphate target subunit-1 (MYPT1) was determined, as a marker of Rho-kinase activity, in
VSMCs by ELISA or Western blotting analysis with anti-phospho MYPT1 antibody. The presence
of actin filament and stress fiber formation was visualized by direct staining with rhodamine-
phalloidin. We also examined the effects of fasudil (10 mg/kg/day, s.c.), a selective Rho-kinase
inhibitor, on MYPT1 phosphorylation and vascular remodeling in uninephrectomized rats treated
with 1% NaCl (in a drinking solution) plus aldosterone (0.75 mg/H, S.C) for 5 weeks. P0.05
was considered statistically significant. In cultured VSMCs, aldosterone (1 nmol/L) increased
MYPT1 phosphorylation with a peak at 90 min (by 1.70.1-fold). Aldosterone-induced MYPT1
phosphorylation was markedly attenuated by pretreatment with eplerenone (10 mol/L), a
selective MR antagonist, or Y27632 (10 mol/L), a specific Rho-kinase inhibitor (n4 6 for
each). Aldosterone also significantly increased VSMC stress fiber formation (at 3 hours). The
aldosterone-induced stress fiber formation was markedly attenuated by pretreatment with
eplerenone or Y27632 (n6 8 for each). Compare with rats treated with salt alone (n6),
aldosterone/salt-treated rats showed hypertension (1263 vs. 1983 mmHg) and increased
medial thickness of the aorta and MYPT1 phosphorylation in aortic tissues (by 1.60.2-fold,
n7). Treatment with fasudil did not alter blood pressure (1933 mmHg), but prevented
aldosterone-induced aortic MYPT1 phosphorylation in aldostreone/salt-treated rats (n7).
Additionally, the aldosterone-induced increase in medial thickness of the aorta was ameliorated
by treatment with fasudil. These data suggest that Rho-kinase is involved in the progression
of aldosterone-induced vascular remodeling, independent of blood pressure changes.
P29
Captopril Prevents Hypertension in Rats Exposed to Intermittent
Hypoxia/Hypercapnia to Simulate Sleep Apnea
Ana Quenia G Silva, Pooja Singh, Nancy L Kanagy; Univ of New Mexico Health Science
Cntr, Albuquerque, NM
Sleep apnea has been associated with systemic hypertension and some studies suggest
elevated plasma renin activity (PRA) contributes to the hypertension. However, other studies in
sleep apnea patients have not observed increased PRA and one clinical study observed that
enalapril actually exacerbated sleep apnea. Thus it is not clear what role PRA and angiotensin
II play in sleep apnea associated hypertension. We simulated sleep apnea in rats to determine
CHBPR ConferencePoster Presentations e51
if this paradigm increases PRA and leads to angiotensin II-dependent hypertension. We tested
this hypothesis by measuring PRA and blood pressure in rats exposed during their sleep (7
hr/day) to 20 short periods every hour of hypoxia/hypercapnia (IH/HC, inhaled O2% 5%,
inhaled CO2% 5%) for14 days. Some rats from each group were instrumented with
telemeters for recording arterial pressure and treated with the angiotensin converting enzyme
(ACE) inhibitor, captopril in their drinking water (1.30.05 mg/kg/day) throughout the 14 days
of exposure to IH/HC. PRA measured with a radioimmunoassay kit was significantly elevated
in the IH/HC rats (19.7 2.6 ng/ml/hr, p 0.05) compared to sham rats (11.3 0.4
ng/ml/hr). In addition, mean arterial pressure (MAP) did not increase in IH/HC rats treated with
captopril in contrast to the increase in untreated IH/HC rats on day 14 (captopril 109 2.5
vs untreated 119 5 mmHg). These data suggest IH/HC exposure increases PRA to contribute
to the development of angiotensin II-dependent hypertension. Therefore blocking the renin-
angiotensin system may be a good first line treatment for sleep apnea patients with
hypertension unless the ACE-inhibitor cough worsens the sleep apnea symptoms.
P30
Chemokine Receptor 2b Blockade Decreases Renal Nf-Kappa B Activity
and Slows the Progression of Hypertension and Renal Damage in
Salt-Sensitive Hypertension
Ahmed A Elmarakby, Jeffrey J Olearczyk, Aarthi Sridhar, Jeffrey E Quigley, David M Pollock,
John D Imig; Med College of Georgia, Augusta, GA
Studies have demonstrated that inflammatory cytokines play a role in hypertension and its
associated renal damage. We have previously shown that urinary MCP-1 excretion increased
in salt-sensitive hypertension. Thus, we hypothesize that MCP-1 and chemokine receptor
activation contributes to the blood pressure elevation and renal injury in salt-sensitive
angiotensin II (ANG) hypertension. Male Sprague Dawley rats were fed a high-salt diet (HS; 8%
NaCl) and osmotic minipumps were implanted to deliver ANG at a dose of 60 ng/min for 14
days. Rats were divided into three groups: HS & ANG/HS & ANG/HS/ CCR2b selective
chemokine receptor antagonist, RS102895 (10 mg/kg/day). Mean arterial pressure (MAP)
increased from (1015 to 13815 mmHg) during the first week in the ANG/HS group.
RS102895 slowed the progression of blood pressure elevation during the first week of ANG/HS
treatment (984 to 11014 mm/Hg). At two weeks, MAP was elevated to 17415 mmHg in
ANG/HS and 14418 mmHg in the RS102895 treated group. Albuminuria was significantly
elevated in ANG/HS group compared to HS rats (237 26 vs. 4 0.6 mg/day) and RS102895
treatment reduced albuminuria in ANG/HS hypertensive rats (111 12 mg/day). Renal cortical
NF-kappa B activity increased in ANG/HS hypertensive rats compared to HS group (0.110.006
vs. 0.080.003 ng activated NF-kappa B/g cortical protein) and RS102895 treatment
lowered NF-kappa B activity (0.080.005 ng activated NF-kappa B/g cortical protein).
ICAM-1 and NF-kappa B mRNA expression also increased in renal cortex isolated from ANG/HS
hypertensive rats and these changes were attenuated with RS102895 treatment using real time
PCR. These data suggest that CCR2b receptor contribute to blood pressure regulation, and renal
injury in salt-sensitive hypertension.
P31
Angiotensin II-Induced Hypertension and Subsequent Renal Pathology is
Attenuated in Mice Lacking Soluble Epoxide Hydrolase
Steven M Weldon, Damian Matera, Jun Xu, Rhonda Chen, Richard Ingraham, Michael
Thibodeau, Jeffrey B Madwed, Alisa K Kabcenell; Boehringer Ingelheim Pharmaceuticals
Inc., Ridgefield, CT
Targeted disruption of the soluble epoxide hydrolase (sEH) gene has been shown to effect basal
blood pressure (BP) in mice (Sinal, 2000). The BP phenotype in these animals is presumed to
be due to an increase in the vasoactive arachidonic acid metabolites, epoxyeicosatrienoic acids
(EETs). We examined whether angiotensin II (ang II)-induced hypertension (HT) and associated
renal pathology would be attenuated in our independently generated sEH knock out (KO) strain
compared to wild type (WT) controls. HT was induced in male (M) and female (F) mice by
infusion of ang II (1 mg/kg/day) for 14 days. Mean BP (MBP) and heart rate (HR) were
continuously recorded via telemetry. Kidney, heart and aortic tissue were collected for gene
expression profiling and renal histopathology at 14 or 26 days after the start of ang II infusion.
Baseline (Day0, D0) MBP was similar across all groups: 1092; 1113; 1112 and 1095
for F-WT, M-WT, F-KO and M-KO, respectively. AngII infusion resulted in a significant,
sustained increase in MBP in all groups. By D14 of angII, there was no difference in MBP
between F-WT and F-KO mice. However, there was a significant separation in MBP observed
between M-WT and M-KO groups; beginning on D5 and sustained throughout the ang II
infusion, 1658 vs. 13410 (p0.012) for M-WT and M-KO, respectively (D14). HR was not
significantly different between any groups at D14. Although renal vascular fibrinoid necrosis
elicited by ang II was similar between M-WT and M-KO at D14, renal cortical infarcts observed
in M-WT at D26 were not present in M-KO indicating renal protection. Female mice did not
exhibit vascular fibrinoid necrosis. Interestingly, microarray data from sEH KO tissues exhibited
suppression of genes involved in calcium regulation. These data are the first to provide
evidence that sEH gene deletion attenuates hypertension and associated renal damage in mice,
possibly through calcium regulation. In addition, the effects exhibited in sEH KO mice may be
gender selective. These data provide additional evidence that sEH may have an intriguing role
in blood pressure regulation and associated renal protection.
P32
Hypertension Caused by Prenatal Testosterone Excess in Female Sheep
Andrew J King, N B Olivier, P.S. MohanKumar, Michigan State Univ, East Lansing, MI; J.S.
Lee, V Padmanabhan, Univ of Michigan, Ann Arbor, MI; Gregory D Fink; Michigan State
Univ, East Lansing, MI
Polycystic ovary syndrome (PCOS), a leading cause of infertility, affects 10% of women of
reproductive age. The etiology and pathophysiology of PCOS are poorly understood. PCOS is
 e52 Hypertension Vol 48, No 4 October 2006

multi-facetted and includes reproductive, metabolic and other aspects of the metabolic hypertensive renal injury, absent in injury-resistant hypertension. This program precedes the
syndrome, including insulin resistance, obesity and hypertension. Exposure to excess emergence of histological injury. This renal injury transcriptional program arises from
testosterone (T) during the prenatal period may predispose individuals to PCOS. The goal of this differential gene expression regulated by components of HNF1, a major renal TF that influences
study was to determine if hypertension occurs in a well-characterized model of PCOS produced expression of a large number of renal genes.
by prenatal treatment of sheep with T. Because abnormal responses to stress may occur in
PCOS, radiotelemetry was used to measure blood pressure over a 24-hour period in conscious,
undisturbed animals. Control (n4) and prenatal T-treated (100 mg T propionate im twice
weekly from days 30 to 90 of fetal life; n4) 2-year-old females were ovariectomized and
replaced with early follicular phase levels of estrogen using an implant. On the same day, the
catheter of a radiotelemetry pressure transmitter (DSI, Inc.) was implanted into the femoral
artery; the transmitter was placed subcutaneously at the inner thigh. Six days later, sheep were P35
housed in small rectangular pens (3 x 4 ft) with free access to food and water. Starting at noon,
a 24-hour recording period was begun. Some key measurements (24-hour averages, mean
Ac-SDKP Mediates Part of the Renal Antifibrotic Effect of ACEi in
SEM) are summarized in the Table. Aldosterone-Salt-Induced Hypertension

Systolic Diastolic Hongmei Peng, Oscar A Carretero, Tang-Dong Liao, Edward L Peterson, Nour-Eddine
Mean pressure pressure pressure Heart rate Rhaleb; Henry Ford Hosp, Detroit, MI
Group (mmHg) (mmHg) (mmHg) (beats/minute)

C-females 90.92.8 115.14.5 79.22.3 73.55.9 We reported that acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) reduced renal fibrosis and cell
T-females 100.52.7* 125.02.8 88.92.8* 82.57.4 proliferation in rats with aldosterone (ALDO)-salt-induced hypertension. Others reported that an
angiotensin-converting enzyme inhibitor (ACEi) prevented renal fibrosis in this model. Recently
* P 0.05 comparing C-females and T-females. Blood pressure differences were greater during the lights-on than during the
lights-off period. Body temperature and activity level did not differ between groups. In T sheep there was marked insulin resistance
we found that the antifibrotic effect of ACEi in the heart was partly mediated by Ac-SDKP in rats
and both total and LDL cholesterol tended to be higher. There was no difference in heart weight between groups. This first-ever use with angiotensin-induced hypertension. Thus we tested whether the antifibrotic effect of ACEi
of radiotelemetric blood pressure recordings in sheep demonstrates that mild hypertension, a risk factor reported in some women in the kidney is mediated by Ac-SDKP in ALDO-salt hypertension. An anti-Ac-SDKP monoclonal
with PCOS, is also a feature of the sheep model of PCOS produced by prenatal T treatment. antibody (mAb) was used to block the effect of Ac-SDKP. SD rats (BW: 310 g) were divided into:
1) sham, 2) ALDO-salt vehicle, 3) ALDO-salt captopril (100 mg/kg/d in drinking water),
4) ALDO-salt captopril mAb (400 g/kg, i.p. every other day), 5) ALDO-salt Ac-SDKP
P33 (800 g/kg/d), and 6) ALDO-salt Ac-SDKP mAb. ALDO (0.75 g/hr) or Ac-SDKP was given
Heart Rat and Blood Bressure Variability in L-NAME Treated NET-Deficient s.c. via osmotic minipump and salt (1% NaCl/0.2% KCl) in drinking water. Treatment lasted for
Mice 6 weeks. As expected, ALDO-salt induced renal fibrosis (hydroxyproline assay), glomerular
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

matrix expansion (periodic acid-Schiff (PAS) staining) and increased phosphorylated p42/44
Volkmar Gross, Michael Obst, MDC, Berlin, Germany; Jens Tank, Med Faculty of the mitogen-activated protein kinase (MAPK) (P-p42/44) (Western blot). ACEi and Ac-SDKP
Charite, Franz Volhard Clinic, HELIOS Klinikum-Berlin, Berlin, Germany; Ralph Plehm, MDC, significantly reduced renal fibrosis and glomerular expansion (p 0.05) but not hypertension
Berlin, Germany; Jens Jordan, Med Faculty of the Charite, Franz Volhard Clinic, HELIOD or renal hypertrophy; while mAb reduced the antifibrotic effects of ACEi or Ac-SDKP on the
Klinikum-Berlin, Berlin, Germany; Friedrich C Luft; MDC and Med Faculty of the Charite, kidney. ACEi and Ac-SDKP also significantly reduced renal P-p42/44, and mAb blunted this
Franz Volhard Clinic, HELIOS Klinikum-Berlin, Berlin, Germany decrease. Phosphorylated p38 or JNK MAPK was not detectable. We concluded that the
antifibrotic effect of ACEi in the kidney is at least partly mediated by Ac-SDKP in ALDO-salt
To characterize the interaction between the blood pressure (BP) buffering effect of nitric oxide hypertension via inhibition of the p42/44 MAPK pathway.
(NO) and the norepinephrine transporter (NET), we blocked NO-generation with N-nitro-L- EFFECT OF MAB ON THE ANTIFIBROTIC EFFECT OF ACEI AND AC-SDKP ON THE KIDNEY
arginine methyl ester (5 mg L-NAME/10 ml tap water) in NET gene-deficient (-/-) mice. ALDO-salt ALDO-salt
Telemetric arterial blood pressure recordings were used for fast Fourier analysis (FFT) of BP ALDO-salt ACEi ALDO-salt Ac-SDKP
Parameters Sham ALDO-salt ACEi mAb Ac-SDKP mAb
and heart rate (HR). NET-/- had lower mean arterial blood pressure (MAP) than NET / mice
during the day (11008 vs. 1121 mmHg) and night (1151 vs. 1241 mmHg). HR was not SBP, mm Hg 1073 1977* 1767* 1844* 1938* 1847*
Renal collagen, 22.71.6 43.11.2* 22.92.6** 33.32.0 27.81.5** 34.22.0#
different between the groups, showed a clear-cut day/night rhythm, and ranged between g/mg dry K
5446 and 6093 beats/min. L-NAME increased MAP in both strains to the same extent ( glomerular 11.12.0 22.21.7* 10.91.3** 20.60.8 10.71.4** 18.22.3#
matrix,
MAP 10 mmHg). BP after acute L-NAME application and selective nNOS and eNOS blockade % of glomerular
were also not different between NET -/- and NET / mice. The absolute HF (8.02.6 vs. area
P-p42/44, fold 1.00.0 5.71.5* 1.50.4** 3.51.2 1.60.4** 4.00.8#
6.130.9 msec2), LF (21.66.0 vs. 16.63.0 msec2), and VLF (34.512.6 vs. 20.65.1 increase
msec2) heart rate variability power values were not different between NET -/- and NET / *: p 0.005 vs sham. **: p 0.05 vs ALDO-salt. : p 0.05 vs ALDO-salt ACEi. #: p 0.05 vs ALDO-salt
mice. In contrast to NET / mice, these parameters increased in NET-/- during L-NAME. BP
Ac-SDKP. Data: mean standard error.
variability in the low frequency range (SBP-LF), thought to reflect sympathetic activity to
resistance vessels, averaged 1.80.2 mmHg2 in NET -/- and 3.10.6 mmHg2 in NET /
mice and increased during L-NAME treatment more in NET / mice than in NET -/- mice
(6.90.9 vs. 3.40.7 mmHg2). Baroreflex sensitivity (BRS-LF) was higher in NET -/- mice than
in NET / mice. This difference between NET -/- mice and NET / mice reached
significance during L-NAME treatment (2.90.5 vs. 1.90.3 msec/mmHg, p0.05). These
results support the hypothesis that NO attenuates baroreflex mechanisms. However, only
L-NAME treatment unmasks the difference between strains. The findings suggest an interaction P36
between NO and NET. Acknowledgement: All animals used in this study were obtained from Cardiac-Specific EP4 Receptor Knock-Out Does not Affect Baseline Cardiac
Autonomic Dysfunction Service, Vanderbilt University, Nashville, TN, USA. Characters 1693 Function

P34
The Transcription Factor Hnf1 Contributes to Initiation of Hypertensive
Renal Injury via Coordinate Regulation of Redox Stress
Renata I Dmitrieva, Cruz A Hinojos, Michael C Braun, Myriam Fornage, Eric Boerwinkle,
Peter A Doris; Univ of Texas HSC Houston, Houston, TX
Redox stress (RS) has been implicated in the pathogenesis of hypertensive renal injury. We
used gene expression arrays to examine renal expression of 32 genes contributing to redox
balance during the development and maintenance of hypertension in SHR lines varying in their
susceptibility to renal injury. Our array data, confirmed by QRTPCR, provides evidence that RS
involves down-regulation of multiple redox radical scavenging genes. By comparing renal-
injury prone SHR-A3 with injury-resistant SHR-B2 and -C animals we observed that
down-regulation was specific to the sub-strain experiencing renal injury and preceded
histopathological evidence of renal injury. A transcriptional mechanism was sought using a
bioinformatics approach to assess the frequency of transcription factor (TF) binding sites in the
regulatory regions of down-regulated radical scavenging genes. A single TF, HNF1, emerged as
a potential mediator of these changes. Expression of each of the three genes encoding subunits
of the HNF1 was lower in the renal injury-prone strain. Western blot analysis of the major HNF1
subunit expressed in the kidney (Tcf2) indicated that loss of one of two alternatively spliced
forms of Tcf2 exclusively in the injury-susceptible strain, SHR-A3. Isoform loss was shown to
be a post-transcriptional event. To support evidence that redox mechanisms of renal injury
might be under control by HNF1, the capacity of HNF1 to bind to 5-regulatory regions of redox
scavenging genes was demonstrated by EMSA. Further evidence of HNF1 regulation of these
genes was obtained by demonstrating conservation of HNF1 binding sites across species
(Consite, Karolinska Institute). Across 35 further genes known to be regulated by HNF1 and
expressed in the kidney, the mean expression level was 2.7 fold-lower in the injury-prone
strain. Our studies have revealed a transcriptional program for renal redox stress in
Jian-Yong Qian, Ed Shesely, Yunhe Liu, Pamela Harding, Margot C LaPointe; Henry Ford
Hosp and Health System, Detroit, MI
Prostaglandin E2 (PGE2) is a pro-inflammatory prostanoid that stimulates cardiomyocyte
hypertrophy in vitro through its EP4 receptor. The role of EP4 in normal heart function is
unknown. Systemic EP4 gene knock out (KO) mice have been reported, but 95% of
homozygotes for the null allele have persistent ductus arteriosus after birth and soon die. Thus
understanding EP4 function in the heart requires cardiac-specific deletion. To generate mice
lacking EP4 only in cardiac myocytes (CM-EP4 KO), we bred 2 lines of transgenic mice: 1) an
EP4 flox/flox mouse which has loxP sites flanking exon 2 of the EP4 gene; and 2) an -myosin
heavy chain-Cre recombinase mouse (to localize expression of Cre to adult cardiac myocytes).
A breeding strategy was developed to generate roughly equal numbers of CM-EP4 KO mice and
wild-type (WT) littermates. CM-EP4 KO and WT mice (male, 1314 week old) had similar
survival rates. CM-EP4 KO and WT mice were subjected to 2D M-mode echocardiography.
There was no difference between CM-EP4 KO mice and WT regarding ejection fraction (KO
78.83.2%, n3; WT 79.51.5%, n5), left ventricular diastolic dimension (KO
3.10.1mm; WT 3.00.1mm) and diastolic posterior wall thickness (KO 0.90.04mm;
WT 0.90.03mm). There was also no difference in heart/body weight ratio (KO
40.31.9; WT 39.10.4 mg/10g BW), consistent with the sonographic posterior wall
thickness data. We confirmed that the EP4 gene was deleted in heart, but not in other tissues.
Organs from CM-EP4 KO and WT mice were divided into two sections from which genomic DNA
and RNA were extracted. PCR primers designed to detect a 346 bp floxed EP4 gene showed
the presence of the gene in the heart, aorta, lung, liver, skeletal muscle and brain of WT
littermates, but a 93% decrease in the gene only in the CM-EP4 KO mouse hearts. Similarly,
an analysis of EP4 mRNA by RT-PCR of hearts showed that expression was significantly
decreased in the heart of CM-EP4 KO mice vs. WT controls. In conclusion, CM-EP4 KO mice
were successfully generated, showing EP4 deletion only in the heart. KO mice had no overt
cardiac phenotypes, as expected. Additional experiments are underway to determine the role
of EP4 in models of hypertrophy and ischemia.

P37
The T-Type Calcium Channel Blocker (CCB), Mibefradil, Attenuates the
Acute Hindleg Edema Induced by the L-Type CCB, Nifedipine, in the
Spontaneously Hypertensive Rat (SHR) as Assessed via Magnetic
Resonance Imaging (MRI)
Terry C Major, Shantanu Dhamija, Serguei Liachenko, Brandy Morenko; Pfizer Inc, Ann
Arbor, MI
Among the calcium channel blockers (CCB), particularly among the dihydropyridines (DHP) like
nifedipine, a common adverse effect is vasodilatory edema. Newer CCBs such as the T-type
CCB, mibefradil, demonstrate antihypertensive efficacy similar to that of their predecessors but
appear to have a reduced propensity to cause edema. Edema formation is currently determined
after long-term clinical trials by measuring patient-reported edema incidence, a fairly
subjective parameter. In this study, we investigated the ability of MRI to measure static water
accumulation in hindleg skeletal muscle of the SHR by the CCBs, mibefradil and nifedipine, both
alone or in combination. Isoflurane-anesthetized SHRs were cannulated in the jugular vein for
CCB delivery and the carotid artery for mean blood pressure (MBP) measurements. After a
1530 minute stabilization period, two baseline T2 relaxation scans in the lower leg muscles
of the SHR were measured by a 7 Tesla Bruker MRI system utilizing multi-echo images and
calculating the integral increase in T2 relaxation (IIT2R;%# of voxels x ave change in
T2/voxels) as a quantitative marker for edema formation. Mibefradil (10 mg/kg IV) and
nifedipine (1 mg/kg IV) both lowered MBP equipotently -975 and -837 mmHg, respectively
compared to vehicle (78 mmHg) . However, the edema formation or IIT2R was significantly
higher with nifedipine (2708 5%;p0.05) than with mibefradil (981171%) or vehicle
(9514%) measured 30 minutes after start of drug infusion. The combination of 1 mg/kg
nifedipine3 mg/kg mibefradil (1N3M) and 1N0.3M lowered MBP -1016 and -644 specimens in order to study sodium transport phenotype and correlate it with genotype in
mmHg, respectively, compared to 1N0M (-723 mmHg) but the IIT2R was significantly
reduced in the 1N3M (1139279%) and 1N0.3M SHRs (1725306), respectively, urine obtained as an aliquot of a 24 hour urine specimen by centrifugation at 150xG and then
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CHBPR ConferencePoster Presentations e53
Systolic blood pressure (SBP) was determined by tail-cuff. Cardiac function was assessed by
echocardiography. Insulin resistance index, the homoeostasis model assessment (HOMA)
score, was calculated from fasting glucose and insulin levels. SBP was significantly lower in
rats given LS (13214 mmHg), AC3174 (15517 mmHg), Cap (16516 mmHg) and
AC3174Cap (16316 mmHg) than in HS rats (2097 mmHg). Left ventricular (LV) weight
/body weight ratio was significantly lower in rats treated with LS, AC3174, Cap, AC3174Cap
than in rats treated with HS. Despite comparable antihypertensive efficacy, LV weight/body
weight ratio in AC3174Cap treated rats was significantly lower than in rats given Cap alone.
Compared with HS rats, LV wall stress was significantly reduced in LS (22%), AC3174 (30%),
AC3174Cap (28%) and Cap (20%) treated rats. Compared with LS treated rats, fasting insulin
levels and HOMA were significantly increased by 3.5 and 3.7 fold, respectively, in HS treated
rats. Treatment with AC3174, Cap and AC3174Cap normalized the insulin levels and HOMA
score. Treatment with AC3174, AC3174Cap and Cap significantly attenuated elevated serum
creatinine levels and improved creatinine clearance rate compared with HS rats. These results
suggest that 4 week AC3174 infusion has anti-hypertensive, insulin sensitizing and renopro-
tective effects.
P40
Measurement of Cellular Physiology in Individual Freshly Exfoliated Human
Renal Proximal Tubular Cells from Urine in Subjects Enrolled in a Genetic
Study of Salt Sensitivity
John J Gildea, Daniel C Huntley, Michael J Surace, Robert M Carey, Robin A Felder; Univ of
Virginia, Charlottesville, VA
We developed a method to obtain living proximal tubular cells (PTC) from freshly voided urine
subjects enrolled in a study of genetic salt sensitivity. PTC were concentrated from 150 mL of

compared to the control SHR group, 1N0M (2539119%). These results show that IIT2R can
be used to quantify peripheral edema with high precision and reproducibility and that T-type
CCBs at equipotent MBP lowering can produce significantly less edema compared to DHPs. In
addition, T-type CCBs can dose-dependently attenuate the peripheral edema induced by DHPs.
This study indicates that T-type CCBs may equalize the pressure across the capillary, reduce
water leakage and reduce vasodilatory edema.
P38
Partial Normalization of Renal Transcriptome in Spontaneously
Hypertensive Rats (SHR) Treated with Multiple Antihypertensive Agents
Sebastiaan Wesseling, Jaap A Joles, Farid Kantouh, Hein A Koomans, Branko Braam; Univ
Med Cntr Utrecht, Utrecht, The Netherlands
covalently attached to a slide using the Cytospin method. PTC were selectively identified using
conjugated Lotus tetragonolobus agglutinin conjugated to a fluorescent dye (Alexa 594-LTA),
which is a specific PTC stain. PTC constituted 10%, n3, of the total cells in the urine of three
out of 10 processed specimens. Cells were found to be viable for 24 hours in culture media
at 37C in a 5% CO2 atmosphere. All cells were then loaded with corona green, a fluorescent
dye that is sensitive to changes in cytoplasmic sodium concentration. Ouabain was used to
selectively block NaKATPase, and the change in intracellular sodium was followed over time
in both PTC and other living cells found in urine. We measured the differential change in
PTC/cuboidal cell corona green fluorescence as an indicator of PTC NaKATPase activity. A
21% increase in fluorescence of PCT after 2 minutes of ouabain treatment indicated that we
were able to inhibit NaKATPase activity and increase intracellular sodium. We suggest that
exfoliated PTC may be used to directly measure cell physiology, in particular sodium transport.
This method may be a useful diagnostic tool for studying aberrant PTC sodium transport which
may be correlated with individual patient SNP genotype.

SHR are a genetic model for essential hypertension (EH). Compared to WKY, SHR have
disturbed NO/O2- balance, dysfunctional Ca-regulation and are sensitive to Ang II signaling. In
patients with EH the leukocyte transcriptome normalized upon antihypertensive therapy. We
hypothesized that the renal transcriptome in SHR will be progressively normalized as more
hypertensive factors are inhibited. SHR (21 wks old) with losartan (Los), Los and tempol (LT),
Los and amlodipine (LA) or Los, amlodipine and tempol (LAT) in food and/or drinking
water were studied vs. untreated SHR and WKY for 3 wks. Renal cortex was processed for rat
7.5k oligonucleotide microarray (n5 6/group). All groups, also WKY, were compared to WKY
with a dye swap. Differential gene expression was defined as absolute log2(GROUP/WKY)0.7.
Systolic blood pressure for WKY, SHR, Los, LT, LA and LAT was 1312, 1935 P1-artificial chromosome (PAC160) containing REN, two upstream (GOLT1A and KISS1), and
(P0.001 vs. WKY), 1533, 1666, 1444 and 1285 mmHg, respectively (P0.01 treated one downstream (ETNK2) gene. hREN expression in these mice is tightly regulated, as is
SHR vs. SHR; N.S. LAT vs. WKY). SHR showed 183 modulated genes vs. WKY of which 56,
97, 125 and 125 genes were normalized by Los, LT, LA and LAT, respectively. kb upstream of the transcription start site which has been reported to markedly enhance
Hierarchical clustering analysis revealed: 1) Los normalized genes with reduced expression; 2)
few genes were oppositely regulated by treatments; 3) several genes were insensitive to any
treatment. Gene Ontology classification was applied to the 183 genes to identify biological
processes. In most processes regulation was bi-directional, but in Na and K ion transport only
reversal of gene induction occurred which was normalized by LA and LAT. Drug
combinations increasingly normalized regulated processes and no processes were totally
insensitive to treatment. Only 14 modulated genes in SHR were insensitive to any treatment:
among these were reduced Cd36 (insulin sensitive, important for cellular fatty acid uptake) and
induced cytosolic epoxide hydrolase 2 (Ephx2; degrades endogenous vasodilators such as EET).
Summarizing, renal cortex of SHR displayed differential gene expression sensitive to Ang II,
Ca2 and, to a lesser extent, superoxide. Genes involved in Na and K transport were sensitive
to multiple treatments. Combining drugs not only normalized BP but also partially corrected the
transcriptome.
P39
The Exenatide Analog Ac3174 Attenuates Hypertension, Renal Dysfunction
and Insulin Resistance in Dahl Salt-Sensitive Rats
P41
The Human Renin Kidney Enhancer Appears Dispensable for Tissue and
Cell-Specific Expression in Transgenic Mice
Xiyou Zhou, Curt D Sigmund; Univ of Iowa, Iowa City, IA
Renin (REN) is the rate-limiting enzyme in the RAS and thus dictates the level of Ang-II.
Transcription of REN is expressed predominantly in renal JG cells where it is tightly regulated
by physiological cues. We previously generated hREN transgenic mice with a 160 Kb
expression of the linked genes. A kidney-specific enhancer of transcription (KE) is located 11
transcription in renin expressing cells. The importance of the KE in vivo remains unclear. We
hypothesize that the KE mediates tissue- and cell-specific expression, and is responsible for
physiological regulation. To test this, we deleted the enhancer sequence from PAC160 using
homologous recombination in bacteria, and then developed several lines of transgenic mice
(termed KE). Lines KE2 and KE4 have a single copy and multiple copies of hREN
integrated, respectively. We used the downstream gene- ETNK2 as an internal control. To our
surprise, deletion of the KE had no effect on tissue-specific expression of hREN. Expression of
hREN protein remained localized to renal JG cells under baseline conditions in KE4, and after
treatment with Captopril in KE2. Captopril (1000 mg/kg/day) significantly induced expression
of hREN in KE2 (10.6-fold) and KE4 (7.4-fold) to an extent similar to the unmodified hREN
gene in PAC160 and to the endogenous mREN gene (5-fold). Ang-II administration (1000
ng/kg/min) which raised BP by 40mmHg decreased hREN expression in KE4 mice (to 30%
of untreated) similar to wildtype PAC160 mice. We also identified another line (KE6)
containing a truncated PAC160 in which the KE as well as additional upstream sequences was
deleted. Whereas expression of hREN in this line was evident in all tissues tested, it was still
expressed renal JG cells. However, initial studies revealed a lack of repression by Ang-II. Our
results suggest that sequences other than the KE, either further upstream or in the proximal
promoter may be necessary for tissue-specific and regulated expression and for retaining
JG-cell specific expression of hREN.

Lisa M Adams, Rayne Fernandez, Alain Baron, David Parkes, Que Liu; Amylin
Pharmeceuticals, San Diego, CA
Glucagon-like peptide-1 (GLP-1) is a gut peptide incretin that enhances glucose-dependent
insulin secretion. Accumulating evidence suggests that activation of GLP-1 receptors also
improves insulin sensitivity and induces vasodilatation and diuresis. Exenatide is a longer-
acting incretin mimetic that exhibits antidiabetic properties in patients with type II diabetes.
AC3174 is an analog of exenatide with similar pharmacologic properties. We hypothesized that
chronic treatment with AC3174 can attenuate salt-induced hypertension, insulin resistance and
renal dysfunction in Dahl salt-sensitive (DSS) rats. DSS rats were fed low salt (LS, 0.3 % NaCl)
or high salt (HS, 8% NaCl) and were treated with vehicle or AC3174 (1.7 pmol/kg/min) via
subcutaneous infusion (Alzet pump) for 4 weeks. Other DSS rats were given captopril (Cap,
2g/L drinking water) or AC3174 (1.7 pmol/kg/min) plus Cap (AC3174Cap) for 4 weeks.
P42
Rats with Ang II-Dependent Hypertension From Human Transgenes Develop
Electrical Remodeling
Robert Fischer, Ralf Dechend, Andrej Gapelyuk, Erdenechimeg Shagdarsuren, Konstanze
Gruner, Andreas Gruner, Petra Gratze, Maren Wellner, Anette Fiebeler, Helios Klinikum,
Franz-Volhard-Klinik, Charite, Berlin, Germany; Dominik N Muller, Max-Delbrueck Cntr,
Berlin, Germany; Alexander Schirdewan; Helios Klinikum, Franz-Volhard-Klinik, Charite,
Berlin, Germany
Rats harboring the human renin and angiotensinogen genes (dTGR) develop hypertension and
cardiac damage. We investigated electrical remodeling in this model. We used non-invasive
 e54 Hypertension Vol 48, No 4 October 2006
cardiac magnetic field mapping (CMFM) to determine whether untreated dTGR or AT1
receptor blocker (ARB)-treated dTGR exhibit electrical remodeling and arrhythmias. We
performed CMFM, ECG, and characterized cardiac damage in 5 and 7 week old dTGR,
losartan (Los)-treated dTGR (30 mg/kg/d), and Sprague Dawley (SD) controls. Heart
weight-to-tibia ratio and systolic blood pressure (BP) increased in dTGR from week 5 to
week 7. Los treatment normalized heart weight-to-tibia ratio and BP. Already at early stage
of cardiac damage (week 5), untreated dTGR showed slightly increased perivascular
(collagen I) and interstitial fibrosis (fibronectin), connective tissue growth factor expression
(CTGF), and monocytes infiltration compared with SD. All differences were much more
prominent at week 7. Los reduced CTGF, fibrosis and inflammation to control levels.
Already at week 5, untreated dTGR showed prolonged de- and repolarization compared to
SD, which was even more pronounced at week 7 (QRS 22.90.5 ms vs. 18.70.8 ms,
QT 1133 ms vs. 821.7 ms, week 7; p 0.01). Parameters reflecting inhomogeneity
of depolarization (inhomogeneity index IHi 24.5 3.7 vs. 10 1.5, p 0.01) and
repolarization (TPeak-TEnd interval 68.6 2.3 ms vs. 54.7 2 ms, p 0.0001) also
increased with progression of cardiac damage. In contrast, Los treatment normalized all
electrophysiological parameters. LV mRNA expression of the potassium channel subunit
Kv4.3 and gap junction protein Cx43 was significantly reduced in dTGR at week 5 and 7
compared to Los and SD. We conclude that electrical remodeling is present early in
angiotensin II/hypertension-induced cardiac damage. ARB treatment reduced fibrosis,
inflammation, and the pathological electrophysiological alterations. Non-invasive CMFM is
a suitable tool to characterize the effects of cardiac electrical remodeling in rats.
P43
In situ Direct Detection System of Reactive Oxygen Species (ros) and Nitric
Oxide (no) in the Kidney
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Naruya Tomita, Tamehachi Namikoshi, Yoshisuke Haruna, Souhachi Fujimoto, Masahito
Oozeki, Shinya Kobayashi, Norio Komai, Tamaki Sasaki, Naoki Kashihara; Kawasaki Med
Sch, Kurashiki, Japan
Increased production of reactive oxygen species (ROS) in hypertension and diabetes may
be one of the important pathways leading to the progression of renal damages induced by
hypertension and diabetes. Especially, an imbalance between ROS and nitric oxide (NO) is
occurring in the glomeruli from the early. Recently, we have developed the unique
technique to determine the production of ROS and NO in glomeruli using confocal laser
microscopy after perfusion with dichlorofluorescein diacetate (DCFH-DA) (a ROS marker)
and diaminorhodamine-4M AM (DAR-4M) (an NO marker). In this study we investigated the
production of ROS and NO in hypertensive model and diabetic model rats. First, we used
Dahl salt sensitive (DS) rats given an 8%NaCl diet (DH-H) or 0.3%NaCl diet (DS-L) for 6
weeks. Systolic blood pressure was significantly elevated in DS-H group at 6 weeks (DS-H:
210 4 mmHg, DS-L: 132 8 mmHg, p0.01). In the DS-L rats little ROS production
and significant NO production were observed in glomeruli at 6 weeks. On the other hand,
accelerated ROS production and diminished bio-available NO were observed in glomeruli
of DS-H rats also at 6 weeks. Next, we used Spague-Dawley rats and induced diabetes by
intravenous injection of streptoztocin (STZ 65 mg/kg). Then, rats whose blood glucose on
day 3 was more than 300 mg/dl were used for further study. At 6 weeks after induction
of diabetes ROS was increased and No was decreased in glomeruli. On the other hand, in
control rats ROS was rarely observed, however, abundant NO production was noticed in
glomeruli. We have developed the method to detect ROS and NO directly in the kidney.
Although further detailed studies may be needed, this method may give a lot of information
concerning the relation between ROS and NO in the pathogenesis of hypertension and
diabetes.
P44
Functional Relationship between Oxidative Stress and Vascular Stiffness in
Humans
Christian Delles, Lukas U Zimmerli, David J McGrane, Univ of Glasgow, Glasgow, United
Kingdom; Alan J McKay, Gartnavel General Hosp, Glasgow, United Kingdom; Vivek L Pathi,
Western Infirmary, Glasgow, United Kingdom; Henry J Dargie, Carlene A Hamilton, Anna F
Dominiczak; Univ of Glasgow, Glasgow, United Kingdom
Background: Oxidative stress is critically involved in the pathogenesis of cardiovascular
disease by promoting endothelial dysfunction and reducing arterial compliance. However,
a direct link between oxidative stress and vascular stiffness has not yet been demon-
strated. Methods: To cover the whole range of oxidative stress and vascular stiffness we
examined 73 patients (age: 6011 years). Of these, 49 patients had severe CAD and
underwent CABG surgery, and 24 underwent surgery for removal of varicose veins but
were otherwise healthy. Vascular superoxide (SO) production was examined by lucigenin-
enhanced chemiluminescence in sections of saphenous vein. Reduced to oxidised
glutathione ratio (GSH/GSSG; whole blood) and thiobarbituric acid-reactive substances
(TBARS; plasma) were measured as circulating markers of oxidative stress in subsets of
patients (n58 and n14, respectively). Compliance of the ascending aorta was
measured in 39 patients (22 CAD and 17 control patients) by MRI (1.5 T Siemens Sonata)
using an ECG gated Fast Imaging with Steady-state Precession sequence from the change
in volume of the aortic segment during the cardiac cycle divided by pulse pressure.
Results: Vascular SO generation (1.000.45 vs 0.760.43 nmol/mg/min; P0.034) and
GSH/GSSG (555352 vs 824384; P0.005) were significantly different between CAD
and control patients, whereas TBARS were similar between the groups (1.0670.242 vs
1.1590.366 nmol/mL; P0.580). Neither GSH/GSSG (P0.496) nor TBARS (P0.213)
were correlated with vascular SO generation. Stepwise multiple linear regression revealed
that age (P0.001) and vascular SO generation (P0.045) were independent determi-
nants of aortic compliance, whereas sex, body mass index, systolic blood pressure and
atherogenic index (the ratio between non-HDL cholesterol and HDL cholesterol) did not
enter the final model (adjusted R20.553). Circulating markers of oxidative stress were not
correlated with aortic compliance. Conclusions: This is the first report revealing a
significant relationship between oxidative stress and vascular stiffness. Detailed pheno-
typing including MRI-based analysis of aortic compliance and measurement of vascular SO
generation is required to uncover this important link.
P45
Withdrawn at Authors Request
P46
In Human Coronary Artery Smooth Muscle Cells (CASMCs) Adenosine
Induces Antimitogenesis via A2B Receptors
Raghvendra K Dubey, Univ Hosp Zurich, Zurich, Switzerland; Nanette J Tomicek, Delbert G
Gillespie, Edwin K Jackson; Univ of Pittsburgh Med Cntr, Pittsburgh, PA
Via A2B receptors, adenosine inhibits growth of human and rat arterial SMCs; however, a
recent study demonstrated that via A1 adenosine receptors adenosine induces growth in
porcine CASMCs. Because in contrast to porcine CASMCs, human CASMCs express A2B
adenosine receptor, we investigated whether unlike porcine CASMCs, adenosine inhibits
growth in human CASMCs. In human CASMCs, fetal calf serum (2.5% FCS) stimulated DNA
synthesis (3H-thymidine incorporation), cellular proliferation (cell number), collagen
synthesis (3H-proline incorporation) and ERK1/2 expression. The adenosine receptor
agonists adenosine and 2-chloroadenosine, but not N6-cyclopentyladenosine, CGS21680
or IB-MECA, inhibited the growth effects of serum, an agonist profile consistent with an A2B
receptor-mediated effect. MRS1754 (selective A2B antagonist), but not 8-cyclopentyl-1,3-
dipropylxanthine (A1 antagonist), SCH58261 (A2A antagonist) or VUF5574 (A3 antagonist),
blocked the growth-inhibitory effects of 2-chloroadenosine, an antagonist profile consis-
tent with an A2B receptor-mediated effect. Our findings strongly support the hypothesis that
adenosine causes inhibition of human CASMC growth by activating A2B receptors coupled
to inhibition of MAP kinase activity. Unlike the findings in porcine CASMCs, our findings
strongly suggest that pharmacological or molecular biological activation of A2B receptors
may prevent vascular remodeling associated with coronary artery disease, hypertension,
atherosclerosis and restenosis following balloon angioplasty.
P47
NAD(P)H Oxidase Activation Induced Reactive Oxygen Species
Accumulation and Enhanced Vasoconstriction in Ren-2 Rat Aortas
Yongzhong Wei, Craig S Stump, Zachary T Resch, Grace M Uptergrove, Shawna A Cooper,
Suzanne E Clark, Univ of Missouri-Columbia Sch of Medicine, Columbia, MO; Carlos M
Ferrario, Wake Forest Univ, Bowman Grey Sch of Medicine, Winston-Salem, NC; James R
Sowers; Univ of Missouri-Columbia Sch of Medicine, Columbia, MO
Activation of the renin-angiotensin system (RAS) plays an important role in development of
cardiovascular diseases, possibly by increasing reactive oxygen species (ROS) generation and
promoting insulin resistance. The TG(mREN-2)27 (Ren2) transgenic rat overexpresses the
mouse renin gene has elevated tissue RAS activity. We hypothesized that Ren-2 rats would
exhibit increased vascular ROS (aorta), and that ROS accumulation is mediated by increases in
NAD(P)H oxidase subunit expression and activity. When Ren2 rats (age 8 10 weeks) were
compared to age-matched Sprague-Dawley (SD) controls, Ren2 rats exhibited significantly
increased aortic ROS as measured by a lucigenin assay (72.6%, P0.05), or by dihydro-
ethidium (DHE) and 4-hydroxy-2-nonenal (4-HNE) staining. Further, NADPH oxidase activity was
significantly increased (82.2%, P0.05) in Ren-2 aorta when compared with SD. When Ren-2
rats were treated with the AT1R blocker valartan (30 mg/kg) for three weeks, ROS level and
NADPH oxidase activity were markedly reduced compared to untreated conditions. NADPH
oxidase subunits (p67phox and Rac1) measured by Western blot were also increased in Ren-2
aorta compared to SD, but valsartan treatment reduced levels in Ren-2. Arterial wall thickness
in Ren-2 rats was also significantly greater than that in SD rats. These findings were associated
with hypertension, increased vasoconstriction in ex vivo aorta preparations, and impaired
insulin-induced Akt activation (ser 307 phosphorylation) in Ren-2 aortas. This study show that
increased NADPH oxidase activity leads to elevated ROS in Ren-2 aortas and that this is
associated with altered insulin signaling and ex vivo vasoconstriction. These observations
suggest that elevated RAS activity contributions to vascular disease via enhanced activation of
NADPH oxidase.

P48
Mechanisms of Oxidative Stress Induced Increase in Salt Sensitivity and
Development of Hypertension in Sprague Dawley Rats
ANEES AHMAD BANDAY, ABDUL BARI MUHAMMAD, MUSTAFA F LOKHANDWALA; Heart and
Kidney Institute, College of Pharmacy, Univ of Houston, Houston, TX
High salt intake produces vascular changes which contribute to development of
hypertension in salt-sensitive individuals. The underlying causes for these alterations are
not elucidated. There is evidence that oxidative stress plays a key role in the pathogenesis
of cardiovascular diseases. We wanted to investigate whether oxidative stress is a
contributing factor for salt sensitive hypertension. Male Sprague Dawley rats were divided
into five groups and received tap water (vehicle), 30mM L-buthionine sulfoximine (BS, an
oxidant), high salt (HS, 1% NaCl), BS plus HS, and BS plus HS with antioxidant tempol
(1mM) in drinking water for twelve days. Compared to vehicle, BS treatment caused
increase in renal tubular malondialdehyde and urinary excretion of 8-isoprostane and
reduced renal glutathione but had no effect on nitric oxide (NO) levels. Phenylephinephrine
(10M) pre-constricted thoracic aortic rings from BS rats showed decreased response to
sodium nitroprusside (SNP) dependent vasorelaxation. Animals treated with HS had
significant decrease in NO levels despite normal oxidative state. Response to SNP was
unaffected but acetylcholine (Ach) showed decreased vasorelaxation in these animals.
Concomitant treatment of rats with BS and HS caused marked increases in oxidative
stress, blood pressure and decreased NO levels. A rightward shift was observed to SNP
and Ach dose dependent vasorelaxation with increase in EC50. In these animals angiotensin
II (Ang II) caused increased and prolonged contraction with significant decrease in EC50. At
cellular level the incubation of vessels from BS rats with SNP showed decreased cGMP
accumulation while HS rats had decreased basal NO systhetase activity. Tempol decreased
the oxidative stress, normalized blood pressure and restored responses to SNP, Ach and
Ang II in BS plus HS rats. In addition tempol also restored NO levels, signaling and NO
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systhetase activity. We conclude that BS increases the oxidative stress and reduces NO
signaling while HS reduces NO levels by decreasing the NO systhetase activity. These
phenomena collectively contribute to reduced activity of both endothelium dependent and
independent vasorelaxants and enhance vasoconstriction leading to development of
hypertension.
P49
Interaction of Apoptotic and Healthy Microvascular Endothelial Cells:
Implications on Inflammatory Responses
Torsten Kirsch, Alexander Woywodt, Joon-Keun Park, Hermann Haller, Marion Haubitz; Med
Sch Hannover, Hannover, Germany
Objective: Circulating endothelial cells have been detected in a variety of vascular disorders.
However, the functional significance of these circulating endothelial cells and their putative
interaction with healthy endothelium remains unknown. The present study aims to evaluate the
response of human microvascular endothelial cells to apoptotic or necrotic EC in an in vitro
model and to delineate molecular pathways of this interaction. Methods and Results: Human
microvascular endothelial cells (HMEC-1) were exposed to their apoptotic or necrotic
counterparts and generation of chemokines and adhesion molecules was determined by ELISA
or quantitative real-time PCR. Adhesion of neutrophils was quantified using adhesion assays.
In addition, the fate of the apoptotic or necrotic EC in the co-culture was determined by
fluorescence microscopy. Exposure of HMEC-1 with apoptotic EC resulted in a temporary
increase of monocyte chemotactic protein 1 (MCP1) and interleukin-8 (IL-8) expression
whereas exposure to lysed (necrotic) cells led to enhanced expression of IL-8 but no increase
in MCP-1 mRNA. Furthermore, adhesion of primary neutrophils to HMEC-1 that were
co-incubated with apoptotic EC was significantly increased compared to HMEC-1 exposed to
lysed cells or untreated controls. Moreover, we observed that both apoptotic and necrotic cells
were localized within the endothelial cells suggesting engulfment of damaged EC by HMEC-1.
Conclusions: Apoptotic EC induce an inflammatory response in microvascular HMEC-1. This
proinflammatory environment triggers increased chemokine production and enhances adhesion
and migration of primary neutrophils. These data suggest that in vivo circulating endothelial
cells may induce vascular inflammation.
P50
Environmental Carbon Monoxide is a Determinant of Oxidative Stress and
Defective Relaxing Responsiveness to Acetylcholine
Frank F Zhang, Pawel M Kaminski, Brian Lamon, Michael S Wolin, Alberto Nasjletti; New
York Med College, Valhalla, NY
Previous studies suggest that carbon monoxide (CO) produced in arterial vessels along with
the antioxidants biliverdin and bilirubin, during metabolism of heme by heme oxygenases,
promotes vasodilatation. However, exogenous CO fosters vascular oxidative stress and
promotes vasoconstriction. This study was designed to test the hypothesis that exposure
to increased levels of environmental CO causes oxidative stress-dependent attenuation of
acetylcholine (Ach)-induced vasorelaxation. Rats were housed in chamber and allowed to
breathe normal air or air enriched in CO (25100 ppm) for 1 hour. Immediately thereafter
the animals were anesthetized and the descending thoracic aorta was dissected out for
immediate assessment of superoxide anion and relaxing responsiveness to Ach (10-9-10-4
mol/L) using vascular rings mounted on a wire-myograph and bathed with oxygenated
Krebs buffer containing 1 mol/L phenylephrine. The level of superoxide anion in the
aorta of rats breathing normal air (115648 CPM/dry tissue ) was exceeded (P0.05) by
that that in the aorta of rats breathing air containing 100 ppm CO (1901145 CPM/dry
tissue). Ach elicited concentration-dependent reduction of isometric tension in aortic rings
taken from air-breathing rats (EC50:0.290.04 mol/L; Rmax:99.00.7% relaxation). The
concentration-response to Ach in aortic rings of animals breathing 50 and 100 ppm CO
was shifted to the right , with attendant increase and decrease (P 0.05) in their
CHBPR ConferencePoster Presentations e55
respective EC50 (0.380.19 and 1.850.44 mol/L ) and Rmax (57.97.9 and
38.64.4% relaxation) values. Importantly, the inclusion of either the antioxidant tempol
(1 mmol/L) or ebselen (1 mol/L) into the bathing buffer restored all indices of
vasorelaxing responsiveness to Ach to values undistinguishable from those in aortic ring
of air-breating rats. Equally important, vasorelaxing responsiveness to a donor of nitric
oxide, NONOate (10-8-10-4 mol/L), was not impaired in rats breathing 100 ppm CO. These
data suggest that supranormal levels of environmental CO brings about impairement of
Ach-induced vasorelaxation, an indicator of endothelial dysfunction, via an oxidative-stress
dependent mechanism which presumably limits the bioavailability of nitric oxide.
P51
Increased ET-1-Mediated Vasoconstriction but not Impaired Vasorelaxation
of Mesenteric Resistance Vessels in Type 2 Diabetic Gk Rats - Interaction
of Endothelin Receptors
Kamakshi Sachidanandam, Alex K Harris, Jim R Hutchinson, Univ of Georgia, Augusta, GA;
Adviye Ergul; Univ of Georgia, Med College of Georgia, Augusta, GA
Diabetes-associated cardiovascular complications oftentimes result from vascular dys-
function, manisfested as hyperreactivity to vasoconstrictors or impaired relaxation to
vasodilators. We previously demonstrated that endothelin-1 (ET-1) is elevated in the
Goto-Kakizaki (GK) model of Type 2 diabetes, causing hyperreactivity of resistance vessels.
However, the relative roles of ET receptors in this response remained unclear. This study
tested the hypothesis that ETA antagonism prevents increased vasoconstriction, whereas
ETB blockade augments hyperreactivity to ET-1. Fourteen week-old GK rats were treated
for 4 weeks with antagonists to either the ETA (ABT-627, 5mg/kg/day) or the ETB receptors
(A-192621, 15mg/kg/day or 30mg/kg/day). Vascular function of third order mesenteric
arteries, reported as sensitivity (EC50-nM) or magnitude (Rmax-% constriction or relaxation)
of responses to vasoactive agents, is outlined in the Table. Hyperreactivity to ET-1
(0.1100nM) was markedly reduced with ETA blockade, while lower dose ETB antagonism
did not worsen vascular function compared to untreated GK rats. However, blockade of ETB
receptors at the higher dose blunted the vasoconstrictor effect of ET-1. Vasorelaxation to
acetylcholine (ACh, 1nM-10M) was similar between groups. Upon prior incubation with
the NOS inhibitor L-NNA (100nM), relaxation to ACh was impaired in ETB antagonist-treated
and untreated GKs, whereas ETA antagonism completely restored vasorelaxation. Thus,
ETA-mediated vasoconstriction is dominant in ET-1-hyperreactivity as well as decreased
relaxation in the presence of NOS inhibition in the GK model. The data suggests that ETB
antagonism does not exacerbate the constriction in resistance arteries. Indeed, at 30
mg/kg/day dose ETB blockade yields similar responses to ETA receptor antagonism by
ABT-627 suggesting an interaction between two receptor subtypes. (n3 8/group;
*p0.05 vs GK)
ET-1 ACh L-NNA/ACh
EC50 Rmax EC50 Rmax EC50 Rmax
GK 0.50.1 15518 1614 902 5722 567
GKABT627 6020 9513* 108 9218 102 1082*
GKA192621 15mg/kg/day 0.30.1 14711 3616 953 329135* 5513
GKA192621 30mg/kg/day 693195* 14424 45 1053 1415 6419
P52
Microcirculatory and Conduit Artery Function Are Associated with Urinary
Albumin in Hypertensive Subjects: A Community-Based Study
Malik A Rauoof, Stephen T Turner, Iftikhar J Kullo; Mayo Clinic, Rochester, MN
Background. Increased urinary albumin is associated with adverse cardiovascular outcomes.
We investigated whether non-invasive measures of microcirculatory and conduit artery
endothelial function are associated with the urinary albumin/creatinine ratio (UACR), a
commonly used index of albuminuria, in hypertensive adults without known coronary heart
disease (CHD) or stroke. Methods. Non-Hispanic white hypertensive adults (n473) belonging
to hypertensive sibships were studied. UACR was measured in the first voided morning urine
sample. Brachial artery ultrasonography was used to assess microcirculatory (reactive
hyperemia, RH) and conduit artery (flow-mediated dilatation, FMD; nitroglycerin-mediated
dilatation, NMD) function. Variables associated with UACR before and after adjustment for CHD
risk factors were identified using linear and ordinal (quartiles of UACR) regression models.
Results. Male sex, diabetes, higher serum creatinine, and lower high-density lipoprotein (HDL)
cholesterol level were associated with higher UACR in univariable analyses. Body mass index,
systolic BP (SBP), and statin use were positively associated with UACR in a borderline
significant manner (P0.10). In stepwise multiple linear and ordinal regression models that
included univariate predictors, diabetes and SBP were positively and HDL cholesterol inversely
associated with UACR. Among brachial reactivity variables, NMD was a significant independent
predictor of UACR in both multivariable linear (P0.0003) and ordinal (P0.001) regression
analyses. RH (P0.022) and FMD (P0.042) were also associated inversely with higher UACR
in ordinal regression analyses. Conclusion. Impaired microcirculatory and conduit artery
function are associated with increased urinary albumin in hypertensive adults. These findings
indicate that increased urinary albumin may be a marker of vascular dysfunction in
hypertension and suggest a potential mechanism underlying the association of this trait with
increased CHD risk.
 e56 Hypertension Vol 48, No 4 October 2006
P53
Endothelial Control of Vascular Remodeling in Hypertension
Aaron Baker, David Ettenson, Michael Jonas, Elazer Edelman; Massachusetts Institute of
Technology, Cambridge, MA
While many studies have focused on the response of isolated endothelial or vascular smooth
muscle cells (vSMCs) to mechanical forces, few studies have examined the dynamic interplay
between these two cell types in the context of vascular remodeling to hemodynamic forces. We
examined the affect of in-vitro mechanical strain and hypertension in-vivo on endothelial cell
modulation of vSMC proliferation. Human umbilical vein endothelial cells grown to confluence
on collagen coated silastic membranes were exposed to cyclic mechanical strain for 24 hrs
(maximal strain of 3% or 5%, 1 Hz). Strain increased the inhibitory properties of endothelial
cells to vSMCs two-fold, endothelial production of soluble perlecan (2.2-fold) and cell-
associated syndecan-1 (3.2-fold) and conditioned media-associated heparan sulfate glycos-
aminoglycans (38%, by ion exchange and size exclusion chromatography analysis). Further,
mechanical strain causes endothelial cells to activate TGF-1 (163.09.1 pg/ml vs. not
detectable) and increase cellular uptake of FGF-2 (30.00.8 pg/ml vs. 19.40.8 pg/ml). Using
inhibitors to p38 MAPK (SB293063) and ERK1/2 (U0126) we showed that activation of both of
these pathways was essential for load-induced HSPG production, TGF-1 activation and FGF-2
uptake. Additionally, we exposed cells to strain in the presence of a neutralizing antibody to
TGF-1 and demonstrated that autocrine TGF-1 signaling was essential for load-induced
HSPG production and sustained p38 MAPK and ERK1/2 activation. Further studies were carried
out comparing 20 week-old spontaneously hypertensive (SHR) rats to wild-type controls.
Immunohistochemical staining on the aortae of these rats revealed increased endothelial HSPG
core proteins, TGF-1 and phosphorylated signaling intermediates, similar to our in-vitro
findings. Prior work has shown that mechanical strain and hypertension stimulate vSMC
proliferation and hypertrophy through several mechanisms. In this context, our findings suggest
a novel, integrated paradigm for understanding endothelial control of vascular remodeling to
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hypertension.
P54
Synergistic Effect of Hypertension and Hypercholesterolemia on Myocardial
Microvascular Permeability Response to Acute Coronary Artery Stenosis
Elena Daghini, Alejandro R Chade, Xiangyang Zhu, Daniele Versari, James D Krier, Amir
Lerman, Lilach O Lerman; Mayo Clinic College of Medicine, Rochester, MN
Background. The initial site of injury in early atherosclerosis is increasingly recognized to be
at the level of myocardial microcirculation. Exposure to hypertension (HT) or hypercholester-
olemia (HC) may aggravate cardiac response to ischemic insults, and their early co-existence
synergistically promotes cardiac events, conceivably as consequence of impaired microvas-
cular function. One of the early features of microvascular damage is represented by barrier
dysfunction, characterized by altered myocardial microvascular permeability. Therefore, this
study was designed to test the hypothesis that the combination of HT and HC would adversely
affect myocardial microvascular permeability in response to an acute coronary stenosis (CAS).
Materials. The effect of acute CAS on myocardial microvascular permeability (determined from
dye time-attenuation curves obtained in the anterior cardiac wall) was explored in-vivo using
electron beam CT in pigs after 12 weeks of high cholesterol diet (HC; n4), renovascular HT
without (HT; n5) or with concurrent HC (HTC; n4), and in a control group (N, n4). Each
animal was studied before and after a 30-minute 90% balloon-induced CAS achieved by
inflating a balloon catheter in the LAD coronary artery. Results. Blood pressure was similarly
elevated in HT and HTC, while cholesterol levels were similarly elevated in HC and HTC. Acute
LAD CAS caused a significant increase in microvascular permeability in its perfusion territory
in all groups (p0.05), which was particularly high in N (275137%), progressively reduced
in HC (5321%) and HT (4418%), and further significantly blunted in HCHT (235%, myograph. Hypersensitivity of GK basilar arteries to ET-1 (0.1100 nM) was significantly
p0.01 vs. N). Conclusion. The current study suggests that an increase in microvascular
permeability may represent an adaptive myocardial response to acute ischemia. However,
exposure to multiple risk factors may progressively attenuate this response, which may reflect
inadequate microvascular response to ischemic insult, and may contribute to the observed
increase in cardiac events.
P55
Effect of Retinoid X Receptor Agonist on Arterial Reactivity and Ventricular
Hypertrophy in Spontaneously Hypertensive Rats
Jun PU, Reji Hosp affiliated to Shanghai Jiaotong Univ Sch of Medicine, Shanghai, China,
Shanghai, China; Ben HE, Yan-Zhou ZHANG; Reji Hosp affiliated to Shanghai Jiaotong Univ
Sch of Medicine, Shanghai, China
Background: It is well recognized that retinoids have potent anti-proliferative and anti-
inflammatory effects. However, the effect of retinoid X receptor(RXR) agonist on endothelial
function and ventricular hypertrophy has not been investigated. The aim of this study was to
assess the effects of long-term RXR agonist administration on high blood pressure, impaired
endothelial function in aorta, and damage observed in heart in spontaneously hypertensive rats
(SHR). Methods: Male SHR (14 weeks old) and age-matched normotensive Wistar Kyoto (WKY)
rats received either the selective RXR agonist LGD1069 (100 mg/kg of diet), 9-cis-retinoic acid
(9-c-RA, 100 mg/kg of diet), or placebo for 6 weeks. At the end of each treatment, arterial
pressure and heart rate were measured, the vascular reactivity of aortic rings was studied, the
heart weight-to-body weight ratio (HW/BW) and left ventricular mass index (LVMI) was
calculated, and collagen deposition in the heart was determined histochemically using
picrosirius red staining. Results: Our results showed a significantly (P0.01) higher HW/BW
ratio, LVMI and collagen deposition in vehicle-treated SHR compared to levels in corresponding
WKY. Systolic blood pressure was significantly higher in SHR (194 7mmHg, P0.01) than
in WKY (128 4 mmHg) at the end of the trial. Treatment with LGD1069 or with 9-c-RA
caused, from the third week on, a significant reduction of the systolic blood pressure of SHR
(P0.05). WKY did not present a significant change in blood pressure during the 6 weeks
treatment of LGD1069 or 9-c-RA (P0.05).Both treatment significantly (P0.05) decreased
LVW/BW ratio and LVMI, and reduced collagen deposition in the heart in SHR. Both treatments
also significantly augmented acetylcholine-induced relaxation in SHR (P0.01). Endothelium-
independent relaxation induced by sodium nitroprusside was not different between the groups.
Conclusion : Our results suggested that activation of RXR may attenuate the development of
hypertensive cardiac hypertrophy and improved endothelial function in SHR.
P56
Increased Cerebrovascular Tortuosity Index is Associated with Increased
Hemorrhagic Transformation in Acute Ischemic Stroke
Adviye Ergul, Mary-Louise Middlemore, Kamakshi Sachidanandam, Vera Portik-Dobos, Anna
Kozak, Susan C Fagan; Univ of Georgia College of Pharmacy, Augusta, GA
Occlusion or rupture of cerebral blood vessels results in acute ischemic stroke and further
breakdown of the blood brain barrier (BBB) increases cerebral injury by the development of
vasogenic edema and secondary hemorrhage known as hemorrhagic transformation (HT). Type
2 diabetes not only is a predictor of poor outcome of stroke but also increases the risk of HT
in stroke patients. Yet, the impact of chronic mild hyperglycemia as seen in majority of Type
2 diabetic patients on cerebral vessel structure and its mechanisms and functional conse-
quences especially in the acute ischemia/reperfusion injury setting remain unknown. The
current study tested the hypothesis that diabetes-induced changes in the cerebral vasculature
increase the risk of hemorrhage and augment ischemic injury. Male Goto-Kakizaki rats, a model
of mild Type 2 diabetes, (260 290 g, n5) or control Wistar rats (260 290 g, n6) were
used. Vascular structure was assessed by measuring cerebrovascular tortuosity index after
visualization of vascular tree by carbon black/latex infusion, which was significantly increased
in the GK model (1.13 0.01 vs 1.34 0.06, P0.01) indicative of increased vascular density
and remodeling. In additional experiments, GK (n5) or control (n6) rats underwent 3 hours
of middle cerebral artery occlusion and 24 h reperfusion. Neurologic function was assessed and
then brain was harvested for infarct size and hemorrhage analyses. Infarct size as defined by
the % of the total hemisphere was significantly smaller in GK rats (8 4 vs 29 5, p0.01).
There was significant intracerebral bleeding and hematoma formation in the ischemic
hemisphere in GK rats as opposed to controls (100 vs 20%, p0.0001). These findings provide
evidence that there is cerebrovascular remodeling and potential neovascularization in diabetes.
While, diabetes-induced neovascularization appears to prevent infarct expansion, development
of immature blood vessels increases the risk for hemorrhage and thereby exacerbates
neurovascular damage due to cerebral ischemia/reperfusion.
P57
Endothelin Promotes Cerebrovascular Dysfunction in Type 2 Diabetes
Mainly Via ETA Receptor Activation
Alex K Harris, Kamakshi Sachidanandam, Jim R Hutchinson, Univ of Georgia, Augusta, GA;
Adviye Ergul; Univ of Georgia, Med College of Georgia, Augusta, GA
Changes in both vascular function and structure may contribute to increased stroke risk as well
as worsened stroke outcome in diabetes. Conduit arteries such as basilar and middle cerebral
artery (MCA) contribute significantly to microvascular pressure and we have previously shown
that type 2 diabetes cause remodeling of MCAs. Since previous studies have demonstrated
enhanced ET-1 induced vasoconstriction and impaired endothelium mediated relaxation of
basilar arteries in streptozotocin induced diabetes, the current study sought to elucidate the
potential vascular roles of the ET receptors in a non-obese, normotensive model of Type-2
diabetes, the Goto-Kakizaki (GK) rat. Fourteen week old GK or control Wistar rats were treated
with antagonists to either ETA (ABT-627, 5mg/kg/d) or ETB (A-192621, 15 mg/kg/d) receptors
for four weeks. Basilar arteries were harvested and vascular function assessed using a wire
reduced by ETA receptor antagonism while ETB blockade had no effect. Paradoxically, ETA
blockade increased ET-1 sensitivity in Wistars. Maximum constriction to ET-1 as well as
acetylcholine (ACh, 1nM-10m) induced relaxation following ET-1 dose response was similar
among groups. Vasorelaxation to ACh following 70% pre-constriction with 5-hydroxytryptamine
(5-HT) was impaired in diabetes. ETA receptor blockade restored relaxation to control values in
the GK animals with no significant effect in Wistars. ETB blockade had no effect in either group.
These findings demonstrate the presence of cerebrovascular dysfunction in diabetes and
suggest that the ETA receptor may exert a greater influence than the ETB in this process.
*p0.05 vs Wis, **p0.05 vs GK
ET ET/Ach Ach
EC50 Rmax EC50 Rmax EC50 Rmax
W 132 10717 174.7 643 5023 537
W ABT 1.8.4* 13616 122 8515 4311 417
W A-192 10497 13929 2210 484* 167103 556
GK 5.42.8* 13927 9353 637 2815 224*
GK ABT 4211** 1229 3816 747 2920 5514**
GK A-192 74 17259 82 6011 7360 2710
P58
Structural Alterations of Subcutaneous Small Resistance Arteries May
Predict Major Cardiovascular Events in Hypertensive Patients
Carolina De Ciuceis, Damiano Rizzoni, Enzo Porteri, Silvia Paiardi, Gianluca E Boari,
Fracesca Zani, Marco Miclini, Maria Lorenza Muiesan, Dept. of Med and Surgical Sciences,
Univ of Brescia, Brescia, Italy; Francesco Donato, Chair of Hygiene, Univ of Brescia, Brescia,
Italy; Massimo Salvetti, Maurizio Castellano, Enrico Agabiti Rosei; Dept. of Med and Surgical
Sciences, Univ of Brescia, Brescia, Italy
Objective Structural alterations in the microcirculation may be considered an important
mechanism of organ damage. An increased media to lumen ratio of subcutaneous small

resistance arteries (M/L) may predict the subsequent development of cardiovascular events in
a high risk population (Rizzoni et al, Circulation 2003). However, it is not presently known
whether structural alterations of small arteries may predict also mortality and/or major
cardiovascular events. Design and Methods Three-hundred and four subjects were included
in the present study. They were 65 normotensive subjects, 112 patients with essential
hypertension, 29 patients with pheochromocytoma, 46 patients with primary aldosteronism, 25
patients with renovascular hypertension, 9 with acromegaly and 18 normotensive patients with
non-insulin dependent diabetes mellitus. All subjects were submitted to a biopsy of
subcutaneous fat. Small resistance arteries were dissected and mounted on an isometric
myograph, and the tunica media to internal lumen ratio was measured (M/L). The subjects were
re-evaluated after an average follow-up time of 6.9 years (0.6 13.5). Results Twelve subjects
died for fatal cardio-cerebrovascular event (FCE), 16 had a major, non fatal cardiovascular
event (stroke or myocardial infarction) (SMI), 20 had a minor cardiovascular event (MCE)
(chronic renal failure, cardiac pacemaker device implant, atrial fibrillation, angina pectoris or
heart failure, claudication intermittens, surgical intervention for aortic aneurism) (overall: 2.3
events % per year), while 256 of them had no cardiovascular event (NCE). A significant
difference was observed in M/L between patients with FCESMIMCE and those with NCE
(M/L: 0.1100.032 vs 0.0880.028, p0.00003) and between patients with FCESMI and
those with NCE (M/L: 0.1050.037 vs 0.0880.028, p0.05). Restricting the analysis to
patients with essential hypertension, a significant difference in M/L between those with
FCESMIMCE and those with NCE (0.1140.022 vs 0.100.033, p0.05) was observed. significantly reduced MMP-2 protein (44 15* vs 201 13 pixels) and activity (12151
Conclusions Our results indicate, in a larger population than that previously investigated, that
structural alterations of small resistance arteries may predict all major cardiovascular events.
P59
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Crosstalk of AT1, AT2 Receptors and Aldosterone in Angiotensin
II-Rgulated Vascular Smooth Muscle Cell Senescence
Masaki Mogi, Li-Juan Min, Jian-Mei Li, Jun Iwanami, Kana Tsukuda, Masaru Iwai,
Masatsugu Horiuchi; Ehime Univ, Graduate Sch of Medicine, Tohon, Ehime, Japan
Recent evidence has revealed a close association between vascular cell senescence and
vascular disorders, and the potential roles of angiotensin (Ang) II on vascular senescence have
been highlighted, although its detailed mechanism regulated by Ang II receptor subtypes is still
unclear. To examine the roles of Ang II receptor subtypes in vascular senescence, we employed
vascular smooth muscle cells (VSMC) prepared from the thoracic aorta of AT1a receptor null
mice (AT1KO) and AT2 receptor null mice (AT2KO). Daily administration of Ang II (10-7 M)
increased senescent cells evaluated with senescence-associated -gal (SA-gal) activity. In
contrast, VSMC prepared from AT2KO showed an acceleration of both proliferation and
senescence compared to VSMC prepared from wild type mice. Moreover, we observed that Ang
II-induced senescence in VSMC prepared from AT1KO was markedly attenuated. Immunoblot
analysis showed 3.2-fold increase in Ki-ras2A expression 72 hours after Ang II stimulation in
VSMC and knocking down of Ki-ras2A gene with small interfering of RNA strongly inhibited the
increase in SA-gal stained cells, suggesting that crosstalk between AT1 and AT2 receptor
could play some antagonistic roles in Ki-ras2A expression. We also observed that Ang II
treatment significantly increased cycline dependent kinase inhibitors, such as p16, p21, p27
and p53. Moreover, we demonstrated that treatment with non-effective doses of aldosterone
(10-12 M) and Ang II (10-10 M) synergistically increased Ki-ras2A activation and senescent VSMC.
Treatment with a selective AT1 receptor blocker (ARB), valsartan, reduced SA-gal stained
cells, Ki-ras2A expression to a basal level and cancelled the effect by aldosterone. These
results suggest that AT1 receptor stimulation increased VSMC senescence via Ki-ras2A
activation, whereas AT2 receptor activation antagonized these effects of AT1 receptor, and that
AT1 receptor and aldosterone signaling synergistically enhanced VSMC senescence.
P60
Veins Are not Arteries: A Story of Remodeling in Hypertension
Catherine Rondelli, Mohammad H Pervaiz, Ralph E Watson, Keshari Thakali, Gregory D Fink,
Stephanie W Watts; Michigan State Univ, East Lansing, MI
Arterial remodeling in hypertension is a process that is adaptive because it provides a stronger
artery that supports higher intra-arterial pressures, but also detrimental in that it contributes to
organ damage. Remodeling occurs in response to mechanical and neurohumoral stimuli. We
hypothesized that veins, which are not exposed to higher pressures in hypertension, would
demonstrate less active remodeling than arteries. We assessed remodeling by measuring
matrix metalloproteinase (MMP) expression and function, vessel morphometry and smooth
muscle alpha-actin staining. Thoracic aorta and vena cava from sham normotensive (systolic
blood pressure 1104 mm Hg) and deoxycorticosterone (DOCA)-salt hypertensive rats (1888
mm Hg) were used in immunohistochemical, western and zymographic assays. Aorta and vena
cava expressed the gelatinases MMP-2, MMP-9 and MT-MMP1; proteins localized to smooth
muscle in aorta, densely in the endothelium and single smooth muscle layer (subendothelial)
of vena cava. Western and zymography analyses validated that MMP-2 was active in all vessels
but more active in aorta vs vena cava (150%). In hypertension, MMP-2 expression and
activity was increased in the aorta (127% control) but not vena cava of the DOCA-salt rat when
compared to sham. Wall thickness was increased in DOCA-salt vs sham aorta (301/023 vs
21814 microns; p 0.05) but medial thickness was not different in vena cava. Moreover, the
smooth muscle layer of the vena cava from DOCA-salt rats was not thicker when compared to
sham, differing from the hypertrophy observed in aorta. These findings suggest that large veins,
at least using these measures, do not undergo vascular remodeling in systemic hypertension
and suggest that elevated pressures or other hemodynamic forces are a primary cause of
remodeling.
CHBPR ConferencePoster Presentations e57
P61
Endothelin B Receptor Antagonism Reduces Cerebrovascular Remodeling in
Diabetes: Potential Interaction among ETA, ETB1 and ETB2 Subtypes
Alex K Harris, Kamakshi Sachidanandam, Jim R Hutchinson, Univ of Georgia, Augusta, GA;
Adviye Ergul; Univ of Georgia, Med College of Georgia, Augusta, GA
Diabetes enhances vascular matrix accumulation, which can further contribute to the
development of vascular dysfunction. It has been demonstrated that endothelin-1 (ET-1) may
contribute to this process by stimulation of collagen synthesis and SMC migration. We have
previously demonstrated that blockade of the ETA receptor attenuates dysregulation of matrix
degrading enzymes (MMP) and blunts collagen deposition and matrix remodeling in Type-2
diabetes. A recent study demonstrated that inhibition of the ETB receptor by pharmacological
or genetic approaches promotes pathological vascular remodeling in a vascular injury model
suggesting that ETB receptors are vasculoprotective. However, the relative role of the ETB
receptor in Type-2 diabetic vascular remodeling processes and its influence on the vascular
matrix remained unknown. Accordingly, the current study tested the hypothesis that
pharmacological inhibition of the ETB receptor augments vascular remodeling of middle cerebral
arteries in Type-2 diabetes. Fourteen week old Goto-Kakizaki rats were treated for 4 weeks
with the ETB receptor antagonist A-192621 (15 or 30 mg/kg/day). High dose receptor blockade
1004* vs 18296 1180 pixels) in middle cerebral arteries while lower dose inhibition had no
significant effect. Morphometric analysis of cross sections demonstrated that similar to MMP-2
protein changes, low dose receptor blockade had no effect on vessel structure. However, high
dose blockade significantly reduced wall:lumen ratio (0.23 .03* vs 0.51 .04) in a manner
similar to that observed with ETA receptor blockade. These data indicate a potentially greater
involvement of the smooth muscle ETB2 receptor in Type-2 diabetes giving rise to ETA-like
effects in the vasculature as opposed to anticipated vasculoprotective effects via activation of
ETB1 receptors on endothelial cells. These findings also point to a potential receptor interaction
among ETA, ETB1 and ETB2 subtypes and may be relevant for the future development and use
of ET receptor antagonists in diabetes. *p0.05 vs GK Untreated
P62
Preeclampsia Activates Neuropeptide-y (NPY) and its Receptors: Possible
Role in Impaired Blood Flow and Ischemic Angiogenesis
Sara P Paiva, Lydia E Kuo, Jason U Tilan, Georgetown Univ, Washington, DC; Ann-Cathryn
Rylander, AstraZeneca, Molndal, Sweden; Ullamari Pessonen, Univ of Turku, Turku, Finland;
Zofia Zukowska; Georgetown Univ, Washington, DC
Hypertension in preeclampsia (PE) is associated with sympathetic hyperactivity and impaired
placental angiogenesis. Early onset PE (34wks) may depend more importantly on defective
angiogenesis along with decreased availability of free VEGF. Late onset PE (34wks) may
depend less on defective placentation and more on maternal vasoconstriction. Both angiogen-
esis and vasoconstriction can be regulated by neuropeptide Y (NPY), a sympathetic co-
transmitter, which acts via a family of receptors (Rs). NPY136 acts via Y1R as vasoconstrictor
and vascular mitogen but - when converted by endothelial dipeptidyl peptidase IV (DPPIV) to
NPY336 - is angiogenic, in part via NO and VEGF, due to activation of Y2 and Y5Rs. We studied
expression of NPY, its Rs and DPPIV in placentas (Real Time RT-PCR), omental fat
(immunocytochemistry) and NPY levels in serum (RIA) from 20 women with early onset PE, 21
with late onset PE, and from 48 controls. NPY and Y2R mRNAs were highly expressed in
placentas from early PE but undetectable in the other 2 groups. Y1R mRNA expression was
highest in placentas from early PE and detectable but similar in the other 2 groups. NPY, Y1,
Y2, Y5, and DPPIV were elevated in omental fat from PE versus normal pregnancies, and serum
NPY levels were 50% higher in late compared to early PE. We hypothesize that NPY contributes
to both early and late PE, by impairing blood flow and placentation. Activation of NPY-Y1R may
enhance maternal vasoconstriction and contribute to hypertension. The expression of NPY/Y2R
in early onset PE may reflect a compensatory mechanism which fails due to impaired
availability of free VEGF, its key angiogenic effector.
P63
Targeted Deletion of Collectrin, a Novel Gene Upregulated after Subtotal
Nephrectomy in Mice, Results in a Urine Concentrating Defect Induced by
Amino Aciduria
Sandra Malakauskas, Duke Univ and Durham VA Med Cntrs, Durham, NC; Timothy Fields,
Duke Univ Med Cntr, Durham, NC; Hui Quan, CIIT Cntrs for Health Rsch, Rsch Triangle
Park, NC; Thu H Le; Duke Univ and Durham VA Med Cntrs, Durham, NC
Chronic kidney disease (CKD) represents a substantial health care burden, yet mechanisms
causing progression of CKD are poorly understood. Collectrin is a novel gene found to be
upregulated after subtotal nephrectomy in mice, a model of CKD. In the adult mouse kidney,
collectrin is expressed in the proximal tubule and collecting duct. To understand its biological
role, we generated a mouse line with targeted deletion of collectrin. We found that, at baseline,
collectrin knockout (KO) mice excrete nearly twice the urine volume of WT, but urine
osmolalities were similar. However, after water deprivation, KO mice have a modest urine
concentrating defect, demonstrated by the inability to achieve maximal urinary concentration
(3880 mosm/kg, n12 WT vs. 3189 mosm/kg, n13 KO; p0.00001). KO mice display a
greater corresponding increase in serum sodium (0.83 mEq/L, n8 WT vs. 6.72 mEq/L, n10
KO; p0.0044). Similarly, after dDAVP administration, KO mice only partially concentrate their
urine (3477 mosm/kg, n5 WT vs. 2498 mosm/kg, n5 KO; p0.0016), suggesting
decreased renal responsiveness to vasopressin. To determine whether the enhanced urine
volume is a result of increased solute or water clearance, we collected 24-hour metabolic data.
KO mice display both an increase in osmolar clearance (7.1 cc/day, n11 WT vs. 11.2 cc/day,
n12 KO; p0.0067) as well as TcH2O, a measure of free water reabsorption (5.9 cc/day,
n11 WT vs. 9.0 cc/day, n12 KO; p0.018). These data suggest that KO mice exhibit an
osmotic diuresis. We further examined the solute composition of the inner medullary regions
 e58 Hypertension Vol 48, No 4 October 2006
of the kidney. We found a trend toward decreased concentrations of solutes in KO mice,
suggesting a mild dilution of their inner medullary gradients. This is consistent with an osmotic
diuresis, in which enhanced urine flow rates can result in a washout of the medullary
gradient. We next determined the causative osmolyte by examining the 24-hour excretions of
solutes known to be regulated or recovered by the kidney. We found that KO mice excrete twice
the amount of amino acids (6.2 mol, n6 WT vs. 12.9 mol, n6 KO; p0.013). Taken
together, we propose that targeted deletion of collectrin results in decreased urine concen-
trating ability due to an osmotic diuresis induced by amino aciduria.
P64
NFAT5 Mediates Osmotic Regulation of Sgk1 Gene Transcription
Songcang Chen, Nada Nekrep, Chris Grigsby, Keith Olsen, David G Gardner; Diabetes Cntr,
San Francisco, CA
We recently reported that elevated extracellular tonicity increases the expression of serum and
glucocorticoid-inducible kinase (sgk1) mRNA and protein (Chen S, et al Hypertension 2004;
43:866). Sgk1 is thought to play an important role in aldosterone-dependent sodium handling
in distal tubular segments. In the present study we show that this increased expression derives
from an increase in p38 MAPK-dependent sgk1 promoter activity. Increased extracellular
tonicity increased activity of a transfected -1400 sgk1-LUC reporter by 5 fold (p.01 vs
control) and this increase was suppressed by the p38 MAPK inhibitor SB203580 or by
co-transfection of a dominant negative mutant of the p38 MAPK-activating kinase MKK6
(p.01 vs. NaCl-treated). Site-directed mutational analysis of the sgk1 promoter identified an
osmoregulatory element (ORE) approximately 324 bp upstream from the transcription start site.
Mutation of 3 bases within this site resulted in complete elimination of the osmotic induction
while having no effect on basal promoter activity. Electrophoretic mobility shift analyses,
carried out with nuclear extracts from IMCD cells and oligonucleotides spanning this ORE,
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
revealed a highly specific DNA protein interaction that was competed with wild type but not
mutant sequence. Immunoperturbation studies demonstrated that the complex was super-
shifted by antibody directed against the nuclear transcription factor NFAT5 but not by antibody
directed against the p50 subunit of NF-B. Co-transfection of sgk1-LUC with an anti-sense
construct or a dominant negative mutant directed against NFAT5, virtually eliminated the
osmotic induction of the promoter. Finally, chromatin immunoprecipitation analysis of IMCD
cells subjected to osmotic stress showed 8-fold increase in association of NFAT-5, as well
as RNA polymerase II, with the sgk1 gene promoter in intact cells. Collectively, these data
identify sgk1 as the target of increased extracellular tonicity, and potentially other stress-
related effectors, in IMCD cells and provide a clear picture of the mechanism underlying this
induction.
P65
Regulation of the Prorenin and Renin Receptor in the Kidney by Salt intake
and Angiotensin II
Jiqian Huang, Chun Xue, John Gildea, Helmy M Siragy; Univ of Virginia, Charlottesville, VA
ATP6AP2 was cloned and found to encode the renin receptor (RR)which binds prorenin and
renin. The deduced 350-amino acid protein has a calculated molecular mass of 39KD.
Bioinformatics computation deduced 4 transcripts and 2 significant proteins of 66 and 39KD
(proRR & RR). The physiologic and pathologic functions of proRR and RR are currently unknown.
In this study we investigated the physiologic factors that may regulate proRR and RR. We
hypothesized that salt intake and angiotensin II(AngII) modulates proRR and RR expression. We
studied renal proRR and RR expression in response to 5 days of normal(NS), low(LS) or high(HS)
salt intake, renal administration of AngII(100ng/kg/min/d) or AT1R antagonist valsartan (Val,10
mg/kg/d). Western blot demonstrated the constitutive expression of RR at 66 and 39KD in rat
renal cortex and medulla (Fig.B), rat mesangial cells (RMCs) (Fig.A & C) and rat proximal tubular
cells (RPTCs) (Fig.D). Using RR siRNA, proRR and RR were knocked down in RMCs (Fig.A). In
the cortex, HS, AngIINS and ValLS enhanced the expression of proRR. In the medulla, AngII
and Val upregulated proRR expression. Similarly LS and HS upregulated, while valsartan
downregulated RR at 39KD in the renal medulla (Fig.B). 48hrs treatment with NaCl(25mM),
Glucose(30mM), AngII(100nM) and AngII plus Glucose upregulated proRR and RR protein in
RMCs (Fig.C) and in RPTCs(Fig.D). These results demonstrate that RR is ubiquitously and
constitutively expressed in the kidney, mainly in RMCs and RPTCs. ProRR(66KD) is the
precursor of RR(39KD). AngII via AT1R is involved in processing proRR to RR. In conclusion salt
intake, glucose and Ang II modulate proRR and RR expression in the kidney.
P66
Aminopeptidase N Down-Regulates Basolateral Na/K ATPase: Evidence of
Novel Signaling via Angiotensin IV and the Angiotensin IV Receptor
Kumar Kotlo, Randal Skidgel, Robert S Danziger; Univ of Illinois at Chicago, Chicago, IL
Most experimental approaches to hypertension have sought to identify proteins and genes
directly linked to salt-sensitivity and development of the hypertensive phenotype. Much less
attention has been paid to the identification of proteins linked to normal adaptation to high salt.
We have reported that aminopeptidase N/CD13 (Nepep), which cleaves an amino-terminal
arginine from angiotensin III (Ang III) to yield the natriuretic hexapeptide angiotensin IV (Ang IV),
exhibits greater renal tubular expression in the Dahl salt-resistant (SR) rat than its Dahl
salt-sensitive (SS) counterpart (Hypertension 43:2825, 2004). In this work, regulation of
basolateral Na/K ATPase by Nepep was determined in LLC-PK1 cells. Transfection of cells with
siRNA to Nepep increased and Nepep over-expression plasmid decreased the surface
expression of the -subunit of Na/K ATPase, measured by biotinylation and Confocal
immunohistochemistry, and oubain-inhibitable activity ATPase activity. In transgenic mice
over-expressing Nepep in epithelial cells, there was decreased expression of basolateral Na/K
ATPase, consistent with cell culture findings indicating that Nepep reduces Na/K ATPase. The
role of Ang IV in the signaling pathway for Nepep mediated down-regulation of Na/K ATPase
was explored. Nepep over-expression increased (p 0.05) intracellular Ang IV abundance,
measured by quantitative immunohistochemistry (QIHC) using a polyclonal Ab to Ang IV. siRNA
to the Ang IV receptor (AT4) inhibited the decrease in Na/K ATPase caused by Nepep
over-expression. Together, these results indicate that Nepep down-regulates basolateral Na/K
ATPase and that the signal is mediated via Ang IV and the AT4 receptor. These results identify
a novel signaling pathway in nephrons that may play a mechanistic role in adaptation to high
salt and serve as a therapeutic target for hypertension.
P67
Role of VEGF in Renal Mechanisms of Hypertension: VEGF Receptor
Inhibition Enhances Dietary Salt-Induced Hypertension in Sprague-Dawley
(SD) Rats
Jian-Wei Gu, Megan Shparago, Wei Tan, Amelia P Bailey; Univ of Mississippi Med Cntr,
Jackson, MS
Clinical evidence links the inhibition of vascular endothelial growth factor (VEGF) to hyperten-
sion. The present study examines: 1) if administration of VEGF receptor inhibitor (SU5416)
enhances dietary salt-induced hypertension in SD rats; and 2) if VEGF or SU5416 directly
affects proliferation of cultured human renal proximal tubular epithelial cells (HRPTEC),
compared to human umbilical vein endothelial cells (HUVEC), and human aortic smooth muscle
cells (HASMC). 3H-thymidine incorporation was determined in those cells following exposure to
VEGF (10 ng/ml), VEGF plus SU5416 (10 mol/L), SU5416 (1, 5, 10, & 20 mol/L), or vehicle
control for 18 hrs. Eight 10 wk old male SD rats received a high sodium diet (HS, 8%) and the
other 8 SD rats received a normal sodium diet (NS, 0.5 %) for 4 wks. After 2 wks of the dietary
program, 4 rats were administrated with SU5416 at 10 mg/kg/d, i.p. or DMSO (vehicle) for 14
days in HS and NS groups. In the end, mean arterial pressure (MAP) was determined in
conscious rats by continuous monitoring through a catheter placed in the carotid artery for 2
days. There were no significant changes in MAP between SU5416 and DMSO-treated rats fed
with NS (126.4 4.1 vs. 115.1 6.3 mmHg; P0.213). In DMSO-treated rats, 4 weeks of HS
did not significantly increase MAP, compared to NS rats (125.9 4.1 vs. 115.1 6.3 mmHg;
P0.233). However, MAP was significantly higher in rats treated with SU5416, as opposed to
those treated with DMSO fed with HS for 4 wks (157.6 3.9 vs. 125.9 4.3 mmHg,
P0.0016). SU5414 treatment further increased total urinary protein in SD rats fed with HS,
compared to the vehicle HS group (53.72.1 vs. 42.5 4.6 mg/d; P0.05). There was a
significant decrease in proliferation of HRPTEC treated with VEGF plus SU5416, compared to
the control (65%, P0.001). VEGF caused a 2-fold increase in proliferation of HUVEC,
compared to the control (P0.001), but its action was completely abolished by SU5416.
Neither VEGF nor SU5416 exerted any effect on proliferation of cultured HASMC. Also, SU5416
caused a dose-related inhibition on proliferation of HRPTEC. These results suggest that VEGF
inhibition enhances dietary salt-induced hypertension and kidney injury, and that VEGF exerts
autocrine and paracrine functions on HRPTEC. (HL51971 & AA013821)
P68
Antioxidant Treatment Reduces Renal Immune Infiltration, Renal TNF,
Renal MCP-1, Renal Damage and Dysfunction in Salt-Sensitive
Hypertension
Niu Tian, Sharkeshia Jordan, R. D Manning, Jr.; Univ of Mississippi Med Cntr, Jackson, MS
Renal free radical (O2.-) production increases and renal infiltration of immune cells occurs in
some forms of experimental hypertension. However, in salt-sensitive hypertension, it is not
clear whether elevations in renal oxidative stress increase renal immune infiltration and renal
cytokines and chemokines leading to renal damage, dysfunction and increased arterial
pressure. The importance of renal oxidative stress in initiating a renal immune response was
determined in Dahl salt-sensitive (S) rats subjected to a high sodium diet with and without
antioxidant treatment. S rats were equipped with arterial and venous catheters and subjected
to a 5-week diet of high Na (8% NaCl) or high NaVit C/E [vitamin C-(1g/L) in the drinking
water and vitamin E-(5000 IU/Kg) in the food]. Mean arterial pressure was measured
continuously throughout the last 10 days. The results at the end of week 5 are shown below
(values are meanSEM; P0.05 comparing Hi Na and Hi NaVit C/E groups). The data show
that antioxidant treatment of high sodium Dahl S rats with vitamin C/E effectively decreased
renal oxidative stress as evidenced by decreased renal O2.- release and increased renal
GSH/GSSG, the reduced-to-oxidized glutathione ratio. As a result, renal macrophage infiltration
(ED1 cells), renal TNF-alpha, and renal MCP-1 decreased. In addition, proteinuria and mean
arterial pressure decreased, and GFR and renal plasma flow increased markedly. In summary,
vitamin C/E treatment in Dahl S rats on high sodium intake decreased renal oxidative stress,
 CHBPR ConferencePoster Presentations e59
renal macrophage infiltration, TNF-alpha and MCP-1, improved renal dysfunction, lessened P71
renal injury and decreased arterial pressure. In conclusion, in Dahl S rats on a high sodium diet, Female Protection from Renal Disease Progression is Associated with
renal oxidative stress plays an important role in mediating increased renal inflammation, which 17Beta-Estradiol (E2)-Mediated Attenuation of the Injury-Induced Increase
is associated with decreased renal hemodynamics, renal damage and increased arterial
in NAD(P)H Oxidase Activity
pressure.
GSH/GSSG ratio cortex 0.3 0.3 2.5 0.8 Wei Zheng, Hong Ji, Ana Paula Dantas, Kathryn Sandberg; Georgetown Univ, Washington,
DC
Renal macrophages 4.90.3 2.30.4
(count/mm2)
Chronic renal disease from several etiologies progresses at a faster rate in men compared to
Renal TNF-alpha (pg/mg) 1.20.06 0.90.05
Renal MCP-1 (OD/g) 1.20.1 0.740.05 women. We found that E2 protects against progressive renal disease induced by renal wrap
Proteinuria (mg/day) 1704 9612 hypertension (RW) and a high salt diet. Compared to male rats (RW-M) and ovariectomized
GFR (ml/min) 1.50.1 3.00.5 females (RW-OVX), estrogen replete females (RW-F & RW-OVXE2) were protected from
Renal plasma flow (ml/min) 3.80.4 7.40.5 indices of renal injury including proteinuria, mean glomerular volume, glomerulosclerosis, and
Mean arterial pressure (mmHg) 1915 1508 tubulointerstitial fibrosis. Progressive renal disease is associated with elevated levels of
reactive oxygen species in the kidney. Objective: To determine if female protection from renal
injury is associated with E2 attenuation in reactive oxygen species generation, we determined,
in RW hypertension, if E2 regulates the activity of renal NAD(P)H oxidase (ox), which is the major
enzyme that generates superoxide in the rat kidney. Methods: Sprague Dawley rats
(n6 8/group) were divided into 2 sham-operated control groups: male (Sham-M) and female
(Sham-F) and 4 RW groups: RW-M, RW-F, RW-OVX and RW-OVX-E2 (0.24 mg E2/60 day pellet).
P69 After 6 wk on a high sodium (4%) diet, NAD(P)H ox activity was measured in renal cortical
Suppression of Renal Immune Cell Infiltration Attenuates Hypertension and homogenates by the lucigenin method and expressed as relative light units/180 sec (RLU);
Kidney Disease in Dahl Salt-Sensitive (SS/Mcw) Rats *p0.05. Results: RW increased NAD(P)H ox activity in both males (1.4-fold*) and females
(2.3-fold*). Compared to females in the same group, both male control (2-fold*) and RW
David L Mattson, Hayley Lund; Med College of Wisconsin, Milwaukee, WI (1.3-fold*) rats had higher enzyme activity. In RW females, ovariectomy increased enzyme
activity by 1.3-fold* while E2 replacement prevented this effect. [RLU: Sham-M, 2840 110;
Previous studies from our laboratory demonstrated that systemic administration of the Sham-F, 1400 160; RW-M, 4080 240*; RW-F, 3200 260; RW-OVX, 4270 320*;
immunosuppressive agent mycophenolate mofetil attenuated the development of hypertension RW-OVXE2, 2730 680.] Conclusions: These results demonstrate that E2-mediated
and kidney disease in the Dahl SS/Mcw rat. The present experiments were performed to protection from RW-induced renal injury is associated with attenuation in RW-induced
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

confirm that observation using a mechanistically different immunosuppressive agent and to increases in NAD(P)H ox activity, suggesting that E2 protects the kidney from progressive renal
determine the histological and immunohistochemical changes which occur in the kidneys of the disease in part, by attenuating the injury-induced increases in renal superoxide. Supported by
vehicle and immune-suppressed rats. Dahl SS/Mcw rats were fed a high salt (4.0% NaCl) diet NIH AG19291.
for three weeks. Rats were treated with a daily injection of vehicle (5% dextrose) or the
immunosuppressive agent tacrolimus (0.25 mg/kg/day, i.p.), a calcineurin inhibitor, during the
period of high NaCl intake (n5/group). High salt mean arterial blood pressure in the rats P73
administered tacrolimus (1523 mmHg) was significantly decreased compared to vehicle- High-Salt Diet Produces Severe Renal Dysfunction Independently of
treated rats (1718 mmHg). Furthermore, the rate of protein (6112 mg/day) and albumin Changes in Arterial Pressure in Spontaneously Hypertensive Rats (SHR)
excretion (209 mg/day) in the tacrolimus-treated rats was significantly lower than the protein
and albumin excretion rate in vehicle-treated rats (18129 and 7428 mg/day, respectively). Luis C Matavelli, Xiaoyan Zhou, Jasmina Varagic, Dinko Susic, Edward D Frohlich; Ochsner
Creatinine clearance and body weight were not different between the groups, averaging Clinical Foundation, New Orleans, LA
0.50.2 ml/min/gkwt and 34714 g, respectively, in the vehicle-treated group. A histological
examination demonstrated marked glomerular damage (fibrotic tissue and collapsed capillary We have previously shown that high dietary salt produced adverse cardiac effects in SHR
structure), blocked tubules in the outer medulla (protein deposition casts), and fibrotic vasa independent of increases in arterial pressure; however, the renal effects are unknown. In the
recta bundles in the kidneys of vehicle-treated rats. Furthermore, immunohistochemical present study we evaluated the role of step-wise increases in salt-loading on renal function,
localization of ED1, a marker of macrophage infiltration, demonstrated ED1-positive cells hemodynamics, and glomerular dynamics in SHR. At 8 weeks of age, male SHR were given 4%
throughout the kidneys of the vehicle-treated rats following the high NaCl intake with the (n11), 6% (n9), or 8% (n11) NaCl diet for the ensuing eight weeks; controls (n11)
greatest infiltration near damaged glomeruli and in the region of the protein casts in the outer received standard chow (0.6% NaCl). Each week, all rats had 24-h urinary albumin (Ualb)
medulla. These experiments indicate that the renal infiltration of immune cells plays an measured. After eight weeks, SHR had systemic and renal hemodynamics determined. The
important role in the development of hypertension and renal disease in Dahl SS/Mcw rats controls, 4%, and 6% salt-loaded SHR were studied further by renal micropuncture. After two
consuming a high NaCl diet. weeks salt-loading, Ualb increased in 6% and 8% salt-loaded SHR. At eight weeks, mean
arterial pressure (MAP) had increased and glomerular filtration rate (GFR) decreased in all
salt-loaded rats. The 6% and 8% salt-loaded SHR decreased renal plasma flow (ERPF) and
increased renal vascular resistance (RVR), left kidney mass index (control3.410.10,
4%3.720.04, 6%3.840.11*, 8%3.930.19* mg/g) and serum creatinine (con-
trol0.470.02, 4%0.560.04, 6%0.720.07*, 8%0.620.04* mg/dl). Although
P70 single nephron glomerular filtration rate was not different (control27.32.57,
4%24.91.1, 6%23.62.2 nl/min), the 4% and 6% salt-loaded rats decreased single
Enhanced Superoxide Activity and its Interaction with Nitric Oxide
nephron plasma flow (SNPF) and increased afferent (RA) and efferent arteriolar (RE)
Modulate Renal Function in Pre-Hypertensive Ren-2 Transgenic Rats resistances. Furthermore, 6% salt-loaded SHR increased glomerular hydrostatic pressure (PG).
In conclusion, salt-excess increased MAP minimally, but progressively deteriorated renal
Libor Kopkan, Tulane Univ Health Sciences Cntr, New Orleans, LA; Monika Thumova, function, hemodynamics, and glomerular dynamics. These findings demonstrate a causal
Zuzana Huskova, Zdenka Vanourkova, Ludsk Cervenka, Cntr for Experimental Medicine, relationship between salt excess and renal injury independent of a minimal increase in MAP.
Institute for Clinical and Experimental Medicine, Prague, Czech Republic; Dewan S Majid; All data are mean 1SEM; *p0.05 vs control.
Tulane Univ Health Sciences Cntr, New Orleans, LA
Control 4% NaCl 6% NaCl 8% NaCl
It has been demonstrated that pre-hypertensive heterozygous transgenic rats (TGR) harboring
Ualb (2nd wk; 0.480.04 0.490.05 0.720.06* 1.240.18*
the Ren-2 renin gene exhibit an excess of superoxide (O2-) production compared to
mg/24h)
aged-matched Hanover-Sprague Dawley rats (HanSD). This study was performed to examine MAP (mm Hg) 1862 1941* 1961* 1972*
the role of O2- and its interaction with nitric oxide (NO) in the regulation of renal hemodynamic GFR (ml/min/g) 1.090.13 0.800.09* 0.510.05* 0.510.07*
and excretory function in pre-hypertensive TGR that may be involved in the development of ERPF (ml/min/g) 3.160.41 2.580.18 1.600.18* 1.670.18*
hypertension in this model. Renal responses to acute infusion of O2- scavenger, tempol (150 RVR (U) 33.44.1 39.83.1 72.713.5* 77.615.5*
g/min/100g BW), and NO synthase inhibitor, nitro-L-arginine methylester (L-NAME, 5 SNPF (nl/min) 101.59.9 75.25.9* 70.35.7*
g/min/100g BW) directly into the renal artery were evaluated in anesthetized male TGR RA (U) 5.640.53 7.850.60* 8.800.86*
(n31) and HanSD rats (n29). Glomerular filtration rate (GFR) and renal plasma flow (RPF) RE (U) 1.600.15 2.200.19* 2.630.26*
PG (mm Hg) 49.20.9 50.70.8 53.61.0*
were estimated by inulin and para-aminohippurate clearances, respectively. Urinary concen-
ration of 8-isoprostane (index of endogenous O2- activity) was determined by enzyme
immunoassay. Although there were no differences in mean arterial pressure and basal renal
hemodynamics and excretory function between pre-hypertensive TGR and age-matched HanSD P74
rats, infusion of tempol alone caused significant increases in RPF and GFR (104 and 122%, Ac-SDKP Exerts Renal Protective Effects in Wild Type and eNOS-/- Mice
respectively; n9) in TGR but not in HanSD (n9). Compared to HanSD, 8-isoprostane with DOCA Salt Hypertension
excretion was significantly higher in TGR (41%) which was significantly attenuated during
tempol treatment. Intra-arterial L-NAME infusion in the kidney caused greater decreases in RPF Saraswati Pokharel, Nour-Eddine Rhaleb, Tang-Dong Liao, Umesh Sharma, Oscar Carretero;
and GFR in TGR (-344 and -224%, respectively; n10) than in HanSD rats (-193 and Henry Ford Hosp, Detroit, MI
-104%, respectively; n10). In TGR (n12), greater hemodynamic effects of L-NAME were
abolished by co-infusion of tempol. Moreover, co-infusion of tempol reversed the anti-diuretic Background: Hypertensive end organ damage is characterized by renal inflammation and
and anti-natriuretic effects of L-NAME. These findings indicate that there was greater proteinuria. In this study, we hypothesized that N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP) inhibits
enhancement of O2- activity during NO inhibition in TGR than HanSD rats. These data suggest hypertension induced nephropathy by decreasing inflammation and increasing nephrin.
that the enhanced O2- activity and its interaction with NO during the pre-hypertensive phase in Methods and Results: We treated 16-week old uninephrectomized wild type (WT) and
TGR modulates renal hemodynamics and excretory function and thus plays a role in the endothelail nitric oxide synthase (eNOS)-/- mice with 1) placebo, 2) DOCA and 3) DOCA
development of hypertension in this transgenic rat model. Ac-SDKP (800 g/Kg/day) for 12 weeks. We measured blood pressure, urine protein, renal
 e60 Hypertension Vol 48, No 4 October 2006
macrophage infiltration and glomerular nephrin expression. DOCA salt increased blood pressure
in both strains, which was unaltered by AcSDKP. In vehicle-treated eNOS-/- mice, the urine
albumin excretion was 65-fold higher than the WT controls. AcSDKP reduced albuminuria in
both strains of mice treated with DOCA-salt (Table 1). Similarly, AcSDKP decreased the number
of the renal cortical macrophages in mice treated with DOCA salt in both strains, though to a
lesser degree in eNOS-/- mice (Table 1). In search of the mechanism by which AcSDKP inhibits
proteinuria, we measured glomerular slit pore protein, nephrin, lack of which causes congenital
nephritic syndrome in humans. Immunofluorescence showed that the nephrin expression in the
mice treated with DOCA salt was lower than the vehicle treated groups and AcSDKP
significantly increased the nephrin expression in both the strains. More importantly, nephrin
expression was significantly lower in vehicle-treated eNOS-/- mice compared to WT mice
(Table 1). This could be responsible for significantly higher amount of proteinuria in eNOS-/-
strain. Conclusion: The antiproteinuric effects of Ac-SDKP may, at least partly, be due to its
ability to decrease renal inflammation and increase nephrin expression in hypertensive mice.
Table 1: Effects of AcSDKP on urine albumin excretion, renal macrophage infiltration and
nephrin expression in WT and eNOS-/- mice
mice Placebo DOCA DOCA AcSDKP
Urine albumin (g/10 gm BW/24 hour)
WT 10.81.7 41.55* 13.53.1
eNOS-/- 693271 1058357 297105
Number of renal cortical macrophages/ high power field
WT 5.71.6 21.93.1* 6.40.7
eNOS-/- 4.50.8 327.3* 209.8
Nephrin expression (% fractional area/ glomerulus)
WT 240.7 123* 184.9
eNOS-/- 92.9 5.81.4* 174.9
*, P0.05 vs. placebo; , P0.05 vs. WT DOCA
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P75
Increased GFR and Renal Excretory Function by Activation of TRPV1 in the
Isolated Perfused Kidney
Jianping Li, Donna H Wang; Michigan State Univ, East Lansing, MI
To test the hypothesis that activation of the transient receptor potential vanilloid type 1 (TRPV1)
channels in isolated perfused kidney leads to natriuresis and diuresis via an increase in
glomerular filtration rate (GFR), recirculating Krebs-Henseleit buffer added with inulin and
lithium was perfused at a constant flow, and perfusion pressure (PP) was pre-adjusted
to185190 mmHg with phenylephrine. Capsaicin (Cap), a selective TRPV1 agonist, in the
presence or absence of capsazepine (Capz), a selective TRPV1 antagonist, CGRP8 37, a
selective calcitonin gene-related peptide (CGRP) receptor antagonist, or spantide II (Spa), a
selective substance P (SP) receptor antagonist, were perfused. Cap (2, 10, 30 M, n5 6)
dose-dependently decreased PP (-8 1, -33 3, -58 2 mmHg, P0.001) and increased
urine flow rate (UFR, 43 4, 89 7, 164 11 l/min/g kidney weight, P0.01), Na
excretion (UNaV, 2.9 0.2, 6.8 0.9, 11.7 1.0 mol/min/g kw, P0.05), GFR (51 5,
172 17, 301 21 l/min/g kw, P0.001), and the release of CGRP (1.4 0.1, 4.3 0.4,
8.0 1.4 pg/g kw, P0.05) and SP (0.31 0.03, 1.02 0.22, 1.48 0.23 pg/g kw,
P0.05) without changing fractional excretion of lithium (FELi ) and fractional distal sodium
excretion (FDSE). CGRP8 37 (0.1 M) or Spa (0.1 M) alone attenuated the effect of Cap on PP
(Cap: -33 3, CGRP8 37 Cap: -20 3, Spa Cap: -22 1 mmHg, P0.05), UFR (89 kidney (37.21.7 vs. unclipped kidney 49.02.1 mmHg; p0.001). ACEI selectively lowered
7, 42 4, 58 6 l/min/g kw, P0.05), UNaV (6.8 0.9, 3.5 0.1, 3.7 0.5 mol/min/g the pO2 in the post-clip kidney (27.70.6 vs. unclipped 47.22.2 mmHg; p0.001)
kw,P0.01), and GFR (172 17, 101 7, 115 9 l/min/g kw, P0.05). However, Capz
(30 M) alone or CGRP8 37 combined with Spa fully blocked the effect of Cap on PP, UFR, UNaV,
GFR, and the release of CGRP and SP. Thus, activation of TRPV1 in the isolated kidney
decreases renal perfusion pressure and increases GFR and water/sodium excretion possibly via
simultaneous activation of CGRP and SP receptors upon their enhanced release, suggesting
TRPV1 plays a key role in modulating renal hemodynamics and excretory function.
P76
Diuresis and Natriuresis Induced by Hypertonic Saline Perfusion of the
Renal Pelvis: Role of TRPVl
Yi Zhu, Donna H Wang; Michigan State Univ, East Lansing, MI
To test the hypothesis that the transient receptor potential vanilloid type 1 (TRPVl) channels
expressed in sensory nerves innervating the renal pelvis mediate diuresis and natriuresis
induced by hypertonic saline (HS) perfusion, sodium chloride at 150, 300 and 600 mM was
perfused into the left renal pelvis (LRP) of anesthetized rats at a rate that did not change renal
perfusion pressure. Mean arterial pressure was not altered by LRP perfusion of HS at any of
these concentrations. LRP perfusion of HS at 600 mM (equal to 1200 mOsm/L), but not at 150
or 300 mM, induced 23 fold increases in urine flow rate (Uflow) and urinary sodium excretion
(UNa) in the contralateral kidney (n5 6, P0.05). The increases in contralateral Uflow and
UNa were blocked by ipsilateral renal denervation or by ipsilateral LRP perfusion of capsazepine
(CAPZ, 24 nM), a selective TRPVl antagonist, or RP67580 (12 M), a selective neurokinin-1
receptor (NK1) antagonist (p0.05). In contrast, LRP perfusion of mannitol at 1200 mOsm/L
had no effect on contralateral Uflow and UNa. Incubation of the renal pelvis in vitro with
capsaicin (CAP, 2.4 nM), a selective TRPV1 agonist, or HS but not mannitol significantly
increased substance P (SP) release (p0.05). Whereas HS-induced increases in SP release
was blocked by CAPZ or RP67580, CAP-induced increases in SP release was blocked by CAPZ
only (p0.05). Taken together, our data indicate that HS perfusion into the renal pelvis
activates ipsilateral TRPV1 and/or NK1 receptors expressed in sensory nerves directly or via
enhanced SP release, leading to contralateral diuresis and natriuresis.
P77
Low Na Intake Suppresses the Expression of Cyp2c23 and the Arachidonic
Acid-Induced Inhibition of ENaC
Peng Sun, Dao-Hong Lin, Dept of Pharmacology,New York Med College,10595, Valhalla, NY;
Tong Wang, Dept of Cellular & Molecular Physiology,Yale Univ of Sch of Medicine,06520,
New Haven, CT; Elisa Babilonia, Zhi-Jian Wang, Rowena Kemp, Alberto Nasjletti, Wen-Hui
Wang; Dept of Pharmacology,New York Med College,10595, Valhalla, NY
We have previously demonstrated that arachidonic acid (AA) inhibits epithelial Na channels
(ENaC) through cytochrome P450(CYP) expoxygenase-dependent pathway. In the present study
we tested the hypothesis that low Na intake suppresses the expression of CYP2C23, which is
mainly responsible for converting AA to epoxyeicosatrienoic acid (EET) in the kidney , and
attenuates the AA-induced inhibition of ENaC. Immonostaining showed that CYP2C23 is
expressed in the Tamm-Horsfall protein (THP)-positive and aquaporin 2 positive tubules. This
suggests that CYP2C23 is expressed in the thick ascending limb (TAL) and collecting duct (CD).
Na restriction significantly suppressed the expression of CYP2C23 in the TAL and CD. Western
blot also demonstrated that the expression of CYP2C23 in renal cortex and outer medulla
diminished in rats on Na deficient diet (Na-D) but increased in those on high Na diet (4%).
Moreover, the content of 11,12 EET decreased in the isolated CCD from rats on Na-D in
comparison to those on a normal Na diet (0.5%). Patch clamp study showed that application
of 510 M AA inhibited the activity of ENaC in the CCD in the control rats. But, the inhibitory
effect of AA to ENaC was significantly diminished in rats on Na-D for 14 days. In contrast,
11,12-EET inhibits ENaC in the CD from rats on Na-D for 14 days with a similar potency as that
in control animals. We also used micropufusion technique to examine the effect of MS-PPOH,
an inhibitor of CYP epoxygenase, on Na transport in the distal nephron. Application of MS-PPOH
significantly increased Na absorption in the distal nephron in control rats but had no significant
effect on Na absorption in rats on Na-D for 14 days. We conclude that low Na intake
downregulates the activity and expression of CYP2C23 and attenuates the inhibitory effect of
AA on Na transport.
P78
Angiotensin Converting Enzyme Inhibition Results in Hypoxia Selectively in
the Post-Clip Kidney in Two-Kidney, One Clip Hypertension
Fredrik Palm, Margarida Mendonca, William J Welch, Christopher S Wilcox; Georgetown
Univ Med Cntr, Washington, DC
We have previously reported a decreased renal venous (RV) oxygen tension (pO2) in the
two-kidney, one clip hypertension (2K1C) model of renovscular hypertension after ACE
inhibition (ACEI). The present study evaluated the renal cortical pO2 and blood flow in both
kidneys and the response to acute ACEI in 2K1C rats. Three weeks after left renal artery
clipping, rats were inactin-anesthetized and both kidneys immobilized in plastic cups.
Thereafter, cortical pO2 (Clark-type microelectrodes) and blood flow (laser-Doppler) were
measured before and after ACEI (enalaprilat; bolus 0.3 mg/kg bw, infusion 0.3 mg/kg bw/h).
RVpO2 was measured at the end of the experiments. The elevated baseline blood pressure was
significantly lowered by ACEI (1626 vs.1076 mmHg; n8; p0.001). The unclipped kidney
displayed hypertrophy compared to the post-clip kidney (1.50.1 vs. 1.10.1 g; p0.001).
The renal cortical blood flow was similar in both kidneys before ACEI (37.63.1 vs. 31.23.0
laser units; ns), but ACEI reduced blood flow significantly only in the post-clip kidney (17.53.4
vs. unclipped 30.74.4 laser units). The cortical pO2 was lower during baseline in the post-clip
accompanied by a corresponding decrease in RVpO2 in the post-clip kidney (37.02.4 vs.
unclipped 52.31.3 mmHg; p0.001). In conclusion, a functional renal artery restriction
reduces baseline cortical pO2 in the post-clip kidney, which is decreased further by ACEI. This
direct demonstration of a sustained reduction in the renal cortical pO2 could be attributed to
reduced renal blood flow, which may be the limiting determinant of oxygen availability in the
post-clip kidney.
P79
Role of Nitric Oxide in Renal Blood Flow Control during Diabetes as
Revealed by Continuous Measurement
Tracy Bell, Michael Brands; Med College of Georgia, Augusta, GA
Vasodilation of the afferent arteriole is thought to be the major cause for the increase in renal
blood flow (RBF) and glomerular filtration rate (GFR) during the early stages of diabetes mellitus.
Previous studies from our lab have implicated an important role for the Nitric Oxide (NO) system
in mediating this response, because giving NOS inhibitors prevented the increase in renal
plasma flow and GFR during diabetes. However, a limitation of these studies is that single point
measurements were taken and may not reflect the time-dependent role of NO. Therefore, in
this study, we have developed a more precise method to measure the role of NO in the chronic
control of RBF during diabetes. We measured RBF continuously, 18 hr/day using a Transonic
flow probe in control (C, n2), diabetic (D, n4), L-NAME (L, n3) and diabetic/L-NAME (DL,
n6) treated rats. Mean arterial pressure averaged 880.8, 911.1, 1151.2 and 1220.7
mm Hg, in the C, D, L, DL groups, respectively, during the L-NAME vehicle control period. At
the onset of diabetes, MAP increased to an average of 1482.1 in the DL group. Renal blood
flow averaged 6.30.1, 4.70.1, 5.90.3 and 5.40.1 in the C, D, L, DL groups,
respectively, during the L-NAME or vehicle control period and, increased immediately by
102.6% of control on day 1 of diabetes in the D group before increasing progressively to
524.7% of control by the end of diabetic week 2. Interestingly, RBF decreased to 813.2 and
831.8% of control in the L and DL groups, respectively, by the end of diabetic week 2.
Glomerular filtration rate increased by 6033.3% of control in the D group, while decreasing
by 7924.5, and 8627.3% of control in the L and DL groups, respectively, by the end of
diabetic week 2. Our model provides a novel method to measure the chronic effects of the NO

on RBF control during diabetes. Furthermore, by using this model we have demonstrated that
NO is required for the immediate increase in RBF.
P81
Activation of Gi Via Calcium Sensing Receptor (CaR) Regulates TNF in
Medullary Thick Ascending Limb (mTAL) Cells
Huda I Abdullah, Paulina L Pedraza, John C McGiff, Nicholas R Ferreri; NYMC, Valhalla, NY
We previously showed that TNF production via CaR activation is PI-PLC-mediated, NFAT-
dependent, and part of a mechanism that regulates ion transport in mTAL cells in a
PGE2-dependent manner. Presently, we determined if activation of Gi in mTAL cells contributes
to CaR-mediated: 1) TNF production in a calcineurin (CN)- and NFAT-dependent manner, and
2) regulation of ouabain-sensitive oxygen consumption, an in vitro correlate of Na transport.
CN activity in response to 1.2 mM Ca2 was inhibited in mTAL cells transiently transfected
with dominant negative CaR, R796W, (control: 0.72 0.29; Ca2: 1.970.69; R796W:
0.990.45; Ca2 & R796W: 0.660.28 pmol/g protein; n4; p0.01), indicating that the
response to extracellular Ca2 is dependent on activation of the CaR. CaR-mediated TNF
production was abolished when cells were pretreated with pertussis toxin (PTX; 100 ng/ml;
n3; p0.001). CN activity induced by CaR also was abolished by PTX (control: 0.720.29;
Ca2: 1.970.69; PTX: 0.690.19; Ca2 & PTX: 0.690.40; n4; p0.01). The
contribution of Gi activation to NFAT-induced TNF production was assessed in cells
co-transfected with either a NFAT cis-reporter construct (2 g/well) or pGV-B2-TNF promoter
construct (0.05 g/well) and a SV40 renilla luciferase promoter construct (0.025 g/well). PTX
inhibited CaR-mediated increases in NFAT- and TNF promoter-luciferase activity (fold induction:
Ca2: 2.740.39; PTX: 1.010.26; Ca2 & PTX: 1.260.22; n5; p0.001); and (Ca2: Diego Healthcare system, La Jolla, CA; Michael G Ziegler, John Ross, Jr., Univ of California
3.530.49; PTX: 1.140.24; Ca2 & PTX: 1.520.06; n3; p0.01), respectively. Inhibition at San Diego, La Jolla, CA; Daniel T OConnor; Univ of California at San Diego & VA San
of Gi also attenuated COX-2-dependent PGE2 synthesis in cells challenged with Ca2 (fold
induction: Ca2: 10.222.88; PTX: 0.780.27; Ca2 & PTX: 2.861.81; n7; p0.001).
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
Moreover, inhibition of ouabain-sensitive oxygen consumption induced by CaR activation was
reversed in the presence of PTX. Collectively, the data suggest that Gi-coupled signaling
contributes to NFAT-mediated TNF production by increasing CN activity, and is part of a
mechanism by which CaR regulates mTAL function. Understanding CaR-mediated signaling
pathways that regulate TNF production in the mTAL will be crucial to defining novel
mechanisms that regulate extracellular fluid volume and salt balance.
P82
Inadequate Regulation of Renal B0AT1 Gene Expression in the Spontaneous
Hypertensive Rat
Maria Joao Pinho, Patricio Soares-da-Silva; Fac. Medicine Porto, Porto, Portugal
We first reported that overexpression of Na-independent type 2 L-amino acid transport (LAT2)
in the spontaneous hypertensive rat (SHR) kidney is organ specific and precedes the onset of
hypertension (Hypertension, 42, 613 618, 2003). B0AT1 is a novel member of the Na-
dependent neurotransmitter transporter family (SLC6), which has a major role in the uptake of
luminal neutral amino acids. The present study evaluated the expression of neutral amino acid
transporter B0AT1 in the rat kidney and intestine. The effect of high salt intake was also
evaluated on the renal expression of B0AT1 in SHR and normotensive Wistar-Kyoto rat (WKY)
at 4 and 12 weeks of age. Animals were fed normal (NS) or high (HS - 1% saline as drinking
water) salt diet for 24 hours. The abundance of transcripts B0AT1 was evaluated by quantitative
real-time PCR, using the specific rat primers (forward primer 5 AAC CAG AAT CAG ACA GGC
TAT 3 and reverse primer 5 AGA ACA CTC CAG GCA CAT 3, position 466 and 606 bp in rat
B0AT1 sequence). Data was normalized to the expression of the constitutively expressed gene
GAPDH. At 4 and 12 weeks of age, the abundance of renal B0AT1 transcript in SHR was 49%
and 32% that in WKY, respectively. However, no significant differences were observed on the
expression of intestinal B0AT1 between SHR and WKY. High salt intake decreased by 30% the
expression of B0AT1 in both 4- and 12-week old WKY and in 4-week old SHR. Surprisingly, HS
intake produced a significant increase (45% augment) in B0AT1 mRNA in 12-week old SHR.
It is concluded that Na dependent B0AT1 amino acid transporter is under-expressed in the
SHR and this is organ specific and precedes the onset of hypertension. Overxepression of the
Na-dependent B0AT1 at the kidney level during HS intake in adult SHR reveals their inability
for adequate sodium handling when hypertension is well established. Supported by grant
POCTI/SAU-OBS/57916/2004.
P83
Nicotine: The Link between Cigarette Smoking and the Progression of
Renal Injury?
Edgar A Jaimes, Run-Xia Tian, Jessica Nigro, Leopoldo Raij; VA Med Cntr and Univ of
Miami, Miami, FL
Cigarette smoke (CS) is the most important cause of preventable morbidity and mortality in the
U.S. Recent clinical studies have suggested that in addition of being a major cardiovascular risk
factor, CS promotes the progression of kidney disease. The mechanisms by which CS promotes
the progression of chronic kidney disease have not been elucidated. The studies reported
herein were designed to: a) determine whether mesangial cells poses nicotine receptors
(nAChRs) and b) determine whether nicotine through activation of these receptors promotes
mesangial cell proliferation and extracellular matrix production via reactive oxygen species
(ROS). Here we demonstrate for the first time, that human mesangial cells (MC) are endowed
with the nAChRs 4, 5, 7, 2, 3, 4 and 5 as assessed by western blot. Studies respectively), as was SI (h2 58%). Familial phenotypic correlations between the SS traits and
performed in other cell types have shown that these nAChRs are ionotropic receptors that
function as agonist-regulated Ca2 channels. At 10 -7 molar, a concentration found in plasma
of cigarette smokers, nicotine induced MC proliferation as assessed by 3H-thymidine
incorporation: Control 931 55 cpm vs nicotine 1318 82 cpm (N3, P 0.05) and
increased synthesis of fibronectin, a critical matrix component involved in the progression of
chronic kidney disease, as assessed by western blot : Control 36 7 relative light units (RLU)
CHBPR ConferencePoster Presentations e61
vs Nicotine 51 9 RLU (n4, P 0.05). In response to protein kinase C (PKC) activation, MC
synthesize ROS via NADPH oxidase (Jaimes KI98). In the current studies we demonstrate that
calphostin C, a PKC inhibitor, as well as DPI and apocynin, two inhibitors of NADPH oxidase,
prevented the effects of nicotine upon MC proliferation and fibronectin production hence
establishing ROS as second messengers of nicotines actions (table). These studies unveil
previously unrecognized mechanisms that indict nicotine, a component of CS, as an agent that
may accelerate and promote the progression of kidney disease via alterations in the glomerular
mesangium.
Nicotine Nicotine Nicotine
Control Nicotine Calphostin C Apocynin DPI
Thymidine Incorporation 100 1382* 857 708 605*
(Percent)
Fibronectin Expression 100 14110* 10230 9627 324*
(Percent)
N 3; * P 0.05 vs Control
P84
Targeted Ablation of the Chromogranin A Gene: Profound Alterations in
Cardiovascular and Renal Physiology
Sushil K Mahata, Univ of California at San Diego & VA San Diego Healthcare System, La
Jolla, CA; Nitish R Mahapatra, Univ of California at San Diego, La Jolla, CA; Sucheta M
Vaingankar, Univ of California at San Diego System, La Jolla, CA; Manjula Mahata, Univ of
California at San Diego, La Jolla, CA; Yusu Gu, Univ of California at San Diego & VA San
Diego Healthcare System, La Jolla, CA
Chromogranin A (CHGA), a 48 kDa secretory pro-peptide, is co-stored and co-released with
catecholamines from secretory vesicles in adrenal medulla and postganglionic sympathetic
axons. It gives rise to the catecholamine release-inhibitory peptide catestatin. CHGA is
over-expressed in essential hypertension, a complex trait with genetic predisposition.
Conversely, the plasma concentration of the catestatin fragment of CHGA is diminished both in
established hypertension and in patients still-normotensive offspring at genetic risk of
developing the disease. These human findings suggest a mechanism whereby diminished
catestatin might increase the risk for hypertension. To gain a better insight into the functions
of CHGA and catestatin in vivo we generated Chga null (Chga-/-) mice. Telemetric and tail-cuff
studies on blood pressure (BP), transthoracic echocardiography, and hemodynamic studies
reveal extreme cardiovascular and renal changes in Chga-/- mice: (i) Elevated systolic and
diastolic BP; (ii) Loss of diurnal BP variation; (iv) Increased left ventricular mass and cavity
dimensions; (iii) higher heart rate, left ventricular pressure, ventricular contractility (max dP/dt)
and end diastolic pressure and lower R-R intervals; (iv) Increased plasma and urinary
catecholamines; (v) increased plasma neuropeptide Y (Npy); (vi) low plasma creatinine and high
GFR. Rescue of elevated BP to normalcy was achieved by either exogenous catestatin
replacement, or humanization of Chga-/- mice with transgenic insertion of a human CHGA
haplotype (Chga-/-:CHGA/). Pithing of the Chga null mice restored the BP to normalcy.
Pithed Chga-/- mice displayed exaggerated BP response to electrical stimulation. Telemetered
Chga null mice also showed inflated BP response to immobilization stress. These findings
suggest involvement of peripheral and central mechanisms in the development of BP. We
conclude from these studies that the loss of the physiological brake catestatin in Chga-/- mice,
coupled with dysregulation of transmitter storage and release, may act in concert to alter
autonomic control of the circulation in vivo, eventuating in hypertension.
P85
Genetic Architecture of the Salt Sensitivity of Blood Pressure and
Correlation with Insulin Resistance in Hypertensive Hispanic Families
Anny H Xiang, Univ of Southern California, Los Angeles, CA; Leslie J Raffel, Cedars-Sinai
Med Cntr, Los Angeles, CA; Miwa Kawabubo, Univ of Southern California, Los Angeles, CA;
Xiuqing Guo, Mark O Goodarzi, Kent D Taylor, Cedars-Sinai Med Cntr, Los Angeles, CA;
Manuel J Quinones, Willa A Hsueh, Univ of California Los Angeles, Los Angeles, CA;
Thomas A Buchanan, Univ of Southern California, Los Angeles, CA; Jerome I Rotter;
Cedars-Sinai Med Cntr, Los Angeles, CA
Salt sensitivity (SS), the blood pressure change in response to changes in salt/water
homeostasis, has been a variable of interest. Blood pressure (BP) and insulin resistance (IR) are
under common genetic control. Whether SS is genetic and its relation with IR, however, have
not been well studied. We assessed the genetic nature of SS and genetic relationship with IR
in 371 non-diabetic offspring from 116 Hispanic families, ascertained through a parent with
hypertension. SS was measured by the Weinberger protocol with SBP sensitivity (SBP SS) and
DBP sensitivity (DBP SS) defined as the blood pressures between end of the salt loading
(day 1) and end of salt depletion (day 2). Three additional phenotypes were derived: blood
pressure between baseline and end of salt loading (SBP load, DBP load), blood pressure
between end of salt loading and baseline of salt depletion (afternoon overnight) (SBP night,
DBP night), and blood pressure beween onset and end of salt depletion. Insulin sensitivity
(SI) was measured by hyperinsulinemic euglycemic clamp. Heritabilities (h2) of these traits were
estimated and phenotypic, genetic, and environmental correlations between SS traits and SI
were examined when h2 was significant. Variance component analysis was used, adjusting for
age, gender and BMI. Results: Several SS traits were significantly heritable (p0.05), including
SBP SS, SBP load, SBP night, and DBP night (h2 18%, 19%, 19% and 33%
SI were -0.06 (p 0.38) for SBP SS, -0.09 (p 0.21) for SBP load, -0.16 (p 0.005)
for SBP night, and -0.12 (p 0.048) for DBP night. When familial correlations were
partitioned, genetic correlations between SBP night, DBP night and SI were significant (rg
-0.56, p0.03 for SBP night, rg -0.43, p0.04 for DBP night), while the environ-
mental correlations were not. Conclusion: These results suggest overnight drop in BP following
salt loading is a better genetic phenotype than traditional salt sensitivity phenotypes. It also
 e62 Hypertension Vol 48, No 4 October 2006
appears that this newly derived trait shares genetic determinants with insulin resistance. The
mechanism underlying this genetic variation and covariation warrant further study.
P86
Identical by Descent Mapping of Hypertension Genes in SHR
Renata I Dmitrieva, Cruz A Hinojos, Eric Boerwinkle, Megan Grove, Myriam Fornage, Peter
Doris; Univ of Texas HSC Houston, Houston, TX
The SHR model of essential hypertension is inbred and was produced by selective breeding.
During inbreeding, it is common to create multiple lines in case fixation of deleterious alleles
causes loss of a line. In fact, SHR is a collection of several inbred lines that can be crossed
without segregation of the hypertensive trait, indicating extensive or complete sharing of
hypertension alleles among SHR lines. Heritable traits are created by underlying genetic
variation that can affect both coding and regulatory sequence. The former can be localized only
imprecisely via mapping studies. However, comprehensive gene expression arrays provide a
platform to identify genes that may harbor regulatory variation. We sought to identify
hypertension alleles in SHR that act via altered expression. We have measured renal gene
expression using Affymetrix arrays in SHR-A3, SHR-B2, SHR-C and WKY (Heid) animals. We
have identified and confirmed those genes and ESTs that are persistently differentially
expressed between WKY and all SHR lines. We have screened these genes for polymorphism
to identify allelic variation and have identified allelic variants that are inherited identical by
descent (IBD) by SHR-A3, -B2, -C and SHR/N, but not by WKY(Heid) and WKY/N. We then
generated an F2 cross between SHR-A3 and WKY (Heid) and determined which of these genes
and ESTs exhibit allelic expression (AE) in F2 individuals. We found evidence of AE for Bhmt,
Ddt, Ela1, Gsto1, Gsst2, Ptprj, AA866432, AA892388 and AA893147. We measured the effect
of the SHR alleles of these genes by contrasting the blood pressure (radiotelemetry) differences
between F2 animals homozygous for WKY and SHR alleles of these strains and found a net
effect of homozygosity for the SHR alleles of 24.7mmHg, or 31% of the trait variance between
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
the strains. This effect arose principally from the 5 genes underlined above. In contrast, the
cumulative effect on blood pressure of differentially expressed genes in which all SHR lines
share the same allele, but in which differential expression is not attributable to AE was minus
5.9mmHg. These 5 genes include plausible blood pressure genes that are IBD in SHR, act via
allelic expression in all SHR lines and are associated with elevated blood pressure.
P87
Substitution of Chromosome 17 Influences Phenotypes Related to the
Metabolic Syndrome in the Lyon Hypertensive Rat
Anne E Kwitek, Med College of Wisconsin, Milwaukee, WI; Sophie Gilibert, Faculte de
Pharmacie - Universite Lyon, Lyon, France; Howard J Jacob, Med College of Wisconsin,
Milwaukee, WI; Jean Sassard, Alain Bataillard; Faculte de Pharmacie-Universite Lyon, Lyon,
France
The Lyon Hypertensive (LH) rat strain is a genetically hypertensive strain associated with high
blood pressure (BP), exaggerated salt-sensitivity, and a metabolic syndrome made of
overweight together with increased plasma lipids. Genetic mapping in a cross between the LH
and the Lyon Normotensive (LN) strains previously showed the existenceof three clusters of
Quantitative Traits Loci (QTLs) related to the metabolic syndrome on rat chromosome 17. The
first cluster was linked to morphological parameters, the second influenced blood pressure and
the third was linked to plasma lipid levels. In order to determine the functional role of these
QTLs we generated a consomic strain LH-17BN in which the LH chromosome 17 has been fully
substituted by a normotensive Brown Norway (BN) one. Male LH-17BN, LH, and BN rats were
phenotypically characterized. Measures included radio telemetric measurements of BP during
normal and elevated salt intake (1% and then 2% in the drinking water) as well as the
determination of renal (creatinine clearance, proteinuria), metabolic (plasma triglycerides,
cholesterol) and morphological parameters. Compared to parental LH rats, the LH-17BN rats
exhibited significant decreases in both body weight and BP and its response to salt load.
Creatinine clearance was increased and proteinuria decreased. Finally plasma triglycerides and
cholesterol were reduced in LH-17BN. Substitution of only BN chromosome 17 completely
normalized triglycerides to the level of the BN parent. The present work confirms that the three
clusters of QTLs found by a linkage analysis in the LH x LN strains can be recapitualted in the
consomic LH-17BN strain. Therefore, these chromosome 17 consomic rats will be useful to
generate overlapping congenic rats in order to approach the genes possibly involved in the BP
and metabolic alterations shown by the Lyon model. Furthermore, the critical QTL region can
be significantly narrowed by SNP haplotype analysis using all three strains.
P88
Comprehensive Analyses of RAAS Polymorphisms on Blood Pressure at
Rest and during Behavioral Stress in Normotensive Youth
Haidong Zhu, Dongliang Ge, Ying Huang, Frank A Treiber, Gregory A Harshfield, Harold
Snieder, Yanbin Dong; Med College of Georgia, Augusta, GA
The renin-angiotensin-aldosterone system (RAAS) is a proteolytic cascade that regulates and
maintains blood pressure (BP). This study aimed to explore the interactive and integrative
effects of multiple RAAS polymorphisms on BP at rest and during behavioral stress in a
normotensive population. A total of 920 young European- (EA) and African American (AA) twins
(age: 12.0 30.0 yr, 45% AAs) were subjected to three 10-minute stress tasks (a social
competence interview, a virtual reality car driving simulation test and a video game). Thirteen
potential functional polymorphisms from genes encoding angiotensinogen (AGT), angiotensin I
converting enzyme (ACE), angiotensin II receptor type 1(AGTR1), and aldosterone synthase
(CYP11B2) were genotyped. We performed multilocus prediction allowing for genetic modifi-
cation effects [gene-gene, gene-gender, gene-ethnicity, and gene-body mass index (BMI)]
using multivariate adaptive regression splines (MARS) and generalized estimating equations
(GEE). Final multivariate models were selected by cross-validation. Single polymorphism
analyses showed modest effects of M235T (AGT) and A-239T (ACE) (p value range:
0.005 0.036), accounting for less than 1% of the total variance of systolic BP (SBP) at rest and
during stress. On the contrary, the best multilocus models revealed multiple independent
genetic modification effects (gene-gene, gene-gender and gene-BMI, p value range: 0.003
0.009), accounting for 2.5% and 7.3% of the total variance for SBP levels at rest and during
stress, respectively. As such, we speculate that the RAAS genetic modifications may contribute
more significantly to the dynamic BP regulation in response to behavioral stress compared to
the static BP value. Given the complexity of BP control, our data support the hypothesis that
multiple RAAS genetic modifications underlie BP elevation. In addition, MARS could be a viable
model to test for the multiple genetic contributions to hypertension.
P89
Transcriptional Regulation of Angiotensinogen Haplotypes in Three
Angiotensinogen-Expressing Human Cell Lines
Matthew E Dickson, Curt D Sigmund; Univ of Iowa, Iowa City, IA
Numerous association studies have correlated single nucleotide polymorphisms (SNPs) in the
angiotensinogen (AGT) promoter with hypertension, and a few studies have reported differential
transcription factor binding to individual SNPs. Most of these studies have been performed in
cultured hepatocyte cell lines, thus ignoring several other important sites of AGT synthesis. In
addition, AGT promoter polymorphisms are inherited in haplotype blocks (17 SNPs), of which
there has been limited analysis. Analysis of naturally occurring haplotype blocks is an important
first step in determining which SNPs differentially regulate AGT expression. We cloned 1.3
kb of the AGT promoter from 11 individuals of ethnically diverse background obtained from the
Coriell Polymorphism Discovery Resource. An analysis of 21 chromosomal clones revealed 8
non-redundant haplotypes that appeared to accurately represent known haplotypes. Luciferase
assays were performed with these 8 haplotypes in 3 human cell lines of diverse origin, each
expressing AGT endogenously (HepG2 derived from the liver, HK-2 derived from the renal
proximal tubule, and CCF derived from astrocytes). Transcriptional assays were performed at
baseline ( the AGT enhancer) and following hormonal stimulation (100 nM dexamethasone,
50 nM testosterone, or 10 nM estradiol). Baseline reporter expression varied 23 fold between
haplotypes within each cell line and was very similar in HepG2 and HK-2 with the most
significant differences resulting from the -20C/A SNP. The expression pattern in CCF was
dissimilar. Gel shift and supershift assays indicated preferential binding of the USF1/2
transcription factors to -20C, especially in the presence of -6A, in all three cell lines. The AGT
enhancer altered baseline expression in HK-2 cells but had minor effects in the other cells.
Hormone treatments did not differentially regulate expression of haplotypes in any of the cell
lines, but treatment with dexamethasone or estradiol caused a haplotype-independent 1.9- or
1.6-fold increase, respectively, in CCF cells. Our data suggest a major effect of the -20 SNP
that may be modified by other SNPs, in particular -6. Some of the transcriptional effects may
also be cell-specific.
P90
Association of the C-344T Aldosterone Synthase Gene Variant with
Essential Hypertension and Related Phenotypes: A Meta-Analysis
Silvia C Sookoian, Tomas Fernandez Gianotti, Instituto de Investigaciones Medicas, A.
Lanari. Universidad de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina; Claudio
Gonzalez, Departamento de Farmacologia, Facultad de Medicina, Universidad de Buenos
Aires, Ciudad Autonoma de Buenos Aires, Argentina; Carlos J Pirola; Instituto de
Investigaciones Medicas, A. Lanari. Universidad de Buenos Aires, Ciudad Autonoma de
Buenos Aires, Argentina
The renin-angiotensin-aldosterone system is an important regulator of blood pressure. Variants
in their component-encoding genes have been associated with essential hypertension and
hypertrophic cardiomyopathy. Among them the CYP11B2 gene (CYP11B2) encoding aldoste-
rone synthase has been extensively studied but its role in hypertension and related phenotypes
remains controversial. Then, we performed a systematically review of the literature by means
of a meta-analysis to evaluate the influence of the CYP11B2 C-344T polymorphism on the
occurrence of arterial hypertension and related phenotypes. From 485 reports, we included 42
observational studies, case control and cohort at baseline. A fixed-effect model was used to
pool data from individual studies. From 4741 essential hypertensive and 5444 control subjects,
we found a significant association between hypertension and the C-344T variant showing a
protective effect of the C allele (OR: 0.819, 95 % CI: 0.743 to 0.902, p 0.0001,n: 10185).
Besides, homozygous CC subjects had lower plasma renin activity (D: -0.109, 95 % CI: -0.213
to -0.006, p0.039, n: 1876) and posterior wall thickness (D: -0.151, 95 % CI: -0.265 to
-0.037, p 0.01, n: 1235). No significant association was found for systolic arterial blood
pressure (D: -0.020, 95 % CI: -0.072 to 0.032, p0.44, n: 7432), diastolic arterial blood
pressure (D: -0.023, 95 % CI: -0.075 to 0.028, p0.37, n: 7432) and plasma aldosterone (D:
0.016, 95 % CI: -0.085 to 0.117, p0.755, n: 1678). Although left ventricular mass (LVM) was
not different in total combined studies (p 0.078, n: 1746), it was lower in Caucasians
homozygous CC (D: -0.106, 95 % CI: -0.207 to -0.006, p 0.04, n: 1591). In conclusion,
homozygous individuals for the -344C CYP11B2 variant are at 20% lower risk of developing
hypertension and may have decreased LVM.
P91
Various Inversions, Deletions, and Reinsertions on Chromosome 12p in
Autosomal-Dominant Hypertension with Brachydactyly - A Fish-Study in
Four Families and One Sporadic Case
Martin Kann, Sylvia Bahring, Max-Delbruck-Cntr for Molecular Medicine, Charite - Univ
Medicine Berlin, HELIOS-Klinikum Berlin-Buch, Berlin, Germany; Michaela Kirsch, Anita
Rauch, Institute of Human Genetics, Friedrich-Alexander-Univ Erlangen-Nuremberg,
Erlangen, Germany; Friedrich C Luft; Max-Delbruck-Cntr for Molecular Medicine, Charite -
Univ Medicine Berlin, HELIOS-Klinikum Berlin-Buch, Berlin, Germany
We described a Turkish family with autosomal-dominant, salt-insensitive primary hypertension,
and type E brachydactyly. Affected persons die of stroke 50 years of age. Genome-wide

linkage analysis disclosed a 10 cM locus on chromosome 12p11.2 (LOD score, 9.29). We
sequenced all known genes within the candidate region, but found no mutations. However,
interphase fluorescence-in-situ-hybridization (iFISH) studies, using a five-clone BAC array
spread across the locus, showed an inversion, deletion, and reinsertion mutation. We now
extended iFISH-analysis to three other families and one sporadic case (all non-Turkish), with
an array of 24 contiguous BAC clones. We confirmed rearrangements at chromosome 12p in
all studied patients. All affected persons had an inversion of the centromeric part of the locus.
However, the inversions lacked a common breakpoint and differed greatly in size. In one
instance, the locus boundaries were extended. No disruptions of any known coding sequences
occurred. Finally, the sporadic case also had a partial deletion of the inverted sequence that
was reinserted in the telomeric part of the locus. The region affected by the rearrangements
does not contain any known genes but does include 5 ESTs that are differentially spliced. We
obtained full-length transcripts of these ESTs by RACE-PCR. These transcripts lack both open
reading frames longer than 89 amino-acids and Kozak sequences. Expression analysis of the
transcripts in affected and unaffected individuals and different tissues, respectively, is
underway. In summary, these findings support a causative role of chromosome 12p
rearrangements in the genesis of the hypertension-brachydactyly syndrome. Since no known
genes are affected by the mutations, possible causes for the syndrome include as yet unknown
genes or non-coding RNA. Initial analysis of ESTs mapped to the rearrangements was
inconclusive. A position effect must also be considered that could alter the interplay between
a regulatory element and its respective gene. We are currently investigating these possibilities.
P92
Reduced Capacity of Heme Oxygenese-1 Induction in Patients with
Coronary Artery Disease
Andrei Brydun, Ryoji Ozono, Koichiro Okuhara, Fujiko Kohno, Tetsuya Oshima, Yuichiro
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
Watari, Kazuaki Chayama, Hiroshima Univ, Hiroshima, Japan; Kazuhiko Igarashi; Tohoku
Univ, Sendai, Japan
Heme oxigenase-1 is a stress-inducible cytoprotective enzyme. There are some data
suggesting that the present of the long GT-repeat sequences (L-alleles) in the HO-1 promoter
region may be associated with susceptibility to the coronary artery disease (CAD). However the
results of the previous studies are controversial and HO-1 mRNA expression has never been
studied in humans. In the present study, we tested the hypothesis that reduced HO-1
expression may impair defense against oxidative stress, leading to progression of CAD. We
evaluated the basal and hemin-stimulated HO-1 expression and GT-repeat polymorphism in
peripheral blood mononuclear cells in the 60 patients with CAD and 30 control subjects, and
investigated the relationship between the HO-1 expression level and markers of oxidative stress
and inflammation including plasma levels of 8-isoprostan, TNF-alpha, and IL-10. There was no
significant difference between the basal HO-1 expression levels of CAD and control groups,
however stimulated HO-1 was significantly (p0.05) suppressed in CAD group compared with
control group. This difference was independent of age, sex, current smoking and the presence
of diabetes mellitus, hyperlipidemia, and hypertension in the multiple logistic regression model.
However, no significant correlation was found between HO-1 expression level, basal or
stimulated, and the markers of oxidative stress and inflammation. Interestingly, measurement
of the stimulated HO-1 expression in the 16 patients before and after CABG showed no
significant difference, suggesting HO-1 upregulation capacity to be an intrinsic factor. LL
genotype carriers showed lower HO-1 mRNA levels after the stimulation with hemin (p0.05).
However we did not observe prevalence of this genotype in the CAD group over the control
group. These results suggest that impaired ability to induce HO-1 may contribute to
development of CAD, although its roles in the control of oxidative stress and inflammation were
not detected. Measurement of the HO-1 mRNA upregulation capacity may better reflect its role
in the pathogenesis of CAD than genotyping.
P93
Regulation of Blood Pressure, Natriuresis, and Renal Thiazide/Amiloride
Sensitivity in PPAR-Null Mice
Patience Obih, Xavier Univ, New Orleans, LA; Adebayo Oyekan; Texas Southern Univ,
Houston, TX
PPAR is a ligand-activated nuclear transcription factor that is expressed in the kidney and
many cardiovascular tissues and recent studies have linked defective fatty acid oxidation with
defective renal function and hypertension including salt-sensitive hypertension. However, salt
handling in these mice is not fully known. This study evaluated thiazide and amiloride
sensitivity and tested whether PPAR KO mice are hypertensive and if they are salt-sensitive.
PPAR KO and WT (129S1/Sv) mice were placed on a normal salt (NS, 0.3% NaCl) or high salt
(8% NaCl, HS) diet for 28 days and mean arterial pressure (MAP) and heart rate (HR)
determined (telemetry). Basal MAP and HR were similar in KO and WT mice (1166 mmHg,
58740 bpm, KO; 1164 mmHg, 55120 bpm, WT) placed on NS. HS diet increased MAP
in WT mice (243 mmHg, p0.05) but not in KO mice. However, HS diet increased
proteinuria in KO mice much more than in WT mice (8- versus 2.5-fold, p0.05). We
hypothesize that ion transport differences might be important in determining rate of NaCl
excretion (UNaV). In response to an acute NaCl-load test (2.5 ml/kg N/S i.p. for 5 hrs), PPAR
KO mice excreted the sodium load faster than WT mice (4.311.11 vs 0.770.31 mol,
p0.05). UNaV was compared in mice pretreated with amiloride (Amil; 200 g/kg) or
hydrocholorothiazide (HCTZ; 3 mg/kg), in vivo measurements of sodium hydrogen exchanger
(NHE) or Na-Cl-cotransporter (NCC) activity, respectively. UNaV was blunted in HCTZ-treated KO
mice (335%, p0.05) mice but increased 15.5 fold in WT mice. In Amil-treated mice, sodium
load-induced increase in UNaV was blunted in KO compared with WT mice (3.3 versus
15.5-fold). These data suggest that PPAR is renoprotective and affects Na transport via NHE
and NCC mechanisms. Thus, despite defective fatty acid oxidation and marked proteinuria,
PPAR null mice are neither hypertensive nor develop salt-sensitive hypertension.
CHBPR ConferencePoster Presentations e63
P94
Heart-Specific Inhibition of Protooncogene C-myc Attenuates Cold-Induced
Cardiac Hypertrophy
Mahajoub Bello Roufai, Hua Li, Zhongjie Sun; Univ Fl Coll Med, Gainesville, FL
Background: The protooncogene c-myc is involved in the regulation of cell growth. Although
increased c-Myc expression is found in hypertrophied hearts, the role of c-Myc in the
development of cardiac hypertrophy has never been determined. The aim of this study was to
test the effect of heart-specific inhibition of c-Myc expression on the development of
cold-induced cardiac hypertrophy (CICH), a natural form of cardiac hypertrophy. We hypothe-
sized that heart-specific inhibition of c-Myc expression attenuates CICH. Methods and
Results: We constructed c-Myc antisense (c-MycAS) plasmid and GFP plasmid driven by a
heart-specific promoter, -MHC. The cell culture study indicated that c-MycAS can selectively
inhibit c-Myc expression and that GFP can selectively express in the rat heart cells. Four groups
of rats were used to test the effect of in vivo inhibition of cardiac c-Myc expression on the
development of CICH. Three groups received an intravenous injection of c-MycAS, GFP, and
buffer, respectively, before exposure to cold (44F), while the last group received buffer and
was kept at room temperature (RT, 78F) to serve as a control. Blood pressure (BP) of the
cold-exposed groups receiving buffer or GFP increased significantly whereas BP of the
c-MycAS group did not increase until 28 days after exposure to cold. Thus, c-MycAS driven by
a heart-specific promoter delayed and attenuated cold-induced hypertension (CIH). The
antihypertensive effect of c-MycAS was probably due to the decreased cardiac output.
Magnetic resonance imaging showed that the in vivo left ventricle wall thickness was
decreased by c-MycAS at day 18 when cardiac c-Myc expression was inhibited by c-MycAS.
Consistently, the cold-induced increase in heart weight was decreased by c-MycAS at day 18.
The heart specificity of -MHC promoter was confirmed by the selective inhibition of c-Myc
expression in the heart and by the selective expression of both GFP mRNA and GFP protein in
the heart. Conclusion: Heart-specific inhibition of c-Myc expression attenuated the develop-
ment of CIH and CICH. The increased c-Myc expression plays a key role in the pathogenesis
of CICH. Thus, heart-specific inhibition of c-Myc expression may provide a new and effective
approach for the control of cardiac hypertrophy.
P95
Genetic and Transcriptomic Analysis of Chromosome 1 Blood Pressure QTL
in the Spontaneously Hypertensive Rat
Fadi J Charchar, Jenny Clemitson, Richard J Dixon, Steve Haines, Laurence Hall, Univ of
Leicester, Leicester, United Kingdom; Andrew Bingham, Bhakti Patel, Univ of Liecester,
Liecester, United Kingdom; Ming Lo, Jean Sassard, Departement de Physiologie et
Pharmacologie Clinique, Lyon, France; Nilesh J Samani; Univ of Leicester, Leicester, United
Kingdom
Introduction and Aims: Chromosome 1 in the rat contains a quantitative trait locus (QTL) with
a major effect on blood pressure (BP). We have previously confirmed the effect of this locus on
BP by the production of reciprocal congenic strains (WKY.SHR-Sa) and (SHR.WKY-Sa) derived
from a cross of the spontaneously hypertensive rat (SHR) with the Wistar-Kyoto rat (WKY).
Furthermore, we constructed a congenic substrain (Sisa1) from the SHR.WKY-Sa strain with
only a 4.3 Mb introgressed region which also exhibited the BP effect. Our aims were to carry
out further genetic dissection of this region and to identify strong positional candidate genes
through transcriptome analysis. Methods: We further fine mapped the QTL region by systemic
construction of two mutually exclusive congenic substrains. Genome-wide microarray expres-
sion profiling in whole kidney was undertaken to identify differentially expressed genes among
the parental SHR, WKY, congenic strains (WKY.SHR-Sa), (SHR.WKY-Sa) and the congenic
substrain Sisa1, at both 6 and 24 weeks of age. Results and Conclusion: We have reduced the
congenic segment to 3Mb region by the construction of two strains (Sisa6000/Sisa9000) that
break down the Sisa1 region. Only Sisa6000 congenic exhibit reduced BP compared to the SHR
animals (166.1 2.5 mmHg, (n 17) vs 174.4 1.4 mmHg (n 20), P0.001). We have
identified 4 hypertension candidate genes (Spondin 1, Homer-2, Thumpd1 and XP 215009)
that mapped within the defined boundaries of the initial BP QTLs on chromosome 1 and that
exhibited significant differential expression between the WKY and SHR genotypes, at both 6 and
24 weeks of age. These differentially expressed genes were confirmed by performing
quantitative RT-PCR. One of these genes, Spon1 maps within the boundaries of our new
minimal congenic region and has not been previously identified as a hypertension candidate
gene. The results of this study justify further investigation of these positional candidate genes
and their potential involvement in BP control in hypertensive rat models and humans.
P96
Assessment of SNP-Based Genotypes in Two Independent Populations:
GRK4 A486V Association with Hypertension
Theodore E Mifflin, Cynthia D Schoeffel, Daniel C Huntley, Univ of Virginia, Charlottesville,
VA; Kelli K Ryckman, Scott M Williams, Vanderbilt Univ, Nashville, TN; Pedro A Jose,
Georgetown Univ, Washington, DC; Christopher Rembold, Robert M Carey, Robin A Felder;
Univ of Virginia, Charlottesville, VA
Background: We determined the contributions of individual gene(s) to the presence and
development of essential hypertension (EH). We investigated 8 separate single nucleotide
polymorphisms (SNPs) and evaluated SNP genotypes (individual and combined) and haplotypes
for association with salt sensitivity (SS). A previous study searched for SNP genotype
correlations in hypertensive persons (HP). Our goal was to evaluate these combined populations
(n122 252) for both genotype/haplotype results that could be related to EH. Methods: We
determined five SNP genotypes of genomic DNA from 122 HP study participants and eight SNP
genotypes from 252 SS study participants. We used standard linkage analysis software to
establish significance of genotypes (and inferred haplotypes) of the separate and combined
data sets. Results: In the HP study, GRK4 A486V was slightly associated with EH (p 0.044).
For the Combined study, GRK4 A486V was significantly associated with EH (p0.0045).
 e64 Hypertension Vol 48, No 4 October 2006
Multidimensional reduction analysis revealed no associations between the eight SNPs within
the individual (SS or HP) studies or combined studies. For the HP study, the odds ratio (OR) for
EH and A486V, OR3.53 (CI 1.259.96, p0.0132). For the combined studies, odds ratio for
EH and A486V, OR1.92 (CI 1.173.16, p0.0092). Conclusion: SNP genotype analysis of
374 persons indicates that GRK4 A486V is significantly associated with EH. A study of
pseudo-haplotypes collectively created using the 8 SNP genotype results revealed none were
associated with EH.
P97
Angiotensinogen Gene Variants do not Affect Ambulatory Blood Pressure in
the General Population
Peter Braund, Martin Tobin, Gail Lavery, Claire Bodycote, Stuart Raleigh, Fadi Charchar,
Paul Burton, Nilesh J Samani; Univ of Leicester, Leicester, United Kingdom
Background Over the last ten years several variants in the angiotensinogen (AGT) gene and its
promoter have been associated with risk of hypertension. However, the data are conflicting. An
important confounder could be the level of salt intake. We hypothesised that if AGT variants
influence risk of hypertension, they should have a discernible effect on blood pressure (BP),
assessed as a quantitative trait, in the general population, especially if salt intake is also taken
into account. We analysed the effect of 8 AGT gene polymorphisms on 24 hour BP measured
by an ambulatory approach in 1533 subjects in whom 24 hour sodium intake was also
assessed. Methods and Results The 1533 subjects comprised parents (aged 40 59 years)
and adult offspring (18 y) from 386 families recruited from the general population as part of
the ongoing GRAPHIC (Genetic Regulation of Arterial Pressure of Humans in the Community)
study. Ambulatory BP was measured using a Spacelabs 90207 monitor. Sodium intake was
estimated from 24 hour urinary sodium excretion. The AGT single nucleotide polymorphisms
(SNPs) genotyped included 4 SNPs in the promoter region (rs5046, rs5049, rs5050, rs5051),
1 SNP in intron 1 (rs2004776), 1 SNP in exon 2 (rs4762), 1 SNP in intron 3 (rs11122575) and
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1 SNP in exon 5 (rs7079). The SNPs were chosen to provide a full coverage of the gene at a
D of 0.8 and to include all SNPs with functional effects or associated with hypertension.
Genotyping was carried using Taqman assays. All analyses took account of familial
relationships and were adjusted for age, age2 and sex as covariates. The minor allele
frequencies for the SNPs ranged from 8.7% (rs11122575) to 40.2% (rs 5051). None of the
SNPs showed a significant association with either ambulatory SBP or DBP analysed either for
the full 24 hours or for day-time and night-time periods. For 24h SBP the 95% confidence
intervals for the per allele BP effect excluded by the study ranged from -0.67 to 0.91 mmHg
for rs5051 to -1.61 to 1.19 mmHg for rs11122575. No significant interactions between salt
intake and AGT SNPs were observed. Conclusion AGT gene variants do not significantly
influence ambulatory BP phenotypes in the general population. Our findings do not support the
concept that variants in the AGT gene are a risk factor for hypertension.
P98
TRPV1 Gene Knockout Abolishes Preconditioning Protection Against
Myocardial Injury in Isolated Perfused Hearts in Mice
Beihua Zhong, Donna H Wang; Michigan State Univ, East Lansing, MI
We have shown that the transient receptor potential vanilloid type 1 (TRPV1) channels play an
important role in protecting the heart from ischemia /reperfusion (I/R) injury via an increase in
substance P (SP) release (Circulation 112(23):36173623, 2005). However, it is unknown
whether TRPV1 is involved in preconditioning (PC)-induced protection of hearts. To test the
hypothesis that TRPV1 expressed in sensory nerves innervating the heart plays a role in
PC-induced protection against myocardial injury and the lack of TRPV1 eliminates such
protection, hearts of gene-targeted TRPV1-null mutant (TRPV1-/-) or wild-type (WT) mice were
Langendorffly perfused in the presence or absence of capsazepine (CAPZ), a selective TRPV1
antagonist, CGRP8 37, a selective calcitonin gene-related peptide (CGRP) receptor antagonist, or
RP67580, a selective NK1 receptor antagonist when hearts were subjected to three 5-mins of
ischemia PC followed by 30 mins of global ischemia and 40 minutes of reperfusion. After I/R,
increased left ventricular end-diastolic pressure (LVEDP) and decreased left ventricular
developed pressure (LVDP), coronary flow (CF), and LV peak positive dP/dt (dP/dt) occurred
in WT, TRPV1-/-, or TRPV1-/- PC compared to WTPC (n8, p0.05). CAPZ, CGRP8 37, or
RP67580 abolished PC induced protection in WT but not TRPV1-/- hearts (p0.05). PC
decreased lactate dehydrogenase (LDH) release from WT but not TRPV1-/- hearts (p0.05).
Radioimmunoassay showed that the release of SP and CGRP after PC was higher in WT hearts
than in KO hearts (p0.05), which was attenuated by CAPZ in WT but not KO hearts. Our data
show that TRPV1 gene deletion abolishes PC induced protection of the heart, indicating TRPV1
plays a key role mediating the beneficial effects of preconditioning against I/R injury through
release SP and CGRP.
P99
Oxytocin Improves Cardiac Healing in Experimentally-Induced Myocardial
Infarction in Rats
Jolanta Gutkowska, Vickram Bissonauth, CHUM, Hotel-Dieu, Montreal, Canada; Rame Taha,
Gilbert Blaise, CHUM, Notre Dame, Montreal, Canada; Marek Jankowski; CHUM, Hotel-Dieu,
Montreal, Canada
Oxytocin (OT), a reproductive hormone also plays a role in cardiovascular regulation, by
mechanisms that include stimulation of the cardioprotective mediators, NO and atrial natriuretic
peptide (ANP). OT also stimulates stem cell differentiation into cardiomyocytes. Based on these
findings, we hypothesized that OT treatment could improve cardiac function in rats subjected
to experimentally-induced myocardial infarction (MI). An infarct in the left ventricle (LV) of
Sprague-Dawley male rats was generated by left coronary artery ligation. OT was then
administered by ALZET pump at a concentration of 25 or 125 ng/kg/h during 3 or 7 days.
Saline-treated MI and sham-operated rats served as controls. Cardiac function was analyzed
by M-mode echocardiography. The infarct size, cell proliferation, apoptosis, and immune cells
were investigated in stained cardiac sections under microscopy. (i) Left ventricular fractional
shortening (FS) was reduced in saline-treated MI rats (19% vs. pre-operative values of
39 42%). This reduction was partially reversed by administration of 25 ng/kg/h OT (FS-33%)
or 125 ng/kg/h (FS-31%). OT also improved ejection fraction and cardiac output. (ii)
Immunohistochemical (IMM) and morpho-pathological analyses indicated OT-mediated in-
crease in proliferating cells in the infarct zone (PCNA staining), augmented neovascularisation
(smooth muscle actin coloration) and reduced apoptosis (caspase-3 staining). (iii) OT treatment
resulted in reduction of neutrophils from 7% of total cell number in the infracted cardiac site
of saline-treated MI rats to 2% and 1.5% in MI rats treated at 25 and 125 ng/kg/h, respectively.
OT treatment markedly decreased (40%) the number of macrophages in the infract zone
compared to saline-treated MI (considered as 100%). (iv) IMM revealed increased staining of
ANP and eNOS in cells localized in the scar zone in response to OT treatment. Real-time PCR
showed OT-dependent reduction of a proinflammatory cytokine TNF mRNA elevated in left
ventricular dysfunction. Therefore, OT treatment reduces inflammation and stimulates angio-
genesis and improves function of the injured heart. In conclusion, OT improves the healing
process in experimentally-induced MI in rats. Supported by CIHR and CHSF
P100
Peroxisome Proliferator-Activated Receptor Activator Attenuates Left
Ventricular Hypertrophy in Salt Sensitive Hypertension
Minori Nakamoto, Yusuke Ohya, Shuichi Takishita; Dept of Cardiovascular Medicine,
Nephrology and Neurology, Univ of the Ryukyus, Okinawa, Japan
In salt-sensitive hypertension, importance of volume overload and local renin-angiotensin
system on the pathogenesis of left ventricular (LV) hypertrophy and heart failure has been
demonstrated, however little is known about the role of peroxisome proliferator-activated
receptor (PPAR) on these LV changes. We thus investigated whether pioglitazone, a PPAR
activator, has beneficial effects on LV hypertrophy in Dahl salt-sensitive (DS) rats, a model for
salt-sensitive hypertension, and compared the effects of pioglitazone to those of angiotensin II
receptor blocker, candsartan. DS rats were fed either low salt (0.3 %: DSL) or high salt (8 %:
DSH) diet from 6 weeks to 16 weeks. Pioglitazone (10 mg/kg per day) or candesartan (4 mg/kg
per day) was administrated with high salt. At 16 weeks of age, LV geometry, cardiac fibrosis,
gene expression of collagen, connective tissue growth factor (CTGF) and transforming growth
factor-1 (TGF-1), and matrix metalloproteinase (MMP) activities were evaluated by
echocardiography, histochemistory, real-time PCR, and zymography, respectively. Blood
pressure (BP) was elevated, heart weight was increased, and LV wall thickness was increased
in DSH (2134 mmHg) compared to DSL (1451 mmHg). Candesartan and pioglitazone
decreased BP in DSH (1813 mmHg and 1914 mmHg, respectively). The LV geometrical
changes and interstitial and perivascular fibrosis, as well as myocardial hypertrophy in DSH
were recovered to the level of DSL by administration of candesartan or pioglitazone. Gene
expressions of TGF-1, collagen type I, collagen type III, and CTGF were significantly increased
in DSH. Candesartan decreased the expressions of all genes, but pioglitazone did those of
TGF-1 and collagen type I. MMP activity was increased in DSH, but the correction of MMP
activity by the two drugs was limited. These results suggest that pioglitazone reduced LV
hypertrophy and fibrosis in salt-sensitive hypertension, as did candesartan. The mechanism for
the beneficial effects may be different between the two drugs, however decrease in BP and
decrease in collagen synthesis may be commonly involved.
P101
Highly Increased Circulating Endothelial Cells in Women with Preeclampsia
Magdalena Grundmann, Alexander Woywodt, Bettina Hollwitz, Torsten Kirsch, Uta
Erdbruegger, Katrin Oehler, Hermann Haller, Marion Haubitz; Med Sch Hannover, Hannover,
Germany
Objective: Preeclampsia is primarily a disorder of the maternal endothelium resulting in
endothelial dysfunction and injury. However, the degree of endothelial damage is difficult to
quantify. As circulating endothelial cells (CEC) have been shown to be a marker of endothelial
injury in patients with different diseases involving the endothelial layer like systemic vasculitis
and thrombotic microangiopathy, we tested the hypothesis that CEC in patients with
preeclampsia are elevated and reflect endothelial cell damage. Methods: Circulating endothelial
cells were measured in 8 patients with preeclampsia and 8 normotensive women before
delivery, 1 day after delivery and 3 - 5 days later. Enumeration of CEC was performed with
anti-CD 146-driven immunomagnetic isolation and subsequent staining with Ulex Europaeus
lectin 1. Results: Women with normal pregnancies had slightly elevated CEC (median 18
cells/ml; p0.05) compared to healthy non-pregnant women (median 8 cells/ml). However,
women with preeclampsia showed highly elevated numbers of CEC (median 100 cells/ml;
range 48 1200; p0.001). Cell numbers declined rapidly after delivery and after 35 days
before discharge both groups had comparable numbers of CEC (women with preeclampsia 28
cells/ml compared to 26 cells/ml). Conclusion: In women with preeclampsia CEC were highly
elevated compared to healthy pregnant women and comparable to CEC in systemic vasculitis.
Our findings demonstrate the severity of endothelial injury in preeclampsia. Numbers seem to
correlate with the severity of the disease. After delivery CEC in women with preeclampsia
declined rapidly and 35 days later values were comparable to those of women without
preeclampsia, indicating a rapid repair of the endothelial damage in preeclampsia.
P102
Time Course of Circulating Endothelial Cells in Patients with a Stroke
Marion Haubitz, Stefan Gerdes, Alexander Woywodt, Torsten Kirsch, Uta Erdbruegger,
Hermann Haller, Karin Weissenborn; Med Sch Hannover, Hannover, Germany
Objective: Circulating endothelial cells (CEC) have been shown to be a marker of endothelial
damage. Elevated levels of CEC could be demonstrated in patients with vascular diseases such
as systemic vasculitis or thrombotic microangiopathy. We now tested the hypothesis that acute
ischemic stroke is associated with endothelial injury and analyzed CEC at different time points

after stroke. Methods: Circulating endothelial cells were measured in 38 patients with the
clincical diagnosis of stroke at admission and 1, 7, 14, 21 and 90 days after the event. 20
patients had an ischemic stroke of the middle cerebral artery, 5 a lacunary infarct and 13 a
thrombembolic event. Enumeration of CEC was performed with anti-CD 146-driven immuno-
magnetic isolation and subsequent staining with Ulex Europaeus lectin 1. Results: At
presentation and at day 1 and 7 patients of all stroke groups had clearly elevated CEC
compared to normal controls (median 32 cells/ml day 0, 36 cells/ml day 1 and 24 cells/ml day
7; normal controls 8 cells/ml, p0.01) with a tendency to lower levels in patients with lacunary
infarction (24 cells/ml day 0 and 1). Values decreased to 16 cells/ml at day 21 and 12 cells/ml
at day 90 but the difference was still significant compared to normal controls (p0.05)
probably due to risk factors involved in endothelial injury. Conclusion: Patients with ischemic
stroke showed elevated levels of CEC indicating the endothelial damage. CEC may enhance the
local inflammatory response. The prolonged decrease of the cell numbers indicates a delayed
endothelial repair in these patients.
P103
Endothelial Progenitor Cells Are Reduced in Refractory Hypertension
Anna Oliveras, Maria J Soler, Hosp del Mar, Barcelona, Spain; Ofelia M Martnez-Estrada,
Cellular Biology Dpt. Facultat de Biologia., Barcelona, Spain; Susana Vazquez, Hosp del Mar,
Barcelona, Spain; Ddac Marco-Feliu, Cellular Biology Dpt. Facultat de Biologia., Barcelona,
Spain; Joan S Vila, Methodologic Assessment to BioMed Investigation. IMIM., Barcelona,
Spain; Josep M Puig, Antonia Orfila, Marisa Mir, Hosp del Mar, Barcelona, Spain; Senen
Vilaro, Cellular Biology Dpt. Facultat de Biologia., Barcelona, Spain; Josep Lloveras; Hosp
del Mar, Barcelona, Spain
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CHBPR ConferencePoster Presentations e65
P105
Duration of Breastfeeding and Aortic Pulse Wave Velocity in Young
Children: The Manchester Childrens Cardiovascular Health Study
Narinder Bansal, Islay Gemmell, Univ of Manchester, Manchester, United Kingdom; Patrick
McElduff, Hunter New England Area Health Service, Newcastle, Australia; Avni Vyas, Peter E
Clayton, Kennedy Cruickshank; Univ of Manchester, Manchester, United Kingdom
Introduction: Breastfeeding is associated with lower blood pressure (BP) in later life. Arterial
stiffness, a known predictor of vascular events, may be a more precise index of vascular health
than blood pressure. However, the effects of breastfeeding on vascular health in young children
are unknown. Breastfeeding is associated with increased serum cholesterol in infancy, and
thus may be associated with decreased arterial distensibility in young children. Aim: We
investigated the effects of duration of breastfeeding, BP, and growth on arterial stiffness as
measured by aortic pulse wave velocity (aPWV) in 2 4 year-old children. Methods: As part of
our ongoing prospective study, 57 children were studied between 2 4 years of age with 70
observations for measures of BP and aPWV, as well as growth (height, weight, skinfold
thickness; suprailiac, triceps and subscapular). A maternal questionnaire, completed at the
visit, provided information on the incidence and duration of breastfeeding. APWV was measured
using two continuous wave Doppler ultrasound probes insonating the aortic arch and
bifurcation. Results: In a multilevel model analysis, after adjustment for age, gender, ethnicity,
weight, length and diastolic blood pressure, duration of breastfeeding was significantly
associated with aPWV in the whole group ( 0.01, p0.02). Conclusion: Our data suggests
that duration of breastfeeding is positively associated with aPWV, and thus increased aortic
stiffness in early childhood. This is consistent with previous findings where increased duration
of breastfeeding has been associated with decreased brachial artery distensibility in early adult
life. This is the first study to examine the vascular effects of breastfeeding in young children
and our continuing investigation will examine how lipid profiles affect this relationship.

Background: Circulating endothelial progenitor cells (EPCs) play a key role for the maintenance
of endothelial homeostasis and may predict cardiovascular (CV) events. It has been found a
correlation between some classical CV risk factors and reduced number and function of EPCs,
but their relationship with hypertension remains unclear. Aim: to investigate whether refractory
hypertension (RHT) determines the number of EPCs. Methods: EPCs (CD34/CD133/
CD45) were isolated from circulating polymorphonuclear cells (PMN) by flow cytometry in 37
RHT and 30 normotensive controls (C). EPCs colony-forming capacity was also determined
quantified in vitro after 7 days in culture (LDL-Dil/lectin). Results: Univariate analyses: EPCs
number was significantly reduced in RHT vs. C (median[percentile 25;75]: 32.5[8.559.5] vs.
50.5 [29.8 82.3] EPC/105 PMN, p0.021). Colony-forming units of EPCs (CFU) were also
reduced in RHT vs. C (median[percentile 25;75]: 142 [33.5288] vs. 518.4[320 715] CFU x
field, p0.001). Age was higher in RHT (meanSD RHT vs. C: 60.711.5yr vs. 37.09.2yr,
p0.001). After adjusting for age, EPCs concentration and CFU number remained significantly
lower in RHT group (p0.046 and 0.001 respectively). Estimated glomerular filtration rate
(by MDRD) was reduced in RHT in comparison to C (62.0 15.9 vs. 72.3 9.5 ml/min/1.73m2;
p0.003) . Otherwise, othere was a significant positive correlation of RHT with dyslipemia,
diabetes mellitus, age, weight, body mass index, fibrinogen, tryglicerides, ferritin, HbA1c,
C-reactive protein and urine albumin/creatinine ratio. By multivariate analyses RHT was the
only independent predictor of EPCs concentration; a RHT patient has 25.3 EPCs/105 PMN less
than a C (p0.02). Moreover, only weight, tryglicerides and being hypertensive predicted CFU
number after culture. After adjusting for those these variables, a RHT has 427.8 CFU x field less
than a C (p0.0001). Conclusion: Circulating EPCs number and culture proliferation are
significantly reduced in patients with refractory hypertension, independently of other known CV
risk factors.
P104
Association of Novel Risk Factors with Reactive Hyperemia and
Flow-Mediated Dilatation in Hypertensive Subjects
Iftikhar J Kullo, Malik A Rauoof, Simone Santos, High-Seng Chai, Stephen T Turner; Mayo
Clinic, Rochester, MN
P106
Controlled Blood Pressure Lowering after Experimental Cerebral Ischemia
Provides Neurovascular Protection
Hazem F Elewa, Anna Kozak, Univ of Georgia and VA Med Cntr, Augusta, GA; Maribeth H
Johnson, Med College of Georgia, Augusta, GA; Adviye Ergul, Susan C Fagan; Univ of
Georgia and Med College of Georgia, Augusta, GA
There is evidence that acutely elevated blood pressure (BP) after stroke is associated with
increased cerebral hemorrhage and edema. Management of hypertension in acute stroke
remains a hotly debated issue since it may lead to an increase in the infarct area due to
hypoperfusion. Purpose: Previous work in our lab has shown that BP lowering with candesartan
1mg/Kg after experimental cerebral ischemia was associated with significant reductions in
infarct size and hemorrhage when compared to saline treated animals (Fagan, 2006). Our goal
was to determine whether this neurovascular protection is mediated by the BP lowering effect.
Methods: Male Wistar rats (280 305 g) underwent 3 hours of middle cerebral artery occlusion
(MCAO). At reperfusion, either saline (n18), hydralazine 1 mg/kg (n8), enalapril 5 mg/kg
(n7) or enalapril 10 mg/kg (n8) were administered intravenously. BP was measured by
telemetry for 2 days before and 24 hours after MCAO. After neurologic function was assessed,
brain tissue was processed for infarct size and hemoglobin content analyses. Results: Mean
arterial BP (MAP) increased from 92 to 124 mmHg immediately upon MCAO and decreased to
112 mmHg after reperfusion, remaining elevated for 24 hours (p0.0001) in the saline group.
Hydralazine reduced MAP (p0.048) and infarct size (53% vs. 30%, p0.0083) and there was
a trend toward decreased hemoglobin content and improved neurologic function. Enalapril
5mg/Kg did not significantly change MAP or other outcomes. Enalapril 10 mg/Kg reduced MAP
(p0.0001) and infarct size (53% vs. 29%, p0.003). There was an intermediate effect on
both hemoglobin content and neurologic function, neither one signficant. The time course of BP
lowering varied with each treatment. Conclusion: Acute blood pressure lowering after
reperfusion in acute ischemic stroke is an effective strategy to achieve neurovascular
protection. The rate, extent and mechanism of blood pressure lowering may determine the
magnitude of protection.
P107
Estimating the Burden of Neurogenic Orthostatic Hypotension in the United
States. A National Population-Based Study

Background. We investigated whether novel risk factors - homocysteine, lipoprotein (a) (Lp(a)),
C-reactive protein (CRP), and fibrinogen - are associated with non-invasive measures of
microcirculatory and conduit artery function in hypertensive subjects without history of
coronary heart disease (CHD). Methods. Brachial artery ultrasound was performed to measure
forearm reactive hyperemia (RH), a measure of microcirculatory function, and flow-mediated
dilatation (FMD), a surrogate of conduit artery endothelial function, in 508 hypertensive adults
(62.29.8 years, 42% men) belonging to hypertensive sibships. The association of each novel
risk factor with RH and FMD was tested in regression models adjusting for conventional CHD
risk factors (age, sex, smoking, systolic BP, total cholesterol, HDL cholesterol, body mass index
(BMI), diabetes), and statin use. We also investigated interactions between novel and
conventional CHD risk factors in predicting RH and FMD. Generalized estimating equations were
used to account for intrafamilial correlation. Results. Greater age, higher BMI, and diabetes
were independently associated with lower RH. Of the novel risk factors, plasma homocysteine
was independently associated with lower RH (P0.039), Lp(a) and CRP were not associated
with RH, and fibrinogen was associated with lower RH in diabetic subjects (P for interaction
0.007). Greater age, male sex, and history of smoking were independently associated with
lower FMD. Higher homocysteine was associated with lower FMD in subjects with systolic BP
140 mm Hg (P for interaction, 0.0008), whereas Lp(a) was associated with lower FMD in
participants on statins (P for interaction 0.024). CRP and fibrinogen were not associated with
FMD. Conclusion. In asymptomatic adults with hypertension, novel CHD risk factors are
associated with microcirculatory and conduit artery function but the association is modified by
interaction with conventional risk factors.
Cyndya Shibao, Carlos Grijalva, Satish R Raj, Italo Biaggioni, Marie R Griffin; Vanderbilt Univ,
Nashville, TN
Background: Neurogenic Orthostatic Hypotension (nOH) is a common condition in adults aged
65 years and older. It is associated with an increased risk of falls, syncope and cerebrovascular
events. Previous studies have evaluated nOH in selected settings, but the national impact of this
condition on hospitalizations has not been examined. Methods: We assessed the burden of
nOH-related hospitalization among US adults, using Nationwide Inpatient Sample (NIS) data for
2003. The NIS is the largest inpatient database in the US, and includes approximately 20% of
all hospitalizations. Records of nOH-related hospitalizations and co-morbidities were identified
with ICD-9-CM discharge diagnosis codes. Records suggesting non-neurogenic causes of nOH
were excluded. Results: In 2003, there were an estimated 80,544 nOH-related hospitalizations
representing 28/100000 US population (95% CI 26 to 30). In 35% of these, nOH was the
primary diagnosis. Hospitalization rates increased with age (Figure), 52% of patients were
females, 76% were emergency admission, and 20% also had a diagnosis of syncope. The
estimated median length of stay was 3.5 days (IQR 1.7 6.7). In the group aged 40 years,
25% had co-existing diabetes mellitus, 4% Parkinson Disease, 0.8% abnormal degeneration of
basal ganglia (including multiple system atrophy), and 0.15% amyloidosis. The overall
in-hospital mortality was 1.1%. Conclusions: nOH is a common condition in hospitalized
patients, especially among elderly and diabetic patients. In light of the increasing incidence of
both diabetes and aging in the US population, the contribution of nOH to morbidity and mortality
deserves further scrutiny.
 e66 Hypertension Vol 48, No 4 October 2006
P108
Dysregulation of the Ubiquitin-Proteasome System in Symptomatic Human
Carotid Atherosclerosis
Daniele Versari, Joerg Herrmann, Mario Gossl, Dallit Mannheim, Katherine Sattler, Fredric B
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Meyer, Lilach O Lerman, Amir Lerman; Mayo Clinic Rochester, Rochester, MN
Background: The ubiquitin-proteasome system is the principal degradation route of intracel-
lular proteins and oxidized proteins, and thus it regulates many cellular processes conceivably
important for atherosclerosis. The aim of this study was to evaluate the activity of
ubiquitin-proteasome system in human carotid artery plaques in relation to oxidative stress and
clinical manifestation of the disease. Methods: Patients undergoing carotid endarterectomy
were divided into asymptomatic (A) and symptomatic (S), in the absence or in the presence of
a cerebrovascular ischemic event within 6 months before the surgery, respectively. Carotid
endarterectomy specimens were collected from 83 A and 94 S. Content of free ubiquitin,
ubiquitin-conjugates and NADPH-oxidase-p67 subunit were evaluated by western blotting and
proteolytic activity of proteasome was assessed by fluorimetric assay. Single and double
immunostaining for ubiquitin-conjugates, nitrotyrosine, smooth muscle alpha-actin and mac-
rophage CD-68 were performed. Results: Symptomatic patients were characterized by a
significantly higher content of ubiquitin-conjugates as compared to A (17.721.36 vs.
10.991.04, respectively; p0.001), despite similar expression of free ubiquitin (1.570.20
vs. 1.320.21, respectively; pn.s.). Moreover, S had lower proteasome activity than A
(5.010.70 vs. 9.411.19 nmol AMC/mg protein/minute, respectively; p0.01). Expression of
p67 was significantly higher in S (2.310.29) than A (1.130.17; p0.01) and a direct
correlation between ubiquitin-conjugates expression and p67 was observed (r0.72;
p0.001). Immunostaining revealed coexpression of ubiquitin-conjugates and nitrotyrosine, a
marker of protein oxidation, as well as accumulation of ubiquitin-conjugates in smooth muscle
cells and macrophages. Conclusions: In human carotid atherosclerotic plaques, increased
oxidative stress might be responsible for accumulation of ubiquitin-conjugates, partly as a
consequence of the inhibition of the proteasome proteolytic activity. The accumulation of
ubiquitin-conjugates in muscle cells and macrophages can in turn induce cell loss, leading to
plaque instability and clinical events.
P109
Comparison of Hydrochlorothiazide and Chlorthalidone upon Blood
Pressure, Endothelial Dysfunction and Cardiorenal Injury in Salt-Sensitive
Hypertension
Ming-Sheng Zhou, Edgar A Jaimes, Ivonne H Schulman, Leopoldo Raij; Nephrology and
Hypertension Section, VAMC, Div of Nephrology and Hypertension and Vascular Biology
Institute, Univ of Miami Miller Sch of Medicine, Miami, FL
The role of thiazide-type diuretics in hypertension is well established, however, whether they
provide CV protection beyond lowering blood pressure (SBP) is still controversial. Dahl salt
sensitive rats (DS) are a paradigm of salt sensitive hypertension in humans. We utilized DS rats
to determine the role of monotherapy with two thiazide-type diuretics, hydrochlorothiazide
(HCTZ) and chlorthalidone (CTL), in controlling SBP and providing cardiac (LVH) and renal
(proteinuria) protection. In addition we investigated in aortas the effects of these agents upon
superoxide (O2-), by lucigenin chemiluminescence, angiotensin type 1 receptor (AT1R) and
monocyte chemoattractant protein-1 (MCP-1), by real-time PCR, as well as NO dependent
relaxation (EDR) to acetylcholine in organ bath. Groups of DS rats were fed either a normal (NS,
0.5% NaCl) or a high (HS, 4% NaCl) salt diet for 6 weeks. In addition, 3 separate groups of HS
rats were given HCTZ 75mg/L, CTL 75mg/L or CTL 37 mg/L in the drinking water. Results: see
table. HS developed hypertension, LVH and proteinuria, accompanied by aortic upregulation of
AT1R (198%) and MCP-1 (145%), increased production of O2- and impaired EDR. HCTZ as well
as the high and the low doses of CTL reduced SBP, LVH and proteinuria. However, both HCTZ
and CTL were ineffective in normalizing or improving AT1R, O2-, MCP-1 or EDR. Compared with
NS, plasma renin concentration (PRC) by RIA in HS was suppressed; HCTZ but not CTL brought
up PRC to values similar to those in NS. We conclude that although thiazide-type diuretics
ameliorate hypertensive baro-injury of the heart and kidney, they fail to concomitantly mitigate
pro-atherogenic changes in the endothelium (AT1R, O2-, MCP-1 and impaired EDR). These
novel studies suggest that neither HCTZ nor CTL provide global CV protection beyond lowering
SBP and provide the scientific basis for the early use of combination therapy, particularly with
inhibition/blockade of RAS, in the treatment of hypertension.
NS HS HS/HCTZ-75 HS/CTL-75 HS/CTL-37
SBP (mmHg) 1384* 1844 1623*# 1654*# 1633*#
LVH (mg/100g 2024* 25413 2186* 2096* 2135*
BW)
Proteinuria 223* 566 425*# 384*# 366*#
(mg/24 h)
PRC 2.00.1* 1.50.2 2.30.2* 1.50.1# 1.30.3#
(ng/ml/hour)
aortic O2- 734129* 1558189 1351116# 1285150# 1487258#
(count/min/mg)
Emax (%) 906* 606 725# 699# 705#
Emax: maximal relaxation, *P0.05, vs HS; #P0.05, vs NS. n6 8
P110
Adding Aliskiren to Ramipril Improves 24-Hour Blood Pressure Control
Compared to Ramipril Alone in Patients with Diabetes and Hypertension
Addison A Taylor, Baylor College of Medicine, Houston, TX; Diethelm Tschoepe, Ruhr Univ
of Bochum, Bad Oeynhausen, Germany; Charles Kilo, Washington Univ Sch of Medicine, St
Louis, MO; Ghionul Ibram, Hui Fang, Andrew Satlin; Novartis Pharmaceuticals Corp, East
Hanover, NJ
Background: Sustained 24-h blood pressure (BP) control is required to maximize cardiorenal
protective benefits of antihypertensive therapy, and stringent BP control is particularly
important in patients with diabetes and hypertension. This study investigated the 24-h
antihypertensive efficacy of the novel oral renin inhibitor aliskiren and the ACE inhibitor ramipril,
alone and in combination, in patients with diabetes and hypertension. Methods: A total of 837
patients with type 1 or 2 diabetes and hypertension (mean sitting diastolic BP 95109 mmHg)
were randomized to once-daily, double-blind treatment with aliskiren 150 mg, ramipril 5 mg
or aliskiren/ramipril 150/5 mg for 4 weeks, followed by forced titration to aliskiren 300 mg,
ramipril 10 mg or aliskiren/ramipril 300/10 mg for a further 4 weeks. Mean changes from
baseline in 24-h ambulatory diastolic and systolic BP (ADBP and ASBP) were assessed at
baseline and Week 8 in a subgroup of 173 patients. Results are presented as least squares
means SEM. Results: Aliskiren (n57), ramipril (n55) and aliskiren/ramipril combination
(n61) reduced mean 24-h ADBP from baseline by 4.30.6, 3.20.6 and 4.80.5 mmHg,
respectively; mean 24-h ASBP was reduced by 6.00.9, 6.51.0 and 8.10.9 mmHg,
respectively. The aliskiren/ramipril combination was superior to ramipril monotherapy in
reducing 24-h ADBP (treatment difference 1.60.8 mmHg; p0.03) and daytime ADBP
(difference 2.81.4 mmHg; p0.04). Statistically significant differences were not found
between groups for reductions in nighttime ADBP, daytime and nighttime ASBP, or ADBP and
ASBP during the early morning BP surge (2124 h post-dosing). All treatments reduced
ambulatory BP regardless of dipper status (extent of nocturnal BP drop), but the aliskiren/
ramipril combination appeared to be more effective in non-dippers (mean reduction in
ADBP/ASBP 6.1/10.8 mmHg; n24) than dippers (mean reduction 4.2/6.8 mmHg; n37).
Conclusions: Aliskiren provides effective 24-h BP control in patients with diabetes and
hypertension. Significant additional ambulatory BP lowering was observed with the aliskiren/
ramipril combination compared with ramipril alone, indicating the benefits of combining
aliskiren with ACE inhibitor treatment.
P111
Tetraethyl Ammonium Causes Vasoconstriction and Impairs
Bradykinin-Induced Vasodilatation via Production of Reactive Oxygen
Species
Tetsuya Ishiki, Yusuke Ohya, Tatsuya Tagawa, Shinichiro Ueta, Syuichi Takishita; Univ of the
Ryukyus, Nishihara cho Okinawa, Japan
Objectives: Bradykinin (BK) stimulates endothelial cells to release several vasodilating factors,
including nitric oxide (NO), prostanoids, and an endothelium-derived hyperpolarizing factor
(EDHF). It has been reported that the vasodilating effect of bradykinin in human forearm
resistance vessels are attenuated by potassium channel blocker; tetraethylammonium chloride
(TEA) but not NO synthase inhibitor, which suggests that hyperpolarization of endothelial cells
would be responsible for vascular effect of BK. However, specificity of TEA in terms of an EDHF
blocker remains unclear. The present study was designed to investigate the contribution of
reactive oxygen species (ROS) and potassium channels on basal vascular tone and BK-induced
vasodilatation in human forearm resistance vessels. Methods and results: Forearm blood flow
(FBF) was recorded with venous occlusion plethysmography in healthy 11 male subjects. All
drugs were intra-arterially infused into the brachial artery. Infusion of TEA (1mg/min) decreased
the basal flow, and pre-infusion of vitamin C (vitC, 25mg/min) reversed this effect. Brachial
artery infusion of BK (5, 15, 45 and 100 pmol/100 mL/min) caused significant forearm
vasodilatation in all subjects (from 3.2 to 13.1 mL/100 mL/min). Pre-infusion of TEA
significantly attenuated BK-induced vasodilatation (from 13.1 to 11.3 mL/100 mL/min). This
TEA-induced effect was recovered by pre-infusion of vitC. Conclusion: These results suggest
that potassium channel blocking and/or depolarization of vascular cells by TEA caused
vasoconstriction and impaired the BK-induced vasodilatation in human forearm resistance
vessels via ROS production. TEA may not be a suitable drug for the evaluation of EDHF.
Alternatively, EDHF action may be impaired by ROS.

P112
In vivo and in vitro Effects of HMG Co-A Reductase Inhibition on
Albuminuria and Albumin Stimulated NAD(P)H Oxidase Activity in the
Proximal Tubule
Adam T Whaley-Connell, E Matthew Morris, Nathan T Rehmer, J. Cipporah Yaghoubian,
Suzanne E Clark, Univ of Missouri-Columbia Sch of Medicine, Columbia, MO; Charles E
Wiedmeyer, College of Veterinary Medicine, Columbia, MO; Melvin R Hayden, Craig Stump,
James R Sowers; Univ of Missouri-Columbia Sch of Medicine, Columbia, MO
Oxidative stress in the kidney and filtration barrier leads to microalbuminuria (MAU) which is
now accepted as a marker for systemic endothelial dysfunction. The development of MAU leads
to subsequent tubulointerstitial dysfunction. Mounting evidence supports an important role for
proximal tubule dysfunction in MAU and there is data suggesting that albumin overload leads
to reactive oxygen species production in proximal tubule cells (PTC) in vitro. HMG-CoA
reductase inhibition (statins) has been shown to reduce albuminuria. Further, statins have been
attributed with affects independent of lipid lowering including modulation of oxidative stress via
regulation small molecular weight G-proteins (e.g. Rac1) and NAD(P)H oxidase (NOX) activity.
Thus, providing a mechanism by which statins may modulate MAU and resultant tubular
dysfunction. To investigate the effects of statins on albuminuria, and albumin handling in the
kidney, Zucker obese (ZO) and Zucker lean rats (4 5 wks) were given daily injections of
Rosuvastatin (10 mg/kg, IP) or vehicle for 21 days. Urine microalbumin normalized to creatinine
was measured after treatment. Further, to assess the effects of statins on tubular handling of
albumin, an immortalized opossum PTC was utilized. NOX activity was determined by
measuring the conversion of Radical Detector (Cayman Chemical) in the absence and presence
of NOX inhibitor apocynin using spectrophotometric (450 nm) techniques. ZO rats developed
proteinuria by 8 9 wks of age that was significantly reduced following statin treatment (70%).
Following incubation with albumin in PTC in vitro, NOX activity was significantly higher than
controls that was abrogated by Rosuvastatin in a dose-dependent manner. Further, treatment
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of PTC with a Rac1 inhibitor, (NSC23766) significantly ameliorated albumin stimulated NOX
activity. These data suggest that statin treatment ameliorates albuminuria and albumin
stimulated oxidative stress by: 1) reducing stimulation of NOX activity in PTC via protein
overload reduction, and 2) by directly inhibiting the cellular mechanism of albumin stimulated
NOX activation, possibly through inhibition of small molecular weight G-proteins such as Rac1.
P113
Morning Rise of Blood Pressure and Subcutaneous Small Resistance Artery
Structure
Carolina De Ciuceis, Damiano Rizzoni, Enzo Porteri, Gianluca E Boari, Maria Lorenza
Muiesan, Caterina Platto, Nicola Rizzardi, Massimo Salvetti, Francesca Zani, Marco Miclini,
Silvia Paiardi, Enrico Agabiti Rosei; Dept. of Med and Surgical Sciences, Univ of Brescia,
Brescia, Italy
It has been previously demostrated that morning rise (MoR) of blood pressure may predict
major vascular events in hypertensive patients (Kario K and coll, Circulation 2003). Structural
alterations of small resistance arteries, as evaluated by the tunica media to internal lumen ratio
of subcutaneous small resistance arteries (M/L) may predict cardiovascular events as well
(Rizzoni D and coll, Circulation 2003). Since an increased M/L may amplify the effect of
hypertensive stimuli, aim of the present study was to evaluate possible relationships between
MR and M/L in a population of hypertensive patients. Design and Methods One hundered
subjects were included in the present study. They were 64 patients with essential hypertension,
8 with pheochromocytoma, 16 with primary aldosteronism, 10 with renovascular hypertension,
and 2 normotensive patients with non-insulin dependent diabetes mellitus. All subjects were
submitted to a biopsy of subcutaneous fat. Small resistance arteries were dissected and
mounted on an isometric myograph, and the M/L was measured. In addition, MoR was
calculated from ABPM according to 6 previously published different methods: MoR 1 and MoR
2 according to Kario K and coll. (Circulation 2003), MoR 3 according to Wizner B and coll
(J Hypertens, 2004; 22,suppl.2), and MoR 4, MoR 5 and MoR 6 according to Bilo G and coll.
(J Hypertens, 2004; 22,suppl.2). Results In the whole population (n100) a statistically
significant correlation was observed between MoR1 and M/L (r0.24, p0.05) and between
MoR1 and internal diameter of subcutaneous small arteries (r-0.25, p0.05), when the
analysis was restricted to patients with essential hypertension, the correlation observed was
even closer (MoR1-M/L: r0.50,p0.001, MoR2-ML:0.32,p0.01, MoR3-ML: 0.25,p0.05,
MoR4-ML: 0.27,p0.05, MoR5-ML: 0.14,pNS, MoR6-ML: 0.25,p0.05). Significant corre-
lations were observed between small artery internal diameter and MoR1 (-0.45,p0.001),
MoR2 (-0.28,p0.05), MoR6 (-0.26,p0.05). Conclusions Our results indicate that subcuta-
neous small artery structure is related to MR, possibly because an altered vascular structure
may amplify blood pressure changes, or, vice versa, because a greater MR may further damage
peripheral vasculature.
P114
mPGES-1 Knockout Mice Have Impaired Ability To Excrete Sodium Load
And Have Increased Blood Pressure During High Salt Intake
Tianxin Yang, Aihua Zhang, Zhanjun Jia; Univ of Utah, Salt Lake City, UT
mPGEES-1, a membrane-associated prostaglandin E synthase, is highly inducible by cytokines,
similar to COX-2. We examined phenotype of mPGES-1 KO mice with respect to salt sensitivity
and blood pressure (Bp) regulation. We monitored daily mean blood pressure (MAP) by
telemetry. An one-wk high salt (HS) diet (8% NaCl in gelled diet plus saline as drinking fluid)
induced a gradual and significant increase in MAP in mPGES-1 -/- mice as compared to /
mice (changes of MAP: 45.7 5.2 vs. 23.4 2.2 mmHg, on day 7, n7, p0.01). mPGES-1
-/- mice had increased sodium balance which was most prominent on days 1 and 2 of HS
loading. Chronic HS treatment induced a 16-fold increase in urinary excretion of PGE2 in /
mice that was completely abolished in -/- mice. Strikingly, following the HS treatment, urinary
NO excretion was increased in / mice (92 31 vs. 361 96 nmol/24 hr, on day 7,
CHBPR ConferencePoster Presentations e67
p0.05) but decreased in -/- mice even to an undetectable level (127.6 33 nmol/24 hr vs.
0, on day 7). The HS treatment induced an 8-fold increase in nNOS mRNA expression in the
inner medulla of / mice as assessed by real time RT-PCR that was abolished in -/- mice.
By immunohistochemistry, faint labeling of mPGES-1 antibody was detected in the collecting
duct (CD) in NS / group, in a sharp contrast to intense labeling in the CD in the HS group.
Following chronic HS treatment, total abundance of mPGES-1 protein as assessed by
immunoblotting exhibited a 2-fold increase in the inner medulla but not in the cortex. The /
and -/- animals received acute loading of 1 mEq of Na via gavages, followed by 12 hr urine
collections for 72 hr. The / mice were able to eliminate the entire sodium load within 24
hours while the -/- mice did so after 48 hours. Following acute sodium loading, the / mice
had remarkable increases in urinary excretion of PGE2 and NO, both of which were completely
blocked in the -/- mice, similar to the chronic setting of HS loading. In primary cultures of /
CD cells, exposure to 550 mOsm/L of NaCl for 24 hours induced a 10- fold increase in PGE2
excretion that was inhibited by 70% in mPGES-1 -/- CD cells. In summary, this study has
elucidated mPGES-1-dependent pathway in determination of natriuretic responses that appears
to require PGE2 regulation of NO production.
P115
Dipeptidyl Peptidase IV in Angiotensin-Converting Enzyme
Inhibitor-Associated Angioedema
James B Byrd, Stephanie Harris, James V Gainer, Chang Yu, Ajai Shreevatsa, Pradeep
Putlur, John Nadeau, Vanderbilt Univ, Nashville, TN; Karine Touzin, Univ of Montreal,
Montreal, Canada; Marshall Summar, Vanderbilt Univ, Nashville, TN; Albert Adam, Univ of
Montreal, Montreal, Canada; Nancy J Brown; Vanderbilt Univ, Nashville, TN
Angioedema is a potentially life-threatening adverse effect of ACE inhibitors (ACEi). Bradykinin
and substance P, substrates of ACE, increase vascular permeability and cause tissue edema
in animal models. We tested the hypothesis that the activity of enzymes involved in the
amino-terminal degradation of these peptides is impaired in individuals with ACEi-associated
angioedema. Thirty-eight subjects with a history of ACEi-associated angioedema (19 male, 19
female; 15 black, 23 white) and 67 ACEi-exposed controls (33 male, 34 female; 37 black, 30
white) were ascertained. Blood was collected for DNA (35 cases, 61 controls) and serum (30
cases, 66 controls). ACE activity, aminopeptidase P activity, aminopeptidase N activity,
dipeptidyl peptidase IV (DPPIV) activity and antigen, and ex vivo degradation half-lives of
bradykinin, des-Arg9-bradykinin and substance P were determined. Polymorphisms in the gene
encoding DPPIV were identified by SSCP using population samples. As expected serum ACE
activity was significantly decreased during ACEi (2.13.1 vs. 51.916.8 nmol/ml/min in the
absence of ACEi, P 0.001), but there was no difference in ACE activity between cases and
controls. There were no differences in APP activity, APN activity, or bradykinin or des-Arg9-
bradykinin degradation half-life between cases and controls in the presence or the absence of
ACEi. In contrast, DPPIV antigen was decreased in cases compared to controls (345.6117.9
vs. 497.3126.6 ng/ml, P 0.001). DPPIV activity was decreased in cases during
angioedema compared to in ACEi-treated (21.25.8 vs. 28.67.6 nmol/ml/min, P 0.041).
The degradation half-life of substance P correlated inversely with DPPIV antigen during ACEi
polymorphism, T24C, in the DPPIV gene accounted for 17.6% of the variability in antigen
(P0.001). Frequency of the 24C allele, associated with low DPPIV concentrations, was
increased in white Americans with a history of angioedema (0.48 in cases vs. 0.17 in controls,
P 0.003), but not in black Americans (0.21 in cases vs. 0.24 in controls). Dipeptidyl
peptidase IV antigen is decreased in individuals with a history of ACEi-associated angioedema.
Genetic or environmental factors that decrease DPPIV activity may predispose individuals to this
adverse effect.
P116
Increased Endocannabinoid Production by High Salt Intake Plays a Role in
Preventing Salt-Induced Increases in Blood Pressure by Activating TRPV1
Youping Wang, Norbert E Kaminski, Donna H Wang; Michigan State Univ, East Lansing, MI
We test the hypothesis that high salt (HS) increases production of anandamide, an
endocannabinoid, which plays a role in preventing salt-induced increases in blood pressure
(BP) via activating the transient receptor potential vanilloid type 1 (TRPV1) channels. Plasma
anandamide levels, analyzed by Liquid Chromatography/Tandem Mass Spectrometry, were
elevated in rats fed a HS diet for 3 wks compared to rats fed a normal salt (NS) diet (4.05
0.47 vs. 2.40 0.31 pmol/ml, p0.05, n6 7). Blockade of TRPV1 by its selective
antagonist, capsazepine (CAPZ, 3 mg/kg, iv), increased mean arterial pressure (MAP) in HS- vs.
NS-treated rats (13 4 vs. 5 2 mmHg, p0.05). A metabolically stable analog of
anandamide, methanandamide (MethA, 0.5, 5, 15 mg/kg, iv), dose-dependently decreased
MAP in HS rats but it had a minimal effect on NS rats (e.g., at 5 mg/kg, HS, 21 3 vs. LS,
6 3 mmHg, p0.05). The MethA-induced decrease in MAP in HS treated rats was
significantly attenuated by CAPZ (3 mg/kg), but fully blockaded by the combination of CAPZ and
SR141716A (3 mg/kg), a selective cannabinoid receptor 1 (CB1) antagonist. Capsaicin (CAP, 10
or 30 g/kg), a selective TRPV1 receptor agonist, or calcitonin gene-related peptide (CGRP, 1
or 5 g/kg), dose-dependently decreased MAP in HS- and NS-treated rats, with a bigger effect
in the former (p0.05). Plasma CGRP levels were higher in HS- vs. NS-treated rats (p0.05).
MethA (0.1 or 10 M) increased CGRP release in mesenteric arteries isolated from HS- vs.
NS-treated rats (p0.05), which was blocked by CAPZ (p0.05). Mesenteric expression of
TRPV1 and RAMP1, a component of the CGRP receptor, was increased in HS- vs. NS-treated
rats (0.41 0.03 vs. 0.28 0.03; 0.54 0.04 vs. 0.41 0.03, p0.05). These data support
the notion that HS stimulates endocannabinoid production of anandamide, which activates
TRPV1 expressed in primary sensory nerves to release CGRP and to decrease BP. Thus,
endocannabinoids may tonically serve as an agonist of TRPV1 to prevent salt-induced increases
in BP.
 e68 Hypertension Vol 48, No 4 October 2006
P117
Caracterization of the Contractile Response Induced by Uridine Adenosine
Tetraphosphate (Up4a) in Rat Aortic Rings
Romulo Leite, A. Elizabeth Linder, R. Clinton Webb; Med College of Georgia, Augusta, GA
The uridine adenosine tetraphosphate (Up4A), a dinucleotide that contains both purine and
pirimidine moieties, described as a novel potent nonpeptidic vasoconstrictor has been shown
to be released from the endothelium upon chemical and mechanical stimulation, suggesting a
potential role as an endothelium-derived contracting factor (EDCF). It has been shown that
Up4A has a potent vasoconstrictor effect in isolated perfused rat kidney but its effect in rat
aorta has not been characterized yet. In this study we characterize the Up4A contractile
response in isolated aortic rings from normotensive rats. Aortic rings were isolated from
Sprague Dawley rats (250 275 g), cleaned and prepared for isometric tension recordings.
Concentration-effect curves to Up4A (10-8 to 10-4M) were performed in aortic rings both in the
presence and in the absence of the endothelium before and after treatment with an ATP
sensitive P2X1 receptor blocker NF279 (10-4M), NOS inhibitor L-NNA (10-4M), Rho Kinase
inhibitor Y-27632 (10-6M) or NADPH oxidase inhibitor apocynin (10-4M). Concentration-effect
curves to tempol (2.5X10-3-1.510-2M) were performed in endothelium denuded aortic rings
contracted with Up4A (10-5M). Up4A contractile response is not tachyphylactic and is
significantly potentiated by endothelium removal or treatment with L-NNA. Treatment with
NF279, Y-27632 or apocynin significantly impaired the Up4A-induced contraction of rat aorta.
Tempol caused relaxation in a concentration dependent manner in endothelium denuded aortic
rings contracted with Up4A (maximum relaxation 39%). These data suggest that the functional
endothelium and nitric oxide are key modulators of the contractile effect of the putative EDCF,
the novel Up4A. The contractile response is mediated mainly by P2X1 receptor and involves
superoxide formation and Rho Kinase pathway activation.
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P118
AT1a Receptor Deficiency Modulates Ang II and Norepinephrine
(NE)-Induced Plasminogen Activator Inihibitor-1 Expression
Nancy J Brown, Jessica Bradford, Zuofei Wang, William Lea, LiJun Ma, Douglas E Vaughan,
Agnes B Fogo; Vanderbilt Univ, Nashville, TN
To determine the role of the AT1 receptor in Ang II-induced PAI-1 expression in vivo, we
measured effects of 4h saline, Ang II(600ng/kg/min), or NE on tissue PAI-1 expression in WT
and AT1a-/- mice (C57Bl/6) in the presence or absence of losartan (1mg/ml). Ang II (125.95.2
vs 103.05.5mmHg, P0.004) and NE (127.04.4mmHg, P0.002) similarly increased SBP
in WT. Losartan decreased SBP during Ang II (108.84.4mmHg) but not NE
(125.37.4mmHg). SBP was lower during Ang II (91.74.5 vs 89.94.6mmHg) and NE
(87.04.5mmHg) in AT1a-/- mice and there was no effect of losartan. Ang II induced
9.9-(P0.001), 4.6-(P0.05), 2.5-(P0.05), and 97-(P0.001) fold increases in PAI-1
expression in heart, aorta, kidney, and liver of WT (Figure). Losartan attenuated Ang II-induced
PAI-1 expression in aorta (1.8-) and liver (9.8-fold, P0.01 vs Ang II alone). Ang II-induced
PAI-1 expression was diminished in heart (P0.05 vs WT) and aorta (P0.05 vs WT), but
enhanced in kidney (9.1-fold, P0.05 vs WT) of AT1a-/- mice. Losartan decreased Ang
II-induced PAI-1 expression in kidney (1.6-fold, P0.05) and liver (11.9-fold, P0.05) of
AT1a-/- mice. NE increased PAI-1 expression in heart (15.6-) and aorta (7.4-fold) but not kidney
or liver of WT, whereas NE increased PAI-1 expression in heart (8.2-fold, P0.001), kidney
(7.7-fold, P0.01) and liver (50.9-fold, P0.001) of AT1a-/- mice; losartan did not affect
NE-induced PAI-1 expression. Ang II induces PAI-1 expression through the AT1 receptor,
including the AT1b receptor in kidney and liver. NE induces PAI-1 expression in vivo. This effect
is modulated in a tissue-specific manner by AT1 receptor deficiency, through an AT1b
receptor-independent mechanism.
P119
Epoxyeicosatrienoic Acids Mediate Flow-Induced Dilation of Bovine Small
Coronary Arteries
Kathryn M Gauthier, Med College of Wisconsin, Milwaukee, WI; William B Campbell; Med
College of Wisconsin, Milwaukee, WI
Epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP) metabolites of arachidonic acid.
In the coronary vasculature, they mediate agonist-induced, endothelium-dependent dilations
that are independent of nitric oxide synthase and cyclooxygenase metabolites. However, the
role of EETs in mediating flow-associated dilations remains poorly defined. Bovine small
coronary arteries (200 300 m) were cannulated, perfused and constricted with the
thromboxane mimetic U46619. Stepwise increases in flow from 35 to 150 ul/min caused
flow-associated increases in diameter (maximal dilation of 518%). Pretreatment with the EET
antagonist, 14,15-EEZE (10 M) inhibited the flow-induced dilation (maximal dilation of
245%). Pretreatment with 14,15-EEZE plus the nitric oxide synthase inhibitor nitro-L-arginine
(30 M) and the cyclooxygenase inhibitor, indomethacin (10 M) blocked the flow-induced
dilations. Similarly, the dilations were blocked by the CYP inhibitor, miconazole (10 M) or
endothelium removal. Intraluminal administration of 14,15-EET to the preconstricted arteries
caused concentration-related dilations (maximal dilation of 81 3%) that were inhibited by
14,15-EEZE (maximal dilation of 32 12%). Perfusates were collected under conditions of low
flow (35 ul/min) and high flow (240 ul/min), extracted and analyzed by liquid chromatography-
electrospray ionization mass spectrometry (LC/MS). LC/MS verified the presence of 11,12- and
14,15-EET under low flow conditions (94 and 121 pg, respectively) which increased during high
flow (463 and 2352 pg, respectively). These results show that flow-induced dilations of bovine
coronary arteries are mediated by an endothelial cell, CYP metabolite that is blocked by
14,15-EEZE. Additionally, flow stimulates the release of 11,12- and 14,15-EET. Thus, in bovine
coronary arteries, flow stimulates EET release which contributes to the regulation of
flow-induced dilation.
P120
Enhanced Arachidonic Acid (AA)-Induced Contractions in Pulmonary
Arteries: Role of 15-Lipoxygenase (LO) and Estrogen
Sandra L Pfister; Med College of Wisconsin, Milwaukee, WI
Of particular interest to our laboratory is the fact that primary pulmonary hypertension affects
females two to four times more often than males. In previous work, we provided the first
evidence that pulmonary arteries from female rabbits had endothelium-dependent contractions
to AA which were enhanced when compared to responses in males. Pharmacological studies
with inhibitors of AA metabolism indicated that a non-specific LO inhibitor blocked the
contractions in females but not males. AA metabolism studies showed increased production of
15-LO derived products in females compared to males. The objective of the present study was
to characterize 15-LO expression in pulmonary arteries and to determine the role of estrogen
on 15-LO expression and AA-induced vascular contractions. Pulmonary artery lysates were
prepared from female and male rabbits and subjected to Western analysis. Results indicated
an increased 15-LO protein expression in females compared to males (n 4). In a separate
study, segments of pulmonary arteries were incubated for either 0, 4 or 24 hours in HEPES
buffer with estradiol (10-5 M) at 37C. Estradiol increased 15-LO protein expression in both
males and females by 4hrs with a greater increase observed at the 24hr time period. To test
the hypothesis that the increased 15-LO expression observed in the presence of estradiol alters
vascular responses, pulmonary arteries incubated with and without estradiol (10-5 M) for 6
hours at 37C were suspended in tissue baths for isometric tension recordings. Concentration-
response curves to AA were performed. Under these incubation conditions, the control vessels
pretreated with vehicle showed little to no contractile response to AA. In contrast, the vessels
treated with estradiol contracted to AA. The response was greater in females compared to
males (maximal contraction, AA 10-5 M, 17 11% vs 37 12%; male vs female, n 3). This
study provides the first evidence that estrogens may regulate 15-LO expression. Furthermore,
15-LO derived products may contribute to the increased incidence of pulmonary hypertension
in females compared to males.
P121
Tempol Acts Directly On Ca2-Activated Potassium Channels to Increase
Open Probability in Smooth Muscle Cells of Mesenteric Arteries
Hui Xu, Gregory D Fink, Willianm F Jackson, James J Galligan; Michigan State Univ, East
Lansing, MI
Large-conductance Ca2-activated potassium (BK) channels modulate vascular smooth muscle
tone. We found previously that tempol, an O2- dismutase mimetic, lowers blood pressure via
activation of vascular BK channels and vasodilation. In this study, we investigated the
mechanisms underlying tempol-induced activation of BK channels in mesenteric arterial
myocytes from sham and DOCA-salt hypertensive rats. Whole-cell patch clamp studies showed
that tempol (0.1 - 3 mM) enhanced peak K currents in a concentration-dependent fashion. In
myocytes from sham rats, tempol reversibly increased K currents at 80 mV from 2.6 0.6
nA to 4.4 0.7 nA (76%, P0.05, n8); this effect was larger in myocytes from DOCA-salt
rats (3.4 0.4 nA to 6.8 0.7 nA, 94% P0.05, n8). Tempol caused a leftward shift in
the activation curve for BK currents in sham and DOCA-salt myocytes. In DOCA-salt myocytes,
the peak K current at 80 mV did not differ from sham myocytes, but TEA (1 mM) and
iberiotoxin (IBTX, 0.1 M, BK channel blockers) caused a larger reduction of K currents in
DOCA-salt (3.2 0.4 nA to 1.2 0.3 nA, 60%) compared to sham myocytes (2.6 0.5 nA
to 1.5 0.4 nA, 40%; n6, P0.05). TEA or IBTX, but not 4-aminopyridine (1 mM, does not
inhibit BK channels), blocked the current activated by tempol. Tiron, another O2- scavenger, had
no effect on K currents. Using inside-out patches (patch potential -80 mV), we found that
tempol caused a 4-fold increase in open probability of BK channels in myocytes from sham and
DOCA-salt rats, but it did not change the mean channel open time (4.3 0.4 vs 4.7 0.5
ms in sham myocytes; 4.7 0.5 ms vs 5.1 0.4 ms in DOCA-salt myocytes, P0.05, n6

patches). Tempol did not change single channel conductance in sham (210 14 pS before
tempol vs 220 15 pS after tempol) or DOCA-salt myocytes (225 10 pS vs 235 11 pS,
n6). Western blots revealed that BK channel -subunit expression was increased by 136
7% in DOCA-salt arteries compared to sham arteries. The data indicate that tempol directly
activates BK channels and this effect contributes to depressor responses caused by tempol.
Upregulation of the BK channel -subunit contributes to the enhanced depressor response
caused by tempol in DOCA-salt hypertension.
P122
Role of Cytochrome P450 Enzymes in Mediating Cardioprotective Effects of
Omega-3 Fatty Acids
Marija Markovic, Gerd Wallukat, Cosima Schmidt, Max Delbruck Cntr for Molecular
Medicine, Berlin, Germany; Friedrich C Luft, HELIOS Franz Volhard Clinic, Berlin, Germany;
Dominik N Muller, Wolf-Hagen Schunck; Max Delbruck Cntr for Molecular Medicine, Berlin,
Germany
Dietary fish oil omega-3 fatty acids such as eicosapentaenoic acid (EPA) protect against cardiac
arrhythmias and cardiac hypertrophy. We tested the hypothesis that alterations in cytochrome
P450 (CYP)-dependent eicosanoid production contribute to these beneficial effects. To address
this question, we (i) determined the EPA metabolite pattern of the cardiac epoxygenase CYP2J2
and (ii) studied the effects of selected metabolites on the contractility of neonatal rat
cardiomyocytes (nRCM). Recombinant CYP2J2 epoxidized arachidonic acid (AA) and EPA with
similar catalytic efficiencies. AA was converted to all four regioisomeric epoxyeicosatrienoic
acids (EETs) without pronounced regioselectivity. In contrast, CYP2J2 showed a high
regioselectivity with EPA and produced 17,18-epoxyeicosatetraenoic acid (17,18-EETeTr) as
the main metabolite (65% of total products). Epoxidation of the 17,18-double bond was
stereoselective and produced the R,S enantiomer with an optical purity of 70%. Incubation of
nRCM with EPA (3.3 M, 30 min) significantly reduced their spontaneous beating rate. The
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same effect was elicited by the CYP-dependent EPA metabolite 17,18-EETeTr. 17,18-EETeTr
was effective already in the nM range (half maximal effect at about 1 nM) and required no
preincubation. Only the R,S but not the S,R-enantiomer of 17,18-EETeTr was effective.
Hydrolysis of the epoxide group caused loss of biological activity. EPA and 17,18-EETeTr
prevented the increase in the beating rate induced by increasing the extracellular calcium
concentration from 1.2 to 5 mM (delta 432 beats per min (bpm) for control vs. 82 bpm for
EPA vs. 23 bpm for 17,18-EETeTr vs. basal rate). The negative chronotropic effect of
17,18-EETeTr was completely reversed by racemic 11,12-EET and 11(R),12(S)EET but not
11(S),12(R)-EET. Our results demonstrate that CYP enzymes convert AA and EPA to metabolites
with opposite effects on the calcium influx and contractility of cardiomyocytes. Moreover, the
effects attributed to the unmetabolized omega-3 fatty acids are actually due to the formation
of CYP-dependent metabolites such as 17(R),18(S)-EETeTr.
P123
PPAR, but not PPAR, Agonist Protects against Salt-Mediated Increases
in Endogenous Carbon Monoxide Production and Blood Pressure in Dahl
Salt-Sensitive Rats
Fruzsina K Johnson, Tulane Univ, New Orleans, LA; William Durante, Univ of Missouri -
Columbia, Columbia, MO; Robert A Johnson; Tulane Univ, New Orleans, LA
Heme oxygenase (HO) metabolizes heme to form carbon monoxide (CO). Increased heme-
derived CO inhibits nitric oxide synthase and abolishes endothelium-dependent vasodilation.
Endogenous CO production is increased in Dahl salt-sensitive rats (Dahl-S) and contributes to
salt-induced hypertension by promoting arteriolar endothelial dysfunction. Peroxisome
proliferator-activated receptor (PPAR) agonists are lipid lowering agents that confer
cardiovascular protection during hypertension and lower blood pressure in Dahl-S. PPAR
agonists are insulin sensitizing agents that confer cardiovascular protection and lower blood
pressure in DOCA-salt hypertensive rats. The current study tests the hypothesis that the PPAR
agonist fenofibrate and the PPAR agonist rosiglitazone prevent the high salt (HS)-diet induced
increase in endogenous CO production and blood pressure in Dahl-S. Male DS were placed on
low (0.3% NaCl, LS) or HS (8% NaCl) diets and subsets received 90mg/kg/day fenofibrate in
the drinking water or 5mg/kg/day rosiglitazone IP. Respiratory CO excretion and blood pressure
were measured weekly via solid-phase gas chromatography and tail-cuff plethysmography,
respectively. CO excretion (HS: 0.570.07 mol/hr per kg), systolic blood pressure (HS:
1324 to 2515 mmHg) and plasma cholesterol levels (LS: 1136 vs. HS: 16310 mg/dL)
increased markedly in HS rats. Treatment with the PPAR agonist fenofibrate lowered plasma
cholesterol levels (1139mg/dL), and attenuated the HS diet induced increase in CO excretion
(0.070.01 mol/hr per kg) and blood pressure (1454 to 1728 mmHg). However, the infusion attenuated the reductions of HR associated with the pressor responses to ET-1. We
PPAR agonist rosiglitazone did not alter metabolic parameters, and did not protect from the
HS diet induced increase in CO excretion (0.480.01 mol/hr per kg) or blood pressure
(1426 to 23711 mmHg). These data show that a PPAR, but not PPAR agonist protects
from HS diet-induced increases in CO production and blood pressure in Dahl-S. Our results
suggest that PPAR agonists might exert their blood pressure lowering effect by normalizing
endogenous CO production in Dahl-S.
P124
Hyperglycemia Impairs Functional Vasodilation via Enhanced Thromboxane
Receptor-Mediated Vasoconstriction in Obese Zucker Rats
Lusha Xiang, Jay S Naik, Sean R Abram, Robert L Hester; Mississippi Univ Med Cntr,
Jackson, MS
Individuals with metabolic syndrome or Type 2 diabetes exhibit impaired exercise performance
and functional vasodilator response. We have shown that arachidonic acid (AA) metabolites are
important in functional vasodilation and there is an increase in thromboxane in Type 2 diabetes.
We hypothesize that hyperglycemia impairs functional vasodilation via increased thromboxane
CHBPR ConferencePoster Presentations e69
receptor-mediated vasoconstriction. We tested this hypothesis by determining if decreasing
blood glucose levels in Obese Zucker rats (OZR) with rosiglitazone (ROZ) would improve the
functional hyperemic response. 11 wk old male lean and OZR (n 5/group) were fed normal
rat chow or rat chow containing ROZ (5mg/kg/day) for 2 wks. Arcade arterioles in the
spinotrapezius muscle were then prepared for microcirculatory studies. Arteriolar diameter was
measured following muscle stimulation and AA application. Experiments were performed in the
absence and presence of the thromboxane receptor antagonist SQ29548 (SQ). OZR exhibited
significantly higher plasma insulin and fasting glucose levels compared with lean controls
(insulin; 302 78 pg/ml vs. 91 14 pg/ml; glucose; 154 7 mg/dl vs. 106 2 mg/dl for
LZR and OZR, respectively). Functional and AA-induced dilation were impaired in OZR. ROZ
normalized plasma insulin and glucose levels and partially restored the vasodilatory responses
in OZR with no effect in lean animals (Fig 1). SQ improved the vasodilator responses in obese
controls with no effect in lean controls or ROZ treated animals (Fig 1). These results suggest
that the impaired functional dilation in OZR is due in part to hyperglycemia-induced
thromboxane receptor-mediated vasoconstriction.
P125
Chronic Infusion of Interleukin-6 May Impair Baroreflex Control of Heart
Rate but Does not Alter Acute Pressor Responses to Endothelin-1 in Mice
Erika I Boesen, David M Pollock; Med College of Georgia, Augusta, GA
Both interleukin-6 (IL-6) and endothelin-1 (ET-1) have been implicated in hypertension and
cardiovascular disease. Although IL-6 stimulates ET-1 production in vitro, it is not known
whether IL-6 contributes to the pressor effects of ET-1 in vivo. We therefore tested whether
acute pressor responses to ET-1 (0.1, 0.3, 1 and 3 nmol/kg i.v. boluses via jugular vein) were
altered in isoflurane-anesthetized mice in the absence of endogenous IL-6 (wild type, WT vs
IL-6 knockout, IL-6 KO; n 8 & 8) or following a 14-day infusion of IL-6 in WT mice (vehicle
or IL-6 at 16 ng/h s.c.; n 8 & 6). Mean arterial pressure (MAP) and heart rate (HR), measured
via a carotid artery catheter, were averaged over the last 8 min of 10 min response periods
following administration of each dose of ET-1, and responses calculated as change in MAP
(MAP) or HR (HR) from pre-ET-1 baseline measurements. Baseline MAP and HR were not
significantly different in WT and IL-6 KO mice. ET-1 dose-dependently increased MAP (P
0.001) and decreased HR (P 0.01) in WT and IL-6 KO mice, but the responses did not differ
significantly between the two genotypes (e.g. at 3 nmol/kg MAP 34 3 & 38 2 mmHg
and HR -67 24 & -48 23 in WT and IL-6 KO mice respectively). Baseline MAP and
HR were not significantly different in vehicle- and IL-6-treated mice. Vehicle- and IL-6-treated
mice displayed dose-dependent increases of MAP in response to ET-1 (P 0.001), but these
responses were similar in both groups (e.g. at 3 nmol/kg MAP 32 4 & 28 3 mmHg
in vehicle- and IL-6-treated mice respectively). There were dose-dependent reductions of HR
(P 0.01), but the reductions were significantly attenuated in IL-6-treated mice (P 0.05;
e.g. at 3 nmol/kg HR -77 26 & -17 20 bpm in vehicle and IL-6-treated mice
respectively). Thus acute pressor responses to ET-1 in anesthetized mice were not altered in
the absence of IL-6 or by chronic infusion of IL-6. The reductions of HR accompanying the
acute pressor responses were also not altered in the absence of IL-6. In contrast, chronic IL-6
conclude that while IL-6 does not appear to alter pressor responsiveness to ET-1, chronic
elevations of plasma IL-6 may lead to impairment of the baroreflex control of HR.
P126
Inducible cAMP Early Repressor Mediates Beraprost-Mediated Growth
Suppression of Vascular Smooth Muscle Cell
Hideki Ohtsubo; Kyushu Univ Graduate Sch, Fukuoka, Japan
cAMP-response element-binding protein (CREB) family transcription factors are composed of
CREB, cAMP response element modulator (CREM) and activating transcription factor-1.
Inducible cAMP early repressor (ICER), an isoform of CREM, is a transcriptional repressor which
has a DNA binding domain but lacks a transcriptional activation domain. Beraprost, a
prostaglangins I2 analogue, is clinically used in the treatment of pulmonary hypertension and
arteriosclerosis obliterans. In this study, we examined the effect of beraprost on platelet-
derived growth factor (PDGF) -induced vascular smooth muscle cells (VSMC) proliferation.
Previously, PDGF has been reported to have an association with proliferative response of
pulmonary artery vascular smooth muscle cells in hypoxia-induced pulmonary hypertension.
 e70 Hypertension Vol 48, No 4 October 2006
Semi-quantitative RT-PCR and Western blot analysis showed that expression of ICER was
increased in beraprost-stimulated VSMC in a time- and dose-dependent manner. The peak
induction was observed after 3 hours of stimulation. The induction of ICER was inhibited by
pretreatment with H89, a protein kinase A (PKA) inhibitor, suggesting that PKA mediates the
ICER expression. Beraprost suppressed PDGF-induced thymidine incorporation in VSMC.
Transfection of short interfering RNA for ICER, but not scramble RNA, reversed the
beraprost-induced suppression of VSMC proliferation by PDGF. In vivo study, overexpression of
ICER by an adenovirus vector attenuated neointimal formation (intima/media ratio) by 50%
(n6, p0.017) in balloon-injured rat carotid artery, compared with overexpression of LacZ.
In ICER transduced artery, TUNEL positive cells were increased and Ki67 positive cells were
decreased. These results suggest that ICER induces apoptosis and inhibits proliferation in
VSMC, and plays a critical role in beraprost-mediated suppression of VSMC proliferation by
PDGF. Therefore, ICER may be an important negative regulator of VSMC growth and effective
therapeutic tool to treat vascular proliferative disease.
P127
Role of Endothelin Receptor Antagonism on Combined Effects of Age and
Salt-Loading on Endothelial Function, Vascular Remodeling and Oxidative
Stress in a Murine Transgenic Model of Endothelial Cell Human
Endothelin-1 Overexpression
Farhad Amiri, Eun A Ko, Danesh Javeshghani, Lady Davis Institute for Med Rsch, Montreal,
Canada; Timothy L Reudelhuber, Clinical Rsch Institute of Montreal, Montreal, Canada;
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
Ernesto L Schiffrin; Lady Davis Institute for Med Rsch, Montreal, Canada
We have previously shown that chronic salt loading in aged transgenic (TG) mice with
endothelial cell overexpression of human prepro-endothelin (ET)-1 exhibit endothelial dysfunc-
tion, increased vascular NAD(P)H oxidase activity and hypertrophic remodeling of mesenteric
resistance vessels as compared to salt-loaded non-transgenic (WT) littermates. We now have
investigated the role of ET receptor antagonism on vascular function and structure, and
oxidative stress in salt-loaded TG and WT mice. Ten-month old male TG and WT littermates
were salt-loaded (4% NaCl), and treated with ETA antagonist (ABT 627, 5mg/kg/day), ETB
antagonist (A-192621, 30mg/kg/day) or ETA/B (Bosentan, 100mg/kg/day) for 4 weeks. BP was
measured by radiotelemetry, mesenteric resistance artery vascular reactivity was studied on a
pressurized myograph, and vascular NAD(P)H oxidase activity by lucigenin chemiluminescence.
Salt-loaded TG had significantly increased BP compared to WT, which was prevented by ETA
and dual ETA/B antagonism but further increased by ETB antagonism. Increased media/lumen
ratio of TG mice was only significantly decreased by dual ETA/B antagonism (P0.01) while no
differences were seen in media cross-sectional area. Impaired maximal relaxation to
acetylcholine (Ach) was significantly prevented with ETA and ETA/B antagonisms (P0.05), while
l-NAME-induced reduction of Ach maximal relaxation was partially prevented by ETA
antagonism, completely prevented by dual ETA/B antagonism and was partially restored by
vitamin C pre-incubation following dual ETA/B antagonism but did not reach levels found in WT
mice. The abolished ET-1 contractile response in salt-loaded TG mice was partially restored by
ETA antagonism and completely prevented by dual ETA/B antagonism. Increased NAD(P)H
oxidase activity of TG mice was further increased by ETB antagonism but was returned to WT
levels by either ETA and ETA/B antagonisms. In conclusion, our results demonstrate the
detrimental role of ETA receptors and the protective effects of ETB receptors on endothelial
dysfunction, altered vascular structure and increased oxidative stress of mesenteric resistance
vessels of chronically salt-loaded aged TG mice with endothelial cell human ET-1 overexpres-
sion.
P128
Effect of Neuropeptide Y (Y1) Inhibition on Differential Neural Control of
Glomerular Capillary Pressure
Kate M Denton, Rebecca L Flower, Gabriela Eppel, Monash Univ, Melbourne, Australia;
Geoff A Head; Baker Heart Institute, Melbourne, Australia
We have identified two morphologically distinct populations of nerves within the kidney, which
are differentially distributed to the renal afferent and efferent arterioles. TYPE I nerves almost
exclusively innervate the afferent arteriole whereas TYPE II nerves are distributed equally on the
afferent and efferent arterioles. We have also demonstrated that TYPE II nerves are
immuno-reactive for neuropeptide Y whilst TYPE I nerves are not. The aim of the current study
was to determine if the renal response to nerve stimulation in the presence of an NPY-Y1
receptor blocker was compatible with the known distribution of TYPE II-NPY positive renal
nerves. In anesthetised rabbits, renal micropuncture studies were performed. Measurements
were made before and after stimulation of the renal nerves (1.5Hz, 8V, 2ms) in the presence
of the NPY-Y1 receptor inhibitor (BIBO3304TF; n6) or vehicle (n6). Renal blood flow fell less
in response to nerve stimulation in the presence of NPY-Y1 receptor inhibition as compared to
vehicle, falling 254% vs 353%, respectively (P0.05). This was due to lesser increases in
both pre-glomerular (759% vs 467%; P0.05) and post-glomerular (487% vs236%;
P0.05) vascular resistance, in the group treated with the NPY-Y1 inhibitor. In conclusion, NPY
contributed to the renal vascular response to nerve stimulation. The renal segmental resistance
pattern of response was compatible with a reduction in the activation of TYPE II NPY containing
nerves. Evidence compatible with our hypothesis that glomerular capillary pressure, and hence
GFR, can be selectively altered by differential activation of separate populations of renal nerves.
P129
Norepinephrine Potentiates the PGE2 Mediated Release of Substance P
from Renal Sensory Nerves
Ulla C Kopp, Michael Cicha, Lori Smith; Univ Iowa Carver Col Medicine & VAMC, Iowa City,
IA
Activation of sensory nerves in the renal pelvic wall increases PGE2 which activates EP4
receptors on the pelvic sensory nerve endings resulting in activation of cAMP/PKA and
Ca-mediated release of substance P (SP). SP leads to increased afferent renal nerve activity
(ARNA) which causes a reflex decrease in efferent renal sympathetic nerve activity (ERSNA) and
natriuresis, a renorenal reflex. In the pelvic wall, sympathetic nerves are close to SP-containing
sensory nerves. Our previous studies showed that reflex increases and decreases in ERSNA
produce parallel changes in ARNA which together with the renorenal reflexes provides a
negative feed back loop. The interaction between ERSNA and ARNA is mediated by
norepinephrine (NE). NE increases and decreases SP release via activation of 1- and
2-adrenoceptors, respectively. Because NE increases PGE2 synthesis by activation of 1
adrenoceptors in the central nervous system and PGE2 is essential for SP release, we examined
whether the NE-induced activation of renal sensory nerves is dependent on intact PG-synthesis.
In anesthetized rats (n9), NE, 10 pM, increased ARNA 2800604, 17772 and 2743
1136%sec (area under the curve of ARNA vs. time) in the presence of pelvic perfusion with
vehicle, indomethacin and vehicle. The reduction produced by indomethacin was 914%. The
EP4 receptor antagonist L161982 produced a similar reduction of the ARNA response to NE,
914% (N9). To examine if the interaction between NE and PGE2 occurred at the peripheral
sensory nerve endings, the effects of NE on SP release was examined in vitro using an isolated
renal pelvic wall preparation. Adding NE at 10 and 50 pM to the incubation bath failed to
increase SP release. NE, 250 pM, resulted in a small increase, from 7.51.3 to 11.72 pg/min
(n7). However, in the presence of a PGE2, 0.03 M (subtreshold concentration for SP
release), NE, 10 pM, resulted in a marked increase in SP release, from 8.51.7 to 19.62.9
pg/min (n7). Likewise, NE, 10 pM, potentiated the release of SP produced by PGE2, 0.03 M.
The NE-induced release of SP (in the presence of PGE2) was reduced by the EP4 receptor
antagonist. Conclusion: NE enhances the PGE2-mediated release of SP by increasing PGE2
synthesis and/or enhancing the EP4 receptor mediated activation of cAMP.
P130
Distinct Populations of Dorsal Root Ganglion Neurons Account for the High
Sensitivity of CGRP Containing Renal Afferent Nerve Fibers
Peter Linz, Claudia Heersink, Gisa Tiegs, Eva Vogel, Kerstin Amann, Karl F Hilgers, Roland
Veelken; Univ Erlangen, Erlangen, Germany
The neurogenic peptide CGRP was recently linked to kidney damage in hypertension. CGRP is
locally released from peptidergic sensory afferent nerve fibers also transducing information to
the central nervous system. The receptors involved in the stimulation of sensory neurons in the
kidney cortex are not well investigated, afferent fibers were mainly studied in the renal pelvis.
We tested the hypothesis that the stimulation of a distinct population of very sensitive dorsal
root ganglion neurons (DRGN) with afferent input from the renal cortex depends on TRPV1
[capsaicine] receptors associated with these fibers. Rat DRGN with axons from the kidney were
identified by retrograde labelling and studied by patch-clamp techniques. DRGN with hind limb
afferents were studied for comparison. Renal DRGN showed significantly higher amplitudes of
acid induced transient and sustained currents than non-renal DRGN; transient: (15.99 5.05
pA/pF vs 0.36 0.17* pA/pF, mean SEM at pH 6) sustained: (3.67 1.13 pA/pF vs 0.64
0.14* pA/pF at pH 6 and 20,04 4,51 pA/pF vs 6,22 1,18* pA/pF at pH 5) (* p 0.05,
n 25). The TRPV1 receptor antagonist capsazepine inhibited the sustained, amiloride the
transient responses suggesting involvement of Acid Sensing Ion Channels (ASIC). In this
respect, small-capacitance renal DRGN (80pF) showed no ASIC- or TRPV1-currents wheras
larger-capacitance renal DRGN (80pF) exhibited strong currents which were significantly
higher than in DRGN with hind limb axons. Confocal microscopy revealed that renal vessels,
tubuli and glomeruli were associated with peptidergic (mainly CGRP) nerve fibers possessing
TRPV1 and ASIC receptors. The kidney cortex is innervated with sensory afferent neurons. A
distinct population of large-capacitance neuronal cells exhibit high sensitivity to stimulation
with protons. These neurons may play a pivotal role for the transduction of afferent input to the
central nervous system, and for the local release of peptides such as CGRP.
P131
The Autonomic Nervous System Contributes to Inflammation and Increases
C Reactive Protein
Cyndya Shibao, Alfredo Gamboa, Andre Diedrich, Ginnie Farley, Italo Biaggioni; Vanderbilt
Univ, Nashville, TN
Inflammation plays a significant role in the pathogenesis and progression of atherosclerosis,
and C- reactive protein (CRP) correlates with cardiovascular morbidity and mortality. To test the
hypothesis that sympathetic activation contributes to this inflammatory response, we measured
CRP in diseases characterized by varying degrees of sympathetic failure or activation: 10
patients with pure autonomic failure (PAF) with virtual absence of sympathetic tone due to
peripheral sympathetic denervation, 10 normal subjects, 6 patients with multiple system
atrophy (MSA) with residual sympathetic tone but loss of central autonomic modulation, 13
patients with hyperadrenergic postural tachycardia syndrome (POTS) but no vascular disease,
and 10 obese subjects known to have increased sympathetic activity. Plasma norepinephrine
levels were 9518, 27432, 16722, 4116 and 25735 pg/ml in PAF, normals, MSA,
POTS and obese, respectively, p0.001). Low frequency blood pressure variability, a measure
of sympathetic vasomotor regulation, was 1.80.5, 40.9, 30.6, 6.31.2 and 9.42.2
mmHg2 in PAF, normals, MSA, POTS and obese, p0.003). Sympathetic activation was
evidenced in obese by greater muscle sympathetic nerve activity (333.9 vs. 191.9 in
normals, p0.007). CRP was lower in PAF patients (0.70.12 mg/dl vs. 0.90.15 and
1.20.26 for controls and MSA) and greater in POTS and obese (2.30.8 and 30.6 mg/dl,

respectively (P0.035, Figure). We found a modest but positive correlation between CRP and
plasma NE (R0.30; p0.04). In conclusion, the sympathetic nervous system activity may
contribute to inflammation and increases C reactive protein levels.
P132
Endothelin B Receptor Activation Mediates the Enhanced Pressor Response
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to Environmental Stress during Endothelin A Receptor Blockade in Dahl
Salt-Resistant Rats
Gerard DAngelo, Jennifer S Pollock, David M Pollock; Med College of Georgia, Augusta, GA
We have previously shown that ETA receptor blockade potentiates stress-induced increases
in arterial pressure and norepinephrine (NE) release, suggesting that ETB receptor
activation contributes to these responses. We therefore tested the hypothesis that ETB
receptor activation mediates the elevated pressor response and NE release during ETA
receptor antagonism. Stress was induced once per week over 3 weeks by restraint and
administration of air jet pulses (3 min) in rats maintained on a normal salt diet (n 79).
In random order, animals were untreated (CON), or given either a selective ETA receptor
antagonist (ABT-627; 5 mg/kg/day in the drinking water) or a mixed ETA/B receptor
antagonist (SB-209670; 2.5 mg/kg i.p., b.i.d.). Blood samples were obtained using
indwelling venous catheters; basal and stress-induced increases in catecholamines were
measured by RIA. In telemetry-instrumented animals, ABT-627 augmented the total
pressor response (area under the curve) to air jet stress (44.03.1 vs. 23.14.2 mmHg
x 3 min, ABT-627 vs. CON, p0.05). Blockade of both ETA and ETB receptors with
SB-209670 reduced the integrated pressor response compared to ETA receptor blockade
alone such that it was not significantly different from CON (34.42.1 mmHg x 3 min,
p0.05 vs. ABT-627). Neither ABT-627 nor SB-209670 had an effect on baseline
epinephrine, but both ETA (38175 pg/ml) and mixed ETA/B (40045 pg/ml) blockade
reduced the stress-induced rise compared to CON (54872 pg/ml; p0.05 vs. ABT-627
and SB-209670). Conversely, baseline NE was significantly elevated by both ABT-627
(30148 pg/ml) and SB-209670 (25923 pg/ml) compared to CON (16924 pg/ml;
p0.05). Selective ETA receptor antagonism heightened the stress-mediated NE release
(66754 vs. 41442 pg/ml, ABT vs. CON; p0.05) but mixed ETA/B receptor blockade had
no effect on the stress-induced response (37660 pg/ml; NS vs. CON; p0.05 vs. ABT)).
These data suggest that stimulation of neuronal ETB receptors amplifies NE release during
behavioral stress. We hypothesize that heightened activation of ETB receptors on
sympathetic nerves during ETA receptor blockade contributes to the augmented pressor
response to acute stress in DR rats.
P133
Endothelial, but not Neural Nitric Oxide Contributes to Blood Pressure
Regulation in Normal Subjects
Alfredo Gamboa, Cyndya Shibao, Andre Diedrich, Ginnie Farley, Sachin Y Paranjape, Italo
Biaggioni; Vanderbilt Univ, Nashville, TN
The increase in blood pressure (BP) induced by systemic nitric oxide synthase (NOS)
inhibition in humans is the result of blockade of endothelially-derived and neurally-derived
nitric oxide, restrained by baroreflex buffering capacity (eNOnNO-Baro). We have
previously estimated the selective contribution of eNO to blood pressure regulation by
measuring the increase in BP induced by iv L-NMMA (NOS inhibitor) in the absence of
autonomic function; we used the ganglionic blocker trimethaphan to eliminate baroreflex
buffering and the contribution of nNO which depends on autonomic interactions. Systolic
BP increased 92 mmHg with 250 g/kg/min iv L-NMMA in autonomically intact
(eNOnNO-Baro), and 252 mmHg in autonomically blocked subjects (selective eNO
blockade, n10, Fig). Baroreflex buffering capacity can be estimated in each subject by
the potentiation of the pressor response to phenylephrine produced by autonomic
blockade. The magnitude of this potentiation was used to predict the increase in BP
L-NMMA should have produced if only the baroreflex was eliminated (eNOnNO). A
PREDICTED BP increase greater than the experimentally OBSERVED would imply a
contribution of nNO to BP regulation. However, we found no significant difference between
the OBSERVED and PREDICTED increases in BP produced by L-NMMA (fig). Our results
suggest that eNO but not nNO contributes significantly to BP regulation in normal humans,
and are in accordance to animal studies that show that eNOS deficient mice have a
significant increase in baseline blood pressure, whereas mice lacking nNOS do not. This
CHBPR ConferencePoster Presentations e71
approach can be used to estimate the contribution of eNO and nNO to BP regulation in
disease states.
P134
Norepinephrine Transporter mRNA is Present in the Heart and Stellate
Ganglion and is Differentially Regulated in Hyperternsion
Erica A Wehrwein, Gregory D Fink, David L Kreulen; Michigan State Univ, East Lansing, MI
Hypertension is associated with a reduction in reuptake of norepinephrine (NE) in the heart and
increased spillover of NE into the cardiac circulation. NE transporter (NET) is responsible for
reuptake of released NE from the neuroeffector junction. Esler has suggested that epigenetic
regulation of NET occurs in human hypertension by repression of transcription by DNA
methylation. The purpose of this study was to determine if altered regulation of NET gene
expression occurred in hypertension in the heart and stellate ganglia. Since NET function and
protein are decreased in the hypertensive heart, it was hypothesized that a reduction of NET
mRNA would also be observed. This study reports the novel finding of NET mRNA in all
chambers of the heart and confirms its presence in the right and left stellate ganglion.
Furthermore, real time PCR results show that the amount of NET mRNA is 35,000-fold higher
in the ganglia than in any heart chamber (p0.05). NET mRNA abundance in heart chambers
does not correlate to a standard index of innervation density (r.22, p0.05). NET mRNA was
reduced across all chambers to varying extents by chemical sympathectomy with
6-hydroxydopamine (32.3%99% reduction, p0.05, for left ventricle, ventricular septum, and
right ventricle), supporting the presence of mRNA in sympathetic nerve terminals. In
uninephrectomized, deoxycorticosterone (DOCA)-salt hypertensive animals, NET mRNA was
unchanged in the left atrium and right ventricle (p0.05) but was significantly higher in the
right atrium (54-fold higher, p0.05) and left ventricle (6.36-fold higher, p0.05). NET mRNA
was unchanged in the right and left stellate ganglia (p0.05) indicating a separation of genetic
regulation of NET mRNA in the heart and ganglia. In summary, this is the first report of NET
mRNA in the heart. It is present in all chambers and is differentially regulated in chronic
hypertension across heart chambers. The increase in NET mRNA in some heart chambers
corresponds to a decrease in NET protein in the same animal model, possibly serving as a
compensation mechanism for the loss of NET protein function in hypertension.
P135
Presence of Growth-Associated Protein 43 and Calcium Sensing Receptor
in Adventitial Neuronal Somata (ANNIES) and Perivascular Nerves of the
Adult Rat Mesenteric Branch Artery
Chandra Somasundaram, Richard D Bukoski, North Carolina Central Univ, Durham, NC;
Debra I Diz; Wake Forest Univ Sch of Medicine, Winston-Salem, NC
We recently demonstrated adventitial neuronal somata (ANNIES) expressing CGRP in the adult
rat mesenteric branch artery (MBA). The growth-associated protein 43 (GAP43) is a neuronal
membrane protein involved in axonal growth and regeneration as well as in the modulation of
synaptic plasticity. Gap43 is reported to present in subsets of sensory and sympathetic neurons
and fibers innervating the mature rat vasculature.The calcium-sensing receptor (CaSR) protein,
a receptor that senses changes in extracellular Ca(2), is also present in perivascular sensory
nerves of mouse MBA. In the present study, we investigated whether GAP43 and the CaSR are
also present in the ANNIES as well as perivascular nerve fibers of adult rat MBA. Segments of
MBA (n 5) were fixed in buffered formalin and immunostained with polyclonal anti-Gap43
and anti-CaSR, using secondary antibodies tagged with alexa fluor 647, as well as the nuclear
stain sytox to identify the ANNIES. Confocal analysis showed that Gap43 was strongly
distributed in 30% of the perivascular nerve fibers whereas the CaSR was present in most
fibers, comparable to CGRP. The CaSR was present in ANNIES (94 6 % of the total number
of ANNIES in a given field; n 4). GAP43 was present in 37 4% of ANNIES (n 4).
Immunoblots from MBA also confirmed the presence of Gap43 protein (n 3). The presence
of GAP43 in ANNIES of the rat MBA indicates that ANNIES show features of sensory neurons
of the dorsal root ganglion (DRG) undergoing responses to stress/injury, given that the
expression of GAP43 in DRG cells is increased in response to injury. The additional finding that
the CaSR is associated with ANNIES suggests these cells may participate in the regulation of
myogenic tone. Support: HL64761 *deceased
 e72 Hypertension Vol 48, No 4 October 2006
P136
Vascular and Neuronal Oxidative Stress is Increased during Chronic ETB
Receptor Activation in Conscious Rats
Melissa W Li, Xiaoling Dai, Janice M Thompson, Stephanie W Watts, Gregory D Fink;
Michigan State Univ, East Lansing, MI
Our lab has shown that 5-day iv infusion of the endothelin ETB receptor agonist sarafotoxin 6c
(S6c) into normal rats causes a significant increase in arterial pressure. Increased oxidative
stress is found in many tissues in hypertension and may be a cause of increased blood
pressure. Since ETB receptors are located in both vascular smooth cells and sympathetic
neurons, the goal of this study was to assess superoxide (O2-, a pro-oxidant molecule) levels
in sympathetic ganglia and blood vessels in S6c-induced hypertension. The splanchnic bed
accounts for a big portion of vascular capacitance and is important in blood pressure regulation;
therefore we examined sympathetic nerves and blood vessels in this region. We speculated that
chronic ETB receptor activation increases neuronal and vascular oxidative stress. All rats were
instrumented with arterial and venous catheters and housed in metabolism cages. Treated (T)
rats (n7) received saline (vehicle, iv) for 2 days (control), then S6c (5 pmol/kg/min) for 5 days
(infusion). SHAM (S) rats (n7) received only saline for 7 days. Mean arterial pressure (MAP)
was recorded at the end of infusion period. Venous and arterial O2- levels were evaluated by
lucigenin-enhanced chemiluminescence and dihydroethidium (DHE)-fluorescence. O2- concen-
trations in inferior mesenteric ganglion (IMG) were measured by DHE-fluorescence. MAP was
increased significantly in T rats (average of 115 2 mmHg) during S6c infusion when
compared to the average of S rats (100 1 mmHg). DHE studies showed that O2- levels of IMG
in T rats were significantly higher than those in S (3.63:1, relative fluorescence). Both DHE and
lucigenin experiments demonstrated increased vascular O2- levels in T rats compared to S (DHE:
1.54 in arteries, 1.94 in veins; Lucigenin: significant increase in arteries (0.067 0.014
nmol/min/mg tissue (T) vs. 0.040 0.002 (S)), non significant in veins (0.168 0.029 vs.
0.116 0.031)). These data indicate that vascular and neuronal oxidative stress in the
splanchnic bed is significantly enhanced during chronic activation of ETB receptors in conscious
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
rats. Neuronal oxidative stress is increased more than vascular oxidative stress, suggesting an
important involvement of sympathetic nerves in S6c-induced hypertension.
P137
Role of AT2 Receptor in Cardiac Function and Remodeling Post-MI in Mice
with Cardiac Overexpression of Angiotensin II
Jiang Xu, Oscar A Carretero, Chun-xia Lin, Edward G Shesely, Henry Ford Hosp, Detroit, MI;
Timothy L Reudelhuber, Clinical Rsch Institute of Montreal, Montreal, Canada; Xiao-Ping
Yang; Henry Ford Hosp, Detroit, MI
Angiotensin II (Ang II) acting on type 1 receptors (AT1) causes hypertension and cardiac
hypertrophy, whereas activation of the AT2 receptor, which stimulates release of kinins and
nitric oxide (NO), may have an anti-hypertrophic and cardioprotective effect. We previously
showed that overexpression of angiotensin II (Ang II) in the heart (Tg-AngII) caused cardiac
hypertrophy and fibrosis and promoted cardiac dysfunction and remodeling post-myocardial
infarction (MI) without a hemodynamic effect. Here we studied whether blockade of AT2
enhances the detrimental effect of Ang II in Tg-Ang II mice. Male 1214-week-old Tg-Ang II
mice and their wild-type littermates (WT) were subjected to MI or sham-MI with or without
AT2-ant (PD123319 20mg/kg/day i. p. via miniosmotic pump) for 8 weeks. Systolic blood
pressure (SBP) was measured by tail cuff. LV ejection fraction (EF) and LV mass were evaluated
by echocardiography. Infarct size (IS), myocyte cross-sectional area (MCSA) and interstitial
collagen fraction (ICF) were studied histopathologically. We found that SBP was similar between
strains and was not influenced by AT2-ant with or without MI. AT2-ant did not affect IS, but
significantly increased cardiac rupture and mortality in Tg-Ang IIAT2-ant compared to Tg-Ang
IIVeh (73.5% vs 54.7%; p 0.05) and to WTAT2-ant (73.5% vs 45.5%, p 0.01). Cardiac
remodeling and dysfunction post-MI were more severe in Tg-Ang II mice and were further
worsened by AT2-ant (Table). However, in both strains with sham-MI, AT2-ant had no effect on
functional and morphological parameters. We concluded that locally produced Ang II worsens
cardiac remodeling and dysfunction post-MI via activation of AT1, whereas AT2 may counteract
AT1 and play a cardioprotective role in the heart.
Sham-MI MI
WT Tg-Ang II WT
Groups Veh AT2-ant Veh AT2-ant Veh AT2-ant
EF (%) 741 752 712 722 443 434 304*
mass 241 231 282* 271* 362 386 505*
(mg/10g
BW)
MCSA 1624 1626 1828* 1844* 2558 2576 31213*
(m2)
ICF (%) 5.50.1 5.40.2 6.40.3** 6.30.1** 7.50.2 7.70.2 8.70.2**
Veh: vehicle; WT: wild-type; AT2-ant: AT2 receptor antagonist; Tg: transgenic; : p 0.01 vs sham-MI within strain
and within treatment;*: p 0.05, **: p 0.01 vs WT within treatment; #: p 0.01 vs Veh within strain.
P138
The Isoproterenol-Induced Increase of Cardiac Ang II Levels Associated
with Heart Hypertrophy is Blunted in Rats Harboring an
Angiotensin-(17)-Producing Fusion Protein [TGR(A17)3292]
Ana Paula Nadu, Anderson J Ferreira, Federal Univ of Minas Gerais, Belo Horizonte, Brazil;
Timothy L Reudelhuber, Clinical Rsch Institute of Montreal, Montreal, Canada; Michael
Bader, Max-Delbruck-Cntr for Molecular Medicine, Berlin, Germany; Robson A Santos;
Federal Univ of Minas Gerais, Belo Horizonte, Brazil
Recently, we have found that chronic infusion of Ang-(17) in Wistar rats induces selective and
marked reduction of Ang II levels in the heart. In addition, we have shown that transgenic rats
(TGR) harboring an Ang-(17)-producing fusion protein [TGR(A17)3292] presented resistance
to heart hypertrophy induced by isoproterenol (ISO). In this study we determine whether this
resistance is associated with changes in the cardiac levels of Ang II. These animals present a
2.5-fold increase of circulating Ang-(17) as compared to normal rats. Heart hypertrophy was
induced in male Sprague-Dawley (SD) and TG rats by daily injection of isoproterenol (2 mg/Kg
i.p, 7 days). Control group received daily injection of vehicle (0.9 % NaCl 0.1 ml/100g i.p, 7
days). Plasma and cardiac Ang II levels were measured by radioimmunoassay. A fluorimetric
method was used to evaluate ACE activity in the heart, lung and plasma and its expression was
assessed by ribonuclease protection assay in the heart and lung. The cardiac Ang II level was
significantly decreased in the TG rats (0.118 0.01 pg/mg protein vs 0.162 0.01 pg/mg
protein in SD rats, n6). In contrast, the plasma Ang II levels were significantly increased in
TG animals. These findings were not associated with changes in ACE activity in the heart, lung
and plasma and in ACE expression in the heart and lung. In keeping with previous findings,
isoproterenol treatment induces an increase in the cardiac Ang II levels without changes in the
Ang II levels in the plasma. Strikingly, cardiac Ang II concentration did not change in
isoproterenol-treated TG rats (0.127 0.01, n6 vs 0.118 0.01 pg/mg protein, n7).
Plasma Ang II was also not altered. These results suggest that attenuation of isoproterenol-
induced hypertrophy in TG(A17)3292 rats is, at least partially, due to a decrease in the cardiac
Ang II levels. In addition, ours results reinforced the hypothesis that increases in Ang-(17)
levels selectively decrease cardiac Ang II levels.
P139
Molecular Mechanisms for the Regulation of Angiotensin Converting
Enzyme 2 by Angiotensin Peptides in Vascular Smooth Muscle Cells
Patricia E Gallagher, Carlos M Ferrario, E. Ann Tallant; Wake Forest Univ Sch of Medicine,
Winston-Salem, NC
Angiotensin converting enzyme 2 (ACE2) catalyzes the conversion of the vasoconstrictor
angiotensin II (Ang II) to the vasodilatory peptide angiotensin-(17) [Ang-(17)]. We previously
showed that treatment of hypertensive rats with the AT1 receptor antagonist olmesartan
increased ACE2 mRNA and protein in the aorta, suggesting that endogenous Ang II tonically
reduces ACE2. We now report that Ang II down-regulates ACE2 mRNA in rat aortic vascular
smooth muscle cells (VSMCs), to reduce the conversion of Ang II to Ang-(17) [from a Control
set at 1 to 0.46 0.04 relative gene expression, n 7, p 0.001]. Although Ang-(17) alone
had no effect on the transcriptional regulation of ACE2, it prevented the Ang II-mediated
reduction in ACE2 mRNA (0.99 0.12 relative gene expression with Ang II and Ang-(17), n
5, p 0.01 compared to Ang II alone). Inhibition of the Ang II-mediated reduction in ACE2
mRNA by Ang-(17) was blocked by the selective Ang-(17) receptor antagonist [D-Ala7]-Ang-
(17), indicating that the response was mediated by the AT(17) receptor. The reduction in ACE2
mRNA by Ang II was also blocked by the mitogen activated protein (MAP) kinase kinase (MEK)
inhibitor PD98059 [0.4 0.06 relative gene expression by Ang II compared to 0.95 0.05
relative gene expression by Ang II and PD98059, n 4]. Treatment of VSMCs with Ang II
increased ERK1/ERK2 activity, which was significantly reduced by pretreatment with Ang-(17).
The regulation of ERK activity by Ang-(17) was blocked by pretreatment with sodium
vanadate, a tyrosine phosphatase inhibitor, or okadaic acid, a serine/threonine phosphatase
inhibitor. Blockade of the Ang II-mediated reduction in ACE2 mRNA by Ang-(17) was also
prevented by pretreatment with either sodium vanadate or okadaic acid. These results suggest
that Ang-(17) activates a MAP kinase phosphatase to inhibit the Ang II-mediated increase in
ERK1/ERK2 activity and the reduction in ACE2 mRNA. Since ACE2 preferentially converts Ang
II to Ang-(17), down-regulation of the enzyme by Ang II constitutes a novel, positive,
feed-forward system. The modulatory role of Ang-(17) in the regulation of ACE2 by Ang II
suggests a complex interplay between the two peptides that is mediated by specific receptors
to activate distinct signaling pathways.
P140
Predominance of Angiotensin Converting Enzyme 2 to Cardiac
Angiotensin-(17) Production in [mRen2]27 Transgenic Hypertensive Rats
Aaron J Trask, David B Averill, Mark C Chappell, Carlos M Ferrario; Wake Forest Univ Sch
Tg-Ang II of Medicine, Winston-Salem, NC
Veh AT2-ant Previous studies have implicated reduced angiotensin-(17) [Ang-(17)] activity as a mecha-
nism facilitating hypertension-induced cardiac hypertrophy. The objective of the current study
243*
was to determine the role of angiotensin converting enzyme 2 (ACE2) in the generation of
593** Ang-(17) from angiotensin II (Ang II) in isolated hearts of 14 male [mRen2]27 transgenic
hypertensive [Tg()] and 20 male Sprague-Dawley normotensive control [Tg(-)] rats. Hearts
mounted on a Langendorff apparatus were perfused with Krebs solution for 60 minutes before
3239* and after addition of Ang II (10 nM). Ang-(17)-forming activity was assessed in the absence
and presence of an ACE2 inhibitor (MLN-4760, 1M) added to half of the hearts. ACE2 activity
9.40.2**# by HPLC and protein content by immunoblot were obtained in representative effluent samples
at the end of the 60-minute recirculation period. Production of Ang-(17) from Ang II was no
different in the hearts of Tg() (377.680.0 pM) and Tg(-) (389.543.2 pM) hearts over the
60-minute recirculation period. In contrast, the production of Ang-(17) from Ang II was
markedly reduced by 81% (70.918.1 pM, p0.01) after ACE2 inhibition only in Tg() rat
hearts. The ACE2 activity found in the Tg() rat effluent correlated significantly (r20.99,
p0.05) with protein content. This study revealed two important facets of Ang-(17)
production and documents for the first time a direct role for cardiac ACE2 in the production of
Ang-(17) from Ang II in the Tg() rats. First, cardiac ACE2 was crucial in the production of
Ang-(17) in Tg() hearts. Second, an active form of ACE2 was released into the effluent of Tg()
hearts. The predominance of Ang-(17)-forming activity by ACE2 in the hypertrophic hearts of
Tg() rats suggests that a deficit in both the endogenous cardiac generation of Ang-(17) and
the metabolism of Ang II contributes to the development of Ang II-dependent cardiac
hypertrophy in Tg() rats. The synthetic capacity of Ang-(17) in counteracting the growth-
promoting actions of Ang II in this renin-dependent model of hypertension is, at the very least,
significantly impaired. The failure of the ACE2 inhibitor to reduce cardiac Ang-(17) formation
in Tg(-) rats suggests that ACE2 is not a primary pathway for Ang II metabolism in normal hearts.

P141
Calcium-Inhibitable Adenylate Cyclase 5 Mediates Renin Release from
Isolated Juxtaglomerular (JG) Cells Induced by Decreased Intracellular
Calcium
Maria C Ortiz Capisano, Pablo A Ortiz, Jeffrey L Garvin, William H Beierwaltes; Henry Ford
Hosp, Detroit, MI
We have previously shown that decreasing intracellular calcium [Cai] in the JG cells increases
both cAMP formation and renin release. We hypothesized that this is due to a compartmen-
talized interaction between [Cai] and the calcium-inhibitable isoform of adenylate cyclase, AC5.
We used primary cultures of JG cells isolated from C-57/B6 mice at 70 80% confluence. The
AC agonist Forskolin (10uM) raised JG cell cAMP 90-fold from 7.6 1.8 to 581 51 pM/mg
(p0.02) and increased renin release from 203 43 to 443 106 ngAngI/ml/hr/mg
(p0.01). Reducing JG intracellular calcium [Cai] with 100 M of the cytosolic calcium chelator
BAPTA-AM stimulated both cAMP by 37.5 8.7 % (p0.05) and renin release by 42% from
175 17 to 249 25 ngAngI/ml/hr/mg (p0.05). The AC inhibitor SQ22,538 (400uM)
completely blocked both BAPTA-stimulated cAMP and renin release. An antibody that
recognizes both AC5 and AC6 (Santa Cruz) localized throughout the cytoplasm of permeabilized
JG cells. Labeling co-localized with renin containing granules (Swant). However, an antibody
specific to the N-terminus of only the AC6 isoform showed no labeling in JG cells. We conclude
that the stimulation of renin induced by lowering [Cai] is due to a permissive dis-inhibition of
the calcium-inhibitable isoform AC5, leading to an increase in cAMP. Supported by NIH RO-1
HL076469.
P142
Novel Expression of ACE2 in Adipose Tissue and Regulation by PPAR
Ligands and by High Fat Diets
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
Carine Boustany, Lisa Cassis; Univ of Kentucky, Lexington, KY
Previous studies demonstrate that adipose tissue expresses all of the components of the
renin-angiotensin system. Recently, angiotensin converting enzyme type-2 (ACE2) was
identified as a homologue of ACE. Both angiotensin (Ang) I and II are substrates for ACE2,
resulting in the production of Ang17, a vasodilating peptide. Thus, ACE2 activity not only
reduces concentrations of the vasoconstrictor AngII, but favors formation of the vasodilator
Ang17. The purpose of this study was to determine whether adipocytes express ACE2, and
to define mechanisms for regulation of ACE2 expression in adipose tissue. ACE2 expression
increased during the course of 3T3-L1 preadipocyte differentiation, and was positively
stimulated by treatment with pioglitazone (PIO) (ACE2/18s rRNA: Vehicle: 1.10 0.10, PIO:
2.70 0.02; P0.05). PIO also increased ACE2 expression in mature 3T3-L1 adipocytes. A
high level of ACE2 mRNA was expressed in white adipose tissue from C57BL/6 mice. To
determine the effect of high fat feeding on adipose ACE2 expression, C57BL/6 female mice
were fed a high fat diet (HF, 45% Kcal as fat) for 20 weeks. Control mice were fed normal
laboratory diet (chow). Mice fed the HF diet had greater body weights (BW) compared to control
(final BW: C57BL/6 chow: 28.4 1.8, C57BL/6 HF: 34.9 2.8 g; P0.05). Surprisingly,
systolic blood pressure (SBP) was decreased in HF fed mice from week 4 - 20 compared to
control (SBP on week 19: C57BL/6 chow: 126.1 9.1, C57BL/6 HF: 109.9 4.0 mmHg;
P0.05). ACE2 mRNA expression was markedly increased in adipose tissue from HF fed mice
(ACE2/18s rRNA: C57BL/6 chow: 0.19 0.01, C57BL/6 HF: 0.51 0.05; P0.05), but was
not altered in the kidney. Elevations in ACE2 mRNA expression in adipose tissue were
accompanied by a heightened expression and activity of PPAR (PPAR/18s rRNA: C57BL/6
chow: 0.51 0.05, C57BL/6 HF: 0.68 0.04, aP2/18s rRNA: C57BL/6 chow: 0.33 0.02,
C57BL/6 HF: 0.75 0.06; P0.05). These results demonstrate a high level of ACE2 expression
in adipose tissue, which is regulated by high fat feeding and by the thiazolidinedione PIO.
Importantly, positive regulation of ACE2 in adipose tissue by PIO may contribute to beneficial
effects of this agent in the treatment of hypertension and the metabolic syndrome. Supported
by NIH HL73085.
P143
Genetic Deletion of the Angiotensin-(17) Receptor Mas Causes a
Differential Fibrotic Profile of Extracellular Matrix Proteins in Both
Myocardium and Av Valves
Elisandra Gava, Robson Augusto Santos, Univ Federal de Minas Gerais, Belo Horizonte,
Brazil; Natalia Alenina, Michael Bader, Max-Delbruck-Cntr for Molecular Medicine, Berlin,
Germany; Laser Antonio Oliveira, Gregory T Kitten; Univ Federal de Minas Gerais, Belo
Horizonte, Brazil
The renin-angiotensin system plays a pivotal role in the biosynthesis of the ECM within the
heart. It has been recently shown that angiotensin (17) [Ang (17)] is an endogenous ligand
for the G protein-coupled Mas receptor. Ang (17) has been reported to modulate a number of
important heart functions, as well as affect ECM biosynthesis. In this study we investigated the
effects of genetic deletion of the Mas receptor on the expression and distribution pattern of
specific ECM proteins in adult and neonatal male mice hearts. Quantification of proteins
(collagen I, III, VI and fibronectin) was performed using immunofluorescence-labeling tech-
niques and confocal microscopy. Picrosirius red staining was used for the histological
assessment of the overall differences in collagen distribution. We observed that the relative
expression of several ECM proteins, i.e., type I collagen, type III collagen and fibronectin, were
significantly increased in the ventricles and atrioventicular (AV) valves of adult KO-mice hearts:
type I collagen (93.33 vs 51.39 in right ventricle WT, 69.00 vs 34.22 in tricuspid valve WT), type
III collagen (100.1 vs 53.27 in right ventricle WT and 86 vs 27.33 in tricuspid valve WT) and
fibronectin (78.40 vs 59.73 in right ventricle WT and 100.9 vs 69.93 in tricuspid valve WT).
Interestingly, the expression of type VI collagen was decreased (27.89 vs 52.78 in right
ventricle WT and 32.11 vs 87.56 in tricuspid valve WT). Neonatal KO-Mas mice hearts
presented similar patterns as observed in adults. No major differences were detected in the
atria. These observations suggest that the Mas receptor is involved in the selective expression
CHBPR ConferencePoster Presentations e73
of specific extracellular matrix proteins within both the ventricular myocardium and AV valves.
The profile observed may contribute to the decreased cardiac performance observed in KO-Mas
mice.
P144
Immune Mechanisms Promote Kidney Injury in Angiotensin II-Dependent
Hypertension
Steven D Crowley, Samantha K Gould, Duke Univ Med Cntr, Durham, NC; Phillip Ruiz, Univ
of Miami Med Cntr, Miami, FL; Robert Griffiths, Thomas M Coffman; Duke Univ Med Cntr,
Durham, NC
The capacity of the renin angiotensin system (RAS) to promote kidney injury is highlighted by
the efficacy of RAS blockade in slowing the progression of chronic kidney disease. Clinical trials
suggest that the degree of renal protection provided by RAS blockade cannot be explained by
BP reduction alone, and may also involve inhibition of injurious cellular effects of angiotensin
II (Ang II). Stimulation of immune responses by Ang II is one potential BP-independent pathway
for tissue damage. To examine this pathways role, we studied the actions of the
immunosuppressive agent mycophenolate mofetil (MMF) on renal pathology during Ang
II-dependent HTN. Wild-type 129SVE mice were uni-nephrectomized and then infused with Ang
II (1000ng/kg/min) for 4 weeks. During the infusion, mice were fed a 6% NaCl diet. MMF
(100mg/kg) or vehicle (Veh) were administered daily by gavage. Ang II infusion increased BP
significantly and similarly in both groups: 23311 mm Hg at 3 weeks in the Veh group and
2428 mm Hg in the MMF group. Despite similar levels of HTN, urinary albumin excretion
tended to be lower in the MMF vs. the Veh group at days 12 (9.42.5 vs. 15.33.9 mg alb/
mg Cr) and 25 (15.43.6 vs. 21.74.4 mg alb/ mg Cr) of Ang II infusion. Moreover, MMF
significantly attenuated renal pathological injury associated with Ang II infusion. At the end of
the 4-week infusion, semi-quantitative scores for the severity of overall kidney pathology were
reduced by 30% in the MMF vs. the Veh group (10.00.6 vs. 14.21.4 arbitrary units (au);
p0.03). Injury scores trended lower with MMF vs. Veh in the glomerular (1.80.3 vs.
2.20.2 au) and vascular (1.70.2 vs. 2.20.5 au) compartments and were significantly
reduced in the tubulointerstitial compartment (6.50.3 vs. 9.80.9 au; p0.005). Further-
more, mononuclear cell infiltrates were significantly reduced in the MMF vs. the Veh kidneys
(2.80.3 vs. 4.40.5 au; p0.04). The extent of tubular casts was also significantly reduced
in the kidneys of the MMF group vs. controls (1.70.2 vs. 2.60.3 au; p0.03), consistent
with an anti-proteinuric effect of MMF. In summary, immunosuppression with MMF reduces
kidney damage in a mouse model of Ang II-dependent HTN. Our findings suggest that Ang II
promotes renal injury by stimulating local immune responses.
P145
Renal Cortical Cox-2 Expression is Differentially Regulated by Angiotensin
II AT1 and AT2 Receptors
Ming-Zhi Zhang, Bing Yao, Hui-Fang Cheng, Su-Wan Wang, Tadashi Inagami, Raymond C
Harris; Vanderbilt Univ, Nashville, TN
Macula densa cyclooxygenase-2 (COX-2)-derived prostaglandins serve as important modula-
tors of the renin-angiotensin system, and cross-talk exists between these two systems. Cortical
COX-2 induction by ACE inhibitors or AT1 receptor blockers (ARBs) suggests that angiotensin
II may inhibit cortical COX-2 via stimulating AT1 receptor pathway. In the present studies, we
determined that chronic infusion of either hypertensive or nonhypertensive concentrations of
angiotensin II attenuated cortical COX-2. Angiotensin II infusion reversed cortical COX-2
elevation induced by ACE inhibitors. However, we found that angiotensin II infusion further
stimulated cortical COX-2 elevation induced by ARBs, suggesting a potential role for an AT2
receptor-mediated pathway when the AT1 receptor is inhibited. Both wild type and AT2 receptor
knockout mice were treated for 7 days with either ACE inhibitors or ARBs. Cortical COX-2
increased to similar levels in response to ACE inhibition in both knockout and wild type mice.
In wild type mice, ARBs increased cortical COX-2 more than ACE inhibitors, and this stimulation
was attenuated by the AT2 receptor antagonist, PD123319. In the knockout mice, ARBs led to
significantly less cortical COX-2 elevation, which was not attenuated by PD123319. PCR
confirmed AT1a and AT2 receptor expression in the cultured macula densa cell line, MMDD1.
Angiotensin II inhibited MMDD1 COX-2, and CGP42112A, an AT2 receptor agonist, stimulated
MMDD1 COX-2. In summary, these results are the first demonstration that macula densa
COX-2 expression is oppositely regulated by AT1 and AT2 receptors and suggest that AT2
receptor-mediated cortical COX-2 elevation may mediate physiologic effects that modulate
AT1-mediated responses.
P146
Renal Artery Systolic Translesional Pressure Gradient Predicts Renin
Activation in Subjects with Renal Artery Stenosis
Michael J Gillespie, Samuel Wu, Frederick M Costello, Robert V Kelly, David Jack, George A
Stouffer; UNCH, Chapel Hill, NC
Background: Angiographic measurements of stenosis severity have had limited success in
determining which patients will benefit from renal artery intervention. We hypothesized that
translesional pressure gradient, measured by a 0.014 inch pressure wire, would better predict
activation of the renin-angiotensin system than would angiography. Methods: We prospectively
studied 50 patients with hypertension who were referred for selective renal angiography.
Subjects had an arterial sample of blood drawn prior to diagnostic images for blinded
determination of plasma renin activity (PRA) (Mayo Clinical Laboratories, Rochester, MN).
Selective renal angiography was performed in an AP projection and angiographic percent
stenosis was determined by quantitative angiography. Systolic translesional pressure gradient
(S-TPG) was measured by comparing pressure distal to the lesion (measured using Smartwire
from Volcano Therapeutics, Rancho Cordova, CA) to aortic pressure with the patient sedated
and supine. Results: The mean age was 63 years (range 32 - 84), 58% were male, the average
( SD) mean arterial blood pressure was 100 mm Hg ( 19 mm Hg) and the average baseline
 e74 Hypertension Vol 48, No 4 October 2006
creatinine was 1.2 mg/dl (range 0.6 - 2.0). The average ( SD) S-TPG was 12 ( 17) mm Hg
with a range from 0 to 72 mm Hg. The mean ( SD) PRA was 4.02 ( 10.52) ng/ml/hr. There
was not a linear correlation between PRA and angiographic stenosis when angiographic
stenosis was evaluated as a continuous variable (r 0.2; p 0.12). There was no statistical
difference in PRA between subjects with 60% stenosis (n 31; PRA 3.84 ng/ml/hr) and
subjects with 60 % stenosis (n 19; PRA 4.32 ng/ml/hr). The mean PRA of subjects with
a S-TPG 5 mm Hg (n 26; PRA 2.92 ng/ml/hr) was significantly lower than that of
subjects with a S-TPG 5 mm Hg (n 24; PRA 5.21 ng/ml/hr) (p 0.02). This difference
remained significant when using a S-TPG cut-off of 10 mm Hg as well (2.91 ng/ml/hr vs. 5.33
ng/ml/hr; p0.04). Conclusion: Systolic translesional pressure gradient is more accurate than
quantitative angiography at identifying renal artery stenosis associated with elevated levels of
renin.
P147
Is Collecting Duct Renin in Normotensive and Hypertensive Rats Regulated
by Aldosterone?
Minolfa C Prieto-Carrasquero, Depts of Physiology and Pharmacology, Ponce Sch of
Medicine; Dept of Physiology and Renal Hypertension Cntr of Excellence, Tulane Univ HSC,
Ponce, PR; Melissa Evans-Vallas, Dept of Physiology and Renal Hypertension Cntr of
Excellence, Tulane Univ HSC, New Orleans, LA; Rudy Ortiz, Div of Natural Sciences, Univ of
California, Merced, CA.; Dept of Physiology and Renal Hypertension Cntr of Excellence,
Tulane Univ HSC, Merced, CA; Hiroyuki Kobori, Dale Seth, L. Gabriel Navar; Dept of
Physiology and Renal Hypertension Cntr of Excellence, Tulane Univ HSC, New Orleans, LA
Collecting duct (CD) renin mRNA and protein levels are upregulated in AngII hypertensive rats
by an AT1 receptor-mediated mechanism. This study was performed to determine if aldosterone
(Aldo) is also involved in the regulation of CD renin. Four groups of rats: 1) Sham operated
(n4), 2) Epleronone (specific Aldo-receptor blocker, Epl, 25 mg/day-21days; n4), 3) AngII
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(minipump infusion, 60 ng/min-28days; n4), and 4) AngIIEpl (n4) were used. After 28
days, SBP measured by tail-cuff method was increased in AngII-infused (2075 mmHg) and
AngIIEpl (2155 mmHg), and normal in Epl (1265 mmHg) and Sham (1365 mmHg) rats.
Compared to Sham: 1) Total kidney AngII content increased 2-fold and 3-fold in AngII-infused
and AngIIEpl-treated; but not in Epl rats. 2) Renin qRT-PCR showed that mRNA levels
decreased in renal cortex of AngII-infused (0.140.1DU) and EplAngII (0.210.0DU) but not
in Epl (0.930.2DU) rats. 3) In contrast, renin transcript in the medulla was suppressed in Epl
(0.30.1DU) and augmented in the AngII-infused (1.50.1DU) and AngIIEpl rats
(1.30.2DU). 4) Renin protein levels assessed by Western blot in kidney cortex decreased in
all groups of rats [Epl (0.70.2 DU), AngII-infused (0.50.2DU) and AngIIEpl (0.60.3DU).
5) However, in the medulla renin protein levels decreased in Epl (0.60.2DU) but increased in
AngII-infused (1.40.2DU) and AngIIEpl (1.60.3DU) rats. In conclusion, while Aldo exerts
a positive influence on CD renin in normotensive rats, Aldo receptor blockade does not prevent
the effect of the elevated Ang II levels in the hypertensive rats.
P148
Nuclear Hormone Receptor LXR Mediates Renin and Early Response Gene
Expressions, and Proliferation of Juxtaglomerular As4.1 Cells: A Unifying
Molecular Mechanism for JG Cell Hyperplasia
Zhiping Zhang, Duke Univ Med Cntr, Durham, NC; Leonard M Anderson, Brigham and
Womens Hosp and Harvard Med Sch, Boston, MA; Fulvio Morello, Duke Univ Med Cntr,
Durham, NC; Sung E Choe, George M Church, Brigham and Womans Hosp and Harvard
Med Sch, Boston, MA; Richard E Pratt, Victor J Dzau; Duke Univ Med Cntr, Durham, NC
The kidney juxtaglomerular cells (JGCs) specializing in renin synthesis and secretion can
undergo hyperplasia yet maintaining their renin-producing phenotype. The molecular mecha-
nism for JGC hyperplasia is unknown. We have previously shown that the nuclear hormone
receptor LXR is a major regulator of cAMP dependent renin gene transcription by interacting
with a promoter element termed the CNRE. Using gene expression profiling, we observed that
c-myc and other genes involved with growth and differentiation are also upregulated by LXR.
Accordingly we hypothesized that LXR may be a transcriptional factor that mediates renin
expression and JGC hyperplasia. In this study, we examined the effects of cAMP activated
LXR on early gene expression, BrdU incorporation and cell growth. A mouse JG cell line As4.1,
stably transduced with mouse LXR, was treated with 8-Br-cAMP for up to 24 hours.
Microarray analysis demonstrated that a subset of 19 genes exhibited an expression profile
similar to that of c-myc, of which, 10 contained a newly identified consensus CNRE motif which
were validated by RT-PCR. The function of the CNRE enhancer element(s) in these genes was
validated using double-stranded oligonucleotide decoys corresponding to the CNRE. Of note,
many of these genes are functionally related to development, cell growth and differentiation.
The BrdU incorporation and cell number were significantly increased in the LXR-expressing
As4.1 cells after 8-Br-cAMP stimulation. These results demonstrate that cAMP activated LXR
may play an important role in the regulation of early response genes and cell proliferation while
stimulating renin expression in JG cells. We thus speculate that the cAMP-LXR pathway might
play a unifying role in the modulation of the JGC renin expression phenotype and in mediating
JGC proliferation, representing a common pathway for both renin synthesis and JGC
hyperplasia.
P149
Impaired Counter-Regulatory Response of Angiotensin Converting Enzyme
2 (ACE2) and Angiotensin-(17) [Ang-(17)] in the Heart of a New
Congenic Model of Hypertension Derived from [mRen2]27 Transgenic Rats
Jewell A Jessup, Patricia E Gallagher, Mark C Chappell, Carlos M Ferrario; Wake Forest
Univ Sch of Medicine, Winston-Salem, NC
In pursuing our hypothesis that hypertension may result from a reduced negative regulatory role
of the ACE2/Ang-(17) axis, we determined the expression and activity of the two vasode-
pressor components of the RAS in adult male mRen2.Lewis congenic hypertensive rats in the
absence and presence of selective blockade of Ang II synthesis (lisinopril, 10 mg/kg/day) or AT1
receptor blockade (losartan, 10 mg/kg/day). The drugs were given in the drinking water for 12
days to rats housed in metabolic cages; their blood pressure was monitored by the tail-cuff
method. Plasma levels of Ang II and Ang-(17) were measured by RIA and ACE, ACE2, AT1, and
mas receptor mRNAs were quantified in cardiac tissue by real-time reverse transcription-PCR.
Although baseline plasma levels of Ang II and Ang-(17) were not different between Lewis
normotensive and mRen2.Lewis hypertensive rats, cardiac ACE2 mRNA and ACE2 activity were
significantly reduced in mRen2.Lewis rats. The efficacious reduction in blood pressure achieved
during 12-day treatment with lisinopril or losartan was accompanied by suppressed plasma
levels of Ang II (81%) and increased plasma Ang-(17) (265%) in mRen2.Lewis rats given
lisinopril while these variables did not change in those medicated with losartan. While both
lisinopril and losartan increased cardiac ACE and ACE2 mRNAs by 55% (p 0.05), the effect
of the treatments on ACE2 gene expression was significantly obtunded compared to those
found in Lewis normotensive rats. Neither lisinopril nor losartan had an effect on ACE2 activity
in mRen2.Lewis rats. In a congenic model of renin-dependent hypertension, our data
documents for the first time an important down-regulation of the ACE2/Ang-(17) vasodepres-
sor axis not just in their natural state, but in response to treatments that either inhibit the
synthesis of Ang II or prevent the coupling of the peptide to the AT1 receptor. Failure of the
counter-regulatory control that is exerted upon the pro-hypertensive and pro-proliferative
effects of Ang II may constitute a fundamental component for the development and evolution
of this experimental form of hypertension.
P150
Resetting of the Renin-Angiotensin-Aldosterone System (RAAS) by Brief
Treatment with Angiotensin Receptor Blocker (ARB) as a Potential
Mechanistic Rationale for Early Hypertension Intervention
Kimiko Ishiguro, Hiroyuki Sasamura, Yusuke Sakamaki, Hiroshi Itoh, Takao Saruta; Keio Univ
Sch of Medicine, Tokyo, Japan
Previous laboratory studies using the SHR, and more recently, the TROPHY clinical study have
suggested that brief pharmacotherapy with an ARB during a critical stage in hypertension
development (prehypertension) may cause a sustained suppression of the later incidence of
hypertension. These findings could cause a major shift in our approach to the treatment and
prevention of hypertension, but the mechanisms are still unclear. In this study we provide
evidence that brief exposure to ARB or Ang II causes a resetting of the RAAS system, with
long-standing effects on the later development of hypertension. (Experiment 1) SHR were
treated briefly from age 3 to 10 weeks with an ARB (candesartan), then taken off treatment for
two months. At age 18 weeks, the rats were administered L-NAME, and the changes in blood
pressure, renal injury, plasma renin/angiotensin/aldosterone, and oxidative stress were
examined. Administration of L-NAME caused both marked hypertension (270 mmHg) and
increases in proteinuria, plasma creatinine, and renal histological changes. However the rats
which had been transiently exposed to ARB not only had a lower blood pressure, but failed to
develop signs of renal injury. L-NAME administration also caused significant increases in PRA,
plasma Ang II, and plasma aldosterone, but these increases were suppressed in the rats
exposed transiently to ARB. The elevation of markers of oxidative stress were also completely
suppressed in this group. (Experiment 2) WKY and SHR were exposed to Ang II from age 4 to
8 weeks, then left untreated for 2 months. Follow-up showed that the blood pressures in the
Ang II-exposed rats remained elevated compared to controls. At age 18 weeks, the rats were
administered L-NAME which resulted in increases in blood pressure, PRA, aldosterone, and
oxidative stress, and these changes were significantly increased in the SHR which had been
previously exposed to Ang II. In conclusion, brief exposure to ARB or Ang II at an early stage
causes a sustained resetting of the RAAS, with long-standing effects on later L-NAME-induced
hypertension and oxidative stress in the SHR. These results could provide a mechanistic
rationale for early and brief therapy with an ARB to prevent the later incidence of hypertension
in individuals at high risk.
P151
IL-6 is Elevated in Patients with Renal Artery Stenosis
Samuel S Wu, Michael J Gillespie, Robert V Kelly, George A Stouffer; Univ of North Carolina,
Chapel Hill, NC
Background: Inflammation as measured by interleukin-6 (IL-6) is elevated in patients with acute
coronary syndromes and following stent placement in coronary interventions. We hypothesized
that IL-6 is elevated in hypertensive individuals with atherosclerotic renal artery stenosis (RAS)
and is increased in response to stent placement. Methods: IL-6 levels were measured in 26
patients with unilateral atherosclerotic RAS undergoing stent placement. IL-6 levels were
determined at four time points: arterial samples were taken immediately prior to renal
angiography and immediately after stent implantation, and venous blood samples were taken
1 day and approximately 2 months later. A control group consisted of 19 patients without
significant RAS (stenosis 20% diameter by angiography) who had a single arterial blood
sample taken immediately prior to renal angiography. High sensitivity (hs) IL-6 was determined
by ELISA, in duplicate and analyzed in a single batch. Results: The mean age was 60 years
(range 47 - 73 years) in RAS group and 66 years (range 57 - 75 years) in the control group.
There were 53% males in the RAS group and 61% males in the control group. The mean
intra-aortic blood pressure was 100 16 mmHg in the RAS group and 104 25 mmHg in
the control group (p .53). Patients with RAS had elevated levels of IL-6 at baseline compared
to the control group, 5.3 3.9 pg/mL (range 2.5 - 8.0 pg/mL) versus 4.0 4.8 pg/mL (range
1.3 - 4.6 pg/mL, p 0.03). IL-6 levels did not change immediately post intervention to 5.6
4.9 pg/mL (range 2.6 - 6.5 pg/mL, p .978), however they were increased significantly 1 day
after intervention to 10.0 6.5 pg/mL (range 5.2 - 13.7 pg/mL, p 0.05). At delayed time
points (average follow-up 2.0 1.3 months), IL-6 levels decreased to 4.3 4.4 pg/mL (range
1.9 - 5.1 pg/mL) which is similar to the control group (p0.32). Conclusion: IL-6 levels are
increased in patients with RAS compared to hypertensive patients without RAS. IL-6 levels
transiently increase following renal artery intervention. Successful renal artery stenting results

in a decrease in IL-6 such that at an average follow-up of two months, IL-6 levels are
comparable to that of the control group.
P153
Intrarenal Aminopeptidase N (APN) Inhibition Augments Natriuretic
Responses to Angiotensin III(Ang III) in AT1 Receptor-Blocked Rats
Shetal H Padia, Nancy L Howell, Brandon A Kemp, Helmy M Siragy, Univ Virginia Sch Med,
Charlottesville, VA; Bernard P Roques, UFR des Sciences Pharmaceutiques/Biologiques,
Paris, France; Robert M Carey; Univ Virginia Sch Med, Charlottesville, VA
Published studies from our laboratory suggest that the renal Ang AT2 receptor mediates
natriuresis and that Ang III (des-aspartyl1-Ang II), not Ang II, may be the preferential AT2
receptor activator. Ang III is metabolized to largely inactive peptide fragments by APN. The
present study addresses the hypothesis that inhibition of APN would augment natriuretic
responses to intrarenal Ang III infusion in AT1 receptor-blocked rats. Sprague- Dawley rats
(N15) received a continuous i.v. infusion of AT1 receptor antagonist candesartan (0.01
mg/kg/min) via osmotic micropump for 24 h prior to the experiment. Candesartan-infused rats
were anesthetized and, following a 30 min control period, received a cumulative renal
interstitial (RI) infusion (2.5 l/min) of Ang III (N8) at 3.5, 7, 14 and 28 nmol/kg/min, each
dose for 30 min, or Ang III combined with APN inhibitor PC18 ( 2-amino-4-methylsulfonyl-
butane-thiol; 25g/min; N7) into one kidney. The contralateral control kidney received a RI
infusion of vehicle (V) at 2.5 l/min. Blood pressure (BP) was monitored by direct carotid artery
catheter and urinary Na excretion (UNaV) was documented separately from infused experi-
mental and non-infused control kidneys for each 30 min period. In kidneys infused with Ang
III alone, UNaV increased from baseline of 0.05 0.01 to 0.07 0.01 mol/min (PNS) at 3.5
nmol/kg/min, 0.07 0.01 mol/min (P0.01) at 7 nmol/kg/min, 0.14 0.01 mol/min
(P0.0001) at 14 nmol/kg/min and 0.11 0.01 mol/min (P0.001) at 28 nmol/kg/min of
Ang III (ANOVA F 3.68; P0.01). UNaV was unchanged (PNS) from control V infused kidneys.
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CHBPR ConferencePoster Presentations e75
of Ang II on collecting duct renin contributes to the increases in medullary Ang I and Ang II
formation. After 25 days of left renal artery clipping, systolic BP in 2K1C (n8) was significantly
increased to 1963 mmHg compared to 1162 mmHg in the sham-operated rats (n4). Ang
I, Ang II, and Ang 17 levels in nonclipped (NC-K), clipped (CK) and sham-operated kidney
medullas, measured by HPLC were: [Ang I (CK10620; NC-K19334 vs. Sham9937
pg/g)], [Ang II (CK19828; NC-K12123 vs. Sham10837 pg/g)], [Ang 17
(CK14.82; NC-K20.53 vs. Sham63.416]. Compared to the sham-operated rats,
semi-quantitative immunohistochemistry of renin showed suppression in juxtaglomerular cells
of NC-K (0.20.0 vs. 1.00.0 relative ratio) and augmentation in CK (1.71.0 vs. 1.00.0
relative ratio). In contrast, renin immunoreactivity in cortical and medullary collecting ducts
increased in both kidneys of 2K1C rats (CK2.81.5 cortex; 2.11.0 medulla; NC-
K4.62.3 cortex; 3.20.8 medulla vs. 1.00.0 relative ratio). Renin protein levels assessed
by Western blot and renin mRNA levels measured by real-time qRT-PCR in kidney medullas of
2K1C rats were also increased (CK1.40.2; NC-K1.50.3 and CK104; NC-
K4.32.6 vs. 1.00.0 relative ratio; respectively). The augmented renin expression in
principal cells from medullas of both kidneys from 2K1C hypertensive rats suggests that renin
up-regulation by Ang II in distal nephron segments is independent from the BP and, may
contribute to higher Ang I formation from proximally delivered angiotensinogen and thus to the
increased medullary Ang II and reduced Ang 17 formation in Ang II-dependent hypertension.
P156
The Renal Renin-Expressing Cell is an Activated Vascular Pericyte
Craig A Jones, Li Pan, Sean T Glenn, Shewanda L Vines, Jianxin Wang, Colleen Kane,
Roswell Park Cancer Institute, Buffalo, NY; Kenneth F Manly, Univ of Tennessee, Memphis,
TN; Kenneth W Gross; Roswell Park Cancer Institute, Buffalo, NY
The renin-angiotensin system (RAS) is known for its role regulating blood pressure by
controlling electrolyte homeostasis and blood volume. In adults, the initiating enzyme, renin, is

In kidneys infused with Ang III combined with PC 18, UNaV increased from a baseline of 0.05
0.01 to 0.15 0.04 mol/min (P0.05) at 3.5 nmol/kg/min, 0.16 0.03 mol/min (P0.05)
at 7 nmol/kg/min, 0.25 0.08 mol/min (P0.01) at 14 nmol/kg/min and 0.32 0.08
mol/min P0.05) at 28 nmol/kg/min of Ang III (ANOVA F 6.2; P0.001). Systemic BP and
UNaV from control kidneys were unchanged by Ang III and/or PC-18.. Ang III combined with
PC-18 induced a greater natriuretic response than Ang III alone (F 16.9; P0.0001). APN
inhibition augmented the natriuretic response to Ang III. These observations indicate that Ang
III is a major agonist of AT2 receptor-induced natriuresis.
P154
Crucial Role of Rho Kinase - Nuclear Factor Kappa Beta Axis in
Angiotensin II-Induced Renal Injury
Yuri Ozawa, Hiroyuki Kobori; Tulane Univ Health Sciences Cntr, New Orleans, LA
released from juxtaglomerular (JG) cells. However, during kidney organogenesis, renin is
expressed throughout the developing renal vasculature. Genetic and pharmacological studies
have shown that a functioning RAS is also required for normal renal development. To
characterize the transcriptional profile of the renin-expressing cell at different stages of
development, transgenic mice expressing green fluorescent protein (GFP) under control of the
renin promoter were used as a source for renin-expressing cells that were collected by
fluorescence- activated cell sorting (FACS). Expression profiles were determined for both the
GFP-positive and the total presorted cell populations using Affymetrix microarrays. Transcripts
exhibiting enrichment or de-enrichment in the GFP-positive cell fraction relative to the
presorted population, were identified. Results for selected transcripts of interest were validated
by real time RT-PCR. Among the highly enriched genes were key markers for activated
pericytes including NG2, RGS5 and PDGFRb. The results support the hypothesis that the
renin-expressing cell found in association with the renal vasculature during kidney organo-
genesis is an activated vascular pericyte.

Others and we reported that nuclear factor kappa beta (NFkB)-dependent upregulation of
monocytic chemotactic protein (MCP) 1 and transforming growth factor beta (TGFb) 1 plays an
important role in the development of renal injury in angiotensin (Ang) II-dependent hyperten-
sion. Recently, a small guanosine triphosphatase, Rho is reported to induce NFkB activity.
However, it has not been established if Rho kinase (a downstream effector molecule of Rho)
inhibitor and/or NFkB inhibitor exerts renoprotective effect in AngII-dependent hypertension.
This study was performed to determine the effectiveness of Rho kinase inhibitor and NFkB
inhibitor in renal injury of AngII-infused hypertensive rats. Male Sprague-Dawley rats,
maintained on a normal diet, received either a sham operation (N7) or continuous AngII
infusion (120 ng/min) subcutaneously via minipumps. The AngII-infused rats were further
subdivided into 3 subgroups (N7 each) to receive one of the following treatments during the
entire period: null, Rho kinase inhibitor (Fasudil, 3 mg/kg/day, intraperitoneously), or NFkB
inhibitor (Parthenolide, 1 mg/kg/day, intraperitoneously). After 12 days of AngII infusion, systolic
BP (2087 vs 1363 mmHg), Rho kinase activity, NFkB activity, renal AngII contents (16025
vs 8414 pg/g), MCP1 mRNA, interstitial macrophage infiltration, TGFb1 mRNA, interstitial
collagen-positive area, urinary protein excretion (436 vs 112 mg/day), and urinary albumin
excretion were significantly enhanced compared to the shams. While Fasudil or Parthenolide
did not alter systolic BP (2228 and 19021, respectively), both treatments completely
blocked AngII-induced enhancement of NFkB activity, renal AngII contents (10311 and
11621, respectively), MCP1 mRNA, interstitial macrophage infiltration, TGFb1 mRNA,
interstitial collagen-positive area, urinary protein excretion (286 and 233, respectively),
and urinary albumin excretion. Importantly, Parthenolide did not alter AngII-induced Rho kinase
activation although Fasudil abolished AngII-induced Rho kinase activation. These data indicate
that Rho kinase - NFkB axis plays a crucial role in the development of AngII-induced renal injury
independently from BP regulation.
P155
Blood Pressure-Independent Up-Regulation of Collecting Duct Renin in
2K1C Goldblatt Hypertensive Rats
Minolfa C Prieto-Carrasquero, Dept of Physiology and Pharmacology, Ponce Sch of
Medicine; Dept of Physiology and Renal Hypertension Cntr of Excellence, Tulane Univ HSC,
Ponce, PR; Fady T Botros, Hiroyuki Kobori, Dept of Physiology and Renal Hypertension Cntr
of Excellence, Tulane Univ HSC, New Orleans, LA; Dulce E Casarini, Nephrology Div, Dept of
Medicine, Federal Univ of Sao Paulo, Sao Paulo, Brazil; Dale Seth, L. G Navar; Dept of
Physiology and Renal Hypertension Cntr of Excellence, Tulane Univ HSC, New Orleans, LA
P157
Long-Term Hyperglucagonemia Induces Early Type 2 Diabetic Phenotypes
in Mice: Role of Glucagon Receptors and Angiotensin II
Xiao C Li, Oscar A Carretero, Martin DAmbrosio, Jia L Zhuo; Henry Ford Hosp, Detroit, MI
Pancreatic bihormones insulin and glucagon are YIN and YANG in the regulation of glucose
metabolism in humans. Clinical studies have shown that most type 2 diabetic patients have
elevated serum glucagon rather than insulin deficiency. Imbalance of insulin and glucagon in
favoring the latter may contribute to persistent hyperglycemia, impaired glucose tolerance,
glomerular hyperfiltration, albuminuria, and glomerular injury. We tested the hypothesis that
long-term glucagon administration can induce early type 2 diabetic phenotypes in mice by
acting on glucagon receptors and interacting with angiotensin II. Four groups of male C57BL/6J
mice (n 5 8 each) were treated with vehicle, glucagon (1 g/h via osmotic minipump),
glucagon plus the glucagon receptor antagonist [Des-His1-Glu9] glucagon (5 g/h via osmotic
minipump), or glucagon plus the ACE inhibitor captopril (25 mg/kg/day, p.o.) for 4 weeks.
Glucagon increased serum glucagon by 129% (basal: 194 47.3 pg/ml; vs. glucagon: 445
45.6 pg/ml, p0.05), systolic pressure (control: 115 3 vs. glucagon: 136 3 mm Hg, p
0.01) without altering heart-to-body weight ratio, elevated fasting blood glucose (control:
74.6 3.0 vs. glucagon: 105.6 5.2 mg/dL, p 0.01), impaired glucose tolerance (p 0.01
vs. control), and increased kidney-to-body weight ratio (control: 0.62 0.02 vs. glucagon:
0.69 0.02, p 0.05) and 24-hour urinary albumin excretion (control: 24.6 2.8 g vs.
glucagon: 51.1 11.9 g, p 0.01). Serum insulin did not increase proportionally, as
expected. Concurrent administration of [Des-His1-Glu9] glucagon or captopril with glucagon
significantly attenuated glucagon-increased blood pressure, fasting blood glucose, kidney-to-
body weight ratio and 24-hour urinary albumin excretion. [Des-His1-Glu9] glucagon or captopril
also improved glucagon-inpaired glucose tolerance and increased serum insulin by 56% (p
0.05) and 75% (p0.05), respectively. These results demonstrate for the first time that
long-term glucagon administration induces early type 2 diabetic phenotypes in mice by
activating glucagon receptors and interacting with angiotensin II, and suggest that glucagon
receptor blockade may be beneficial in treating type 2 diabetic cardiovascular and renal
complications.
P158
Differential Effects of ETA vs ETB Receptor Antagonism on Oxidative Stress
and Total Antioxidant Status in Type-2 Diabetes

Collecting duct renin may provide a possible pathway for the formation of angiotensin (Ang) I
leading to higher medullary Ang I and Ang II levels in Ang II-dependent hypertension despite
the suppression of juxtaglomerular renin. We recently reported that renin mRNA and protein
levels are up-regulated via AT1 receptors in the collecting ducts of chronic Ang II-infused rats.
However, it has not been determined whether the augmentation of collecting duct renin is due
to a direct action of Ang II or a blood pressure (BP)-dependent effect, or if this positive feedback
Mostafa M Elgebaly, Vera Portik-Dobos, Kamakshi Sachidanandam, David Rychly, Daniel
Malcom, Jimmie Hutchinson, Maribeth H Johnson, Adviye Ergul; Univeristy of Georgia,
Augusta, GA
Endothelin (ET-1), which contributes to increased oxidative stress in several models of
hypertension, is chronically elevated in diabetes. However, role of ET-1 in increased oxidative
 e76 Hypertension Vol 48, No 4 October 2006
stress and the regulation of the antioxidant status in type 2 diabetes is less clear. The goals
of this study were to test the hypotheses that: 1) oxidative stress markers are increased and
total antioxidant capacity is decreased in diabetes, and 2) activation of ETA receptors mediates
oxidative stress whereas ETB receptors display opposing effects. To achieve these goals,
circulating indices of oxidative stress, total antioxidant status (TAS) and 8-isoprostane (iso), as
well as total nitrotyrosine (TNY) levels in mesenteric resistance vessels were measured in
control and mildly hypertensive diabetic Goto-Kakizaki (GK) rats treated with vehicle, ETA
antagonist (ABT627, 5 mg/kg/day), or ETB receptor antagonist (A192621, 15 or 30 mg/kg/day,
low and high dose, respectively) for 4 weeks. Results summarized in the Table (mean SEM,
n510) indicate that 8-iso is slightly elevated in GKs and blockade of ETB receptors with low
A192621 augments oxidative stress in both groups (p0.001). At the tissue level, protein
nitration is increased in diabetes (p0.001), and ET receptor antagonism with ABT627 lowers
TNY levels (p0.01) with no effect of ETB blockade. On the other hand, there is a significant
interaction of disease and treatment on TAS levels with low A192621 increasing antioxidant
capacity in controls but decreasing in GKs. These findings suggest that although circulating
markers of oxidative stress are not significantly different, resistance arteries display increased
protein nitration as a result of increased oxidative stress in diabetes. Furthermore, ET-1
contributes to free radical generation and ET receptor antagonism with ABT627 or A192612
displays differential effects depending on dose and disease state.
TAS (mM) 8-iso (pg/ml)) TNY (pixels)
Wistar Vehicle 0.350.06 901173 57874
A192621 low 0.340.07 2145491 45724
A192621 high 0.600.15 558211 39435
ABT627 0.450.02 641242 22275
GK Vehicle 0.470.03 2059551 732114
A192621 low 0.530.03 3392368 864292
A192621 high 0.380.05 572177 760221
ABT627 0.420.04 790177 60361
. vasodilation contributes to the enhanced pressor response. Animals were restrained and
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P159
Visceral Fat Deposition and Prevalence of Hypertension among United
States Adults: Findings from the IRAS Family Study
Capri G Foy, Fang-Chi Hsu, Wake Forest Univ Sch of Medicine, Winston-Salem, NC; Steven
Haffner, Univ of Texas Health Sciences Cntr, San Antonio, TX; Jill M Norris, Univ of
Colorado Health Sciences Cntr, Dever, CO; Jerome I Rotter, Univ of California, Los Angeles,
Sch of Medicine, Los Angeles, CA; Yii-Der I Chen, Cedars-Sinai Med Cntr, Los Angeles, CA;
Lynne E Wagenknecht; Wake Forest Univ Sch of Medicine, Winston-Salem, NC
Objective: The purpose of this study was to investigate the relationship between abdominal
adiposity on prevalent hypertension (HTN), independent of total adiposity, among a biethnic
sample of adults. A secondary purpose entailed investigating the roles of insulin sensitivity (SI)
and C-reactive protein (CRP) as potential mediators of this relationship. Research Methods and
Procedures: The IRAS Family Study is an epidemiologic study investigating the genetic and
metabolic determinants of adiposity and insulin resistance among African-American and
Hispanic-American families recruited from three clinical sites. Visceral adipose tissue (VAT) and
subcutaneous adipose tissue (SAT) were obtained by computed tomography scan of the
abdomen at the L4/L5 level under a standardized scanning and reading protocol. HTN,
measured via standardized protocol, was defined as SBP 140 mmHg, DBP 90 mmHg, or
current pharmaceutical treatment. We examined the relationship among VAT, SAT, and
prevalent HTN in generalized estimating equation models adjusted for BMI, age, ethnicity,
gender, and fasting glucose status among 1582 participants. Separate models added SI and
CRP as additional covariates. All continuous variables were standardized. Significant VAT (and
SAT) by gender interactions prompted stratified analyses. Results: The sample was comprised
primarily of women (N 925; 59%), and Hispanic-Americans (N1095; 69%). The mean age
(SD) of the sample was 41.3 (13.9; range 18 - 81) years, with a mean BMI (SD) of 28.7 (6.0)
kg/m2. Prevalent HTN was observed in 21% and impaired glucose status in 21%. In women,
VAT was significantly associated with HTN (OR 1.57 p 0.0002), after adjustment for
covariates, and remained significant after adjustment in separate models for SI (OR 1.47,
P0.01), and CRP (OR 1.46, p 0.02). In men, only a significant effect of BMI (OR1.86,
p0.006) was observed. SAT did not have an independent effect on HTN in either
gender.Discussion: Although BMI is associated with HTN among both men and women, an
independent relationship may exist between VAT and HTN among women, but not men. The
increased odds of HTN associated with increased VAT was not explained by insulin resistance
or CRP levels, suggesting that other factors may be implicated.
P160
Smaller Increase in Blood Pressure with Weight Gain in Men with Higher
Compared with Lower Cardiorespiratory Fitness
Christopher L Gentile, Jeb S Orr, Brenda M Davy, Kevin P Davy; Virginia Polytechnic
Institute and State Univ, Blacksburg, VA
We tested the hypothesis that the increase in blood pressure with weight gain would be smaller
in men with higher compared with lower cardiorespiratory fitness (CRF). Ten healthy men
(age242, BMI 240.8) were overfed by 1000 kcal/d over 8 weeks until a 5 kg results suggest that a HF diet causes an inward remodeling of the cerebral vasculature with a
weight gain was achieved. Resting (sphygmomanometry) and ambulatory (Spacelabs 90207)
blood pressure (BP), body composition (DEXA), and abdominal fat distribution (CT scans) were
measured in men above and below the median level of VO2max at baseline and following
weight gain. As intended, CRF was different in the two groups (49.31.6 vs.38.91.3
ml/kg/min, P0.001). At baseline, body weight (74.35.5 vs. 74.14.6 kg, P0.494) and
both systolic (1233 vs. 1193 mmHg; P0.285) and diastolic (643 vs 703 mmHg;
P0.078) BP were not different, whereas total body fat (17.41.8 vs. 24.61.6%; P0.009)
and AVF (47.57.8 vs. 84.715.7 cm2; P0.033) were greater in men with higher compared
with lower CRF (H-CRF and L-CRF, respectively). Following weight gain, body weight (5.10.2
vs. 4.80.3 kg; P0.228) and body fat (3.20.9 vs. 3.60.5 kg; P0.369) increased
similarly in the two groups. However, there was a smaller increase in the percent of total
abdominal fat gained as AVF (-4.117.1 vs. 51.419.1 %; P0.032); the absolute increase
in AVF tended to be smaller in H-CRF (8.56.5 vs. 24.19.4 cm2, P0.104). As hypothesized,
there was a smaller increase in systolic (22 vs. 72mmHg; P.031) and diastolic BP (-22
vs. 41mmHg; P0.006) in H-CRF compared with L-CRF, respectively. The increases in
nighttime systolic BP followed a similar trend (23 vs. 82 mmHg; P0.079). In the pooled
sample, CRF was inversely correlated with the increases in resting systolic (r-0.65; P0.022)
and diastolic BP (r-.86; P0.001), the absolute increase in AVF (r-0.56, P0.045), and the
proportion of total abdominal fat gained as AVF (r-0.76, P0.006). The increase in AVF was
correlated with diastolic BP (r0.62, P0.029), but the relation between CRF and diastolic BP
was only minimally impacted by controlling for the increase in AVF (r-0.79, P0.006). These
findings suggest that CRF exerts a modulatory influence on the increase in BP with weight gain
that is independent of changes in AVF.Supported by NIH HL62283 and HL67227.
P161
Impaired Beta-Adrenergic-Mediated Vasodilation Contributes to Augmented
Cardiovascular Responses to Stress in Obese Zucker Rats
Gerard DAngelo, James D Mintz, John E Tidwell, David M Pollock, David W Stepp; Med
College of Georgia, Augusta, GA
Clinical studies have demonstrated that the pressor response to acute stress is larger in obese
vs. lean individuals due to a greater increase in total peripheral resistance. Using telemetry-
instrumented rats, we tested the hypotheses that the pressor response to behavioral stress is
greater in obese (OZR) vs. lean Zucker rats (LZR) and that reduced -adrenergic-mediated
subjected to acute pulsatile air jet stress (3 minutes), followed by a post-stress period of 20
minutes in the restrainer; -adrenergic blockade was achieved with propranolol (PRP; 5 mg/kg,
i.v.) given 15 minutes before the start of air jet stress. Baseline (24-hour) mean arterial
pressure (MAP) was modestly, yet significantly elevated in OZR (1225 vs. 1002 mmHg,
OZR vs. LZR, p0.05); baseline heart rate (HR) was not different. The integrated pressor
response (area under the curve; AUC) to acute stress was greater in untreated OZR (41.26.1
vs. 21.23.3 mmHg x 3 min, OZR vs. LZR, p0.05). During the 20 minutes after air jet stress,
MAP of LZR declined to a sub-baseline level throughout, while that of OZR failed to recover. PRP
produced a significant increase in MAP in LZR but not OZR, despite causing a significant
reduction in HR in both. -adrenergic blockade had no effect on the integrated pressor
response to stress in either LZR or OZR, but significantly attenuated the post-stress recovery
of MAP in LZR only (post-stress AUC: -100.148.1 vs. 49.013.5 mmHg x 20 min, untreated
vs. PRP, p0.05). In anesthetized animals, depressor responses to isoproterenol were
significantly lower in OZR vs. LZR, both before and after autonomic ganglion blockade, due
primarily to smaller increases in mesenteric vascular conductance. Conversely, increases in
cardiac output were significantly greater in LZR, despite no difference in the tachycardic
response. These data suggest that -adrenergic stimulation causes a greater reduction in total
peripheral resistance in lean vs. obese animals. We conclude that -adrenergic-mediated
vasodilation facilitates blood pressure recovery following stress, and that this pathway is
compromised in an animal model of morbid obesity.
P162
Cerebral Vessel Remodeling in Rats Fed a High Fat Diet
Christian Deutsch, Med College of Georgia, Augusta, GA; Vera Portik-Dobos, Adviye Ergul,
Univ of Georgia College of Pharmacy, Augusta, GA; Anne M Dorrance; Med College of
Georgia, Augusta, GA
Hypertension and elevated fasting blood glucose and plasma insulin develop in rats fed a high
fat (HF) diet. In humans these factors increase the risk of cerebral ischemia, yet their combined
effects on cerebral vessels has not been studied. We hypothesized that the cerebral vessels
from rats fed a HF diet would undergo inward remodeling with an increase in wall stiffness and
collagen deposition. Three-week-old male Sprague Dawley rats were fed a HF diet for 10
weeks after which middle cerebral artery (MCA) structure was assessed. MCA matrix
metalloproteinase (MMP)-2 activity and MMP-13 expression were measured as well as aortic
collagen 1 and MMP-13 expression. The rats fed the HF diet were hypertensive (1612
vs1372mmHg HF vs control p0.05) and had increased fasting blood glucose (1554
vs1235mg/dl HF vs control p0.05) and plasma insulin levels (23263 vs 6011pM HF vs
control p0.05). Despite having an increased body weight (4085 vs 3534g HF vs control
p0.05), the MCAs from the rats fed the HF diet had smaller lumen and outer diameters than
those from control rats (Table 1), this occurred without a change in the wall/lumen ratio
suggesting eutrophic remodeling. There was a leftward shift in the stress/strain curve for the
MCAs from the rats fed the HF diet and an increase in the -coefficient, a measure of wall
stiffness. MMP-2 activity was increased in the MCAs from the rats fed the HF diet and there
was a trend toward a reduction in MMP-13 expression. MMP-13 expression was reduced in the
aorta of the rats fed the HF diet and collagen 1 expression was increased. Together, these
concomitant increase in vessel stiffness that appears to be due in increased collagen
deposition. These changes may result in an increased stroke risk in these rats. Table 1. Lumen
and outer diameters at 100mmHg intralumenal pressure. * significantly different from
control (p0.05) n315
Outer Inner MMP-13 MMP-13
Diameter Diameter - expression MMP-2 expression Collagen 1
(m) (m) Coefficient (MCA) activity (Aorta) expression
HF 2716.06* 2327.16* 2.860.14* 248115 715.6* 2043436* 96954.4*
Control 2907.41 2547.80 1.560.10 959434 384.3 3363179 50223.2

P163
Hypergricemia Induced Hypertensive Action in Insulin Resistant Dahl Salt
Sensitive Rats
Takefumi Mori, Yoshimi Yoneki, Satoshi Endo, Susumu Ogawa, Chun-hua Jin, Kazuhiro
Nako, Div of Nephrology, Endocrinology and Vascular Medicine, Tohoku Univ Graduate Sch
of Medicine, Sendai, Japan; Sadayoshi Ito; Tohoku Univ Graduate Sch of Medicine, Sendai,
Japan
High blood pressure is often observed in diabetes and metabolic syndrome but the mechanism
involved is not fully investigated. Present study was designed to investigate whether sucrose
intake would induce hypertension in a model of metabolic syndrome and whether sodium-
glucose transporter (SGLT) would involve in sucrose-induced hypertension. 8 week-old male
Dahl salt-sensitive rats were treated with 10% sucrose in drinking water under 0.5% salt diet.
Rats were divided to three groups, 1) Telemetry group; blood pressure was monitored with
radio-telemetry methods for 14 days. 2) Phloridzin group; 0.4g/kg/day of SGLT inhibitor
phloridzin at 40% solution with propylene glycol (PG) was administered subcutaneously for 14
days. 3) Vehicle group; PG was administered subcutaneously as a vehicle control. Blood
pressure for Phloridzin and Vehicle group was measured with tail cuff method. Rats were
housed in a metabolic cage for urinary sampling. Mean blood pressure elevated and reached
to a level of significance at 6th day after sucrose intake and sustained until 14th day
(162mmHg increase from baseline, n6) in Telemetry group. SGLT inhibition with phloridzin
significantly attenuated the blood pressure elevation with sucrose intake for 14 days (n6).
Urinary protein and creatinine clearance did not change in either group. Urinary glucose
excretion was significantly increased from 263 mg/gCr at baseline to 23793 mg/gCr after
14days in Phloridzin group but not in other groups. Fasting blood glucose level (146mg/dl
increase, n6) increased in Vehicle group but not in Phloridzin group. HOMA-R increased after
sucrose intake in both groups but no difference was observed between groups (n6). We
conclude that increase in blood glucose stimulate sustained hypertension. The results indicate
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
that SGLT were involved in hypertensive action of blood glucose in hypertension with insulin
resistant Dahl salt sensitive rats.
P164
Rosiglitazone Reduces Hypertension in Fructose-Fed Rats by Increasing
Nitric Oxide Bioavailability
Michael D Nyby, UCLA/VA GLAHS, Sepulveda, CA; Karolin Abedi, Victoria Smutko, VA
GLAHS, Sepulveda, CA; Michael L Tuck; UCLA/VA GLAHS, Sepulveda, CA
The insulin sensitizer, rosiglitazone (RGT) is known to reduce hypertension in insulin resistant
conditions. The mechanism for this action of RGT is not completely known. This project was
designed to determine if RGT reduces blood pressure and improves vascular function by
enhancing nitric oxide bioavailability in the fructose-fed rat model of hyperinsulinemia. Rats
were fed either a control diet (CONT), a high (60%) fructose diet (FFR), or a high fructose diet
containing 30mg/kg rosiglitazone (FFRRGT) (n8 in each group). Systolic blood pressure
(SBP) was monitored weekly by tail cuff. After 8 weeks on the diets, the rats were sacrificed
and blood, aorta and heart were collected for analysis. Mesenteric artery segments were
removed for vascular reactivity studies. Mesenteric artery wall/lumen ratio, measured by
videoimaging, was 0.470.06 in CONT, and was increased in FFR to 0.690.05 (P0.01 vs.
CONT), and reduced in the FFRRGT to 0.530.05 (P0.03 vs. FFR). Nitric oxide-dependent
vasorelaxation to BHT920, an alpha-2 adrenergic agonist, was suppressed in mesenteric artery
segments from FFR (P0.05 vs. CONT), but not different from CONT in those from FFRRGT.
Plasma insulin was measured by ELISA, H2O2 by amplex red assay, and nitrite by fluorimetric
assay. SBP and plasma results are given below: (Table values are mean SEM. H2O2 plasma
hydrogen peroxide. *P0.05 vs. CONT; **P0.05 vs. FFR.) Real-time quantitative RT-PCR
showed that aortic and heart tissue eNOS mRNA expression in FFRRGT was significantly
increased over that observed in CONT (1.640.29-fold, P0.05, and 1.510.12-fold,
P0.05, respectively). These results show that RGT decreased oxidative stress in FFRRGT,
and increased eNOS expression and plasma nitrite concentrations. Collectively, these results
suggest that RGT reduces blood pressure and prevents vascular dysfunction in FFR by
improving nitric oxide bioavailability in part by upregulating expression of eNOS in vascular
tissue.
SBP (mmHg) Insulin (ng/ml) H2O2 (micromol/L) Nitrite (nmol/L)
CONT 123.21.2 2.110.41 9.50.4 37.34.4
FFR 154.13.4* 3.230.61* 19.63.3* 17.64.8*
FFRRGT 126.62.4** 1.700.28** 12.71.4** 33.03.2**
P165
Angiotensin At1a Receptors Are Critical in the Cardiovascular Response to
a High Fructose Diet
Vera Farah, Boonshoft Sch of Medicine of Wright State Univ, Dayton, OH; Tatiana S Cunha,
Univ of Campinas Faculty of Dentistry of Piracicaba, Piracicaba, Brazil; Mary Key, Janaina P
Aguiar, Mariana Pazzine, Khalid M Elased, Mariana Morris; Boonshoft Sch of Medicine of
Wright State Univ, Dayton, OH
We have shown previously that a high fructose diet in mice induces a moderate increase in
blood pressure and an increase in plasma angiotensin II (Ang II). To extend these findings, we
determined the cardiovascular effects of a high fructose diet in mice lacking Ang AT1a
receptors. Male Ang AT1a knockout (AT1aKO) and wild type (AT1aWT) mice with carotid
telemetric catheters were fed a diet containing 60% fructose. Fructose produced no changes
in body weight, plasma glucose or plasma insulin. There was impaired glucose tolerance (i.p
glucose load) in both fructose-fed groups. The fructose diet increased mean arterial pressure
(MAP) in AT1aWT. The change was seen only during the dark (active phase) (8% increase) as
observed in C57Bl mice. However, in AT1aKO, there was an unexpected decrease in MAP
which occurred during both light (24% decrease) and dark (13% decrease) periods. MAP levels
CHBPR ConferencePoster Presentations e77
were 1174 mmHg in fructose fed AT1aWT as compared 823 mmHg in the AT1aKO fructose
group. There was a decrease in circulating Ang II levels, from 17.55 to 50.2 pg/ml (a 70%
decrease) in the AT1aKO fructose group. In the AT1aWT fructose group there was trend toward
an increase, from 82 to 186 pg/ml (125% change). The changes in plasma Ang II may
mediate the MAP effects, the increase in the AT1aWT as compared to the decrease in AT1aKO.
Ang converting enzyme type 1(ACE 1) was also measured using a mass enzyme proteolytic
assay. Results showed that there were no significant differences between the groups showing
a lack of correlation between the changes in peptide levels with ACE activity. Results document
the importance of Ang AT1a receptors in mediating the cardiovascular effects of a high fructose
diet. The depressor responses observed in fructose fed ATKO mice could be due to the
reduction in plasma Ang II levels or to change in other vasoactive peptide forms.
P166
PPAR- Knock Down and Hyperglycemia Downregulate Insulin Signaling in
VSMC from Normotensive and Hypertensive Rats
Nihar R Pandey, Karim Benkirane, Farhad Amiri, Ernesto L Schiffrin; Lady Davis Institute for
Med Rsch, Montreal, Canada
Peroxisome proliferator-activated receptor (PPAR)- is a target in the treatment of diabetes and
hypertension, and has been shown to improve insulin sensitivity in humans and to reduce
hypertensive vascular complications in experimental models. However, the precise mechanism
for the beneficial effects of PPAR- remains unclear. We investigated the underlying molecular
mechanism responsible for PPAR--associated insulinomimetic signaling by using VSMC from
mesenteric arteries of WKY and SHRSP rats. Passaged VSMC grown in euglycemic (5.5 mmol/L)
or hyperglycemic (25.0 mmol/L) conditions, were treated with/without PPAR- targeted siRNA
(5 nmol/L), PPAR- antagonist (GW-9662, 10 mol/L) or rosiglitazone (10 mol/L) in the
presence/absence of insulin (100 nmol/L). Western blot analysis revealed that hyperglycemia,
GW-9662 and PPAR--siRNA significantly (P0.005) decreased insulin-stimulated insulin
receptor (IR)-, Akt and glycogen synthase kinase (GSK)-3 phosphorylation while it
significantly (P0.05) increased phosphotyrosine phosphatase (PTP)-1B expression in both
WKY and SHRSP VSMC, however the effects were more pronounced in SHR-SP especially under
high glucose. Rosiglitazone induced a trend to an increase in insulin-mediated effects on IR-,
Akt and GSK-3 phosphorylation in both WKY and SHRSP VSMC, while it decreased
insulin-induced PTP-1B expression under hyperglycemic conditions. In conclusion, these data
suggest that PPAR- inhibition results in decreased insulin signaling, under eu- and
hyperglycemic conditions and particularly when associated with hypertensive state, in a IR-
phosphorylation-dependent manner.
P167
Vascular but not Metabolic Insulin Resistance in Aging: Role of the
Akt/eNOS Pathway
Ivonne H Schulman, Ming-Sheng Zhou, Edgar A Jaimes, Leopoldo Raij; Univ of Miami Miller
Sch of Medicine and Veterans Affairs Med Cntr, Miami, FL
Physiologic actions of insulin serve to couple regulation of metabolic and hemodynamic
homeostasis. Insulin receptor activation leads to phosphorylation of Akt. Phosphorylated Akt
(p-Akt) directly phosphorylates eNOS, resulting in vasodilation, and promotes glucose uptake
via Glut-4 in muscle and adipose tissue. The metabolic and hemodynamic actions of insulin do
not completely overlap since eNOS inhibition abolishes vasodilation but reduces glucose uptake
by only 30%. Endothelial dysfunction, hypertension, and insulin resistance are part of the
metabolic syndrome, which increases in prevalence with aging. We investigated whether the
hemodynamic and metabolic actions of insulin are coupled in groups (n5) of aged 24-month
old (24mo) vs. young 3-month old (3mo) Sprague Dawley rats. Using the hyperinsulinemic
euglycemic clamp, the rate of glucose infusion (mg/kg/min) necessary to maintain equivalent
plasma glucose (100mg/dl) was similar in 24mo compared to 3mo, as was fasting glucose
(946.0 vs. 806.9 mg/dl; meanSEM), suggesting that whole-body insulin sensitivity was
not impaired. Intra-arterial systolic blood pressure was higher in 24mo than 3mo (1335
vs.1104; P0.005). Endothelium-dependent [insulin and acetylcholine (Ach)] and indepen-
dent (SNP) relaxation was measured in aortas from parallel groups of 24mo and 3mo rats.
Vascular relaxation to insulin (Emax 8.94.3% vs. 34.93.9%; P0.002), but not to Ach or
SNP, was impaired in 24mo compared to 3mo rats and did not improve after superoxide
dismutase. L-NAME abolished insulin-mediated relaxation in 3mo but not 24mo rats. In aortas
obtained after insulin-clamp, phosphorylated Akt (6.20.7 vs. 10.70.8; p-Akt/actin;
P0.003) and phosphorylated eNOS (1.50.2 vs. 3.10.3; p-eNOS/actin; P0.001), by
western blot, was lesser in 24mo compared to 3mo rats. In summary, vascular insulin
resistance in 24mo rats was associated with impaired activation of Akt/eNOS by insulin, but
intact activation of the Ca2-calmodulin pathway by Ach, and was independent of whole-body
insulin sensitivity, suggesting selective dissociation between metabolic and vascular insulin
signaling in aging.
P168
Renal Epoxyeicosatrienoic Acid Induction by Fenofibrate Attenuates the
Abnormal Renal Functions and Hypertension in Obese High Fat Rats
Mong-Heng Wang, Hui Huang, Yiqiang Zhou, Hsin-Hsin Chang, Jing-Feng Wu, Med College
of Georgia, Augusta, GA; John R. Falck; Univ of Texas Southwestern Med Cntr, Dallas, TX
We previously reported high fat (HF) diet causes obesity-induced hypertension in male rats,
which is associated with decreased expression of renal cytochrome P450 (CYP) epoxygenase
and decreased synthesis of its product, epoxyeicosatrienoic acids (EETs). Since EETs affect
sodium reabsorption in renal tubules and have renal vascular dilation effect, this study is
designed to examine the effects of fenofibrate, a pharmacological EET inducer, and
N-methanesulfonyl-6-(2-proparyloxyphenyl)hexanamide (MSPPOH), a selective EET inhibitor,
on renal hemodynamics and sodium retention in HF rats. Comparing with control HF rats,
fenofibrate (30 mg/kg/day, p.o., for 2 weeks)-treated HF rats showed lower mean blood
 e78 Hypertension Vol 48, No 4 October 2006
pressure (102 5 vs 120 6 mmHg, n6, P0.05), renal vascular resistance (8.7 1.2
vs 12.5 1.5 mmHg/ml/min, n6, P0.05), and glomerular filtration rate (1.2 0.1 vs
1.65 0.2 ml/min, n6, P0.05), whereas higher renal blood flow (11.7 2 vs 9.6 1.3
ml/min). In addition, fenofibrate treatment decreased cumulative sodium balance (an index for
sodium retention) in HF rats (-0.84 0.02 vs 1.05 0.03 mEq, n3, P0.05). The treatment
of MSPPOH (20 mg/kg/day, iv, for 2 weeks) reversed the renal hemodynamics and cumulative
sodium balance of fenofibrate-treated HF rats back to the levels of control HF rats. Moreover,
fenofibrate caused a 3 fold increase in renal cortical EET production (236 34 vs 82 13
pmol/mg/min, n4, P0.05), whereas fenofibrate plus MSPPOH treatment decreased EET
production to 142 pmol/mg/min. Western blot analysis showed that fenofibrate induced the
expression of CYP2C23 (the major epoxygenase) in both renal cortex and microvessels, and the
induction effect of fenofibrate was blocked by MSPPOH. Conclusions: Fenofibrate causes the
increased expression of CYP2C23 and EET production in both renal microvessels and tubules,
which can cause renal vascular dilation and reduced sodium retention, consequently
contributing the improvement of abnormal renal function and hypertension in obese HF rats.
P169
Measures of Liver Function Are Associated with Blood Pressure Variation
Xiuqing Guo, Leslie J Raffel, Belle Fang, Cedars-Sinai Med Cntr, Los Angeles, CA; Anny H
Xiang, USC, Los Angeles, CA; Manuel J Quinones, UCLA, Los Angeles, CA; Kent D Taylor,
Cedars-Sinai Med Cntr, Los Angeles, CA; Howard N Hodis, USC, Los Angeles, CA; Willa A
Hsueh, UCLA, Los Angeles, CA; Thomas A Buchanan, USC, Los Angeles, CA; Jerome I
Rotter; Cedars-Sinai Med Cntr, Los Angeles, CA
The liver plays a crucial role in the metabolic syndrome (MS), while insulin resistance (IR) is a
known determinant of blood pressure (BP) variation. As we previously observed significant
phenotypic and genetic correlations between IR and liver enzymes, we examined the
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
relationship between liver function and BP in 635 non-diabetic offspring from 157 Hispanic
American families, ascertained through a proband with hypertension. Insulin sensitivity was
measured by fasting insulin, as well as by glucose infusion rate (GINF) and sensitivity index (SI),
assessed during a hyperinsulinemic euglycemic clamp. Liver functions measured were alkaline
phosphatase (AP), total protein (TP), albumin (ALB), total and direct bilirubin (TB, DB), lactic
dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT).
Other risk factors included in analysis were lipids (TC, HDL, TG, ApoAI, ApoAII, and ApoB), age,
sex, BMI, whratio, and carotid intima-media thickness (IMT). Given the correlated nature of IR
measures, lipid traits, and liver functions, we first conducted principle component analyses
individually for IR, lipids, and liver function. One principle component accounted for 97% of total
IR variance (IR P1 0.13*fasting insulin 0.97*GINF 0.20*SI), one principle component
accounted for 84% of total lipid variance (LIP P1 0.11*TC 0.29*TG - 0.08*HDL -
0.02*APOAI 0.03*APOAII 0.95*APOB), and 4 principle components accounted for 86% of
total liver function variation. These principle components were used, together with the other
risk factors, in SBP and DBP modeling. Stepwise regression methods were used for variable
selection and significance of the predictors was confirmed by mixed model analysis, which
takes into account familial correlations among family members. Two of the 4 liver function
principle components (0.05*AP 0.02*TP 0.36*ALB 0.69*TB 0.60*DB 0.03*LDH investigated whether administration of tempol reversed the developed cardiac hypertrophy in
0.14*AST 0.09*ALT and 0.88*AP - 0.002*TP - 0.04*ALB - 0.31*TB 0.34*DB 0.04*LDH
- 0.05*AST - 0.13*ALT) were found to be significant predictors for SBP, as were IMT,
IR PRIN1, sex, and BMI. For DBP, the predictors were: age, sex, and IR PRIN1. These
findings indicate that liver function is related to SBP, independent from IR, in non-diabetic HA
families.
P170
2-Adrenoceptor Protein Expression, but not mRNA Expression, Relates to
Obesity
Kazuko Masuo, Urbain Tchoua, Nora Straznicky, Gavin Lambert, Florentia Socratous, Baker
Heart Rsch Institute, Melbourne Vic, Australia; Tomohiro Katsuya, Hiromi Rakugi, Toshio
Ogihara, Osaka Univ Graduate Sch of Medicine, Suita City, Osaka, Japan; Murray Esler;
Baker Heart Rsch Institute, Melbourne Vic, Australia
Objective: The 2 (ADRB2)- and 3-adrenoceptor polymorphisms relate to obesity. In the
present study, we examined the relationships between ADRB2 protein and mRNA levels in buffy
coats, ADRB2 genotypes and obesity (BMI) to clarify further mechanisms by which ADRB2
polymorphisms relate to obesity. Methods: [Study 1] We measured the 2 (ADRB2, Arg16Gly,
Gln27Glu) and 3 (Trp64Arg)-adrenoceptor polymorphisms in 67 normotensive Australian men
with a wide range of BMI (19.6 40.4 kg/m2) after 12 hours fasting. Since the Gly16 allele
of ADRB2 gene is associated with obesity (28.16, P0.017), we compared ADRB2 protein
concentration, ADRB2 mRNA expression of white blood cells (buffy coats) and whole body
plasma norepinephrine (NE) spillover in lean vs obese men, and between Arg16/Arg16,
Arg16/Gly16 and Gly16/Gly16 genotypes. ADRB2 protein expression was analysed by Western
blotting. Real time-PCR (RT-PCR) was used to determine receptor mRNA levels. [Study 2]
ADRB2 protein and ADRB2 mRNA expressions between the 3 genotypes of Arg16Gly in
overweight or obese subjects (n23) was investigated before and after significant (10%)
weight loss of 12 weeks. Results: [Study 1] The Western blotting showed that obese subjects
and those carrying the Gly16/Gly16 genotype had significantly greater ADRB2 protein
expression compared to lean subjects (P0.01) or those carrying the Arg16/Arg16 genotype
(P0.05). However, ADRB2 mRNA expression by RT-PCR was similar between lean,
overweight and obese groups and between 3 genotypes. Whole body NE spillover were greater
in the obese group (P0.05) and in the subjects carrying the Gly16 allele (P0.01) compared
to the lean group or those carrying the Arg16/Arg16 genotype. [Study 2] The protein expression
was significantly decreased with significant weight loss, being greater in the subjects carrying
Arg16/Arg16 compared to those carrying Gly16/Gly16, whereas mRNA expression did not
change. Whole body NE spillover decreased after significant weight loss in the group carrying
the Arg/Arg genotype. Conclusions: The 2-adrenoceptor polymorphisms associated with
heightened sympathetic nerve activity and high 2-adrenoceptor protein expression are linked
to obesity in the Australian male population.
P171
A Gene Polymorphism of Novel Z-Disc Protein, Myospryn, is Associated
with Left Ventricular Hypertrophy and Diastolic Dysfunction
Hironori Nakagami, Tomohiro Katsuya, Ryuichi Morishita, Yasushi Kikuchi, Osaka Graduate
Sch of Medicine, Suita, Japan; Hiroshi Akasaka, Sapporo Med Univ, Sapporo, Japan; Hiromi
Rakugi, Yasufumi kaneda, Toshio Ogihara; Osaka Graduate Sch of Medicine, Suita, Japan
Recent understanding of stretch sensing in muscle would address to Z-disc elements in
cytoskeleton. Recently, the novel Z-disc protein, named myospryn, was reported to be localized
on Z-disc of striated muscle cells where it co-localized with the sarcomeric -actinin. Although
biomechanical forces due to the activation of stress pathways has been denoted to play a
critical role in the progression of left ventricular diastolic dysfunction, there is no clinical
evidence of association between Z-disc protein and cardiac dysfunction under pressure
overload. Thus, in this study we analyzed the association between its polymorphism and
cardiac function. To examine the association between myospryn polymorphism and cardiac
function, we selected 27 SNPs in the coding lesion of human myospryn gene leading to amino
acid substitution by searching the NCBI and Ensemble databases. The sequencing of 50 DNA
samples from Japanese volunteers showed that 16 SNPs of them were linkage disequilibrium
with fairly frequency (minor allele frequency 0.413). The screening of its polymorphism
(K2906N) in a rural Japanese population resulted in the association with cardiac diseases in
general checkment (genotype AA:6.9 1.4 %, AC:5.4 1.0 %, CC:5.1 1.4 %, p0.0082).
In further analysis of the interaction between K2906N polymorphism and cardiac function of
patients, we found evidence that K2906N polymorphism was associated with the marker of left
ventricular cardiac dysfunction, the ratio of the peak velocity of early diastolic filling wave and
the peak velocity of atrial filling (A/E) (genotype AA:1.27 0.06, AC:1.10 0.04, CC:1.08
0.07, p0.0278), and left ventricular wall thickness (genotype AA:10.85 0.34 mm,
AC:9.99 0.25 mm, CC:10.05 0.29 mm, p0.0324), measured by echocardiography.
These results suggest that K2906N polymorphism would be clinically associated with left
ventricular hypertrophy and diastolic dysfunction independent on known parameter, such as
blood pressure. Although the precise mechanism of the association remains to be elucidated,
our results strongly suggest that myospryn can work with mechanical sensor as a member of
striated muscle complex in hypertensive patients.
P172
Effects of Superoxide Dismutase Mimetic, Tempol on Developed
Hypertensive Cardiac Hypertrophy in Genetic Hypertensive Rats (shr-sp)
Tomoko Shinzato, Yusuke Ohya, Syuichi Takishita; Univ of the Ryukyus, Nishihara-cho,
okinawa, Japan
Objectives: Increase in oxidative stress has been reported in left ventricular (LV) tissues from
various models of cardiac hypertrophy. However, contribution of oxidative stress to the
maintenance of hypertensive cardiac hypertrophy remains unclear. The superoxide dismutase
mimetic, tempol has been used as an anti-oxidant in various animal models. We thus
adult stroke-prone SHR (SHR-SP). Method: We used 32 week-old SHR-SP with or without
preceded oral administration of tempol (1mol/L) for 8 weeks, and Wistar Kyoto rats (WKY). We
evaluated BP by tail-cuff method and LV morphology by echocardiography. In isolated LV, we
evaluated fibrosis and appearance of -smooth muscle actin-positive fibroblast (myofibroblast)
by histochemistry; mRNA levels of collagen type I, collagen type III, transforming growth
factor-1 (TGF-1), and angiotenisin II type1 receptor (AT1R) by real time PCR; protein
expression of connective tissue growth factor (CTGF) by western blot; and matrix metallopro-
tease (MMP) activities by zymogaphy. We determined urine 8-hydroxydeoxyguanosine (8-
OHdG) as a marker of systemic oxidative stress. Results: LV from untreated SHR-SP showed
concentric hypertrophy (increased heart weight, increased wall thickness, and decreased LV
diameter), compared with WKY. Interstitial and perivascular fibrosis, number of myofibroblast,
and myocyte diameter were increased in LV from untreated SHR-SP. mRNA levels of collagen
type I, collagen type III, TGF-1 and AT1R, and protein level of CTGF were increased in LV from
SHR-SP. Tempol decreased urine 8-OHdG level, but did not decrease BP. Tempol improved
above morphological alterations in LV with decrease in mRNA levels of collagen type I and
AT1R, and decrease in protein level of CTGF. Tempol did not significantly change mRNA level
of collagen type III or TGF-1, or activities of MMPs. Conclusion: In adult SHR-SP, tempol
reversed hypertensive LV remodeling with regression of interstitial and perivascular fibrosis and
decrease in myocyte size. Oxidative stress may be involved in mechanism for the progression
or maintenance of hypertensive LV hypertrophy at least in part, independently from
hypertension.
P173
Protein Kinase C Alpha Deficiency Protects against Angiotensin II- Induced
Myocardial Hypertrophy and Heart Failure
Alexander Kranzhofer; Med Sch Hannover, Hannover, Germany
Introduction: Protein kinase C isoform alpha (PKC-) plays a crucial role in pressure-induced
heart failure. Since PKC- has been implicated in the signal transduction of angiotensin II (Ang
II) we tested the hypothesis that PKC- is important in Ang II- induced myocardial hypertrophy
and cardiac dysfunction. Methods: PKC- deficient mice were generated from a SV129
background. Male PKC- deficient and wild type (WT) mice aged 1214 weeks were used for
experiments. Ang II was infused continuously at a dose of 150 ng/kg body weight/day by
implanted osmotic minipumps for 4 weeks. Blood pressure was assessed by tail-cuff
measurements. Hemodynamic parameters were analyzed in-vivo by intracardial catheteriza-
tion. Hearts were further analyzed by histology and immunohistochemistry. Results: No blood
pressure increase was observed in the Ang II treated animals. Ang II resulted in myocardial
hypertrophy in WT but not in PKC- deficient mice (WT 4.78 0.15 vs. PKC- deficient mice
4.14 0.31 heart/body weight ratio (mg/g); p0.01). Hemodynamic analysis showed that Ang
II lead to a reduced cardiac output and decreased contractility in WT animals compared to

baseline (p0.05). In contrast, PKC- deficient mice were protected against these changes.
Interstitial as well as perivascular fibrosis was also different between two genotypes. The
expression of fibronectin was significantly increased in the WT-Ang II vs. PKC- deficient-Ang
II mice. Conclusions: Our Results suggests that PKC-alpha is an important mediator of Ang
II-induced myocardial hypertrophy and dysfunction.
P174
The Isoproterenol-Induced Impairment of Heart Function and Remodeling
Are Prevented by the Nonpeptide Angiotensin-(17) Analogue AVE 0991
Anderson J Ferreira, Thauana L Oliveira, Maria C Castro, Alvair P Almeida, Carlos H Castro,
Elisandra Gava, Gregory T Kitten, Robson A Santos; Federal Univ of Minas Gerais, Belo
Horizonte, Brazil
The aim of this study was to evaluate the effects of AVE 0991 (AVE), a nonpeptide compound
that mimics Ang-(17) actions, on cardiac remodeling. Heart hypertrophy and heart failure were
induced by isoproterenol (ISO) (2 mg/kg i.p., 7 days) in male Wistar rats. To assess the role of
NO in the effects of AVE (0.58 mol/kg) in rat hearts treated with ISO, another group of rats
was treated with L-NAME (10 mg/kg). At the end of the 7-day period, the hearts were perfused
according to the Langendorff method to evaluate cardiac function. The left ventricles wet
weights was normalized for body weight and expressed as muscle mass index (mg/g). Left
ventricles serial sections were stained with hematoxilin-eosin for cell morphometry and with
collagen-specific Massons trichrome for detection of fibrosis. Immunofluorescence-labeling
and confocal microscopy were used to investigate the distribution and deposition of collagen
types I, III, VI, and fibronectin. AVE completely blocked the ISO-induced hypertrophy quantified
by myocyte diameter measurement (Control: 10.60 0.08 m; ISO: 14.60 0.11 m;
ISOAVE 11.22 0.08 m, n5). In addition, AVE markedly attenuated the increase of matrix
proteins induced by ISO as illustrated for collagen III (Figure). AVE treatment also abrogated the
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
CHBPR ConferencePoster Presentations e79
Lewis (L) and congenic (SCL-10) rats. Cardiac function was assessed by electrocardiography,
echocardiography and a Millar catheter in left ventricular (LV) cavity. LV MMP and TIMP levels
were measured by Q-RT-PCR and Western blot analyses. MMP and TIMP activities were
assessed by zymography and reverse-zymography, respectively. In L, D and SCL-10 rats, heart
weight/body weight (g/g) were 3.730.06, 4.450.04 and 3.350.05 x 10-3, respectively,
suggesting LV hypertrophy (LVH) in D group. The ST duration was longer in D group as
compared to L group, but normalized in SCL-10 rats. The fractional shortening and ejection
fraction were decreased in D group as compared with L group, but normalized in SCL-10
groups. LV wall was thickened in D group as compared to L group, but normalized in SCL-10
groups. The levels of MMP were higher and TIMP were lower in D as compared to L group, but
normalized in SCL-10 rats. The results suggest that cardiac dysfunction and LVH in DSS rats
were associated, in part, due to the increased MMP and decreased TIMP levels. The congenic
transfer of TIMP ameliorates LVH and cardiac dysfunction in DSS rats.
P177
Angiotensin-(17) Reduces COX-2 in Cardiac Fibroblasts
LaTronya T McCollum, Patricia E Gallagher, Carlos M Ferrario, E. Ann Tallant; Wake Forest
Univ Sch of Medicine, Winston-Salem, NC
Cardiac remodeling is characterized by an inflammatory response in which cytokines induce
cyclooxygenase 2 (COX-2) to promote production of inflammatory prostaglandins. Angiotensin
II (Ang II) plays a critical role in cardiac remodeling by upregulating COX-2 to promote
inflammation and fibrosis. Angiotensin-(17) [Ang-(17)], also present in the heart, is
generated endogenously from angiotensin I (Ang I) or Ang II. Since Ang-(17) often opposes the
physiological effects of Ang II, the aim of this study was to determine whether Ang-(17)
reduces the inflammatory response(s) associated with remodeling in cardiac fibroblasts.
Treatment of neonatal rat cardiac fibroblasts with 100 nM Ang-(17) caused a significant
time-dependent reduction in COX-2 mRNA. COX-2 mRNA was decreased maximally by 57.8%

decrease in systolic tension and dT/dt and exacerbated the vasodilatation induced by ISO.
The vascular and anti-hypertrophic effects of AVE were completely blocked by L-NAME. On the
other hand, the beneficial effects of AVE on heart function were not influenced by NOS
blockade. These results show a marked cardioprotective role of AVE on ISO-induced cardiac
remodeling which is partially dependentEmax: maximal relaxation, *P0.05, vs HS; #P0.05,
vs NS. n6 8 on NO release.
P175
ACE2 Overexpression Inhibits Hypoxia-Induced Collagen Production by
Cardiac Fibroblasts
Justin L Grobe, Shant Der Sarkissian, Yuan Lihui, Jillian M Stewart, Mohan K Raizada,
Michael J Katovich; Univ of Florida, Gainesville, FL
Cardiac remodeling is a key risk factor for the development of heart failure in the chronic phase
following myocardial infarction. Our previous studies have shown an anti-remodeling role of
angiotensin converting enzyme 2 (ACE2) in vivo during hypertension, and that these protective
effects are mediated through increased circulating levels of angiotensin-(17). In the current
study, we examined the protective effects of ACE2 gene delivery to cultured cardiac fibroblasts
during exposure to hypoxia and reperfusion. Neonatal rat cardiac fibroblasts were isolated from
five day old Sprague Dawley rats, grown to confluence, and infected by a lentivirus delivering
the murine ACE2 gene under transcriptional control by the elongation factor 1 alpha promoter.
Infection resulted in a dose-dependent increase in ACE2 activity. This increased ACE2 activity
resulted in significant attenuation of hypoxia-reperfusion induced collagen production by the
fibroblasts. Cytokine production, specifically transforming growth factor beta (TGFb), by these
cells was also significantly attenuated by ACE2. In a subsequent experiment using murine ACE2
under transcriptional control by the hypoxia response element (HRE) promoter, it was
determined that hypoxia-induced ACE2 expression also significantly attenuated collagen
production by the cultured fibroblasts. These results indicate that 1) ACE2 prevents
hypoxia-induced collagen production by cultured fibroblasts, 2) that this prevention is likely
mediated by decreased expression of cytokines such as TGFb, and 3) that ACE2 can mediate
its protective effects even if expression is temporally limited to after the onset of hypoxia. We
conclude that hypoxia-induced ACE2 expression, limited to cardiac fibroblasts, may represent
a novel paradigm for in vivo therapy following acute ischemia.
following a 2-hour treatment with Ang-(17) and the reduction continued for up to 8 hours
(23.7% reduction). [D-Ala7]-Ang-(17), a specific Ang-(17) receptor antagonist, blocked the
down-regulation of COX-2 mRNA by the heptapeptide, indicating that the response was
mediated by a selective AT(17) receptor. RNA interference technology was used to silence mas,
a G protein-coupled Ang-(17) receptor, to determine whether mas mediates the effect of
Ang-(17) in cardiac fibroblasts. The Ang-(17)-mediated reduction in COX-2 mRNA was
blocked completely by a small interfering RNA (siRNA) against the mas receptor. In contrast,
siRNA against lamin, a structural protein, had no effect on the down-regulation of COX-2 mRNA
by Ang-(17) (51% reduction following a 2-hour treatment). Importantly, COX-2 mRNA was
significantly reduced in the hearts of rats with myocardial infarctions that were infused with
Ang-(17) for 8 weeks [0.25 0.05 relative gene expression with Ang-(17) infusion
compared to 1.02 0.08 relative gene expression with saline infusion, n 5, p 0.05].
These data demonstrate that Ang-(17) reduces COX-2 mRNA, through activation of the AT(17)
receptor mas. We and others previously showed that Ang-(17) reduces collagen production in
isolated cardiac fibroblasts. These results suggest a role for the heptapeptide as an
anti-inflammatory agent to reduce collagen deposition and cardiac remodeling following
cardiac injury.
P178
Specific [Beta1]-Adrenergic Receptor Silencing with SiRNA Lowers High
Blood Pressure and Improves Heart Functions in Myocardial Ischemia
Anne-Sophie Arnold; Univ of South Florida, Saint Petersburg, FL
-blockers are widely used and effective for treating hypertension, acute myocardial infarction
(MI) and heart failure, but they present many side effects mainly due to antagonism of
2-adrenergic receptor (AR). Currently available -blockers are at best selective but not
specific for 1 or 2-AR. To specifically inhibit the expression of the 1-AR, we developed a
small interfering RNA (siRNA) targeted to 1-AR. Three different sequences of 1 siRNA were
delivered into C6 2B cells with 90 % efficiency. Real-time RT-PCR experiments showed that
one of the three sequences reduced the level of 1-AR mRNA by 70 %. The siRNA was highly
specific for 1-AR inhibition with no overlap with 2-AR. To test it in vivo, systemic injection
of 1 siRNA complexed with liposomes resulted in efficient delivery into heart, lung, kidney,
liver and spleen, and effectively reduced the 1-AR expression in the heart without altering
2-AR, at the mRNA as well as the protein levels. 1 siRNA significantly lowered blood
pressure of spontaneously hypertensive rats (SHR) for at least 12 days and reduced cardiac
hypertrophy following a single injection. Pretreatment with 1 siRNA three days before
induction of MI in Wistar rats significantly improved cardiac functions, as demonstrated by
dP/dt and electrocardiogram following the MI. The protective mechanism involved reduction of
cardiomyocyte apoptosis in the 1 siRNA treated hearts. This study demonstrates the
possibility of using siRNA for treating cardiovascular diseases and may represent a novel
blocker specific for 1-AR.

P176
Congenic Expression of Tissue Inhibitor of Metalloproteinase Mitigates
Hypertension in Dahl-Salt Sensitive Rats
Walter Rodriguez, Neetu Tyagi, Univ of Louisville Sch of Medicine, Louisville, KY; Alan Deng,
Univ of Montreal Sch of Medicine, Montreal, Canada; ASO Adeagbo, Irving G Joshua, Suresh
C Tyagi; Univ of Louisville Sch of Medicine, Louisville, KY
Although congenic translocation of a segment from chromosome 10 from Lewis rat, containing
an extracellular proteinase inhibitor gene, decreases blood pressure in Dalh-salt sensitive (DSS)
rats, the relationship between the levels of matrix metalloproteinase (MMP), tissue inhibitor of
metalloproteinase (TIMP), and cardiac function was unclear. To test the hypothesis that cardiac
hypertrophy in DSS, in part, due to higher MMP and lower TIMP levels as compared to Lewis,
cardiac function and levels of MMP and TIMP were determined in 16 weeks male DSS (D),
P179
Selective Anti-Proliferative and Apoptotic Effect of Moxonidine on Cardiac
Fibroblasts in Culture
Suhayla Mukaddam-Daher, Safa Bentaeibi, Pierre-Alexandre Paquette, Ahmed Menaouar,
CR-CHUM, Hotel-Dieu Hosp, Montreal, Canada; Gaetan Thibault, IRCM, Montreal, Canada;
Jolanta Gutkowska; CR-CHUM, Hotel-Dieu Hosp, Montreal, Canada
Regression or prevention of hypertension-induced left ventricular hypertrophy (LVH) by
antihypertensive therapy is associated with favorable treatment outcomes. Chronic treatment
with moxonidine, a sympatholytic antihypertensive compound that binds preferentially to
imidazoline I1-receptors, results in blood pressure reduction and regression of LVH. We have
shown that in vivo moxonidine treatment of adult spontaneously hypertensive rats for 4 weeks,
results in prevention of LVH, associated with lower left ventricular DNA synthesis and increased
DNA fragmentation, occurring 1 week after treatment, and normalized by 4 weeks. However,
 e80 Hypertension Vol 48, No 4 October 2006
the cellular target of moxonidine was not identified. Therefore, studies were performed to
investigate the effect of moxonidine on left ventricular neonatal and adult fibroblasts and
myocytes in culture. Western blotting revealed the presence of imidazoline receptors in
membranes of both cell types with higher density in fibroblasts. Cells were cultured for 24 h
with norepinephrine (NE, 10-4 M) in the presence and absence of moxonidine (10-6 & 10-5 M).
DNA synthesis measured by 3H-thymidine incorporation revealed that NE-induced uptake
(100%) was concentration-dependently inhibited (p0.01) by moxonidine in adult (8112 and
5930%) and neonatal fibroblasts (7410 and 6717%). Compared to untreated fibroblasts,
NE and moxonidine, each up-regulated the apoptotic mitochondrial protein, Bax (4 and 3 fold)
and the effect of both treatments was additive (7 fold). Moxonidine reversed NE-induced
inhibition of DNA fragmentation in neonatal fibroblasts. In contrast, moxonidine tended to
increase NE-treated myocyte protein synthesis measured by 3H-leucine incorporation and did
not affect DNA fragmentation, despite mild potentiation of Bax. Moxonidine did not affect
anti-apoptotic protein Bcl-2 in both cell types. These results demonstrate a selective growth
control effect of moxonidine on fibroblasts mediated by inhibition of DNA synthesis and
stimulation of DNA fragmentation. The cell-selective action is consistent with a cardioprotective
effect of moxonidine in hypertensive heart disease. Supported by the Canadian Institutes of
Health Research and the Heart and Stroke Foundation of Quebec
P180
Novel Anti-Inflammatory Mechanisms of Ac-SDKP in High Blood
Pressure-Induced Target Organ Damage
Umesh C Sharma, Saraswati Pokharel, Pamela Harding, Saman Rasoul, Nour-Eddine Rhaleb,
Oscar Carretero; Henry Ford Health System, Detroit, MI
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Background: High blood pressure (HBP) is an important risk factor for cardiac, renal and
vascular dysfunction. Excess inflammation is the major pathogenic mechanism for HBP-
induced target organ damage (TOD). Ac-SDKP, a tetrapeptide specifically degraded by
angiotensin converting enzyme, reduces inflammation, fibrosis and TOD induced by high blood
pressure. Hypothesis: In this study, we hypothesize that Ac-SDKP exerts its anti-inflammatory
effects by inhibiting 1) activation and migration of macrophages and 2) release of pro-
inflammatory cytokine, TNF- by activated macrophages. Methods: Macrophages were
obtained by culturing freshly isolated bone marrow stem cells in giant cell tumor conditioned
medium. Myocardial macrophage activation in DOCA salt-treated hypertensive mice was
detected by Western blotting of Mac-2 (galectin-3) protein. Macrophage migration was
measured in a modified Boyden chamber. TNF- release by activated macrophages was
measured by enzyme-linked immunossays (ELISA). Results: Ac-SDKP decreased macrophage
colony stimulating factor (M-CSF)-dependent macrophage migration in a dose-dependent
manner [Absorbance: vehicle, 1.870.29; Ac-SDKP (10nM), 0.650.04; Ac-SDKP (1000 nM),
0.600.02]. This inhibitory effect was blocked by anti-AC-SDKP monoclocal blocking antibody.
In vivo, Ac-SDKP reduced the infiltration of activated macrophages into the myocardium of
DOCA salt-treated hypertensive mice (Mac-2, densitometric units: DOCA, 1.30.62;
DOCAAc-SDKP, 0.640.28, P0.05). In addition, Ac-SDKP significantly decreased the
secretion of TNF- by peritoneal macrophages stimulated with bacterial LPS (pg/ng of cell
protein: LPS, 7048.8; LPSAc-SDKP, 63512, P0.05). Conclusion: This study reveals
novel and previously unknown mechanisms of the anti-inflammatory property of Ac-SDKP.
Ac-SDKP can be a new therapeutic option for the treatment of HBP-induced inflammation and
TOD.
P181
The Kinin B1 Receptor Contributes to the Cardioprotective Effect of ACEi
and Angiotensin Receptor Blockade
Jiang Xu, Oscar A Carretero, Ying Sun, Edward G Shesely, Nour-Eddine Rhaleb, Henry Ford
Hosp, Detroit, MI; Michael Bader, Max-DelbrUeck-Cntr for Molecular Medicine, Berlin-Buch,
Germany; Xiao-Ping Yang; Henry Ford Hosp, Detroit, MI
Activation of the kinin B2 receptor contributes to the cardioprotective effect of angiotensin-
converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARB), whereas the role
of B1 remains unclear. We previously showed that mice lacking B1 (-/-) had increased LV mass
and chamber dilatation, indicating that B1 exert a physiological role in maintaining cardiac
structure and function. We further hypothesized that kinins acting on B1 partially contribute to
the cardiac beneficial effect of ACEi or ARB. Female B1-/- mice and wild-type controls
(C57BL/6J, B1/) were subjected to sham MI or MI by ligating the left anterior descending
coronary artery. Three weeks after MI, each strain was treated with vehicle, ACEi (ramipril, 2.5
mg/kg/day in drinking water) or ARB (valsartan, 40 mg/kg/day in drinking water) for 5 weeks.
LV ejection fraction (EF) and LV diastolic dimension (LVDd) were evaluated by echocardiogra-
phy. Infarct size was studied histologically. SBP and EF did not differ between strains in
sham-MI and decreased similarly after MI in both B1/ and B1-/-. Infarct size was similar
among groups. However, B1-/- had a greater LV mass and LVDd either at baseline or after MI.
ACEi and ARB significantly increased EF and decreased LVDd and these effects were
diminished in B1-/- (Table), although SBP decreased to a similar degree in both strains. Our
data suggest that kinins acting on the B1 receptor may exert a cardioprotective role and
contribute to the cardiac beneficial effects of ACEi and ARB.
Vehicle ACEi ARB
Groups B1 / B1 -/- B1 / B1 -/- B1 / B1 -/-
EF (% change)a -6.97.3 -7.16.0 39.86.5# 17.95.4*# 43.713.8# 16.44.0*#
LVDd (% Change) 3.12.1 3.72.6 -14.42.4# -3.11.5* -15.92.2# -6.82.2*#
#: p 0.05 vs vehicle within strain;*: p 0.05 between strains in the same treatment; a: % change after
treatment.
P182
The human D5 Dopamine receptor mediates Big KCa channel activity in
human coronary artery smooth muscle cells
Aruna R Natarajan, Georgetown Univ Hosp, Washington, DC; Guichun Han, Richard White,
Med College of Georgia, Augusta, GA; Pedro Jose; Georgetown Univ Hosp, Washington, DC
Coronary artery disease is a leading cause of mortality and results from impaired vasodilatation.
We have previously shown expression of D1-like (D1 and D5) dopamine receptors in human
coronary artery smooth muscle cells (HCASMC). To study the effects of D1-like receptor
stimulation on the Calcium and voltage-activated Big KCa channels in HCASMC, cells were
purchased from Cambrex and grown to Passage 5 6. Probability of opening of large-
conductance calcium- and voltage- activated channels Big KCa channels (NPo) were studied by
single-cell patch clamp technique in these cells in response to D1-like receptor agonists and
antagonists. We have confirmed our earlier reports that Fenoldopam (1M) and SKF 21987
(1M), both D1-like receptor agonists, cause greater than 10-fold increase in Big KCa channel
activity (n 3) in untransfected HCASMC, and in cells transfected with D1 antisense, D1
scrambled, and D5 scrambled oligonucleotides. However, this response was abrogated in cells
transfected with D5 antisense oligonucleotides. (n 3 for each group of cells) Downregulation
of D1 and D5 protein expression was shown in cells transfected with D1 and D5 antisense
oligonucleotides respectively. The Big KCa channels were blocked with the D1-like receptor
antagonist, SCH 23390 (10M), and inside-out patches characterized the Big KCa channel in all
patches. Thus D5 receptors are mainly responsible for the dopaminergic activation of Big KCa
channels in HCASMC, and may be implicated in coronary vasorelaxation.
P183
Plasminogen Activator Inhibitor-1 as a Predictor of Postoperative Atrial
Fibrillation Following Cardiopulmonary Bypass
Mias Pretorius, Brian S Donahue, Chang Yu, Dan M Roden, Nancy J Brown; Vanderbilt Univ
Med Sch of Medicine, Nashville, TN
Background: Postoperative atrial fibrillation (AF), leading to significant morbidity and prolon-
gation of hospital stay, complicates 20% to 40% of surgical procedures requiring cardiopul-
monary bypass (CPB). This study tests the hypothesis that inflammatory markers predict the
development of postoperative AF. Methods and Results: Two hundred and fifty-three adult
patients undergoing elective cardiac surgery requiring CPB were prospectively enrolled. Blood
samples were obtained for measurement of inflammatory markers immediately after separation
from CPB and administration of protamine. Sixty-seven patients (26.5%), without AF within 7
days prior to surgery, developed postoperative AF. Patients who developed postoperative AF
were significantly older (P0.001), more likely to have a remote history of AF (P0.001) and
tended to be more likely to have had valve surgery (P0.082). Plasminogen activator
inhibitor-1 (PAI-1, 17.21.2 ng/mL versus 14.60.7 ng/mL, P0.014), interleukin(IL)-6
(380.6151.1 pg/mL versus 174.816.9 pg/mL, P0.019) and N-terminal prohormone brain
natriuretic peptide (248.263.2 pg/mL versus 182.030.6 pg/mL, P0.028) concentrations
were significantly higher in the blood of patients who developed postoperative AF. Logistic
regression identified age (P0.001), remote history of AF (P0.002), valve surgery (P0.076)
and PAI-1 (P0.057) as independent predictors of postoperative AF. The Chi-squared
Automatic Interaction Detection (CHAID) model indicated that age was the primary determinant
for the development of postoperative AF (17% in age 67.3 years versus 49% in age 67.3
years). Within younger patients (age 67.3 years) with no remote history of AF, PAI-1 antigen
concentration next determined risk of AF (13% if PAI-128.5ng/mL versus 46% if PAI-1
28.5ng/mL). There was a significant correlation of PAI-1 antigen concentration with CPB time
(P0.008), age (P0.005), body mass index (P0.001) and IL-6 (P0.001). Conclusion: An
elevated postoperative PAI-1 antigen concentration is an independent predictor for develop-
ment of postoperative AF following CPB. Studies are needed to determine whether drugs that
reduce PAI-1 concentrations could reduce the risk of AF.
P184
Xanthine Oxidase as a Contributor to Venous Oxidative Stress in
Hypertension
Theodora Szasz, Gregory D Fink, Stephanie W Watts; Michigan State Univ, East Lansing, MI
Reactive oxygen species generated by enzymes like xanthine oxidase (XO) modulate vascular
function and may contribute to hypertension pathogenesis. We are investigating a potentially
distinct role for veins in these processes. We have previously shown that basal superoxide
production is higher in the rat vena cava compared to aorta (V 0.080.15, A 0.040.006
nmol*min/mg tissue). Moreover, in isometric contractile experiments, the xanthine/xanthine
oxidase system induced a concentration-dependent contraction that was potentiated in vena
cava compared to aorta (contraction to 3*10-2 U/ml XO, 200 M xanthine: A 17.57.5, V
19641 % of initial adrenergic-mediated contraction). Allopurinol (100 M) reduced maximal
contraction to endothelin-1 (100 nM) in the vena cava, but not in the aorta (control V
55368 to allopurinol V 38444, control A 1437 to allopurinol A 1369 % of initial
adrenergic-mediated contraction). Thus, we hypothesized that XO protein expression and/or
activity in veins is higher compared to arteries, a difference that may participate in the oxidative
stress of hypertension. Western analysis of thoracic aorta and vena cava homogenates from
sham normotensive and DOCA-salt hypertensive rats showed increased XO protein expression
in the vena cava compared to aorta in both groups, but no difference between sham and DOCA
(SA 11416, SVC 24215, DA 12424, DVC 29138 densitometry units).
Immunohistochemically, XO stained throughout the wall of the vena cava, while it was only
detected in the endothelial and adventitial layers of the aorta, with no change of pattern
between sham and DOCA sections. XO activity was measured spectrophotometrically as
allopurinol (100 M)-inhibitable uric acid formation in the presence of xanthine (200 M) and
oxonic acid (1 mM). Vena cava had increased XO activity compared to aorta in both groups.
Importantly, while the XO activity was similar in DOCA and sham aorta, it was significantly
increased in the DOCA vena cava compared to sham (SA 17.77.1, DA 18.84.6,

SVC 44.19.4, DVC 101.414.8 nmol uric acid/g protein). These data suggest that limited. The present study was conducted to understand the molecular mechanism regulating
venous rather than arterial XO-mediated oxidative stress may exert its deleterious effects on
vascular function during mineralocorticoid hypertension.
P185
The Existence of a 5-HT Synthetic and Metabolism System in Rat
Peripheral Arteries
Wei Ni, Theodora Szasz, Keith Lookingland, Stephanie W Watts; Michigan State Univ, East
Lansing, MI
We have reported a 5-hydroxytryptamine transporter (5-HTT)-dependent uptake and release of
5-HT in peripheral arteries. Arterial 5-HTT can modify contraction as evidenced by potentiation
of 5-HT-induced contraction by the 5-HTT inhibitor fluvoxamine in arteries from DOCA-salt rats.
It is unknown whether the source of arterial 5-HT depends only on extracellular import or
whether 5-HT could be synthesized in arteries, nor do we know whether peripheral arteries
regulate 5-HT concentration by metabolizing 5-HT. We hypothesized that peripheral arteries
synthesize and metabolize 5-HT by testing the existence of the key enzyme in 5-HT synthesis,
tryptophan hydroxylase (TPH) and monoamine oxidase A (MAO A), which metabolizes 5-HT to
5-hydroxyindole acetic acid (5-HIAA). Moreover, we examined whether TPH and MAO A exist
in arteries from DOCA-salt hypertensive rats. The presence of TPH mRNA in rat aorta (RA) and
superior mesenteric arteries (SMA) was determined by real time RT-PCR (TPH CT:
RA30.60.8, SMA26.61.6; GAPDH CT: RA25.71.3, SMA23.10.1). TPH protein
was localized in the cytosol of freshly dissociated RA and SMA smooth muscle cells and in
endothelium and smooth muscle of arteries from SHAM and DOCA rats by TPH antibody. MAO
A protein was similarly expressed in RA from SHAM and DOCA rats by western analysis.
However, there was denser staining for MAO A in the endothelium in RA from DOCA compared
to SHAM rats, which might contribute to the reduced basal level of 5-HT in RA from DOCA rats
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
(ng/mg protein, arterial 5-HT, SHAM0.310.07, DOCA0.170.03) as the endothelium is
the barrier between free circulating 5-HT and smooth muscle cell. Measurable 5-HIAA in aorta
suggests that MAO A is functional (ng/mg protein, SHAM0.410.14, DOCA0.520.11). In Alexa L Mundy, Corinne C Widmer, Martin Kretz, Julia Rasi, Elvira Haas, Matthias Barton;
conclusion, we observed the existence of TPH and MAO A, which suggests a 5-HT synthetic and
metabolism system exists in peripheral arteries under normal and hypertensive conditions.
Thus we begun to lay the groundwork for a serotonergic system in arteries uptake, release,
synthesis and metabolism. Further understanding of an arterial serotonergic system in
physiological and pathological situations is important to our knowledge of 5-HT as a modifier
of arterial resistance and blood pressure.
P186
Role of Nitric Oxide and Superoxide in Increased Afferent Arteriole
Myogenic Response in Mice with Chronic Heart Failure
Hong Wang, Jeffrey L Garvin, Xiao-Ping Yang, Jiang Xu, Yun-He Liu, Oscar A Carretero;
Henry Ford Hosp, Detroit, MI
Heart failure (HF) increases afferent arteriole (Af-Art) myogenic constriction and is associated
with diminished responses to endothelium-dependent vasodilatation. We hypothesized that this
increased Af-Art myogenic response is mediated by reduced nitric oxide-dependent dilatation
and/or increased accumulation of superoxide (O2-) anion in HF. Mice with HF (ejection fraction,
33.74.5%) and control mice (ejection fraction, 79.20.8%) were studied 4 to 5 weeks
post-myocardial infarction. Af-Arts were microdissected and perfused at intraluminal pressure
from 60 to 140 mm Hg. Nitro-L-arginine methyl ester (L-NAME) (100 mol/L) was added to the
arteriolar perfusate to inhibit nitric oxide following the first pressure-response curve and
maintained throughout the second pressure-response curve. To study the role of O2-, the O2-
dismutase mimetic tempol (100 mol/L) was added to the arteriolar perfusate after the initial
pressure-response curve. L-NAME decreased basal Af-Art diameter by 11.4 0.3% in control
and 6.7 0.4% in HF mice. L-NAME also enhanced Af-Art myogenic constriction in control but
not HF. Tempol did not change basal Af-Art diameter in either control or HF, but significantly
reduced myogenic constriction in HF mice and had no effect in control (table). We concluded
that increased Af-Art myogenic response in mice with HF may be due to increased O2-, which
scavenges nitric oxide and leads to increased vascular tone.
PRESSURE-INDUCED CHANGES IN AF-ART DIAMETER AS % CHANGE
FROM 60 MMHG
Pressure (mm Hg) 80 100 120 140
control (n5) 0.20.7% -5.30.6% -9.50.1% -10.80.1% Fibroblast growth factor-binding protein (FGF-BP1) is a secreted protein that chaperones
control L-NAME 0.60.3% -9.40.7%* -16.40.7%* -16.90.4%* fibroblast growth factors (FGFs) stored in the extracellular matrix milieu, thereby enhancing
HF (n6) -0.60.3% -7.70.5% -17.50.9% -19.01.2% FGF-dependent biochemical and biological events. FGFs play a major role in cancer,
HFL-NAME -0.40.5% -7.80.5% -17.11.1% -18.20.9% angiogenesis, tissue homeostasis and development. Studies with FGF-2-null mice revealed
control (n4) 0.40.6% -4.91.1% -10.10.8% -10.20.8%
control Tempol 0.60.7% -5.10.9% -10.10.6% -10.20.5%
HF (n4) -0.20.3% -8.91.3% -21.13.5% -20.93.2% effects of FGF-BP1 in vivo, we generated transgenic mice with conditional expression of
HFTempol -1.30.4% -5.20.8%# -11.61.6%# -12.81.6%# FGF-BP1 under a tetracycline regulatable promoter (tet-off system). FGF-BP1 expressing mice
*
p0.01 L-NAME vs. vehicle; #p0.05 tempol vs. vehicle.
P187
Regulation of Natriuretic Peptide Receptor-A Gene (Npr1) Transcription by
Cooperative Interactions of Ets-1 and p300
Prerna Kumar, Kiran K Arise, Kailash N Pandey; Dept of Physiology and Hypertension and
Renal Cntr of Excellence, Tulane Univ Health Sciences Cntr & Sch of Medicine, New
Orleans, LA
Activation of natriuretic peptide receptor-A (NPRA) produces the second messenger cGMP,
which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis.
Studies to elucidate the molecular regulation of Npr1 (coding for NPRA) gene transcription are
CHBPR ConferencePoster Presentations e81
Npr1 gene transcription which would provide new insights into strategies to control NPRA-
dependent signaling in pathophysiology of hypertension and cardiovascular disorders. Npr1
promoter-reporter deletion constructs were transiently transfected in mouse mesangial cells
(MMCs) and transcriptional activity was measured by dual luciferase assay. To see the effect
of transcription factors cotransfections were done along with the promoter constructs.
Functional deletion analysis of Npr1 promoter defined a 400 base pairs (bp) region as basal
promoter having Sp1, Lyf-1, p300, c-ETS, and GATA 1/2 transcription factor binding sites.
Further deletion of the basal promoter defined a 90 bp fragment as core promoter, having two
Ets-1 and a p300 binding motifs, which is essential in controlling transcription. Electrophoretic
mobility shift assay, supershift and mutational analyses confirmed that endogenously
expressed Ets-1 binds to Ets-1 consensus sites. Site directed mutagenesis of the two Ets-1
binding sites decreased >90% Npr1 promoter activity. Furthermore, deletion of Ets-1 and p300
binding sites significantly reduced the functional activity (p0.001) of Npr1 promoter.
Overexpression of Ets-1 and p300 showed synergistic effect in upregulation of Npr1 promoter.
There was significant activation in Npr1 core promoter activity by almost 16-fold (p0.001),
thus confirming the involvement of these transcription factors in mediating gene expression. In
conclusion, Npr1 gene expression underlies a complex regulation in which Ets-1 activates and
functions cooperatively with p300 to promote Npr1 gene transcription. Results from these
studies will greatly help to understand the role of Npr1 gene in control of hypertension and
cardiovascular regulation.
P188
High-Fat Diet Modulates Angiotensin-Mediated Vasoconstriction: Role of
Nox2/gp91phox
Dept of Internal Medicine, Med Policlinic, Univ Hosp Zurich, Zurich, Switzerland
A major source of vascular ROS is the endothelial Nox2 (gp91phox)-containing NADPH oxidase.
The aim of our study was to investigate in different vascular beds of C57BL/6 (CTL) and NOX2
knock-out mice (Nox2-/-) the effect of high fat diet (42% calories from fat, for 15 weeks) on
angiotensin II (Ang II)-mediated vasoconstriction. Rings of thoracic aorta, carotid, femoral, and
renal artery were exposed to either Ang II (100 nM) or the alpha1 adrenergic agonist
phenylephrine (1100 nM, PE) in the presence of L-NAME (0.3 mM) to exclude effects of NO.
Ang II-induced contractions were greatest in femoral artery (687%, p0.05 vs aorta, carotid
and renal artery), followed by renal artery (467%, p0.05 vs aorta and carotid artery), carotid
artery (31% p0.05 vs aorta), and aorta (0.40.2%). In high-fat diet fed mice, Ang
II-induced contraction increased in the aorta, but decreased in the carotid artery (both p 0.05
vs standard chow); there was no change in contractility in the femoral or renal artery. In
Nox2-/-mice, vasoconstriction to Ang II was increased in aorta and carotid artery (3.5 and
2.1-fold, respectively, p0.05, vs CTL), but similar to controls in femoral and renal arteries. In
aorta and carotid artery effects of high-fat diet were observed in arteries of Nox2-/- mice
suggesting that Nox2-derived vasodilator ROS are activated in the response to Ang II upon
high-fat feeding. PE-induced contractions were unaffected by either Nox2-deficiency or
high-fat feeding. These data demonstrate that consumption of a high-fat diet induces
heterogeneous changes in the response to Ang II in different vascular beds and that
Nox2-derived ROS have a vasodilatory role in Ang II-induced vasoconstriction in the aorta and
carotid artery. In Nox2-deficiency this effect is attenuated by a high-fat diet.
P189
Regulation of Blood Pressure and Vascular Tone by an Angiogenic
Modulator, an Fgf-Binding Protein
Elena Tassi, Kevin McDonnell, Sung Eun Kim, David P Kodack, Krissa Gibby, G. Ian
Gallicano, Michael D Johnson, Glenn Solis, Yifan Chen, William Welch, Christopher S Wilcox,
Anton Wellstein; Georgetown Univ, Washington, DC
hypotension in the knock-out mice and decrease in vascular contractility. To investigate the
are viable, display a ubiquitous transgene expression and exhibit increased angiogenesis, as
assessed in a subcutaneous matrigel plug assay. Quite remarkably, induction of the FGF-BP1
transgene in telemetrically monitored mice caused a striking rise in mean arterial blood
pressure by 30 5 mm Hg within 2 days (p0.001). Treatment of mice with the antioxidant
superoxide dismutase mimetic Tempol inhibited the FGF-BP1-induced hypertensive as well as
angiogenic phenotype, suggesting reactive oxygen species as significant regulators of the
effect of FGF activation. In addition, in vivo measurement of cremaster arteriole contractility of
showed a dramatic approximately 100-fold sensitization to vasoconstrictors, such as angio-
tensin II (Ang II) after FGF-BP1 induction (pA2- values: control 5.8 0.3; FGF-BP1 7.8 0.1).
At the molecular level, we found an enhancement of FGF-2-induced downstream signal
transduction pathways (p38, JNK, ERK1/2) in the presence of Ang II in kidney epithelial cells,
making this crosstalk a likely mechanism of interaction of angiogenic and vascular control
factors. We conclude that angiogenesis and blood pressure regulation are intimately
intertwined and that this transgenic model may help shed some light on the underlying
mechanisms.
 e82 Hypertension Vol 48, No 4 October 2006
P190
Downregulation of Thioredoxin and Increase of Xanthine Oxidase Activity
in AII-Induced Hypertension in Follitropin Receptor Knockout Mice
Talin Ebrahimian, Ernesto L Schiffrin, Lady Davis Institute for Med Rsch, SMBD Jewish
General Hosp, McGill Univ, Montreal, Canada; Rhian M Touyz; Kidney Rsch Cntr, Ottawa
Health Rsch Institute, Ottawa, Canada
Increased intracellular reactive oxygen species (ROS) contributes to hypertension. The role of
several ROS-scavenging systems in hypertension is unclear. Thioredoxin (Trx) is a key regulator
of cellular redox balance, interacting with its endogenous inhibitor, Trx-interacting protein
(Txnip). Moreover estrogen is known to have protective effects against oxidative stress, we
hypothesized that in the absence of estrogen, downregulation of Trx contributes to increase
oxidative stress in Angiotensin II (Ang II)-induced hypertension. We used estrogen-deficient
follitropin receptor knockout (FORKO) female mice. Svj129 (WT), heterozygous () or FORKO
(-/-) female mice were infused with 400 ng/kg/min Ang II for 14 days. Systolic blood pressure
measured by tail cuff, similar in basal conditions between groups, increased similarly by Ang
II in WT and mice (1668mmHg vs 1215mmHg in WT and 1599mmHg vs
11313mmHg in , P0.0001, n9), increased further in -/- mice (1767mmHg vs NAADP. However, both IP3 (1M) and Rya (2 M), the agonists of IP3R and RyR/Ca2 release
1155mmHg, P0.0001, n9). Cardiac Trx expression, evaluated by western blot was
significantly decreased by Ang II (42%) in -/- but not in wt and mice (P0.05, n5).
Accordingly, Txnip tended to be increased only in -/- mice. Ang II had no effect on cardiac O2-
level, evaluated by dihydroethidine fluorescence (P0.05, n6), as well as on NAD(P)H
oxidase activity (P0.05, n6), evaluated by chemiluminescence, however it increased
significantly (3 fold) the cardiac xanthine oxidase activity in -/- mice (P0.05, n4) but not in
WT and mice. Moreover, cardiac hypertrophy expressed as a ratio of cardiac mass on tibia
length, was increased (37%) by Ang II in -/- group (P0.0009, n6), and not in WT and
animals. This was associated with an increased of ERK 1/2 phosphorylation (2 fold) in -/- mice
(P0.05, n5) and not in other groups. These findings suggest that downregulation of cardiac
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
Trx by its inhibitor and the increase of xanthine oxidase activity seem to be involved in the
development of Ang II- induced cardiac hypertrophy and hypertension in FORKO mice.
P191
cGMP-Dependent Protein Kinase Promotes Smooth Muscle Relaxation by
Phosphorylating and Stabilizing RGS2
Xiaoguang Sun, Patrick Osei-Owusu, Thomas H Steinberg, Kendall J Blumer; Washington
Univ Sch of Medicine, Saint Louis, MO
The nitric oxide (NO)-cGMP-PKG pathway plays an important role in vascular tone and blood
pressure regulation, the mechanisms of which are incompletely understood. RGS2 (regulator of
G-protein signaling 2), a GTPase-activating protein for Gq, is critical for blood pressure
homeostasis, and has been suggested to serve as an effector protein in the NO-cGMP-PKG
pathway that promotes vascular relaxation. Thus, we tested the hypothesis that RGS2 functions
as an NO-cGMP effector to block vasoconstriction induced by vasoactive peptides in aortic
smooth muscle cells. We observed that 1) vasopressin-triggered Ca2 transients through Ca2
release from intracellular stores, Ca2 entry through CCE (capacitative calcium entry), and
NCCE (non-capacitative calcium entry) were all augmented in smooth muscle cells of RGS2-/-
mice; 2) Ca2 transients through Ca2 release from intracellular stores, and through CCE were
inhibited by cGMP analogs in wild type but not in RGS2-/- cells; 3) vasopressin-triggered DAG
production was also inhibited by cGMP analogs in wild type but not in RGS2-/- cells. 4) cGMP
analogs attenuated degradation of the phosphorylable RGS2, but not the RGS2(S46A, S64A)
mutant lacking PKG phosphorylation sites. These observations suggest that activation of
cGMP-PKG pathway stabilizes RGS2 through phosphorylation of the protein. We conclude that
the NO-cGMP-PKG pathway uses RGS2 as a novel downstream effector to promote vascular
relaxation by attenuating vasoconstrictor-triggered Ca2 signaling in vascular smooth muscle
cells.
P192
Increased Ca2 Sensitization due to Enhanced Rho-Kinase Signaling
Resulting in Reduced Myosin Phosphatase Activity in Small Mesenteric
Arteries of Angiotensin II-Induced Hypertensive Rats
Rob H Hilgers, Joseph Todd, Jr, R C Webb; Med College of Georgia, Augusta, GA
Background - The phosphorylation of myosin light chain (MLC) maintains the contracted state
of vascular smooth muscle. Dephosphorylation results in relaxation and is determined by the
activity of myosin phosphatase (MP). In a process called Ca2 sensitization, Rho kinase
phosphorylates MP to inhibit its activity and maintain contraction. We tested whether a
decreased contribution of MP results in an increased contractility of small 4th-order mesenteric
arteries (MA) during early onset of hypertension. Methods and Results - Ca2 sensitivity was
assessed as the relation between changes in tension and [Ca2]i in depolarized MA (120
mmol/L K) exposed to stepwise increases in Ca2ex (0 - 5 mmol/L). Ca2 sensitivity was
compared between MA of normotensive (NT) and angiotensin II-induced (14-days) hypertensive
(HT) rats. Under control conditions maximal contractions induced by increasing Ca2ex were
larger in MrA of HT compared to NT rats. These contractions were more attenuated by the Rho
kinase inhibitor Y-27632 (0.1 mol/L) during HT compared to NT. The MP inhibitor calyculin
A (50 nmol/L) caused a greater Ca2 sensitization in MrA of NT compared to HT rats.
Rho-kinase (ROCK I) mRNA and protein expression levels were significantly higher in MA of
HT rats compared to NT. In stimulated (120 mmol/L K for 3 min) MA, the degree of
phosphorylation of the MP subunit (MYPT1) was greater during HT. Conclusions - This is the
first study demonstrating enhanced Ca2 sensitization during hypertension at the level of
resistance-sized arteries. The mechanism likely involves enhanced RhoA/Rho kinase signaling
due to decreased MP activity, resulting in enhanced contribution of MLC kinase to contraction.
P193
Characteristics of a New Lysosomal Ca2 Release Channel and its
Activation by NAADP in Bovine Coronary Arterial Smooth Muscle
Fan Zhang, Li Chen, Fan Yi, Pin-Lan Li; Virginia Commonwealth Univ, Richmond, VA
Recent studies in our laboratory and by other have indicated that nicotinic acid adenine
dinucleotide phosphate (NAADP) produced Ca2 release via a two-pool mechanism. There are
some pharmacological evidences that lysosome-associated Ca2 release may be the first pool
in NAADP-mediated Ca2 release. The present study attempted to demonstrate and charac-
terize the functional lysosomal Ca2 release channels using purified lysosome from bovine
coronary artery smooth muscle. A modified method of Percoll gradient ultracentrifugation was
used in the lysosomes isolation and purification from coronary artery. Lysosome fraction was
reconstituted into lipid bilayer with a protocol as we described previously. Biophysically, the
amplitude of recorded Ca2 channel current was clearly voltage-dependent from -40 to 40
mV under symmetrical 300 mM cesium in cis and trans solution, and the conductance of this
channel was 145 pS. NAADP (1 - 100 nM) significantly increased the open probability (NPo) of
these reconstituted channels from 0.0063 0.0019 of control to 0.0519 0.0158 of 100nM
channel respectively, had no effects on the NPO of these lysosomal NAADP-sensitive Ca2
release channels. These results for the first time indicate that the lysosomal NAADP-sensitive
Ca2 release channels are present in coronary arterial smooth muscle and these channels
represent another type of intracellular organelle Ca2 release channels different from RyR and
IP3R Ca2 release channels on the sarcoplasmic reticulum.
P194
Angiotensin Type 2 Receptor Mediated Vascular Relaxation and Nitric
Oxide Production during Pregnancy
Amanda Stennett, Raouf Khalil; Brigham and Womens Hosp, Boston, MA
Background: During normal pregnancy significant cardiovascular changes occur, including
upregulation of the RAAS; however, systolic blood pressure is reduced during pregnancy
compared to nonpregnant females. AngII activates not only AT1R to induce vascular contraction,
but also AT2R to stimulate relaxation. Although the role of AT1R in vascular contraction is
well-documented, the role of AT2R in vascular relaxation, especially during pregnancy, is not
clearly understood. The aim of this study was to determine whether AT2R mediated vascular
relaxation is enhanced during pregnancy. Methods: Aortae were harvested from female virgin
(12 wk) and timed pregnant (day 18 20 of gestation) Sprague-Dawley rats. Isometric
contraction/relaxation was measured in aortic strips, which were stimulated with an AT2R
agonist (CGP42112A); an AT1R blocker (Losartan) and AngII; or, an AT2R blocker (PD123319)
and phenylephrine (Phe). NO production from aortic strips was measured with 4-amino-5-
methylamino-2,7-difluorescein (DAF-FM), an NO-sensitive fluorescent dye, after stimulation
with CGP42112A, Losartan and AngII, or acetylcholine (Ach) for 45 min. Tissue homogenate of
thoracic aorta was used for immunoblot analysis. Results: Studies in isolated aortic strips
reveal that treatment with PD123319 results in greater Phe-stimulated contraction in pregnant
rats (1.10.2 vs. 0.80.1 g/mg tissue). In aorta treated with CGP42112A, relaxation was
greater in pregnant rats compared to virgin (2111% vs. 11% after Phe pre-contraction). NO
production after Ach stimulation was greater in tissue from pregnant rats (0.1990.03 vs.
0.1570.02 mg-1). In aorta stimulated with Losartan and AngII, NO production was greater in
pregnant rats when compared to nonpregnant controls (0.1670.01 vs. 0.1380.02 mg-1).
Additionally, the amount of vascular eNOS was greater in pregnant tissue, compared to virgin
controls, as determined by Western blot analysis (0.74 vs. 0.48 normalized to actin intensity).
Conclusion: These results suggest that vascular relaxation and NO production are mediated,
in part, by AT2R, and this may be increased during pregnancy. This AT2R mediated relaxation
may play a role in the decreased blood pressure observed during normal pregnancy.
P195
Cooperation between Hypoxia and Adenosine in Regulation of Angiogenesis
Sergey Ryzhov, Jennifer L McCaleb, Anna E Goldstein, Igor Feoktistov, Italo Biaggioni;
Vanderbilt Univ, Nashville, TN
Because hypoxia stimulates angiogenesis and increases extracellular adenosine concentra-
tions, we evaluated the potential contribution of adenosine to hypoxia-induced angiogenesis. In
the absence of adenosine (adenosine deaminase added to culture media), hypoxia stimulated
vascular endothelial growth factor (VEGF) secretion from human microvascular endothelial cells
HMEC-1, but not interleukin-8 (IL-8). In contrast, the stable adenosine analog NECA stimulated
both VEGF and IL-8 secretion. Furthermore, VEGF secretion increased 1.90.04-fold with
NECA (10 mol/L) and 1.70.1-fold with hypoxia (5% O2), but 3.80.1-fold when these two
stimuli were combined. Thus, adenosine acts in a cooperative fashion with hypoxia to stimulate
VEGF, and induces IL-8 secretion not stimulated by hypoxia alone. In vitro, migration of human
vein endothelial cells was significantly greater in response to conditioned media from HMEC-1
simultaneously activated by NECA and hypoxia, compared to hypoxia or NECA alone. In vivo,
antagonism of adenosine receptors with caffeine produced a 46% reduction of angiogenesis in
a mouse ischemic hindlimb model (Figure). Thus, adenosine not only contributes to the overall
effect of hypoxia but also has additional actions in the regulation of angiogenic factors resulting
in greater stimulus for angiogenesis.

P196
Inhibition of the Soluble Epoxide Hydrolase Results in Increased Activation
of Janus Kinase (JAK2)/ Signal Transducers and Activators of
Transcription (STAT) Pathway in Hypertensive Goto-Kakizaki Rats (GK)
Jeffrey J Olearczyk, Med College of Georgia, Augusta, GA; In-Hae Kim, Bruce D Hammock,
Univ of California, Davis, CA; John D Imig, Mario B Marrero, Amy Banes-Berceli; Med
College of Georgia, Augusta, GA
Type II diabetes leads to renal and cardiovascular complications that are exacerbated by the development
of hypertension. Soluble epoxide hydrolase enzyme (sEH) inhibitors have been successfully employed in
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animal models of hypertension with beneficial effects. One inhibitor, AUDA, inhibits sEH but has also been
reported to have partial PPAR agonist activity. PPAR activation has been shown to effect activation and
expression of members of the JAK/STAT pathway, the activity of which has been implicated in the
development of vascular disease and hypertension. Here, we hypothesized that treatment with AUDA
would provide both renal and vascular protection in an animal model of hypertension and diabetes. GK,
a non-obese model of type II diabetes, were infused with angiotensin II (ANG, 65 ng/min) and fed a high
salt diet (HS) to induce hypertension. Rats were treated for 2 weeks with either vehicle or sEH inhibitor
(AUDA, 25 mg/L, n3). ANG/HS increased mean arterial pressure from 118 2 to 182 20 and 187
6 mmHg for vehicle and AUDA treated GK rats, respectively. Hypertensive GK rats exhibited a significant
decrease in non-fasting blood glucose compared to GK control animals (148 19 mg/dL vs. 239 49
mg/dL, respectively) and was not altered with AUDA treatment (171 29 mg/dL). Hypertensive GK rats
had a 17 fold increase in urinary albumin excretion, an indicator of renal damage, which was inhibited
to control levels with AUDA (22.0 5.0 vs. 6.3 2.6 mg/d, respectively). Endothelium-denuded thoracic
aorta demonstrated a significant decrease in phosphorylated JAK2 (pJAK2) in GK compared to Wistar
controls (52 16% vs. 101 21% phosphorylated/total protein, respectively). pJAK2 levels were further
decreased in hypertensive GK rats (15 6% phosphorylated/total protein). Interestingly, treatment with
AUDA increased pJAK2 levels to those seen in the Wistar control group (84 23% phosphorylated/total
protein). These changes in pJAK2 are specific rather than global as mitogen activated protein kinase did
not show similar changes. Taken together, these data support the hypothesis that AUDA treatment
protects the kidney from damage via its actions on sEH and could also improve vascular signaling by
activation of JAK/STAT in the vasculature of hypertensive GK rats.
P197
Microvascular Rarefaction in the Derma of Hypertensive Patients
Damiano Rizzoni, Enzo Porteri, Silvia Paiardi, Dept. of Med and Surgical Sciences, Univ of
Brescia, Brescia, Italy; Luigi Rodella, Chair of Human Anatomy, Univ of Brescia., Brescia,
Italy; Carolina De Ciuceis, Gianluca E Boari, Dept. of Med and Surgical Sciences, Univ of
Brescia, Brescia, Italy; Rita Rezzani, Chair of Human Anatomy, Univ of Brescia, Brescia,
Italy; Fracesca Zani, Marco Miclini, Dept. of Med and Surgical Sciences, Univ of Brescia,
Brescia, Italy; Francesca Ricci, Rossella Bianchi, Chair of Human Anatomy, Univ of Brescia,
Brescia, Italy; Enrico Agabiti Rosei; Dept. of Med and Surgical Sciences, Univ of Brescia,
Brescia, Italy
It has been previously demonstrated that the development of hypertension in animal models is associated
with functional and anatomical microvascular rarefaction. Data in humans are scarce, and mainly limited
to the dorsum of the fingers, evaluated by intravital capillary videomicroscopy, in patients with borderline
or essential hypertension (Antonios TF and coll, Hypertension 1999). Objective Our aim was to evaluate
microvessel density (MVD), in the derma of normotensive subjects and hypertensive patients, using an
immunohistochemical method. Design and Methods Twenty subjects were included in the present
study. They were 6 normotensive subjects, 10 patients with essential hypertension, 3 with primary
aldosteronism, and one patient with phaeochromocytoma. All subjects were submitted to a biopsy of the
cutis and subcutaneous fat from the gluteal or the anterior abdominal region. Small resistance arteries
(internal diameter 100300 um) were dissected and mounted on an isometric myograph and the media
to lumen ratio was measured (M/L), according to Mulvanys technique. MVD (vessels with internal
diameter 100 um) were evaluated after staining the derma with anti-CD31 antibody and measuring %
of CD 31 stained area by automated image analysis. Results were as follows (*p0.05, **P0.01 vs
Normotensives). A statistically significant inverse correlation between M/L and %CD 31 stained area was
observed in normotensive subjects and hypertensive patients (n 20, r -0.56, p0.01). Conclusion
A significant microvascular rarefaction has been observed in the derma of hypertensive patients,
compared with normotensive subjects. Microvascular rarefaction resulted correlated to greater values of
M/L, an indicator of structural alterations in small resistance arteries.
M/L %CD 31 stained area
Normotensives n 6 0.0690.011 3.001.08
Essential hypertensives n 10 0.0940.013** 2.050.67*
All hypertensives n 14 0.0910.014** 2.050.62*
CHBPR ConferencePoster Presentations e83
P198
Chronic Inhibition of the GLUT4 Facilitative Glucose Transporter Causes
Potentiated Vascular Smooth Muscle Reactivity via Rho Kinase
James L Park, Brittany L Irey, Frank C Brosius, III, Kevin B Atkins; Univ of Michigan, Ann
Arbor, MI
We have demonstrated that vascular GLUT4 expression in DOCA-salt hypertensive animals is
decreased and correlates with hypertension-related potentiated vascular smooth muscle
(VSMC) contraction and that over-expression of vascular GLUT4 can prevent the potentiated
VSMC reactivity. Paradoxically, acute (30 min) GLUT4 antagonism in aortas from normal mice
inhibits VSMC contraction to norepinephrine (NE). However, long-term effects of GLUT4
inhibition in normal arteries are unknown. We hypothesized that chronic inhibition of GLUT4 in
normal VSMC causes potentiated VSMC reactivity similar to that in hypertension. To test this
hypothesis, the GLUT4 antagonist, indinavir (Ind), was used to inhibit GLUT4 in mouse aortic
explants. We demonstrated previously that Ind (25 uM) is a specific inhibitor of GLUT4-
mediated glucose uptake in VSMC cells. Thoracic aortas from male C57Bl/6J mice were
incubated in culture medium containing 25 uM Ind or vehicle (PBS, pH 1.0) at 37 C. After
24 hours, contractility to NE, expressed as a percent KCl contraction, was measured in
endothelium-denuded rings with or without Ind (25 uM). The NE EC50 did not change, while
maximal NE VSMC reactivity significantly increased from 150% without Ind to 240% with
Ind. Rho kinase inhibition with Y27632 (1 uM) did not significantly alter the NE EC50 or the
maximal contraction in the 24 hr vehicle-treated aortas but prevented the potentiated VSMC
contraction in the 24 hr Ind-treated aortas. We also observed increased phosphorylation of the
regulatory subunit of myosin phosphatase (MYPT) after 24 hr Ind treatment. These data are
evidence of increased Rho kinase activity in Ind-treated aortas, since MYPT is a substrate for
Rho kinase, and its phosphorylation reduces myosin phosphatase activity. A time course in
cultured mouse aortic VSMC showed increased MYPT phosphorylation as early as 2 hours after
GLUT4 inhibition, peaking at 4 hours and remaining elevated after 24 hours. These data
suggest GLUT4 may play an important role in regulating VSMC contractility by modulating Rho
kinase. Alterations in GLUT4 expression or activity, as has been observed in experimental
models of hypertension, may contribute to the vascular changes observed in hypertension.
P199
Chronic Exposure to Intermittent Hypoxia Augments ET-1 Vasoconstriction
by Increasing nPKC-Dependent Calcium Sensitization
Kyan J Allahdadi, Nancy L Kanagy; Univ of New Mexico Health Science Cntr, Albuquerque,
NM
We reported previously that simulating sleep apnea by exposing rats 7 hr/day to intermittent
hypoxia/hypercapnia (IH/HC) elevates plasma endothelin-1 (ET-1) and causes hypertension.
Blood pressure in IH/HC rats is acutely normalized with an ETA receptor antagonist.
ET-1-mediated constriction in isolated IH/HC arteries is greater than in Sham arteries but vessel
wall [Ca2] only increases in Sham arteries. The non-selective PKC inhibitor GF-109203x (3
M) reduces ET-1-mediated constriction in IH/HC arteries but not in Sham arteries without
affecting vessel wall [Ca2]. These data suggest PKC contributes to augmented ET-1-mediated
constriction in IH/HC arteries independent of [Ca2]. We hypothesized that one or more novel
PKC isoforms (nPKC) mediate the augmented ET-1 constriction because nPKC require only
diacylglycerol (DAG) for activation whereas classical PKC isoforms (cPKC) require both DAG and
Ca2. To test this hypothesis, denuded mesenteric arteries were exposed to increasing
concentrations of ET-1 (10-8-10-10 M) in the presence or absence of a cPKC inhibitor (Go6976,
1 M) or an nPKC inhibitor (Rottlerin, 0.3 M). The cPKC inhibitor did not affect ET-1-mediated
vasoconstriction in either IH/HC or Sham arteries. However, the nPKC inhibitor profoundly
decreased ET-1-mediated vasoconstriction in IH/HC but not Sham arteries (Figure). Further-
more, membrane localization of the nPKC isoform, PKC, appears to be greater in unstimulated
IH/HC than Sham arteries suggesting IH/HC leads to activation of this PKC isoform. These
observations support our hypothesis that increased ET-1-mediated constriction following IH/HC
exposure is mediated by activation of nPKC.
P200
c-Src Mediates Angiotensin II but not Endothelin-1-Induced Activation of
MAP Kinases in Vascular Smooth Muscle Cells
Alvaro Yogi, Univ of Sao Paulo, Sao Paulo, Brazil; Glaucia E Callera, Univ of Ottawa, Ottawa,
Canada; Rita C Tostes, Univ of Sao Paulo, Sao Paulo, Brazil; Rhian Touyz; Univ of Ottawa,
Ottawa, Canada
Objectives: Endothelin-1 (ET-1) and angiotensin II (Ang II) are multifunctional peptides that can
modulate vascular tone, cell growth, apoptosis, cell migration and extracellular matrix
deposition. These molecules mediate their action through activation of G protein-coupled
 e84 Hypertension Vol 48, No 4 October 2006

receptors (GPCR) leading to activation of complex intracellular signaling pathways including the
phosphorylation of mitogen-activated protein kinases (MAPKs). Since these receptors lack
intrinsic kinase activity we sought to determine whether ET-1 and Ang II activate MAPKs
through c-Src dependent pathways. Methods and results: In our study vascular smooth
muscle cells (VSMC) from mice (8 to 10 weeks old) with different levels of disruption in the
c-Src gene (c-Src and c-Src-/-) and wild-type (c-Src/) were used. Cells were stimulated with
ET-1 (10-7 M, 2 to 30 minutes) or Ang II (10-7 M, 2 to 10 minutes). Protein was extracted and
western blots were performed for total and phosphorylated forms of ERK 1/2 (extracellular
signal-regulated kinase), SAPK/JNK (Stress-activated protein kinase/c-Jun N-terminal kinase)
and p38MAPK. Both peptides induced phosphorylation of ERK 1/2 in VSMCs from c-Src/
mice, with a maximal response within 2 minutes of stimulation (P0.001 vs vehicle). In VSMCs
from c-Src and c-Src-/-, ET-1, but not Ang II (P0.01 vs c-Src/), induced the same level
of ERK 1/2 phosphorylation. Similar results were observed when we studied ET-1 and Ang
II-mediated activation of SAPK/JNK and p38MAPK. Conclusion: Our findings demonstrate that
whereas MAP kinase activation by Ang II is c-Src-sensitive, ET-1-mediated actions do not rely
on c-Src activation. These data indicate differential activation of MAP kinase signaling by Ang
II and ET-1 in VSMCs and suggest that different ligands to GPCR can activate similar signaling
pathways through unrelated mechanisms. These processes may contribute to unique actions
of Ang II and ET-1 in VSMCs.
P201
Pharmacological Endothelin Receptor Interaction Occurs in Veins but not
Arteries
Keshari Thakali, James J Galligan, Gregory D Fink, Stephanie W Watts; Michigan State Univ,
East Lansing, MI
In mineralocorticoid hypertension, arteries exhibit reduced contraction to endothelin-1 (ET-1),
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
kinase), the regulation of ubiquitination of different target proteins will be discussed and effects
of siRNA mediated knockdown of SOCS-1 will be detailed. These results identify a novel
mechanism in the transduction and regulation of a mechanical signal in VSMC.
P204
Novel Reciprocal eNOS Regulation by Protease-Activated Receptors
Involving Gq, G12/13 and Rho/Rho Kinase
Hiroyuki Suzuki, Temple Univ Sch of Medicine, Philadelphia, PA; Evangeline D Motley,
Meharry Med College, Nashville, TN; Kunie Eguchi, Haruhiko Ohtsu, Sadaharu Higuchi,
Satoru Eguchi; Temple Univ Sch of Medicine, Philadelphia, PA
Protease-activated receptors (PARs), such as PAR-1 and PAR-2 have been implicated in the
regulation of endothelial nitric oxide (NO) production. We hypothesized that PAR-1 and PAR-2
distinctly regulate the activity of endothelial NO synthase (eNOS) through the selective
phosphorylation of a positive regulatory site, Ser1179 and a negative regulatory site, Thr497. We
used selective synthetic peptide ligands for these receptors to see if they phosphorylated eNOS
in bovine aortic endothelial cells (BAECs). The PAR-1 ligand, TFLLR, phosphorylated eNOS at
Thr497. It had no effect on Ser1179 phosphorylation or cyclic GMP (cGMP) production. In contrast,
the PAR-2 ligand, SLIGRL, phosphorylated Ser1179 with no noticeable effect on Thr497. SLIGRL
stimulated cGMP production that was blocked by L-NAME. Neither eNOS phosphorylation nor
cGMP production was observed by the PAR-4 agonist, AY-NH2. Thrombin has been shown to
transactivates PAR-2 through PAR-1. Thus, thrombin stimulates eNOS phosphorylation at both
sites as well as cGMP production in BAECs. Importantly, SLIGRL-induced Ser1179 phosphory-
lation and cGMP production were inhibited by a Gq inhibitor, YM-254890. YM-254890 also
blocked thrombin-induced cGMP production and eNOS phosphorylation at Ser1179 but not
Thr497. By contrast, TFLLR-induced Thr497 phosphorylation was selectively inhibited by a
Rho-kinase (ROCK) inhibitor, Y27632 and overexpression of p115RhoGEF RGS domain, a

while veins remain responsive to ET-1. Also, when exposed acutely to ET-1, arteries but not
veins completely desensitize to ET-1. We hypothesized that maintained venous contraction to
ET-1 in hypertension and reduced ET-1 desensitization in veins occurs because of ETA and ETB
receptor interaction. To assess pharmacological endothelin receptor interaction, ET-1 (10
pM-100 nM)-induced contraction in rings of rat thoracic aorta and thoracic vena cava was
compared in the presence of vehicle, ETA receptor blockade (atrasentan, 10 nM), ETB receptor
blockade (BQ-788, 100 nM) or dual ETA and ETB receptor blockade (atrasentan BQ-788)
(Table 1). The ability of atrasentan to inhibit aortic ET-1-induced contraction was not different
in the presence or absence of functional ETB receptors. In vena cava, atrasentan caused a
significantly larger rightward shift in estimated EC50 values for ET-1-induced contraction when
functional ETB receptors were absent (Table 1), suggesting that pharmacological ET receptor
interaction occurs in veins, but not arteries. In freshly dissociated aortic and venous vascular
smooth muscle cells, ETA and ETB receptors co-localized to the membrane (verified by cadherin
staining). Our data suggest that though pharmacological ET receptor interaction occurs in veins,
this interaction may be independent of ET receptor location as ETA and ETB receptors co-localize
in both arteries and veins. Thus, pharmacological endothelin receptor interaction may account
for maintained venous contraction in hypertension, and supports a role for venous involvement
in hypertension. Table 1. Estimated EC50 (-log M) values for ET-1-induced contraction in aorta
and vena cava in the presence and absence of functional ETB receptors.
Aorta EC50 Aorta Fold Vena Cava EC50 Vena Cava
Condition (-log M) Shift (-log M) Fold shift
Vehicle ETBreceptors 8.2 6.7 8.1 2.3
Atrasentan (10 nM) 7.3 7.8
BQ-788 (100 nM) -ETBreceptors 8.3 6.4 8.5 8.1*
Atrasentan BQ-788 7.5 7.6
*represents a statistically significant difference from ETB receptors (p0.05)
P202
Withdrawn at Authors Request
P203
Mechanical Stress Modulates SOCS-1 Expression In Human Aortic Vascular
Smooth Muscle Cells
Marc Dangers, Hermann G Haller, Inna Dumler; Med Sch Hannover, Hannover, Germany
Elevated blood pressure leads to artery remodeling, cardiac hypertrophy, and cardiac failure,
most likely via changes in functional behavior of vascular smooth muscle cells (VSMC) in
response to mechanical stretch imposed on the vessel wall. The underlying molecular
mechanisms are, however, not fully understood. We have studied primary human VSMC using
a model for in vitro multilateral stretch. Since the JAK/STAT pathway is known to be activated
in stretched cardiomyocytes and can contribute to proliferative events, we investigated
negative modulators of this pathway, namely members of the suppressor of cytokine signalling
(SOCS) family. The expression of SOCS-1 mRNA and protein levels was significantly inhibited
by cyclical mechanical stretch (1 Hz, 15% elongation). Surprisingly, this appeared not to be
caused by an increase in JAK/STAT pathway activation, as measured by phosphorylation of the
Janus kinases JAK1/2 and Tyk2. This implies a regulation of SOCS-1 expression in mechanical
stress which differs from the negative feedback loop regulation of a typical cytokine response.
SOCS-1 mRNA expression inhibited by mechanical stretch was restored by pre-treatment of
cells with RGD peptides in a dose dependent fashion, indicating a contribution of integrins to
the observed effect. Blockage of the interaction of integrins with another cell surface adhesive
receptor, namely the GPI-anchored urokinase-type plasminogen activator receptor (uPAR) by
specific peptides also suggested a role for uPAR in the stretch mediated inhibition of SOCS-1.
No increase in methylation of the SOCS-1 promotor after mechanical stress could be detected
in VSMC, although this mechanism has been shown to attenuate SOCS-1 mRNA expression in
other cell types. Since SOCS-1 recruits ubiquitin ligase not only to members of the JAK/STAT
pathway, but also to other proteins implied in mechanotransduction (e.g. focal adhesion
selective G12/13 inhibitor. Thus, TFLLR but not SLIGRL stimulated phosphorylation of MYPT, a
ROCK substrate in BAECs. From these data, we conclude that PAR-1 and PAR-2 distinctly
regulate eNOS activity through Gq and G12/13/Rho-kinase, respectively, delineating the novel
signaling pathways by which proteases regulate eNOS activity.
P205
Dendroaspis Natriuretic Peptide Binds to the Natriuretic Peptide Clearance
Receptor: Pharmacological Characterization and Therapeutic Implications
Douglas G Johns, Zhaohui Ao, Gerald E Hunsberger, Bradley J Heidrich, Taylor Graham,
Lisa Payne, Quinn Lu, Nambi Aiyar, Stephen A Douglas; Glaxo Smith Kline, King Of Prussia,
PA
Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which, like the
atrial, brain, and C-type natriuretic peptides (ANP, BNP, CNP) causes vasodilation and lowers
blood pressure. Unlike the other natriuretic peptides, DNP resists degradation by neutral
endopeptidase (NEP). The natriuretic peptide clearance receptor (NPR-C), in addition to NEP,
removes natriuretic peptides from the circulation. Whether DNP interacts with human NPR-C
has not been determined. The purpose of this study was to test the hypothesis that DNP binds
to NPR-C with a pharmacological profile similar to that of endogenous natriuretic peptides.
Human recombinant NPR-C, and the guanylate-cyclase-coupled natriuretic peptide receptors
GC-A and GC-B were expressed in CHO cells using BacMam technology. 125I-ANP and 125I-CNP
were used as radioligands for GC-A/NPR-C and GC-B, respectively. The recombinant receptors
displayed single-site, saturable binding to radioligands (NPR-C KD 50pM Bmax 450fmol/mg
protein; GC-A KD 104pM Bmax 498fmol/mg protein; GC-B KD 436pM Bmax 478fmol/mg
protein). ANP, BNP, CNP and the putative NPR-C antagonists AP811 and cANP(4 23) displaced
[125I]-ANP from NPR-C with nanomolar affinity (Table 1). DNP displaced [125I]-ANP and [125I]-CNP
from NPR-C with Kis of 2.40.5 and 1.60.3nM, respectively, which represents the first direct
demonstration of binding of DNP to human NPR-C. DNP showed high affinity for the GC-A
receptor (Ki 0.12.03nM) and no affinity for GC-B (Ki 1000nM). DNP was 75-fold more potent
than ANP at stimulating cGMP production in GC-A expressing cells (EC50 DNP 0.160.01nM,
ANP 12.20.3nM, P0.05), but did not stimulate cGMP in GC-B- or NPR-C-expressing cells,
illustrating selectivity of DNP for GC-A. These results are the first evidence of DNP binding to
human NPR-C. Antagonism of NPR-C represents a novel therapeutic approach to augment the
actions of DNP in cardiovascular diseases such as hypertension and heart failure.
BINDING AFFINITY OF NATRIURETIC PEPTIDES AND ANALOGS AT
NPR-C
Ligand Ki ([125I]-ANP) displacement
ANP 30.010.0pM
BNP 20.01.0pM
CNP 1.00.1nM
DNP 2.40.5nM
AP811 3.32.3nM
cANP(423) 1.00.1nM
P206
Calcitonin Gene-Related Peptide Inhibits High Glucose-Induced Reactive
Oxygen Species Production in Vascular Smooth Muscle Cells
Jing Liu, XiaoBo Cao, Joana Dado, Khurshed Katki, Valorie Chiasson, Sheldon C Chaffer,
Donald J DiPette, Scott C Supowit; Texas A&M Univ System Health Science Cntr, Scott &
White Hosp Rsch Cntr, Temple, TX
Recent studies suggest that calcitonin gene-related peptide (CGRP), a potent vasodilator
sensory neuropeptide, protects against diabetes-induced end organ damage through the
inhibition of increased oxidative stress observed in this setting. The present study was
undertaken to assess the mechanism of this putative anti-oxidant activity of CGRP against high

glucose-evoked reactive oxygen species (ROS) generation in rat aortic smooth muscle cells
(VSMCs) using DCF fluorescence and lucigenin chemiluminescence methods. High glucose (25
mM) treatment (12 h) of VSMCs markedly increased ROS production (1.9 0.1-fold, p0.05)
compared to cells treated with normal glucose (3.5 mM). The enhanced ROS generation was
significantly attenuated by CGRP in a concentration dependent manner (10-10-10-8M). CGRP
treatment also attenuated high glucose-induced translocation of Rac1 and p47 phox, two
critical membrane-bound components of NADPH oxidase in VSMCs, almost to the same degree
as apocynin (decreased 1.8 0.2-fold). This antioxidant effect of CGRP was mimicked by
dibutyl-cAMP (decreased approximately 2-fold) and completely blocked by pretreatment with
H-89, a protein kinase A inhibitor, and CGRP(8 37), a CGRP receptor antagonist and
overexpression of a constitutively-active Src.Moreover, we showed that CGRP attenuated high
glucose-evoked ROS generation via stimulation of Csk thereby reducing Src activity. Therefore,
this study demonstrates, for the first time, that CGRP inhibits high glucose stimulation of
oxidative stress in VSCMs through a c-AMP/PKA-dependent mechanism that reduces Src-
stimulated ROS production via activation of Csk. Thus, CGRP may play a protective role as an
endogenous anti-oxidant in diabetes-induced vascular injury.
P207
G-Protein Coupled Receptor Kinase 4 (GRK4) Polymorphisms Block
Receptor Recruitment to Cell Membranes
John J Gildea, Univ of Virginia, Charlottesville, VA; Junichi Yatabe, Midori Sasaki, Fukushima
Med Univ, Fukushima City, Japan; Pedro A Jose, Georgetown Univ, Washington, DC; Robin
A Felder; Univ of Virginia, Charlottesville, VA
Background: GRK4 polymorphisms are associated with salt sensitivity and hypertension in
humans due to blocking of membrane expression of D1R and AT2R in renal proximal tubule
(RPT). To test our hypothesis that GRK4SNPs cause this phenotype, CHO-K1 cells that do not
express D1R, AT2R, or GRK4 were transfected with tagged and non-tagged exogenous
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receptors. Methods: We determine D1R-and AT2R cell surface recruitment with or without D1R
stimulation with fenoldopam (FEN, 1 M, 30 min), by surface biotinylation-dot blot and 3D
confocal microscopy, in CHO-K1 cells transfected with: (1) D1R-EYFP with and without GRK4
wild type (WT) or triple polymorphic GRK4 (65L/142V/486V); (2) N-terminal 3xHA epitope-
tagged D1R with and without GRK4 and (3) untagged D1R and 3xHA epitope-tagged AT2R.
Results: D1R were expressed at the membrane and then internalized following FEN (1 M, 30
min): 614% reduction in membrane expression (MSD, n4, p0.05 vs vehicle, ANOVA).
In the presence of GRK4WT, there was a decrease in D1R membrane expression (725%,
n4, p0.05 vs vector). FEN caused a 3.20.2 fold (n4, p0.05 vs vehicle) increase in D1R
membrane. Triple polymorphic GRK4 decreased D1R membrane expression to values similar
to GRK4WT (674%, n4, p0.05 vs vector) but prevented FEN to increase (1.20.1-fold Vascular Smooth Muscle Cells
increase, n4, p0.05 vs FEN/GRK4WT) D1R membrane expression. Confocal microscopy
also showed that triple polymorphic GRK4 caused miscolocalization of D1R to lipid bodies.
Similar to D1R, HA-tagged AT2R was recruited to the cell surface by FEN (3.30.3-fold, n4,
p0.05 vs vehicle) and co-expression of triple polymorphic GRK4 blocked AT2R membrane
recruitment (1.50.2-fold increase, p4, p0.05 vs FEN/GRK4WT). Conclusion: GRK4 Japan
variants block the cell surface recruitment of D1R and AT2R in CHO-K1 cells, similar those
observed in human RPT cells. Impaired D1R and AT2R function may be, in part, responsible for
the sodium retention in hypertension.
P208
Extracellular Cyclic AMP-Adenosine Pathway Inhibits Glomerular Mesangial
Cell Growth via A2B Adenosine Receptors
Raghvendra K Dubey, Univ Hosp Zurich, Zurich, Switzerland; Delbert G Gillespie, Zaichuan
Mi, Edwin K Jackson; Univ of Pittsburgh Med Cntr, Pittsburgh, PA
Here we determined whether exogenous and endogenous cAMP is metabolized to adenosine
in glomerular mesangial cells (GMCs) and whether cAMP-derived adenosine modulates growth
(DNA synthesis, collagen synthesis, cell number) and ERK1/2 activity in cultured GMCs. Addition
of exogenous cAMP to GMCs increased extracellular levels of AMP, adenosine, and inosine in
a concentration-and time- dependent manner. This effect was attenuated by blockade of total
phosphodiesterase (IBMX), ectophosphodiesterase (high concentration of DPSPX), or ecto-5-
nucleotidase (AMPCP). Treatment of GMCs with forskolin increased cAMP production and
extracellular levels of adenosine, and these effects were blocked by inhibition of adenylyl
cyclase (2,5-dideoxyadenosine). Treatment with exogenous cAMP and forskolin inhibited cell
growth and antagonism of A2 (KF17837) or A1/A2 (low concentration of DPSPX), but not A1
(CPA), adenosine receptors blocked the growth inhibitory effects of exogenous cAMP, but not
the growth inhibitory effects of 8-bromo-cAMP (stable cAMP analogue). The growth-inhibitory
effects of exogenous cAMP and forskolin were enhanced by the combined inhibition of
adenosine deaminase [EHNA] and adenosine kinase (iodotubercidin). Downregulation of A2B
receptors with antisense, but not sense and scrambled, oligonucleotides abrogated cAMP and
forskolin induced adenosine formation and the inhibitory effects of cAMP and forskolin on GMC
growth. In conclusion, the extracellular cAMP-adenosine pathway exists in GMCs and
attenuates cell growth. Pharmacological augmentation of this pathway could abate pathological
glomerular remodeling in renal disease.
P209
Functional Interaction between 1- and 2-Adrenoreceptors in Murine
Mesenteric Veins but not Arteries
Alexandra Hlavacova, James J Galligan; Michigan State Univ, East Lansing, MI
Mesenteric veins (MV) are much more sensitive to the constrictor effects of -adrenergic
receptor (-AR) agonists and sympathetic nerve stimulation than are the adjacent mesenteric
arteries (MA). In addition, constrictions caused by -AR agonists in veins desensitize more
slowly than responses in arteries. We investigated the mechanisms underlying these
differences by measuring -AR agonist-induced constrictions, in vitro, of mesenteric arteries
CHBPR ConferencePoster Presentations e85
(MA) and veins (MV) from C57Bl/6 mice. Prazosin (0.1 M), an 1AR antagonist, inhibited
norepinephrine (NE) constrictions in MA and MV. UK14,304, an 2AR agonist, did not constrict
MA or MV. However, the 2AR antagonists, yohimbine (1 M) and idazoxan (1 M) produced
rightward shifts of NE concentration-response curves in MV but not MA. BRL44408 ( 2AAR
antagonist, 0.1 M,) or imiloxan (2B antagonist 1 M) did change NE concentration-response
curves in MV. In contrast, MK912 (0.01 M, 2CAR antagonist) caused a rightward shift in NE
concentration-response curves in MV. This finding suggests that 2CARs indirectly contribute
to responses in MV. We next tested whether stimulation of 2AR would facilitate MV
constrictions caused by the 1AR agonist phenylephrine (PE). UK14,304 (0.1 M) did not
constrict MV itself but it did cause a leftward shift in PE concentration-response curves. We
investigated whether Gi/Go proteins play a role in the 2AR component of MV responses to NE
by incubating tissues with pertussis toxin (PTX, 2 g/ml, 2 hrs at 37C). NE concentration
response curves were not altered by PTX treatment. As 2AR activate calcium channels in
vascular smooth muscle, we reduced extracellular calcium from 2.5 to 0.25 mM. This
treatment inhibited NE-induced constrictions in MV but not MA. Overall, these data indicate that
there is a functional interaction between 1ARs and 2CARs in MV but not MA. The interaction
requires extracellular calcium entry but is independent of PTX-sensitive G-proteins. The
functional interaction likely contributes to the high sensitivity of MV to the constrictor effects
of NE and sympathetic nerve stimulation. This interaction also contributes to the reduced
desensitization of MV to AR stimulation compared to MA.
P210
Downregulation of Angiotensin II Type 1 Receptor by Liver X Receptor in
Ikuyo Imayama, Kyushu Univ Graduate Sch of Med Sciences, Fukuoka, Japan; Dan Patton,
Univ of Toronto, Toronto, Canada; Keita Inanaga, Hideki Ohtsubo, Kyushu Univ Graduate Sch
of Med Sciences, Fukuoka, Japan; Kenji Sunagawa, Toshihiro Ichiki; Kyushu Univ, Fukuoka,
Atherosclerosis is considered to be a combined disorder of lipid metabolism and chronic
inflammation. Recently, ligands of the Liver X receptors (LXRs), a nuclear hormone receptor,
have been reported to inhibit atherosclerosis through the regulation of specific genes, involved
in both lipid metabolism and inflammation. Angiotensin (Ang) II is also an important player in
atherogenesis. It is well known that Ang II accelerates atherogenesis through activation of the
Ang II type 1 receptor (AT1R). To further examine the relation between these two major factors
in atherosclerosis, we examined the effect of a LXR ligand on AT1R expression in vascular
smooth muscle cells (VSMCs). TO-901317 (TO), a synthetic LXR ligand, was found to decrease
AT1R mRNA and protein expression, with peak reductions at 6 hours and 12hours of incubation
respectively. This suppression of AT1R mRNA and protein were observed in a dose dependent
manner. The reduction of AT1R expression by TO was seen under cycloheximide exposure,
indicating that de novo protein synthesis is not required in the downregulation. Furthermore,
althought preincubation of VSMCs with TO for 30minutes had no effect on Ang II-induced
extracellular signal-regulated kinases (ERK) phosphorylation, it was found that preincubation for
6hours markedly suppressed the phophorylation of ERK by AngII. These results indicate that TO
attenuates Ang II signaling by reducing the number of AT1R but not by a direct inhibition of
AT1R signal cascade. The stability of AT1R mRNA was not affected by TO which suggests that
TO inhibits AT1R gene transcription. This conclusion is further supported by the finding that
AT1R gene promoter activity, measured by luciferase assay, was decreased following exposure
to TO. Subsequent examination using the deletion mutants of the AT1R gene promoter region
had revealed that the promoter region between -61bp and1bp is essential for the
LXR-induced suppression. This is the first study clarifying the role of LXR in the regulation of
AT1R gene expression. These results indicate that the suppression of AT1R may be one of
important mechanisms in the anti-atherogenic effects of LXRs.
P211
Downregulation of Protein Kinase C Inhibits Perivascular Sensory Nerve
Ca2-Sensing Receptor Signaling
Emmanuel M Awumey, North Carolina Central Univ, Durham, NC; Debra I Diz, Wake Forest
Univ, Winston Salem, NC; Allyn C Howlett, North Carolina Central Univ, Durham, NC; James
W Putney, Jr.; NIEHS, National Institutes of Health, Durham, NC
The Ca2-sensing receptor (CaSR), expressed in the perivascular sensory network, is
implicated in Ca2-induced relaxation of isolated, phenylephrine-contracted mesenteric
arteries and may be the missing link between Ca2 intake and vascular function. We have
previously demonstrated that expression of the full length dorsal root ganglia CaSR as an EGFP
fusion protein in HEK293 cells leads to extracellular calcium (Ca2e)-induced transient
increases in intracellular calcium concentration ([Ca2]i). This response undergoes desensiti-
zation with repeated applications of Ca2e. In the present study, we determined the effects of
PKC downregulation on changes in [Ca2]i in cells stably expressing the receptor. Treatment of
cells with 100 nM PMA for 24 hrs reduced the CaSR-mediated Ca2i peaks of fura-2-loaded
 e86 Hypertension Vol 48, No 4 October 2006
cells in response to 1 mM Ca2e by 49.9 5.2%, and to 2 mM Ca2e by 40.5 6.5%
compared to control ( n 5; p 0.002). Pre-incubation of cells with 100 nM each of
Ca2-specific PKC inhibitors, Go 6976, Ro 31 8220 and Ro 32 0432 for 20 min reduced initial
Ca2i peaks by 49.0 3.0, 45.1 0.7 and 30.1 2.7, respectively (p 0.008; n 3).
Stimulation of the receptor with Ca2e increased phosphorylated PKC(Ser 657) levels in cell
membranes over 1 hr (55.2 6.8 % increase at maximum desensitization, n 3; p 0.05).
The results indicate a role for PKC in sensory nerve CaSR signaling and suggest that
Ca2e-induced reduction in Ca2i mobilization is due to feedback inhibition of signaling
proteins. Thus, Ca2e control of myogenic tone may be limited by rapid desensitization of CaSR
function via PKC phosphorylation events. Supported by NIH Grants 2R01 HL064761 06 and
5UH1HL059868 07
P212
The Role of Mesangial Cell Protein Kinase C in Transcription Regulation of
Prorenin/Renin Receptor in Hyperglycemic State
Jiqian Huang, Chun Xue, Helmy M Siragy; Univ of Virginia, Charlottesville, VA
In the diabetic milieu mesangial cells are transformed into a sclerotic phenotype by the direct
effect of high glucose including enhanced expression of autocrine growth factors. Some of the
involved mechanisms that mediate the toxic effect of high glucose.includes increased activity
of protein kinase C (PKC) isozymes such as PKC-, -I, -II, -, - and -. In this study we 1.030.11 G, both n18) between HT and NT cells with DiA labeling. Bath application of
hypothesized that in the presence of hyperglycemia, renin receptor (RR) expression is mediated
by PKC isotypes cPKC and nPKC. Rat mesangial cells (RMCs) were treated with 30mM glucose
for 2 weeks and combined glucose and cPKC inhibitor, chelerythrine or glucose and nPKC
inhibitor, rottlerin for 6 hrs. Western blot analysis was utilized to monitor changes in ProRR
(66KD) and RR (39KD). Both ProRR and RR were found to be constitutively expressed and
upregulated in response to high concentration of glucose. Chelerythrine had no effect on
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glucose-induced ProRR expression but increased RR expression in dose dependent manner. In
contrast, Rottlerin upregulated ProRR expression and had no effect on constitutive and
glucose-induced RR expression. Our data suggest that nPKC isoform is involved in the
transcriptional regulation of ProRR while cPKC isoforms plays a role in processing ProRR to RR.
The identification of the role of different PKC isotypes in RR regulation will help better
understanding of pathophysiology of diabetic kidney disease.
P213
The Novel Cannabinoid Receptor GPR55 Binds Atypical Cannabinoids but
Does not Mediate Their Vasodilatory Effects
Douglas G Johns, David J Behm, Zhaohui Ao, Debbie Walker, Michael Parsons, Shobha
Senadhi, Matthew T Goserud, Dion A Daniels, Michelle Riddick, Penelope C Staton, Paula
Green, Weike Bao, Nambi Aiyar, Tian-Li Yue, Andrew J Brown, Alastair D Morrison, Stephen
A Douglas; Glaxo Smith Kline, King Of Prussia, PA
Atypical cannabinoids, such as abnormal cannabidiol (abn-cbd), bind to a non-CB1/CB2 binding
site and elicit vasodilation via an as yet unidentified receptor which has been termed the CBx
receptor. Recently, the orphan G-protein coupled receptor GPR55 was reported to bind
cannabinoid moieties, making this receptor a possible candidate for the CBx receptor. The
purpose of this study was to test the hypothesis that GPR55 is an atypical cannabinoid-binding
receptor that mediates vasodilation to atypical cannabinoids. [35S]GTPS binding to human
GPR55-expressing HEK293 cell membranes was used to determine receptor activation in
response to abn-cbd and O-1602, purported CBx-selective agonists with no CB1/CB2 activity.
Abn-cbd and O-1602 increased GTPS activity with EC50 values of 1.40.5nM and
1.81.1nM, respectively. Abn-cbd and O-1602 did not increase GTPS activity in membranes
from cells transfected with empty vector, CB1, or CB2 receptor indicating selectivity for GPR55.
Furthermore, the CB1/CB2-selective agonist WIN-55,212 did not augment GTPS activity in
GPR55-expressing cells (EC501,000nM). Mice deficient in GPR55 (GPR55 KO) and age-
matched wild-type (WT) controls were used to determine whether GPR55 mediates dilation to
abn-cbd and O-1602. In WT endothelium-intact isolated mesenteric resistance arteries
(100m diameter), abn-cbd and O-1602 reversed phenylephrine contraction (EC80 concentra-
tion) with micromolar potency (IC50 3.00.6M, 2.80.3M). In GPR55 KO arteries, the
vasodilatory responses to abn-cbd and O-1602 were not different from those seen in WT mice
(IC50 abn-cbd 2.60.5M, P0.6; O-1602 2.20.4M, P0.3). The contraction to phenyl- conscious recordings of blood pressure (MAP) and heart rate (HR). After 1 wk of recovery, ICV
ephrine was also not different between WT and KO mice (WT: EC50 25588 and 516154nM,
respectively, P0.2; Rmax 16511 and 1799% 60mM KCl, respectively P0.4, n5) (AdsiNox2 1002, n14 vs AdGFPi 1044 mmHg, n8) or HR (AdsiNox2 57412, n14 vs
suggesting a lack of basal GPR55 activity in these vessels. These data suggest that GPR55 is
an atypical cannbinoid receptor, as it binds the atypical cannabinoids abn-cbd and O-1602.
However, it does not appear to mediate vasodilation to these agents in mice. Therefore, the
receptor responsible for atypical cannabinoid-induced vasodilation remains to be characterized.
P214
Chronic Hypertension Enhances the Post-Synaptic Effect of Baclofen in the NTS
Weirong Zhang, Myrna Herrera-Rosales, Steve Mifflin; Univ. of Texas Health Science Cntr,
San Antonio, TX
Microinjection of the GABAB receptor agonist baclofen into the nucleus tractus solitarius (NTS)
increases arterial pressure, heart rate and sympathetic nerve discharge. This baclofen-induced
pressor response is enhanced in several animal models of hypertension. Whether this is due to an
alteration in the relative contribution of pre- and/or post-synaptic GABAB receptors to the
baclofen-induced pressor response is not fully understood. We hypothesized that an alteration in the
post-synaptic component of the NTS neuronal response to baclofen contributes to the enhanced
baclofen-induced pressor response observed in chronic hypertension. Using whole-cell recordings,
we investigated the influence of chronic hypertension on post-synaptic effect of baclofen on
baroreceptor neurons on NTS slices from sham-operated normotensive (NT) and unilateral
nephrectomized, renal-wrap hypertensive (HT) Sprague-Dawley rats. DiA labeling of the aortic nerve
was used to identify 2nd-order neurons. After four weeks, arterial blood pressure was 1537 mmHg
in HT rats (n9) and 933 mmHg in NT rats (n8) (p0.05). There was no difference in resting
membrane potential (54.50.7 vs 53.30.6 mV, both n18) and input resistance (1.070.11 vs
baclofen induced outward currents and decreased input resistance in NTS neurons. The EC50 of
baclofen effect was significantly greater in NT rats (9.13.2 M, n5) than HT rats (3.00.5 M,
n7, p0.05). At 10 M baclofen, HT cells (n10) had a greater outward currents than NT cells
(n12) (32.64.2 vs 16.32.3, p0.01). The reduction of input resistance during application of
baclofen (measured as the slope of I-V curve) was greater in HT cells (n6) than in NT cells (n7)
(477% vs 565%, p0.05). The results suggest that in renal-wrap HT there is an enhanced
post-synaptic response to baclofen in in 2nd-order baroreceptor neurons. This enhanced inhibition
could alter NTS neuronal responses to GABA and baroreflex function in HT.
P215
Overexpression of ACE 2 in the Rostral Ventrolateral Medulla Causes a
Long-Term Decrease in Blood Pressure in the SHR
Masanobu Yamazato, Carlos Diez-Freire, Yoriko Yamazato, Mohan K Raizada; Univ of
Florida, Gainesville, FL
The rostral ventrolateral medulla (RVLM) in the brainstem is a relay point in providing
supra-spinal excitatory input to sympathetic preganglionic neurons and thus is critical in the
regulation of sympathetic activity. An increase in sympathetic activity as a result of
hypersensitivity to angiotensin II (Ang II) in the RVLM has been implicated in the development
and establishment of hypertension. Angiotensin converting enzyme 2 (ACE2), a newly
discovered member of the renin-angiotensin system, has been shown to be central in
maintaining the balance between vasoconstrictor activity of Ang II with vasoprotective action of
angiotensin 17 in the peripheral system. However, the role of ACE2 in central cardiovascular
regulation in general and in the RVLM in particular is yet to be investigated. Thus, our objectives
in this study were to determine the expression of this enzyme in the RVLM and to investigate
its participation in cardiovascular functions in the spontaneously hypertensive rat (SHR).
Immunohistochemical analysis showed that ACE2 is predominantly localized in the RVLM
neurons of both normotensive Wistar-Kyoto rat (WKY) and SHR. Western blot analysis revealed
40% decrease in ACE2 in the RVLM of SHR compared to WKY. Lentiviral-mediated gene
transfer of ACE2 was employed to determine if decrease in ACE2 in the RVLM is associated with
hypertensive state. SHRs were instrumented with radio-telemetry transducers and bilateral
RVLM guide cannula. This was followed by delivery of either lenti-GFP or lenti-ACE2 (3105
TU in 250 nl) into functionally confirmed RVLM. Lenti-ACE2 injected SHR showed a long-term
decrease in mean arterial pressure (1423 mmHg in lenti-GFP vs 1268 mmHg in
lenti-ACE2). This decrease in mean arterial pressure was associated with a decrease in heart
rate (300 13 beats/min in lenti-GFP vs 281 2 beats/min in lenti-ACE2). Immunohistochem-
ical examination of the brainstem following termination of the experiment confirmed both the
injection site and neural overexpression of ACE2 in the RVLM. These observations demonstrate
that overexpression of ACE2 overcomes its intrinsic decrease in the RVLM and reverses
hypertension in the SHR.
P216
Adenoviral-Mediated Silencing of Nox2 Expression in Forebrain
Circumventricular Organs Blunts the Cardiovascular and Dipsogenic
Responses to Central Angiotensin-II
Jeffrey R Peterson, William Kutschke, Xin Tian, Ram V Sharma, Robin L Davisson; Univ of
Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA
We have previously demonstrated a role for superoxide production by a Rac1-dependent
NAD(P)H oxidase (Nox) in the pressor, bradycardic, and dipsogenic responses to intracerebro-
ventricular (ICV) administration of angiotensin-II (Ang-II). The Nox homologues are differentially
expressed in various cardiovascular control regions of mouse brain, with Nox2 being
abundantly expressed in forebrain circumventricular organs (CVOs) (Infanger et al., FASEB J
2006, 20:1190A). Here we hypothesized that adenoviral-mediated delivery of Nox2 siRNA
(AdsiNox2) to forebrain CVOs would silence Nox2 expression in these brain regions, preventing
the physiologic responses to ICV Ang-II. Adult C57BL/6 mice underwent CVO gene transfer with
AdsiNox2 or a control siRNA vector (AdGFPi), and were implanted with radiotelemeters for
Ang-II (200ng, 200nl)-mediated changes in MAP, HR and drinking were recorded. Baseline MAP
AdGFPi 57919 bpm, n8) did not differ between groups. Following ICV Ang-II, the peak
change in MAP was significantly attenuated in animals treated with AdsiNox2 compared to

controls (71, n10 vs 133 mmHg, n6, p0.05). Importantly, of the AdsiNox2 treated
animals that did show a slight pressor response to ICV Ang-II, the duration of this response was
significantly blunted, as indicated by a lesser area under the pressure-time plot for 15 min
following ICV Ang-II (3.00.8x105, n5 vs 5.20.5x105 mmHgsec, n6, p0.05).
Furthermore, the peak bradycardia to ICV Ang-II was also reduced in mice treated with
AdsiNox2 (HR -4912, n10 vs -10512 bpm, n6, p0.05), as was the dipsogenic
response (total time spent drinking over 15 min, AdsiNox2 308, n12 vs AdGFPi 9012
seconds, n8, p0.001). Finally, western analysis revealed decreased Nox2 protein levels
within these forebrain regions of AdsiNox2-treated animals, confirming in vivo brain silencing
of Nox2. This study establishes that Nox2-containing NAD(P)H oxidase is a key component of
Ang-II signaling in the brain, and is crucial for the physiologic responses to central Ang-II.
P217
ACE2 in the Brainstem is Regulated by Angiotensin-II Receptors
Teresa Obr, Univ of Iowa, Iowa City, IA; Eric Lazartigues; Louisiana State Univ Health
Sciences Cntr, New Orleans, LA
We previously reported the presence of angiotensin converting enzyme type 2 (ACE2) in
brainstem nuclei involved in the central regulation of blood pressure (BP) such as the nucleus
of the tractus solitarius (NTS) and the rostral ventrolateral medulla (RVLM). Here, we tested the
hypothesis that angiotensin-II receptors blockade would modulate ACE2 expression in these
brain regions. Non-transgenic (NT) and transgenic mice (n6 per group) with brain-selective
overexpression of AT1a receptors (NSE-AT1) were instrumented with radiotelemeters for
continuous BP monitoring. Baseline BP was recorded for 1 week before subcutaneous
implantation of osmotic pumps containing either saline, the AT1 receptor blocker losartan (10
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
mg/Kg/day) or the AT2 receptor antagonist PD123319 (10 mg/Kg/day). Mice were infused and
BP recorded for 2 weeks post implantation. In losartan-treated mice, mean BP decreased
significantly (NT:-103 mmHg; NSE-AT1:-123 mmHg; P0.05) by 2 weeks but remained
unchanged in the saline groups (NT:-12 mmHg; NSE-AT1:-23mmHg; P0.05). On the
other hand, AT2 blockade increased BP only in NSE-AT1 (125 mmHg; P0.05) mice. Using
quantitative real-time PCR (relative units), we observed that basal ACE2 mRNA expression was
increased in the NTS (NT:10.1; NSE-AT1:1.30.2; P0.05) and decreased in the VLM
(NT:10.1; NSE-AT1:0.70.1; P0.05) of NSE-AT1 mice but was similarly decreased by
losartan (NT:0.50.1; NSE-AT1:0.70.1 and NT:0.60.1; NSE-AT1:0.40.1, respectively,
P0.05) in both genotypes. On the contrary, AT2 blockade increased ACE2 expression in these
regions (NT:1.90.4; NSE-AT1:1.50.2 and NT:1.90.3, respectively, P0.05). These data
suggest that both AT1 and AT2 angiotensin receptors regulate ACE2 expression in brainstem
regions controlling BP, confirming the involvement of brain ACE2 in the central regulation of
cardiovascular function and its possible role in neurogenic hypertension.
P218
Silencing Brainstem Angiotensin AT1a Receptor Reduces Angiotensin II
Induced Hypertension
Yanfang Chen, Wenfeng Zhang, Mariana Morris; Dept of Pharmacology & Toxicology, Wright
State Univ Boonshoft Sch of Medicine, Dayton, OH
This study was designed to investigate the possible role of angiotensin (Ang) AT1a receptor in
brainstem dorsal nucleus vagus (DNV) and nucleus tractus solitarii (NTS) in Ang II induced
hypertension. C57BL/6 mice were surgically implanted with radiotelemetric probes for
recording the blood pressure (BP) and heart rate (HR). After one-week recovery the baseline BP
and HR was recorded (day 0). Mice were divided into four groups (n 4 6/group) for
subcutaneously minipump infusion of saline or Ang II (600 ng/kg/min) and bilateral NTS/DVN
microinjection (200 nl) of adenovirus (Ad) control (Ad-lacZ) or Ad mediated inference small
hairpin RNA for AT1a (Ad-AT1a-shRNA). Injection site and gene transferring were verified by
histologically checking the expression of marker gene lacZ, which was also encoded in the
Ad-AT1a-shRNA vector. The efficiency and specificity of Ad-AT1a-shRNA on silencing AT1a
receptor were determined in our previous in vitro and in vivo experiments. Results showed that
chronic infusion of Ang II slowly increased BP with the significant change seen on day 10.
Ad-AT1a-siRNA treatment postponed (increase seen on day 14) and reduced (- 43%) the
development of Ang II induced hypertension. There were no changes on HR and drinking
volume among groups. This study suggests that Ang II induced hypertension is developed
partially through AT1a receptor in NTS/DVN region.
CHBPR ConferencePoster Presentations e87
P219
Role of the Median Preoptic Nucleus in Chronic AngII Induced
Hypertension
Trasida Ployngam, John P Collister; Univ of Minnesota, St. Paul, MN
The median preoptic nucleus (MnPO), a nuclear group situated in the lamina terminalis,
receives afferent input from the subfornical organ that has been shown to be necessary in
mediating the full hypertensive response to angiotensin II (AngII) administration. In addition,
intravenous angiotensin II administration has been shown to cause activation of a number of
neurons in both the dorsal and ventral part of MnPO. Taken together, we hypothesized that the
MnPO is necessary for the full hypertensive response observed during chronic Ang II induced
hypertension. To test this hypothesis, male Sprague Dawley rats were subjected to either sham
(SHAM) or electrolytic lesion of both the dorsal and ventral part of the MnPO (MnPOx). During
the same surgery, rats were instrumented with venous catheters, and radiotelemetric
transducers for the intravenous administration of AngII and the measurement of blood pressure
and heart rate, respectively. Rats were then given a week recovery period. After 3 days of saline
control infusion (7 ml 0.9% NaCl/day), AngII was intravenously infused at a rate of 10 ng/kg/min
in both SHAM and MnPOx animals for 10 consecutive days, and followed by 3 recovery days.
A 0.4% NaCl diet and distilled water were provided ad libitum. At the end of the experiment,
rats were perfused and the extent of MnPO lesion was determined histologically in each rat.
By day 7 of Ang II infusion, MAP had increased 417 mmHg in SHAM rats (n2). MAP of
partial MnPOx rats (90 % MnPO ablated; n2) increased similarly to SHAM (4110 mmHg),
but MAP of complete MnPOx rats (90% MnPO ablated; n2) had only increased 152
mmHg. This trend continued through day 10 of AngII treatment. These results support the
hypothesis that the MnPO is necessary for the chronic hypertensive response to AngII
administration.
P220
ACE2 Inhibition in the Nucleus of the Solitary Tract Reduces Baroreceptor
Reflex Sensitivity
Debra I Diz, Ellen N Tommasi, Carlos M Ferrario, E. Ann Tallant, Patricia E Gallagher; Wake
Forest Univ Sch of Medicine, Winston-Salem, NC
Angiotensin converting enzyme 2 (ACE2), a novel enzyme of the renin-angiotensin system with
high catalytic activity for the conversion of angiotensin II (Ang II) to angiotensin-(17)
[Ang-(17)], is present in the dorsomedial medulla of rats as determined by the detection of
ACE2 mRNA. Moreover, ACE2 represents the major Ang-(17)-forming enzyme in solubilized
membranes prepared from this brain region. Previous studies in normotensive rats showed that
injection of [D-Ala7]-Ang-(17), an Ang-(17) receptor antagonist, into the nucleus of the
solitary tract (nTS) reduced baroreceptor reflex sensitivity by as much as 60%. The object of
the current study was to determine whether ACE2 contributes to the generation of Ang-(17)
in the dorsal medial medulla, by measuring baroreflex sensitivity during inhibition of ACE2
activity. Baroreflex control of cardiac interval was assessed in anesthetized male Sprague-
Dawley rats (SD, 10 - 12 week old; n 8) before and after bilateral injection of an ACE2
inhibitor (12 pmol/120 nL MLN4760; gift from Millenium Pharmaceuticals). ACE2 inhibition in
the nTS significantly reduced mean arterial pressure (92 5 vs. 79 4 mm Hg; p 0.05).
Baseline baroreceptor reflex sensitivity averaged 1.2 0.3 msec/mm Hg, whereas ACE2
inhibition substantially attenuated baroreflex sensitivity (0.3 0.1 msec/mm Hg; p 0.05).
By 90 - 120 minutes after bilateral injection of MLN4760, baroreflex sensitivity had returned
to values not different from baseline. Blunting of the baroreflex by the ACE2 inhibitor was the
same as that produced by bilateral injection of the Ang-(17) receptor antagonist [D-Ala7]-
Ang-(17) (144 fmol/120 nL; 0.3 0.1 msec/mm Hg; n 5). These results reveal a critical
role for ACE2 in the central control of blood pressure by regulation baroreceptor reflex
sensitivity, presumably through the conversion of Ang II to Ang-(17). Support: HL51952,
GM64249.
P221
Intracisternal Injection of Angiotensin-(17)-Producing Fusion Protein
Plasmid Lowers Blood Pressure in Hypertensive (mren2)27 Rats
Maria A Garcia-Espinosa, Patricia E Gallagher, Wake Forest Univ Health Sciences,
Winston-Salem, NC; Detlev Ganten, Charite-Univ Medicine, Berlin, Germany; Carlos M
Ferrario, Wake Forest Univ Health Sciences, Winston-Salem, NC; Timothy L Reudlehuber,
Clinical Rsch Institute of Montreal, Quebec, Canada; Debra I Diz; Wake Forest Univ Health
Sciences, Winston-Salem, NC
The transgenic (mRen2)27 rat (TGR) that overexpresses a murine Ren2 gene develops fulminant
hypertension at an early age despite low concentrations of renin in plasma and kidney. Hypothalamic
levels of angiotensin (Ang) II and Ang-(17) are elevated markedly in this strain and intracerebroventricular
(ICV) administration of a specific Ang-(17) monoclonal antibody increases mean arterial blood pressure
(MAP) further in these rats. Medullary tissue Ang II is also elevated in TGR, but medullary Ang-(17) is
lower than in control rats. We used an engineered fusion protein (EFP) that releases Ang-(17) to increase
the peptide specifically in the intracerebral compartment. TGR (40 week old heterozygous) rats were
cannulated for chronic MAP monitoring in conscious rats prior to and after receiving Ang-(17) EFP (n
9) or a control plasmid without the peptide (C-EFP; n 4) by injection into the 4th ventricle. We detected
a progressive decrease in the blood pressure during the first three days following the injection with a
gradual recovery to the resting MAP by day seven. The maximal fall in MAP was at day two (28 7 mm
Hg) in the Ang-(17) EFP-treated rats compared with 7 1 mm Hg in the C-EFP group (p 0.02). Thus,
replacement of Ang-(17) via gene transfer into the 4th ventricle leads to a significant fall in MAP in TGR.
These data highlight the counter-regulatory role of Ang-(17) in blood pressure homeostasis in an animal
model with elevated brain Ang II.
 e88 Hypertension Vol 48, No 4 October 2006

P222 2.1 ml, LF: 40.4 2.0 ml). The present findings suggest that sympathetic-regulatory neurons
Differential Inhibitory Effect of Translin on Action Potential Frequency in of the RVLM contribute to the elevated blood pressure in obese rats.
Neurons from Shr Brain

Adolfo E Cuadra, Mohan K Raizada; Univ of Florida, Gainesville, FL P225

Differential inhibitory effect of translin on action potential frequency in neurons from SHR brain
The rostral-ventrolateral medulla (RVLM) provides supra-spinal excitatory input to sympathetic
preganglionic neurons and thus plays a central role in neural control of arterial blood pressure.
This view is further strengthened by observations that increased sympathetic activity is
associated with hypertension. We have hypothesized that altered expression of one, or a set of
genes in the RVLM, leads to increased sympathetic activity in hypertension. In previous
expression profile analyses we demonstrated that the expression of translin is significantly
decreased in the SHR RVLM compared to normotensive WKY rat RVLM. Translin is a
transcriptional/post-transcriptional regulatory protein that resides in both the cytoplasm and
nucleus and associates with a number of other regulatory proteins. Here, we investigated if
translin is involved in regulation of neuronal activity and whether it exerts a differential effect
on WKY and SHR neurons that may underlie the increased sympathetic activity observed in the
SHR. The peptide, NH3-MSVSEIFVELQGFLA-COOH corresponding to the first 15 amino acid
residues of translin (TSN-P1) was used to investigate this hypothesis. TSN-P1 was injected into
the pipette solution of impaled neurons during current clamp recordings and changes in action
potential frequency (APF) were compared between WKY and SHR neurons. TSN-P1 caused a
time-dependent decrease in APF with differential sensitivity in WKY and SHR neurons. While
110nM TSN-P1 failed to show any significant effect on APF, at 100 nM it caused a significant
decrease in APF in WKY neurons. A maximal decrease of 36 7% (n9) was observed in 20 Peak Increases
minutes. In contrast, 1nM and 10nM TSN-P1 caused respective 57 6% (n8) and 43 8
% (n12) decreases in APF after 20 min in SHR neurons. Treatment of either neuron type with MAP (mmHg)
vehicle or scrambled peptide did not alter neuronal firing compared to control untreated
neurons. These observations show that neurons from SHR are more sensitive to TSN-P1 by
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
Mechanisms Mediating Sodium-Induced Hypertension in the PVN of Wistar Rats
Alex Gabor, Jr., Frans H Leenen; Ottawa Heart Institute, Ottawa, Canada
Sympathoexcitatory and hypertensive responses to central infusion of Na rich aCSF are
enhanced by aldosterone and mediated by MR and ENaC activation leading to the production/
release of brain ouabain and AT1-receptor stimulation. Where and how these mechanisms/
factors interact in the CNS has not yet been resolved. All are present in the PVN, and we
postulated that they form a functional pathway within the PVN. One week following sterotaxic
cannulation of the PVN, infusion (300nl/min) of Na rich aCSF (0.7M Na) for 15 min alone or
after aldosterone infusion (300nl/min) for 5 min in the PVN of conscious Wistar rats was
performed. Blockers were administered to evaluate functional pathways mediating the effects
of Na rich aCSF. Data are meanSE (n 4 6/group). *p0.05, vs. baseline; ^ p0.05, vs.
vehicle. The data indicates that aldosterone enhances the response to Na rich infusion in the
PVN. Both benzamil (to block ENaC) and fab fragments (to block ouabain) block only this
enhancement, whereas losartan blocks all responses to Na in the PVN. The results suggest
that in the PVN, Na increases BP and HR via AT1-receptor stimulation. MR activation enhances
this response via ENaC and local ouabain release.
aCSF (0.7M Aldosterone aCSF
Aldosterone Na) (0.7M Na)
01 142* 193*
HR (bpm) 205 536* 7410*
Ang II (90ng) Ouabain (80ng)

producing comparable inhibition with 10 100 fold lower concentrations. The data suggest that
decreased expression of translin may be linked to an increase in neuronal activity in the SHR. Losartan Losartan
Peak Increases Vehicle (80ug) Vehicle (80ug)
MAP (mmHg) 151* 20.4^ 161* 20.5^
HR (bpm) 234* -25^ 286* 108^
P223
Sympathoexcitatory Effect of Oxidative Stress in the Brain Mediate Arterial # p0.05, vs. aldosterone aCSF (0.7M Na) vehicle
Pressure Elevation in Salt-Sensitive Hypertension Aldosterone aCSF (0.7M Na) Aldosterone Veh. aCSF
pretreated with (0.7M Na) pretreated with
Megumi Fujita, Ai Nagae, Katsuyuki Ando, Toshiro Fujita; The Univ of Tokyo, Tokyo, Japan
Peak Increases Vehicle Benzamil Fab Fragments Losartan Vehicle Benzamil Losartan
Objective: Central sympathetic activation is one of the mechanisms of salt-induced rise in arterial MAP (mmHg) 211* 122*^ 102^ 11^ 121*# 111* 30.4^
pressure (AP). Recently, oxidative stress has been proposed to play an important role in HR (bpm) 7417* 558*^ 375^ 88^ 436*# 3315* 182^
salt-sensitive hypertension. We have suggested that oxidative stress in the brain may modulate
sympathetic regulation of AP. Thus, this study was aimed to investigate whether oxidative stress in
the brain might be related to the development of salt-sensitive hypertension through sympathoex- P226
citaion. Design and Methods: We used 9 to 10-week-old male Dahl salt-sensitive (Dahl-S) rats, Role of Kynurenine Aminotransferase-1 Gene within RVLM on the
which were fed with normal (0.6%) or high (8%) salt diet for 4 weeks. In urethane-anesthetized and Regulation of Blood Pressure and Sympathetic Nerve Activity in Metabolic
artificially ventilated rats, renal sympathetic nerve activity (RSNA) was recorded concomitantly with
AP and heart rate (HR), when tempol, a membrane-permeable superoxide dismutase mimetic, was
Syndrome
infused into the lateral ventricle. Changes in each parameter were compared between high
Satoru Ito, Kazutoshi Komatsu, Yoshiharu Yajima, Nihon Univ Sch of Medicine, Tokyo,
salt-loaded and normal salt-loaded rats. In addition, production of superoxide anion was measured
Japan; Yoko Iizuka, Takanari Gotoda, Toshiro Fujita, Univ of Tokyo, Tokyo, Japan; Kazuyoshi
in the isolated hypothalamus by lucigenin chemiluminescence method. Results: Basal mean AP and
Tsukamoto, Kouichi Matsumoto, Satoshi Saito; Nihon Univ Sch of Medicine, Tokyo, Japan
urinary norepinephrine level of high salt-loaded rats were significantly elevated than those of normal
salt-loaded rats. Intracerebroventricular tempol decreased AP, HR and RSNA in a dose-dependent
Kynurenic acid (KYN) is an endogenous glutamate antagonist. Kynurenine aminotransferase-1
fashion. These reductions in high salt-loaded rats were significantly greater than those in normal
(KAT-1) catalyzes the conversion of kynurenine to KYN. Recently, it has been reported that a
salt-loaded rats (AP: -38.74.9 % vs -10.63.3 %; p0.01; HR: -10.32.8 % vs -2.00.7 %;
missense (E61G) mutation exists in the KAT-1 gene of the spontaneously hypertensive rat
p0.05; RSNA: -21.53.7 % vs -6.51.5 %; p0.01 ), associated with significantly increased
(SHR), which is an experimental model of metabolic syndrome. This missense mutation
superoxide in the hypothalamus. Also, even in high salt-loaded rats whose AP was normalized by
decreases activity of KAT-1 in central nervous system including rostral ventrolateral medulla
hydralazine hydrochloride, responses to intracerebroventricular tempol were not normalized. NADPH-
(RVLM) of SHR. We reported previously that bilateral injections of KYN into RVLM decrease arterial
augmented superoxide levels in the hypothalamus was also significantly higher in high salt-loaded rats
pressure (AP) in SHR but not WKY. These results indicated that the reduced KAT-1 activity within
than in normal salt-loaded rats. Conclusions: In salt loaded Dahl-S rats, a salt-sensitive hypertension
RVLM due to the missense mutation may play a key role in the development and maintenance of
model, increased superoxide in the brain, possibly via activation of NADPH oxidase, might mediate arterial
hypertension in SHR. To determine how exogenous expression of the KAT-1 gene (wild type KAT-1
pressure elevation through central sympathoexcitatory effect.
gene of WKY) in the RVLM can affect AP in SHR, adenovirus vectors encoding either KAT-1
(AdKAT-1) or beta-galactosidase (AdLacZ) were transfected bilaterally into RVLM of SHR. The local
expression of KAT-1 mRNA in the RVLM was confirmed by Northern blotting. At days 7, 14 and 21
P224 after gene transfer, the expression of KAT-1 in the RVLM was increased in the AdKAT-1 SHR, but
Neurons of the Rostral Ventrolateral Medulla Contribute to Obesity-Induced not in the AdLacZ SHR. AP and HR was decreased in AdKAT-1, but not AdLacZ SHR under conscious
state. Sympathetic index was decreased and baroreflex sensitivity was increased at day 7 in
Hypertension
AdKAT-1, but not AdLacZ SHR under conscious state. Microinjection of KYN into the RVLM at day
7 decreased AP less in the AdKAT-1 SHR, while microinjection of glutamate into the RVLM increased
Sean D Stocker, Julye M Adams; Univ of Kentucky, Lexington, KY
AP similarly in both groups. Urinary excretion of catecholamines was also decreased in AdKAT-1
SHR. Insulin resistance measured by Insulin tolerance test tended to be decreased but not
Activation of the sympathetic nervous system contributes to obesity-induced hypertension. In
significantly in AdKAT-1 SHR. These results suggest that KAT-1 gene transfection in the RVLM
the present study, we sought to determine whether sympathetic-regulatory neurons of the
decreases AP and sympathetic nerve activity in conscious SHR. These changes may be mediated
rostral ventrolateral medulla (RVLM) contribute to elevated blood pressure in obese rats. Male
by normalization of KAT-1 activity in the RVLM of SHR. Since excitatory amino acid neurotrans-
Sprague Dawley rats (350 425 g) were placed on a moderately high fat diet (32% kcal as fat,
mission in the RVLM is considered important in central cardiovascular regulation, the reduced KAT-1
n24) or a low-fat diet (LF, 10.6% kcal as fat, n7). After 14 weeks, rats fed the moderately
activity in the RVLM due to the missense mutation should contribute to maintenance of elevated AP
high fat diet segregated into obesity-prone (OP) and obesity-resistant (OR) groups based on
and sympatheic nerve activity observed in metabolic syndrome.
their body weight (OP: 869 25 g, OR: 709 8, LF: 702 33 g; P0.05). Baseline mean
arterial blood pressure (MAP) was significantly elevated in the OP rats versus the OR and LF
rats (OP: 1082 mmHg, OR: 100 2 mmHg, LF: 973 mmHg; P0.05). Inhibition of the
RVLM with bilateral microinjection of the GABAA receptor agonist muscimol (200 pmol per 100 P227
nl) decreased MAP to similar levels across the three groups (OP: 491 mmHg, OR: 502 Anti-Oxidant Effect of Atorvastatin in the Brain Contributes to the
mmHg, LF: 491 mmHg; P0.01), but the magnitude of this decrease was significantly Atorvastatin-Induced Sympatho-Inhibitory Effects in Stroke-Prone
greater in the OP versus the OR and LF groups (OP: -58 2 mmHg, OR: -49 1 mmHg, LF: Spontaneously Hypertensive Rats
-48 3 mmHg; P0.01). These differences in MAP cannot be explained by changes in
vascular reactivity, as the EC50 was similar across groups in response to multiple intravenous Takuya Kishi, Yoshitaka Hirooka; Kyushu Univ, Fukuoka, Japan
injections of phenylephrine (OP: 12.2 1.5 g/kg, OR: 15.0 2.7 g/kg, LF: 13.8 2.0
g/kg) or norepinephrine (OP: 1.4 0.1 g/kg, OR: 1.4 0.2 g/kg, LF: 1.4 0.1 g/kg). Statins have reported to increase endothelial nitric oxide synthase (eNOS) expression and activity
Blood volume was not different among OP, OR, and LF rats (OP: 45.0 2.2 ml, OR: 43.9 independent of cholesterol-lowering effects. It is also suggested that statins have beneficial sympatho-

inhibitory effects in hypertesion and heart failure. Recently, we demonstrated that long-term treatment
with atorvastatin had sympatho-inhibitory effects with upregulation of eNOS in the brain in stroke-prone
spontaneously hypertensive rats (SHRSP), which is the hypertensive model with the increase in
sympathetic nerve activity. Furthermore, we also demonstrated that reactive oxygen species (ROS) in the
rostral ventrolateral medulla (RVLM) of the brain stem, where the vasomotor center is located, was
significantly increased in SHRSP and that ROS in the RVLM contribute to sympatho-excitatory effects in
SHRSP. Therefore, the aim of the present study was to determine whether atorvastatin reduces oxidative
stress in the RVLM of SHRSP associated with sympatho-inhibitory effects. SHRSP and Wistar-Kyoto (WKY)
rats were treated with or without oral administration of atorvastatin (50mg/kg per day) for 30 days. Systolic
blood pressure and heart rate were measured using the tail-cuff method. As the parameter of the
sympathetic nerve activity, urinary norepinephrine excretion was measured for 24 hours before and after
the treatment with atorvastatin. Treatment with atorvastatin significantly decreased blood pressure and
urinary norepinephrine excretion in SHRSP, but not in WKY (1996 vs 1735 mmHg, 1.410.05 vs
1.070.04 g, p0.05, n5). Thiobarbituric acid-reactive substances (TBARS) levels in the RVLM
tissue obtained using the punch-out technique were used as measures of oxidative stress. Treatment with
atorvastatin decreased TBARS levels in the RVLM of SHRSP (0.750.04 vs 0.570.03 mol/g wet wt,
p0.05, n5), but not in WKY. These results suggest that atorvastatin reduces oxidative stress in the
RVLM of SHRSP, which might contribute to the sympatho-inhibitory effects of atorvastatin in SHRSP. We
think that this anti-oxidant effect of arorvastatin in the brain will contribute to the new-treatment for
hypertension and heart failure.
P228
Increased c-Fos Immunoreactivity in Medullary and Forebrain
Cardiovascular Nuclei in Acute and Chronic Renal Wrap Hypertension
J. T Cunningham, Myrna Herrera-Rosales, Maurice L Penney, Michele A Martinez, Steve
Mifflin; Univ. of Texas Health Science Cntr, San Antonio, TX
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Renal wrap hypertension is characterized by sodium sensitivity, increased sympathetic outflow and
differential alterations in baroreflex regulation of sympathetic nerve discharge and heart rate. The present
study used staining for c-Fos immunoreactivity to determine if more neurons are synaptically activated in
brainstem and forebrain cardiovascular control regions in this form of hypertension. Hypertension was
produced using a unilateral nephrectomy, renal wrap procedure. Sham operated controls and 1 wk and
4 wk hypertensive rats were implanted with arterial catheters and mean arterial pressure was measured
2 days later in the conscious, unrestrained rat. Mean arterial pressures were 103 2 mmHg for sham
(n 6), 138 4 for 1 wk renal wrap (n6), and 159 6 for 4 wk renal wrap (n6). After
measurement of arterial pressure, rats were perfused and brains sectioned coronally (40 um) and the
sections processed for c-Fos immunoreactivity using a commercially available primary antibody
(Oncogene AB-5; 1:30,000). Renal wrap hypertension was associated with increased c-Fos staining in the
nucleus of the solitary tract (NTS; sham 3 1; 1 wk 12 3; 4 wk 36 3; P 0.01) with 4 wk renal
wrap significantly higher than 1 wk renal wrap. Similar increases were observed in the rostral ventrolateral
medulla (RVLM; sham 2 1; 1 wk 9 2; 4 wk 17 2; P 0.01). In the caudal ventrolateral medulla
(CVLM), c-Fos staining was significantly increased in rats with hypertension but there were no differences
between 1wk and 4 wk groups (sham 3 1; 1 wk 6 0.4; 4 wk 7 1; P 0.01). c-Fos staining
in the paraventricular nucleus of the hypothalamus (PVN) was significantly increased after 4 wk renal wrap
(sham 19.6 4; 1 wk 31.2 5; 4 wk 50.6 8; P 0.05). Changes in c-Fos staining were not
observed in the area postrema or the supraoptic nucleus. Renal wrap hypertension was associated with
increased c-Fos staining in NTS and RVLM that were related to the level of blood pressure. The failure to
observe similar changes in the CVLM could be related to baroreflex resetting at this site or reflect
convergence of NTS inputs to this region. The results indicate that sustained hypertension results in an
increased number of synaptically activated neurons in the major nuclei associated with blood pressure
regulation.
P229
Local Production of Angiotensinogen in the SFO Results in Elevated ANG
Production and Increased Water Intake
Khristofor Agassandian, Koji Sakai, Robin L Davisson, Martin D Cassell, Curt D Sigmund;
Univ of Iowa, Iowa City, IA
It is well known that heightened activity of renin-angiotensin system in the brain plays a role in
pathogenesis of hypertension. However, the regulation and localization of Ang-II production in the
brain remains unclear. Previous studies using reporter genes indicate that glial cells and some
neurons synthesize angiotensinogen (AGT), whereas renin (REN) is expressed primarily in neurons.
To examine the consequences of neuronal REN and neuronal and glial AGT we generated double
transgenic mice expressing human AGT from its endogenous promoter (which targets the correct
population of neurons and glia) and human REN from a neuron-specific promoter. The double
transgenic mice exhibit an increase in arterial pressure (121.46.0 vs. 96.53.5 mmHg, n7,
P0.004), and baseline water intake (5.20.5 vs. 1.80.1 mL/day/10g BW, n16, P0.001)
which is AT1 receptor dependent. Immunohistochemistry using antisera directed against Angioten-
sin-I/II (ANG) revealed the primary site of ANG synthesis to be the subfornical organ (SFO). The SFO,
situated on the ventral surface of the fornix, is one of the AT1 receptor-containing circumventricular
organs involved in the regulation of fluid and electrolyte balance and plays an important role in
regulating thirst. Immunoelectron microscopy of the SFO from non-transgenic mice identified
endogenous ANG immunoreactivity (IR) in neurons and dendrites, with the intensity of the IR
increasing toward the ventricle. In comparison with non-transgenic mice, the neurons and dendrites
in the SFO of double transgenic mice showed a dramatic increase of ANG IR, again with the highest
level in the cells facing the ventricle. Stereotaxic injection of an adenovirus expressing cre-
recombinase (Adcre) directly into the SFO in a model expressing a floxed version of hAGT
(providing an opportunity for cell-specific ablation of the gene in the presence of cre-recombinase)
resulted in a significant decrease ANG IR in the SFO. Functionally, SFO-specific injection of Adcre
caused a significant decrease in water intake (from 6.60.7 pre Adcre to 3.80.6 mL/day/10g BW
post Adcre, n8, P0.01). We conclude that de novo expression of AGT in the SFO is an important
mediator of local ANG production and plays a role in cardiovascular homeostasis.
CHBPR ConferencePoster Presentations e89
P230
AT1a Receptors Predominantly Mediate Pressor, Cardiac Hypertrophic, and
Left Ventricular Responses to Long-Term Angiotensin II Infusion in Mice
Xiao C Li, Oscar A Carretero, Yun-He Liu, Jiang Xu, Xiao-Ping Yang, James Yang, Edward
Shesely, Jia L Zhuo; Henry Ford Hosp, Detroit, MI
The current view holds that arterial pressure and cardiac effects of angiotensin II (Ang II) are mediated by
three different receptors, AT1a, AT1b, and AT2. The AT1b receptor is thought to play a compensatory role
when AT1a is absent, whereas the role of AT2 is to oppose AT1a. We questioned whether the AT1b receptor
would assume the role of the AT1a receptor after the latter is genetically deleted. Age-matched, adult male
wild-type (Agtr1A/) and AT1a receptor-deficient mice (Agtr1A-/-) (n 79 each group) were treated
with vehicle, Ang II (40 ng/min, i.p. via osmotic minipump), or Ang II plus the AT1 blocker losartan (10
mg/kg/day, p.o.) for 2 weeks. Cardiac hypertrophy and left ventricular (LV) function were assessed by
echocardiography before and 2 weeks after treatment. As expected, wild-type mice had increased systolic
blood pressure (basal: 113 3 vs. Ang II: 168 6 mmHg; p0.001), LV mass (basal: 25.7 1.4 vs.
Ang II: 32.4 2.1 mm3/10g; p0.05) and LV dimension area (basal: 1.70 0.01 vs. Ang II: 2.2 0.2
mm2; p0.05), heart weight-to-body weight ratio (basal: 0.52 0.03 vs. Ang II: 0.71 0.06; p0.001),
aortic dimension (basal: 1.50 0.01 vs. Ang II: 1.63 0.05 mm2; p0.01), cardiac output (basal: 9.0
0.5 vs. Ang II: 13.2 1.6 ml/min/10g; p0.05), and systolic posterior wall thickness (PWT) (basal:
1.20 0.01 vs. Ang II: 1.35 0.01 mm, p0.05) in response to 2-week Ang II infusion. All of these
pressor and cardiac responses to Ang II were prevented by concurrent losartan administration. By
contrast, Agtr1A-/- mice had lower basal systolic blood pressure (p0.001) and LV wall thickness
(p0.005), and higher ejection fraction (p0.05), stroke volume (p0.005) and cardiac output
(p0.001). Although Ang II slightly increased systolic blood pressure and LV wall thickness (p0.05),
there were no significant changes in other cardiac or LV indices 14 days after Ang II infusion in Agtr1A-/-
mice. Losartan, which blocks remaining AT1b receptors, did not significantly alter overall cardiac responses
to Ang II in Agtr1A-/- mice. These results suggest that AT1a receptors overwhelmingly mediate pressor and
cardiac LV responses to Ang II, and AT1b receptors play only a minor (if any) role in blood pressure and
cardiovascular control when the AT1a receptor is absent.
P231
Telmisartan and Insulin Affect Angiotensin II Type 1 and Type 2 Receptor
Expression in Vascular Smooth Muscle and Vascular Endothelial Cells
Michael D Nyby, VA Greater Los Angeles Healthcare System/UCLA, Sepulveda, CA; Kyle
Douglas, Karolin Abedi, Hirofumi Makino, VA Greater Los Angeles Healthcare System,
Sepulveda, CA; Michael L Tuck; VA Greater Los Angeles Healthcare System/UCLA,
Sepulveda, CA
Vascular cells express angiotensin II type 1 receptors (AT1R) and angiotensin II type 2 receptors
(AT2R). AT1R is associated with cell proliferation, vasoconstriction, oxidative stress, and
inflammation. AT2Rs actions are thought to oppose many of the actions mediated by AT1R.
AT2R activation has been associated with vasorelaxation and antiproliferation. High insulin
concentration induces proliferation of vascular smooth muscle cells and apoptosis of vascular
endothelial cells. This study determined if AT1R and AT2R expression in vascular cells was
altered by insulin alone, and with captopril, an ACE inhibitor, and with telmisartan, a AT1R
blocker. Rat aortic smooth muscle cells (RASMC) and endothelial cells (RAOEC) were grown in
culture until confluent. Cells were exposed to insulin at concentrations of 0.1 and 1.0 mU/ml
for 48 hours. Captopril or telmisartan was also added to some cultures at a concentration of
10-6 M. RNA was extracted, reverse-transcribed and used in real-time quantitative PCR. In the
RASMC, AT1R mRNA expression was enhanced by 1.0 mU/ml insulin (1.30.2 fold of control
), and significantly reduced by telmisartan to 0.640.27-fold of control (P0.05 vs. insulin
alone). Captopril did not reduce expression of AT1R in these cells. AT2R mRNA expression was
decreased to 0.840.24 of control by 0.1 mU/ml insulin in RASMC, and was upregulated by
both captopril (1.50.4-fold of control, P0.05) and telmisartan (1.90.5-fold increase,
P0.05). In RAOEC, AT1R mRNA expression was not affected by insulin, but AT2R mRNA
expression was increased by 0.1 and 1.0 mU/ml insulin to 4.01.3 and 4.30.9-fold
increases above control, respectively (P0.05 for both). Captopril alone increased AT2R mRNA
expression by 2.40.5-fold in RAOEC. Telmisartan increased the expression of AT2R mRNA by
1.80.3-fold alone, and by 4.51.0-fold and 5.21.6-fold in combination with 0.1 and 1.0
mU/ml insulin, respectively (P0.05 for both). These results suggest that AT2R expression in
vascular cells may influence insulins effects on proliferation. Also, these results suggest that
the beneficial vascular effects of telmisartan are the result of increased expression of vascular
AT2R as well as blockade and downregulation of vascular AT1R.
P232
High Fructose Diet Enhances Losartans Hypotensive Effect
Danielle Senador, UNIFESP, Sao Jose dos Campos, Brazil; Vera Farah, Wright StateUniv,
Dayton, OH; Morris Mariana, Wright State Univ, Dayton, OH; Maria Claudia Irigoyen; InCor,
Sao Paulo, Brazil
Previous work from our laboratory documented the role of angiotensin AT1a receptors in
mediating the pressor effects of a high fructose diet. The objective of this study was to
determine the influence of pharmacological blockade of Ang AT1 receptors on the cardiovas-
cular and metabolic effects of a high fructose diet. Male C57BL mice were fed a 60% fructose
diet for 10 weeks. Telemetric probes were inserted into the carotid artery at week 8 and
losartan (30 mg/kg/day) was given in the drinking water beginning at week 9. The blood
pressure signal (recorded at 5 kHz) was and submitted to autoregressive spectral analysis (SA).
Pulse interval (PI) and systolic arterial pressure (SAP) were analyzed in time and frequency
domains (low frequency, LF, 0.11.0 Hz; high frequency, HF, 15 Hz). Fructose produced
impairment in glucose tolerance (ip glucose load). Losartan treatment decreased mean arterial
pressure (MAP) with a greater response in animals on the fructose diet. MAP was reduced 15
% (1005 vs. 847 mmHg) in the fructose group as compared to 5% (1019 vs. 966
mmHg) in the controls. There were no alterations in heart rate. Autonomic balance was
analyzed using autoregressive spectral methods. Results showed that the losartan treatment
 e90 Hypertension Vol 48, No 4 October 2006
decreased the SAP variance and the LF component in both groups. However the fructose group
had a greater decrease in both parameters (reduction in variance of: 42% vs. 27%; reduction
in LF of: 50% vs. 27%). In conclusion, results demonstrate that Ang AT1 receptors are critical
in mediating the pressor response to a high fructose diet. They demonstrate the importance of
interactions between dietary consumption of sugars and antihypertensive drug therapy.
P233
Increased Tachyphylaxis to Angiotensin II in Hypertensive Rat Aortic Rings
Does not Involve Receptor Internalization via Caveolae
A. Elizabeth Linder, Romulo Leite, R. Clinton Webb; Med College of Georgia, Augusta, GA
Angiotensin II (Ang II) type I (AT1) receptor activation is generally followed by receptor
internalization (RI). We have previously observed that tachyphylactic contractile responses to
Ang II are prevented by caveolae disruption and inhibition of RI induced by methyl--
cyclodextrin (CD) in rat aorta. It has been shown increased Ang II internalization in hypertension.
Decreased number of caveolae has been shown to be associated with vascular diseases such
as hypercholesterolemia and sexual dysfunction. We hypothesized that tachyphylaxis to Ang II
is increased in hypertensive animals due to increased caveolae-independent RI pathway.
Endothelium-denuded rat aortic rings from normotensive and hypertensive [Ang II (60
ng/Kg/day)-treated for 14 days] rats were exposed to increasing concentrations of Ang II (1 nM
to 1 M) to generate two cumulative concentration-effect curves (CEC I and CEC II). A 90-min
interval separated CEC I and CEC II. CEC II was performed after a 60 min pre-incubation with
vehicle or CD (10 mM), a drug that depletes cholesterol from the membrane disrupting
caveolae. Ang II induces tachyphylactic contractile responses in rat aortic rings from
normotensive and hypertensive rats. When CEC II after vehicle is expressed as % of CEC I, we
clearly demonstrate that tachyphylaxis is increased in rat aortic rings from hypertensive rats.
Whereas CD prevented the tachyphylactic contractions to Ang II in rat aorta from normotensive
animals, it had no effect on vessels from hypertensive rats. Our data indicate that the increased
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tachyphylactic responses to Ang II in hypertensive aortic rings may be associated with
increased RI via a caveolae-independent pathway.
P234
eNOS Transfection Prevents Angiotensin II-Induced Vascular Hypertrophy
through Selective Inhibition of the Rho/ROCK Pathway
Hiroyuki Suzuki, Sadaharu Higuchi, Haruhiko Ohtsu, Kunie Eguchi, Hidekatsu Nakashima,
Sudhir Dhobale, Temple Univ Sch of Medicine, Philadelphia, PA; Gerald D Frank, Vanderbilt
Univ Sch of Medicine, Nashville, TN; Evangeline D Motley, Meharry Med College, Nashville,
TN; Satoru Eguchi; Temple Univ Sch of Medicine, Philadelphia, PA
Enhanced angiotensin II (AngII) actions are frequently associated with endothelial dysfunction, which is
characterized as decreased nitric oxide (NO) availability. Although endothelial NO synthase (eNOS) is
believed to antagonize vascular dysfunction induced via the AT1 receptor, the exact signaling mechanisms
remains controversial. Therefore, we investigated a possible signal cross talk between eNOS and AT1
along with their impacts on vascular hypertrophy in vascular smooth muscle cells (VSMCs) infected with
adenovirus encoding the eNOS gene. In VSMCs infected with adenovirus vector encoding eNOS or its
phosphorylation site mutant eNOS[S1179A], basal G kinase activity was enhanced. AngII rapidly
stimulates phosphorylation of eNOS at Ser1179 through AT1. This is accompanied with enhanced
cGMP production and G kinase activity. AngII was unable to stimulate cGMP production in VSMCs
infected with eNOS[S1179A] adenovirus. In contrast, AngII-induced VSMC hypertrophy was
markedly inhibited by eNOS and by eNOS[S1179A]. This is accompanied with selective inhibition of
the Rho/Rho-kinase(ROCK) cascade. RhoA activation, a ROCK substrate, MYPT phosphorylation and
myosin light chain phosphorylation induced by AngII but not EGF receptor transactivation or ERK
activation by AngII were attenuated by eNOS overexpression. From these data, we conclude that
under eNOS gene transfer, sustained basal level NO production and the resultant G kinase activation
appear to be sufficient to prevent AngII-induced hypertrophy by selective inhibition of the Rho/ROCK
pathway. Also, the AngII/AT1 system could positively couple to eNOS via Ser1179 phosphorylation to
produce more NO. These data demonstrate a novel molecular mechanism of eNOS regulation and
the importance of a sustained basal level of eNOS/PKG activity in preventing vascular remodeling.
P235
Roles of Gq, G12/13 and G Protein-Independent Mechanisms in Growth
Promoting Signal Transduction of Angiotensin II
Haruhiko Ohtsu, Sadaharu Higuchi, Kunie Eguchi, Hiroyuki Suzuki, Hidekatsu Nakashima,
Hidekatsu Nakashima, Satoru Eguchi; Temple Univ, Philadelphia, PA
Angiotensin II (AngII) and its G protein-coupled receptor, AngII type-1 receptor (AT1), play critical roles in
mediating cardiovascular diseases such as hypertension. It is widely believed that AngII promotes these
diseases by inducing vascular remodeling that involves hypertrophy and/or hyperplasia of vascular smooth
muscle cells (VSMCs). Although it is well-known that Gq plays an important role in intracellular Ca2
elevation and protein kinase C activation via the AT1 receptor, the role of heterotrimeric G proteins in
mediating VSMC hypertrophy induced by AngII remains controversial. We and others have shown that
AngII has two major growth-promoting signal transduction pathways through the AT1 receptor such as
EGFR/ERK and the Rho/ROCK pathway in VSMCs. This study examines the requirement of G protein on
these two AngII-pathways that lead to vascular remodeling. Intracellular Ca2 elevation induced by AngII
(100 nM) but not by PDGF (50 ng/ml) was completely inhibited by pretreatment with a selective Gq
inhibitor, YM-254890 (10 min, 1 M). In addition, EGF receptor transactivation as detected by its Tyr1068
phosphorylation as well as ERK activation induced by AngII, but not Ca2 ionophore, A23187, was
inhibited by YM-254890 in VSMCs. Alternatively, YM-254890 markedly but partially inhibited AngII-
induced phosphorylation of MYPT, a substrate of ROCK, in VSMCs. Thus, AngII also activates G12/13 as
selectively measured with a pull-down assay using GST-TPR fusion protein. Stimulation of quiescent
VSMCs with AngII (100 nM) for 72 hours resulted in an increase of cellular protein (2024.7 %, Pierce
BCA assay) and cell volume (1671.9 %, Beckman Coulter counter), but did not affect proliferation or cell
death as determined by MTS assay (Promega). YM-254890 completely inhibited these hypertrophic
effects of AngII in VSMCs. Also, G protein-independent AT1 receptor agonist, [Sar1, Ile4, Ile8] AngII, did not
stimulate phosphorylation of EGFR and MYPT or hypertrophy in VSMCs. Taken together, these results
suggest that Gq plays a major role in EGFR/ERK and Rho/ROCK pathways leading to vascular hypertrophy
induced by AngII, whereas G12/13 partially participates in the Rho/ROCK pathway in VSMCs.
P236
Reduced Renal AT2 Receptor in Diabetes is Reversed by Inhibition of AT1
Receptor or NANPH Oxidase
Chun Xue, Robert M Carey, Helmy M Siragy; Univ of Virginia, Charlottesville, VA
Earlier studies suggested decreased AT2 and increased AT1 receptors activities in diabetes.
Since AT2 receptor (AT2R) seems to antagonize the functions of the AT1 receptor (AT1R),
restoration of this receptor functions could be beneficial. In this study we hypothesized that in
streptozotocin (STZ) induced diabetes rat model there is down-regulation of AT2R in the kidney
that is reversed by inhibition of AT1R and NADPH oxidase. We first monitored changes in renal
AT2R mRNA in normal and streptozotocin induced diabetes rats for 12 weeks. Renal AT2R
mRNA and protein expression were monitored 6 weeks after induction of diabetes and in
response to 1 week treatment with insulin (4units/kg/d), the AT1R blocker valsartan
(10mg/kg/d) or the NADPH oxidase DPI (0.5mg/kg/d). AT2R mRNA expression was reduced
significantly in diabetic animals throughout the study period (Figure A). Insulin treatment,
valsartan and DPI enhanced AT2R mRNA and protein expression (Figure B). Immunostaining for
the AT2R was localized mainly in glomeruli and tubules. The intensity of the immunostaining
was decreased in diabetes, but reversed with insulin, Valsartan or DPI treatments. These
results demonstrate that renal AT2R is down-regulated by hyperglycemia, AT1R or NADPH
oxidase activities. This study suggests that in diabetes the cause of the reduction of the AT2R
is multifactorial and related to increased AT1R and NAPDH oxidase activities.
P237
The GABA Receptor Trafficking Protein, GABARAP, Binds to the AT1
Receptor Carboxy-Terminus
Julia L Cook, You-E He, Ryan T Naquin, Richard N Re, Jawed Alam; Ochsner Clinic
Foundation, New Orleans, LA
The objective of is this study was to identify brain proteins which bind to the carboxy-terminus
of the rat AT1a receptor. We have been the first to report that the cytoplasmic C-terminus of the
G-protein coupled receptor, AT1, is cleaved and traffics into the nucleus where it may regulate
gene expression. The present study was designed to identify partners that bind to and
chaperone the cleavage product into the nucleus. A yeast two-hybrid screen of a mouse brain
cDNA library was carried out to identify proteins that interact with the C-terminus of the AT1R
receptor (amino acids 306 359). Of 700,000 clones screened, 40 clones interacted with AT1R
as judged by growth on selective media. Sequence analysis revealed that 18 of the 40 clones
encoded GABARAP or the related protein GABARAP-L1. The AT1R yeast two-hybrid interaction
was verified by AT1R-GST fusion protein in vitro pull-down assays. Mammalian two-hybrid
assays and in vivo co-immunoprecipitation studies established that the interaction also exists
in mammalian cells. The C-terminus of GABARAP is known to bind to the subunit of the
pentameric GABA A receptor and to mediate trafficking of the GABA receptor, via microtubule
arrays, to the plasma membrane. The N-terminus of GABARAP possesses a tubulin-binding
motif. We speculate that GABARAP may be involved in AT1R protein trafficking, either in the
biosynthetic pathway or the internalization pathway. We are the first laboratory to identify and
report interaction of the carboxy-terminus of the AT1R with GABARAP. GABARAP has been
shown to influence cell-surface expression, membrane clustering properties, and activation of
the GABA A receptor by GABA. GABARAP may perform similar functions for the AT1R. Moreover,
GABARAP may serve as a link for co-regulation of the AT1R with the GABA receptor. GABARAP

may also serve to traffic the AT1R cleavage product along the microtubule pathway and into the
nucleus.
P238
Role of Reactive Oxygen Species and Phospho-Caveolin in Activation of
EGF Receptor and Akt in AT1 Receptor Signaling Endosomes in Vascular
Smooth Muscle
Masuko Ushio-Fukai, Lian Zuo, Satoshi Ikeda, Nikolay Patrushev, R. Wayne Alexander;
Emory Univ, Atlanta, GA
Angiotensin II (Ang II) promotes growth and hypertrophy primarily through the G-protein coupled
receptor (GPCR) Ang II type 1 receptor (AT1R) in vascular smooth muscle cells (VSMCs). We
showed that the tonic phase of Ang II signaling correlated with the internalization of the Ang
II-AT1R complex, indicating the existence of an intracellular domain of active AT1R signaling.
Major outputs of the AT1R are dependent upon transactivation of the EGF receptor (EGF-R),
which is mediated through cSrc, cAbl, caveolin-1 (Cav1) and reactive oxygen species (ROS)
derived from NAD(P)H oxidase. Evidence suggests that activated EGF-R and GPCRs continue to
generate signals from endosomes. Biochemial cell fractionation shows that Ang II stimulation
rapidly promotes AT1R internalization from the membrane fraction into the EEA1-positive early
endosomes within 5 min. EGF-Rs are present basally in both membrane and endosome
fractions, and AT1R appearance in endosomes is cotemporaneous with the tyrosine phosphor-
ylation of EGF-R at pY1173 and pY1068 and its downstream Akt phosphorylation in this
intracellular domain, which continues at least for 30 min. Of note, Cav1, a marker protein of
caveolae, is also found in both membrane and endosome fractions basally. Cav1 is tyrosine
phosphorylated by Ang II within 5 min, which is inhibited by the Src inhibitor PP1 and ROS
inhibitor, N-acetylcysteine (NAC) and cAbl siRNA. A functional role of Src-, cAbl- and
ROS-dependent tyrosine phosphorylated Cav1 (PY-Cav1) is demonstrated by the observation
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that PP1, NAC, cAbl siRNA and overexpression of mutant Cav1 (Y14F) prevent AT1R
internalization, EGF-R transactivation and Akt phosphorylation in endosomes. In contrast, Cav1
(Y14F) has no effects on AT1R trafficking into the Cav1-enriched lipid rafts membrane fractions.
In summary, these results suggest that PY-Cav1 plays an important role in regulating Src-,
cAbl- and ROS-dependent AT1R internalization into endosomes, which appear to be a major
intracellular compartment enabling the development of redox-dependent, EGF-R-mediated
tonic phase of active AT1R signaling in VSMCs.
P239
Physiological Angiotensin II Activation of ERK: To Transactivate or not to
Trasactivate the Epidermal Growth Factor Receptor, That is the Question
Bradley T Andresen, Lindsay B Keever, Crisanto S Escano, Jr; Georgetown Univ,
Washington, DC
Activation of the Mek-ERK pathway has been shown to be involved, in some instances, in
vasoconstriction including Ang II-induced vasoconstriction. This occurs through ERK-mediated
phosphorylation of the smooth muscle specific 20 kD myosin light chain, similar to myosin light
chain kinase. There are multiple proposed mechanisms for Ang II-mediated ERK phosphory-
lation/activation. In thoracic aortic smooth muscle cells, Ang II signals to ERK through an
inside-out transactivation of the epidermal growth factor receptor (EGFR); however, this, and
previous studies, suggest this may not be the case in all cell types, including the renal
vasculature. Since Ang II primarily constricts the renal and mesenteric vascular beds, the
molecular mechanisms of ERK activation may have physiological consequences. We hypoth-
esize that inhibition of Mek, and consequently ERK, but not the EGFR, will reduce Ang
II-mediated changes in systolic blood pressure. In vivo experiments were conducted in WKY
rats with two periods: control and experimental. In both periods, 2.5 g/kg Ang II was
administered via the jugular vein within 30 seconds. No experimental treatment significantly
altered baseline blood pressure. Vehicle and 20 mg/kg AG1478, an EGFR inhibitor shown to
function in vivo, did not significantly alter the change in systolic blood pressure between the
two periods; however, 15 mg/kg PD98059, a Mek inhibitor shown to function in vivo,
significantly reduced Ang II-mediated increase in systolic blood pressure by 33 10% (paired
t-test p 0.044, n 3, mean SEM). Furthermore, confocal imaging of renal smooth muscle
cells demonstrates that Ang II directs phospho-ERK to actin filaments and the nucleus, whereas
EGF treatment directs phospho-ERK predominantly to the nucleus. To conclude, in thoracic
aortic smooth muscle cells, Ang II activates ERK through the EGFR; however, in renal
microvascular smooth muscle cells, Ang II activates ERK through a non-EGFR-mediated
pathway. This difference in signaling mechanisms accounts for the altered phospho-ERK
localization in the renal smooth muscle cells, and for PD98059, not AG1478, to lower Ang
II-mediated changes in systolic blood pressure.
P240
Signal Transduction of the Renin/ Prorenin Receptor
Jan H Schefe, Mario Menk, Jana Reinemund, Karin Effertz, Patricia Ruiz, Thomas Unger,
Heiko Funke-Kaiser; Charite - Universitatsmedizin Berlin, Berlin, Germany
A human renin/ prorenin receptor (RER), which is involved in brain development and probably
cardiac and renal end-organ damage, has recently been cloned. Since little is known about the
molecular properties of the RER, we analysed its function and signal transduction cascade. We
found an ubiquitous and intracellular expression pattern of the human RER. Consistently, we
observed several transcriptional start sites and a high promoter activity of human RER. We
could identify the transcription factor PLZF (promyelocytic zinc finger protein) as direct protein
interaction partner of the RER by yeast two-hybrid screening and coimmunoprecipiation (coIP).
CoIP experiments also indicated homodimerization of the RER. Upon activation of the RER by
renin, PLZF is translocated into the nucleus, represses transcription of the RER itself, thereby
creating an extraordinary short negative feedback loop, but activates transcription of
PI3K-p85alpha . siRNA against the RER abolished these effects. A PLZF cis-element in the RER
promoter was identified by site-directed mutagenesis and EMSA. In addition, renin stimulation
CHBPR ConferencePoster Presentations e91
caused a 6-fold recuitment of PLZF to this promoter region as shown by chromatin-
immunoprecipitation (ChIP). Our data demonstrate the existence of a novel signal transduction
pathway involving the ligand renin, RER and the transcription factor PLZF.
P241
Cox-1 Isoenzyme is Involved with Angiotensin II-Induced Endothelial
Dysfunction in Murine Mesenteric Small Arteries
Agostino Virdis, Rocchina Colucci, Matteo Fornai, Corrado Blandizzi, Stefano Taddei, Mario
Del Tacca, Antonio Salvetti; Univ of Pisa, PISA, Italy
Angiotensin II (Ang II) induces endothelial dysfunction in small arteries by reducing nitric oxide
(NO) availability and increasing oxidative stress. We assessed whether cyclooxygenase (COX)-1
and COX-2 participate in the Ang II-induced endothelial dysfunction in murine mesenteric small
arteries. Two groups of DBA/1N male mice (n8, age: 12 weeks) were treated with Ang II (600
ng/kg/min, s.c.) or physiological solution (controls). After 2 weeks, endothelial function of
mesenteric small arteries (by pressurized myograph) was assessed by acetylcholine (ACh,
0.001100 M)-induced relaxation. NO availability and ROS production were evaluated by
repeating Ach under L-NMMA (100M) and ascorbic acid (Vit C, 10mM), respectively. Ach was
tested also in the presence of SC-560 (1 M; COX-1 inhibitor), DFU (1 M; COX-2 inhibitor)
and SQ 29548 (1 M; TP-receptor antagonist). The expression of mRNA encoding COX
isoforms in mesenteric vessels was analysed by RT-PCR. Ang II increased systolic blood
pressure (144.94.7 vs 94.03.6 mmHg; p0.01). In controls, maximal relaxation to ACh
(96.61.1%) was blunted by L-NMMA (62.91.8%; inhibitory effect: 33.62.5%; p0.001),
and unmodified by SC-560 (96.80.7%), DFU (95.41.4%) or Vit C (96.01.9%). In Ang
II-treated mice, relaxation to ACh was blunted (58.91.1%; p0.001 vs controls), less
sensitive to L-NMMA (48.11.2%; 10.80.6% inhibition; p0.001 vs controls), not affected
by DFU (65.72.9%), but potentiated, although not normalized, either by SC-560 (82.61.4%;
p0.001) or SQ 29548 (81.01.0%; p0.001) and, similarly, by their combination
(78.41.3%; p0.001). In the presence of SC-560, inhibition of L-NMMA on Ach was
enhanced (63.70.8%; 18.72.7% inhibition; p0.01 vs L-NMMA Ach alone). Response
to ACh was normalized by Vit C (97.72.9%). Vasodilation to sodium nitroprusside was similar
between groups. RT-PCR analysis showed that Ang II reduced COX-2, while it enhanced COX-1
expression. In small mesenteric vessels, the reduced NO availability and oxidant excess
secondary to Ang II administration is associated to an enhanced expression and activity of
COX-1 isoform, most likely producing contracting endoperoxides, while COX-2 seems not to be
involved.
P242
Withdrawn at Authors Request
P243
AT1a Versus AT1b Receptor-Mediated Intrarenal Accumulation of Angiotensin
II In AT1a Receptor-Deficient Mice
Jia L Zhuo, Yuan Shao, Xiao C Li; Henry Ford Hosp, Detroit, MI
We have previously shown that the rat kidney accumulates exogenous angiotensin II (Ang II)
during Ang II-induced hypertension via an AT1 receptor-mediated mechanism. However, we do
not know which isoform of the AT1 receptor mediates renal Ang II accumulation, nor whether
the AT1b receptor will assume the role of the AT1 receptor when AT1a is absent. We tested the
hypothesis that mice lacking the AT1a receptor are unable to accumulate Ang II in the kidney
due to the absence of AT1a receptor-mediated endocytosis. Adult male wild-type (Agtr1A/)
and AT1a receptor-deficient (Agtr1A-/-) mice (n 79 each group) were treated with vehicle,
Ang II (40 ng/min, i.p. via osmotic minipump), or Ang II plus the AT1 receptor antagonist losartan
(10 mg/kg/day, p.o.) for 2 weeks. In wild-type mice, Ang II induced systemic hypertension
(control: 114 2 mmHg vs. 168 4 mmHg, p0.001), increased kidney-to-body weight ratio
(control: 1.18 0.03 vs. 1.29 0.03, p0.01), 24 hour urine output (control:1.16 0.05
vs. Ang II: 2.73 0.15 ml, p0.01), urinary sodium excretion (control: 179.8 15.8 vs.
249.6 9.9 mol, p0.05), and kidney Ang II levels (control: 218 8 16.2 vs. 1098.5
43.1 pg/100 mg, p0.001). Concurrent administration of Ang II with losartan significantly
attenuated these responses to Ang II in wild-type mice. Agtr1A-/- mice had lower basal systolic
pressure (p0.001), smaller kidney (p0.001), higher 24 hour urine and urinary sodium
excretion (p0.01) and basal kidney Ang II levels (p0.01). Ang II increased systolic pressure
to a much lesser extent than in wide-type (control: 90 2.4 vs. Ang II: 101 3.5 mmHg,
p0.05), but had no effect on kidney-to-body weight ratio, 24 hour urine and urinary sodium
excretion, as well as kidney Ang II levels. Interestingly, concurrent administration of losartan
with Ang II restored systolic pressure to basal levels and decreased kidney Ang II levels by 30%
in Agtr1A-/- mice compared with Ang II alone (p0.05). These results not only demonstrate a
predominant role of AT1a receptors in overall renal responses to long-term Ang II administration,
but also suggest that AT1b receptors may assume a minor role in blood pressure control and
renal accumulation of Ang II in the absence of AT1a receptors.
 e92 Hypertension Vol 48, No 4 October 2006
P244
Withdrawn at Authors Request
P245
Epidermal Growth Factor Receptor and Extracellular-Regulated Kinase 1/2
are Necessary for Angiotensin II-Mediated Restoration of Vascular
Relaxation in Middle Cerebral Arteries of Sprague-Dawley Rats on High
Salt Diet
Scott T McEwen, Julian H Lombard; Med College of Wisconsin, Milwaukee, WI
Angiotensin II (ANG II) suppression with high salt (HS) diet leads to impaired vascular relaxation
that can be restored by continuous i.v. infusion of a subpressor dose of ANG II acting at the AT1
receptor. The present study identified two protein kinases that are downstream from the AT1
receptor and are required for this response. Male Sprague-Dawley rats were fed a HS diet (4%
NaCl) before receiving one of the following i.v. infusions for 3 days: (1) ANG II (5 ng/kg/min);
(2) Epidermal Growth Factor (EGF; 2 g/hr); (3) EGF AG1478 (EGF receptor tyrosine kinase
inhibitor; 20 g/hr); or (4) ANG II AG1478. Middle cerebral arteries (MCA) were isolated and
cannulated, and responses to acetylcholine (ACh), reduced PO2 (35 45 mmHg), and iloprost
were measured by television microscopy. Vasodilation in response to these stimuli was lost in
rats on HS diet, but was restored by either ANG II or EGF infusion. Co-infusion of AG1478
prevented the restoration of vasodilator responses by ANG II or EGF. HS rats were also
co-infused with ANG II and one of the following inhibitors for 3 days: (1) cycloheximide (protein
synthesis inhibitor; 5 g/hr); (2) wortmannin (PI3K inhibitor; 2 g/hr); (3) PD98059 (ERK 1/2
pathway inhibitor; 10 g/hr); or (4) SB203580 (p38 inhibitor; 10 g/hr). The restored relaxation
to these vasodilator stimuli was eliminated when ANG II was co-infused with cycloheximide or
PD98059. Wortmannin attenuated ACh-induced dilation, but had no effect on hypoxia- or
iloprost-induced dilation. SB203580 did not affect ANGII-induced restoration of vasodilator
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responses. These results indicate that both the EGF receptor tyrosine kinase and ERK 1/2 are
necessary for the protective effect of ANGII to restore vascular relaxation in MCA of rats on HS
diet.
P246
Angiotensin II-Independent Extracellular-Signal-Related Protein Kinases
Activation by Prorenin in Human Vascular Smooth Muscle Cells
Mariyo Sakoda, Atsuhiro Ichihara, Yuki Kaneshiro, Tomoko Takemitsu, Matsuhiko Hayashi,
Keio Univ Sch of Medicine, Tokyo, Japan; Fumiaki Suzuki, Gifu Univ, Gifu, Japan; Tadashi
Inagami; Vanderbilt Univ Sch of Medicine, Nashville, TN
A recent study demonstrated that renin increases transforming growth factor-1 and matrix
proteins through prorenin/renin receptors in rat mesangial cells. The present study was
designed to determine the effects of prorenin on extracellular-signal-related protein kinases
(ERK) in human vascular smooth muscle cells. Total and phosphorylated ERK in human vascular
smooth muscle cells were determined by Western blot analyses. Recombinant human prorenin
increased phosphorylated ERK in dose- and time-dependent manners but did not change total
ERK. Maximum increases in phosphorylated ERK were obtained by a 20 min-incubation with
2 nmol/L prorenin. This increase in phosphorylated ERK was significantly greater than that
obtained by 1 mol/L angiotensin II and was not altered by adding 10 mol/L of the
angiotensin-converting enzyme inhibitor imidapril or the angiotensin receptor antagonist
losartan (n 6 experiments). However, 100 mol/L of the handle region decoy peptide, concentrations (130, 140, and 150 mmol/L) supplemented with 10% FBS for 48 hrs. The
which inhibits the binding of prorenin to prorenin/renin receptor, significantly reduced the ERK
activation by prorenin (n 6 experiments). In addition, blockade of the human vascular smooth
muscle cell prorenin/renin receptor by siRNA significantly decreased the ERK activation by
prorenin (n 3 experiments). These results suggest that prorenin stimulates ERK phosphor-
ylation through receptor-mediated, angiotensin II-independent mechanisms in human vascular
smooth muscle cells.
P247
Vitamin E Effects on Leukocyte-Endothelial Cells Interactions in Doca-Salt
Hypertensive Rats
Fernando S Carneiro, Univ of Sao Paulo, Sao Paulo, Brazil; Glaucia E Callera, Univ of
Ottawa, Ottawa, Canada; Heraldo P Souza, Stephen F Rodrigues, Augusto C Montezano,
Dorothy Nigro, Zuleica B Fortes, Maria H Carvalho, Rita C Tostes; Univ of Sao Paulo, Sao
Paulo, Brazil
Objectives: Increased vascular oxidative stress, decreased acetylcholine (ACh) vasodilation and
altered leukocyte-endothelial cell interactions in DOCA rats are associated with activation of the
endothelin system via ETA receptors. Since reactive oxigen species (ROS) play a key role in
endothelial dysfunction and leukocyte behavior, we hypothesized that vitamin E (VE) amelio-
rates impaired endothelium-dependent relaxation and leukocyte-endothelial cells interactions
in DOCA hypertension. Methods and results: DOCA and uninephrectomized (UniNX) control
rats were treated with VE (200 mg/Kg/day) or vehicle during five weeks. VE treatment
normalized the increased superoxide anion generation, evaluated by lucigenin, as well as the
impaired ACh relaxation in DOCA aortae (% relaxation, DOCA: 72.36.0 vs UniNX: 99.23.5,
DOCA VE: 91.44.8). Intravital microscopy analysis, used to estimate the number of rolling and
adherent leukocytes (number of cells in 100m of vessel/during 10 min.) in the venules of the
internal spermatic fascia, showed that VE normalized the decreased rolling (DOCA: 9917 vs
UniNX: 2106, DOCA VE: 18416) and attenuated the increased adhesion (DOCA: 15.82.6 endothelial function through its receptor. In this study we aimed to test the vasculo-protective
vs UniNX: 4.01.0, DOCA VE: 8.81.4) in DOCA rats. Flow citometry, used to evaluate cellular
adhesion molecules (CAMs) expression, showed decreased L-selectin (arbitrary units, UniNX:
22.61.9 vs DOCA: 15,72.6) and increased CD18 (UniNX: 22.82.06 vs DOCA: 45.411.2) type (WT, N6) mice of the same age were used for the experiments. Arterial blood pressure
protein expression in DOCA leukocytes. VE normalized CD18 (DOCA VE: 23.62.3), but not
L-selectin expression (DOCA VE: 11.10.9). VE also normalized increased eNOS and ICAM-1
gene expression in mesenteric vessels from DOCA rats (p0,05). Conclusion: ROS play a
direct role on the impaired vascular reactivity and leukocyte behavior in DOCA hypertension.
ROS effects may be mediated by decreased NO bioavailability and by changes in CAMs
expression.
P248
High Salt Diet Causes Hypertension and Impairs Renal VEGF Signaling
System in Dahl Salt-Sensitive (SS) Rats
Jian-Wei Gu, Wei Tan, Megan Shparago, Amelia P Bailey; Univ of Mississippi Med Cntr,
Jackson, MS
The molecular mechanisms of salt-sensitive hypertension are not clear. Recent clinical
evidence links inhibition of vascular endothelial growth factor (VEGF) to hypertension. However,
there are no animal studies available for examining the existence of impaired renal VEGF
signaling in salt-sensitive animal models. We used Dahl SS rats to determine whether a high
salt diet impairs the renal VEGF signaling system related to development of salt-sensitive
hypertension. Four, 8-wk-old male Dahl SS rats received a high sodium diet (HS, 4%) and 4
Dahl SS rats received a low sodium diet (LS, 0.3 %) for 5 weeks. In the end, mean arterial
pressure (MAP) was determined in conscious rats by continuous monitoring through a catheter
placed in the carotid artery. MAP was significantly higher in the HS than the LS group (177.9
3.7 vs. 109.4 2.9 mmHg, P0.001). There was a significant increase in urinary albumin
secretion in the HS group, compared to the LS group (22.3 2.6 vs. 6.1 0.7 mg/d;
P0.001). Total tissue protein was extracted from the whole kidneys. ELISA (R&D Systems)
demonstrated that renal VEGF protein levels significantly decreased in HS group, compared to
LS group (41.9 3.5 vs. 58.0 6.2 pg/mg total protein; P0.01). Interestingly, total urinary
excretion of sFlt-1, an endogenous VEGF inhibitor, was significantly higher in HS than LS group
(3182 330 vs. 636 99 pg/24 h urine; P0.01). There were no significant changes in
plasma levels of VEGF and sFlt-1 (an endogenous VEGF inhibitor) between HS and LS groups.
These findings suggest that a high salt diet impairs the renal VEGF signaling system related to
the development of hypertension in Dahl SS rats. The study also demonstrated a unique animal
model for studying the role of the VEGF signaling system in renal mechanisms of salt-sensitive
hypertension. (NIH/P01 HL51971 & NIH/R21 AA013821)
P249
Increased Physiologically Relevant Sodium Concentrations Stimulate NF B
Activation and Na, K-ATPase Activity in Cultured Human Umbilical Vein
Endothelial Cells
Jian-Wei Gu, Megan Shparago, Wei Tan, Amelia P Bailey; Univ of Mississippi Med Cntr,
Jackson, MS
Chronic high salt intake increases cardiovascular morbidity and mortality both by its influences
on blood pressure and by pressure-independent effects on the target organs. The vascular
endothelium plays a key role in the short- and long-term regulation of the cardiovascular
system and is the source of many factors that influence blood flow as well as blood pressure.
More recent studies suggest that renal NF B activation is related to oxidative stress in
salt-sensitive hypertensive animals. The present study determines whether increased physio-
logically relevant sodium concentrations directly stimulate NF B activation and Na,
K-ATPase activity (a parameter of substrate oxidation rate) in cultured human umbilical vein
endothelial cells (HUVEC). HUVEC were cultured in M199 media having different sodium
different sodium concentrations in the media were adjusted by a fixed sodium bicarbonate
content and additional sodium chloride. At the end of the experiment, total cellular protein and
nuclear protein were extracted. The binding activity of NF B p65 proteins was determined by
electrophoretic mobility shift assay (EMSA). Na, K-ATPase activity was determined by
ouabain (0.5 mmol/L)-sensitive Rb86 (as a tracer for K) uptake (cpm/min/mg total cell protein),
and the relative activity was expressed as % change over the control. 140 and 150 mmol/L of
sodium in the media caused a 23% (P0.05) and 45% (P0.01) increase in the binding
activity of NF B p65 proteins of cultured HUVEC, compared to the low sodium group (130
mmol/L), respectively. Increased physiologically relevant sodium concentrations stimulate Na,
K-ATPase activity in a dose-dependent manner in HUVEC (P0.01). The relative Na,
K-ATPase activities were 67.7%, 100%, and 155% in HUVEC exposed to sodium concentra-
tions of 130, 140, and 150 mmol/L, respectively. These findings support the concept that
increased physiologically relevant sodium concentrations can have a direct cellular effect on
NF B activation in the vascular endothelium, which may be related to increased substrate
oxidation rate due to increased Na, K-ATPase activity. (NIH/HL51971 & NIH/AA013821)
P250
Genetic Deletion of the Angiotensin-(17) Receptor Mas Increases Blood
Pressure and Impairs in vivo Endothelial Function in Fvb/n Mice
Mihail Todiras, Max-Delbruck Cntr for Molecular Medicine, Berlin, Germany; Marina M
Moura, Andrea S Haibara, Federal Univ of Minas Gerais, Belo Horizonte, Brazil; Ping Xu,
Michael Bader, Natalia Alenina, Max-Delbruck Cntr for Molecular Medicine, Berlin, Germany;
Robson A Santos; Federal Univ of Minas Gerais, Belo Horizonte, Brazil
Angiotensin-(17) [Ang-(17)] is reported to produce vasodilation in many vascular beds. We
have recently shown that Mas-deficient mice presented a blunted vasorelaxation of aortic rings
in response to Ang-(17). These observations suggested that Ang-(17) may modulate
role of Mas by evaluating the effect of its genetic deletion on the in vivo endothelial function.
Three month old Mas-knockout mice on FVB/N background (Mas-KO, N7) and FVB/N wild
was recorded by a catheter placed into abdominal aorta through the femoral artery. After an
initial period (12 hours) of blood pressure recording the animals were anesthetized
(ketamine/xylazine) and a catheter was introduced into the descending aorta through the

carotid artery. Endothelial function was evaluated by bolus intra-aortic administration of
acetylcholine. Strikingly and in contrast to previous reports in Mas-deficient mice on C57BL/6J
background, mean arterial pressure of non-anesthetized FVB/N Mas-KO mice was significantly
elevated (122 1.9 mmHg vs 109 1.6 mmHg in age-matched WT mice, p0.05). In
addition the response to acetylcholine was markedly decreased in Mas-KO mice (200 ng/Kg:
-8.2 1.5 mmHg vs.-15.2 2.1 mmHg in WT). These results suggest that the Ang-(17)-Mas
axis has a previously unsuspected role in blood pressure control which may be related to an
important vasculo-protective effect through modulation of endothelial function.
P251
A Novel Regulatory Mechanism of Nox 4 Expression in Endothelial Cells:
Role of MicroRNA-210
Guo Zhang, Pin-Lan Li; Med College of Virginia, Virginia Commonwealth Univ, Richmond, VA
MicroRNAs have been now emerging as one of the important mechanisms regulating gene
expression in a variety of mammalian cells. The present study was designed to test the
hypothesis that microRNAs importantly regulate the expression and function of Nox 4, a major
catalytic subunit of NAD(P)H oxidase in human coronary arterial endothelial cells (HCAECs). Two
human cardiovascular cell specific microRNAs, microRNA-210 and microRNA-25 were tested,
which are capable of binding to Nox 4 gene sequence. Using stem-loop real-time quantification
RT-PCR, we found that microRNA-210 was enriched in HCAECs, while the abundance of
microRNA-25 was very low. When HCAECs were incubated with angiotensin II (100 nM) and
homocysteine (150 nM), two well-known NAD(P)H oxidase inducers, microRNA-210 levels in
these cells increased by 67% and 79%, accompanied by an 80% or 3-flod increases in Nox
4 mRNA levels, respectively. There were no changes in microRNA-25 detected under these
conditions. Antisense oligonucleotide (40 nM) of microRNA-210 was found to induce a dramatic
decrease in microRNA-210 levels, but increase in Nox 4 mRNA, while sense oligonucleotide (40
nM) had no effect. These results indicate that microRNA-210 in HCAECs is an endogenous
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
inhibitor of Nox 4 gene expression in HCAECs and that this microRNA may be responsible for
the response of Nox 4 expression to different stimuli. (Supported by NIH Grants HL57244,
HL70726, and HL075316).
P252
Hyperhomocysteinemia Stimulates Endogenous Carbon Monoxide
Production to Promote Endothelial Dysfunction in Rats
William Durante, Univ of Missouri, Columbia, MO; Fruzsina K Johnson, Tulane Univ, New
Orleans, LA; Kelly J Peyton, Kannan Sankaranarayanan, Univ of Missouri, Columbia, MO;
Robert A Johnson; Tulane Univ, New Orleans, LA
Heme oxygenase-1 (HO-1) catalyzes the degradation of heme to biliverdin, iron and carbon
monoxide (CO). Recent studies from our laboratory indicate that vascular HO-1-derived CO
attenuates flow-induced dilation and contributes to endothelial dysfunction in salt-
sensitive hypertension and diabetes by inhibiting nitric oxide synthesis. Hyperhomocys-
teinemia (HHcy) is a well-established risk factor for cardiovascular disease that promotes
endothelial dysfunction; however, the mechanism underlying this impairment is not fully
known. Since we recently reported that homocysteine directly stimulates HO-1 expression
in vascular cells, the current study tests the hypothesis that HO-1-derived CO formation is
increased and contributes to endothelial dysfunction in an experimental model of HHcy.
Male Sprague-Dawley rats received L-methionine (1g/kg/day for 4 weeks) in drinking
water to induce mild HHcy. Respiratory CO excretion was measured weekly via solid-phase
gas chromatography. Endothelial function was examined in isolated Krebs buffer
superfused first-order gracilis muscle arterioles with constant midpoint (80mmHg), but
altered endpoint pressures to establish graded levels of intraluminal flow (0 50L/min).
Respiratory CO excretion (max0.170.03 vs 0.010.02mol/hr per kg) and mean
arterial pressure (1509 vs 1228mmHg) were significantly increased in L-methionine-
treated animals. Stepwise increases in flow resulted in significant dilation
(max121m) in arterioles from control animals, whereas increases in flow constricted
(max-103m) arterioles from L-methionine-treated rats. Acute in vitro pretreatment
with a HO inhibitor, chromium mesoporphyrin (15mol/L), restored flow-induced dilation
in L-methionine-treated rats and abolished the difference between the two groups of
animals (max 91 vs 101m). In conclusion, this study demonstrates that CO excretion
and blood pressure are increased, and flow-dependent dilation is converted to constriction
in rats with HHcy. In addition, it shows that a HO inhibitor fully restores endothelium-
dependent dilation in HHcy. These results suggest that HHcy may promote hypertension
and arteriolar endothelial dysfunction by enhancing endogenous CO production.
P253
Heme Oxygenase Inhibition Causes Renal Vasoconstriction in Nitric Oxide-
Deficient Diabetic Kidney
Francisca Rodriguez, Miguel G Salom, Barbara Bonacasa, Santiago Cuevas, Francisco J
Fenoy; Univ of Murcia, Murcia, Spain
Renal vascular and tubular structures contain heme oxygenase (HO) proteins which convert
heme to biliverdin, free iron and carbon monoxide (CO). Nitric Oxide (NO) synthesis inhibitors
increase renal CO production, sustain CO induced vascular relaxation and intensify the
vasoconstriction induced by HO inhibitors. All this data suggest that the renal vasodilatory
mechanisms mediated by HO are particularly facilitated when NO levels are previously
disminished. Therefore, the renal vascular responses associated to HO inhibition could become
relevant in conditions associated with impaired endogenous NO bioavailability, such us
Streptozotocin (STZ) induced type-1 diabetes mellitus. To test this hypothesis we studied in
anesthetized Sprague Dawley rats the effects of Stannous Mesoporphyrin (SnMP 40 umol/kg,
iv), a HO inhibitor, on renal hemodynamic after two weeks of STZ (65 mg/Kg iv) or vehicle
injection. In additional experiments, renal cortical NO levels were determined in vivo, by using
double pulse differential voltammetry a highly specific and selective technique in diabetic
CHBPR ConferencePoster Presentations e93
(n5) or vehicle treated rats (n3). Compared to vehicle (n7), diabetic animals (n5)
showed comparable BP (114 4 vs 118 3, mmHg ) (P 0.05), and renal blood flow (RBF)
values (7.5 2 vs. 8.9 1 ml/min/g) (P 0.05), but higher blood glucose (420 49 vs 88
4.4 mg/dL), kidney weight /body weight (BW) ratio (1.00 0.03 vs. 0.65 0.03, %) and
glomerular filtration rate (GFR) (10558 435 vs 3634 575 uL/min/g BW ), (P0.05 all
instances). The administration of the HO inhibitor did not modify RBF (8.7 1 ml/min/g) or GFR
(3535 354 L/min/g BW) in vehicle, but greatly reduced GFR (5948 147L/min/g BW )
and RBF values (5.8 2 ml/min/g) in STZ hyperglycemic rats (p0.05). Importantly, STZ
infused animals exhibited a significant deficit in NO cortical levels (183 117 nmol/L)
compared to vehicle treated rats (479 113 nmol/L). We conclude that HO activity contributes
to the maintenance of renal hemodynamic in the diabetic NO deficient kidney. These data are
consistent with a HO derived product being involved in mechanisms mediating glomerular
hyperfiltration in diabetes.
P254
Gene Ablation Bach-1 Leads to Suppression of Atherosclerosis in
Apolipoprotein-E and Bach1 Double Knockout Mice
Yuichiro Watari, Ryoji Ozono, Yoshiyuki Yamamoto, Yoko Yano, Andorei Brydun, Tetsuya
Oshima, Kazuaki Chayama, Hiroshima Univ Graduate Sch of BioMed Sciences, Hiroshima,
Japan; Kazuhiko Igarashi; Tohoku Univ Graduate Sch of Medicine, Sendai, Japan
Bach1 is a novel transcriptional factor governing cytoprotective programs including heme
oxygenase-1 (HO-1). Mice lacking Bach-1 gene shows increased activity of HO-1 in
cardiovascular system, increased phagocytic activity of macrophage, increased expression
of macrophage chemotactic factor-1, suppression of vascular smooth muscle proliferation,
and suppression of neointimal formation after in vivo vascular injury. However, the overall
role of Bach1 in the atherosclerosis is poorly understood. In the present study, we
investigated the effect of Bach1 ablation on the development of atherosclerosis by
comparing ApoE-kockout (KO) mice and ApoE/Bach1 double KO mice. The homozygous
ApoE/Bach1 double KO (ApoE-/- and Bach1-/-) mice were generated by intercrossing
ApoE-KO and Bach1-KO mice, both have been back-crossed onto C57BL/6J background
for at least 12 generations. There was no difference in the birth rate, fertility, growth rate
between of Apo-E/Bach1 double-KO and ApoE-KO mice. After mice were fed high
cholesterol diet for 8 weeks, there was no significant difference in body weight, blood
pressure, and serum levels of total cholesterol between the double-KO and ApoE-KO mice.
The Western blot analysis revealed that the HO-1 protein expression in the aorta, was
markedly increased in double-KO mice than in ApoE-KO mice. The atherosclerotic plaque
formation in the thoracic and abdominal aorta, visualized by oil-red-O staining, was
reduced by 38% in Apo-E/Bach1 KO mice compared with Apo-E KO mice (P0.01). These
results suggest that atherosclerosis was inhibited in ApoE/Bach1 double KO. Bach1
appears to negatively control the activity of antiatherosclerotic defense mechanism
including HO-1 system, limiting the maximum survival level against stress. Inhibition of
Bach1, conversely, may be a novel therapeutic strategy to treat atherosclerotic diseases.
P255
Increased Sensitivity to the NO-Independent Soluble Guanylyl Cyclase
Stimulator BAY 412272 in Aorta from eNOS -/- Mice
Cleber E Teixeira, Fernanda B Priviero, Saiprasad M Zemse, Rob H Hilgers, R. Clinton Webb;
Med College of Georgia, Augusta, GA
OBJECTIVE: In this work, we investigated the mechanisms by which endogenous nitric
oxide (NO) affects the vasorelaxation elicited by the nonNO-based soluble guanylyl cyclase
(sGC) stimulator BAY 412272 in wild-type (WT) mice, as well as endothelium and
neuronal NO synthase knockout mice (eNOS -/- and nNOS -/-, respectively). METHODS:
Endothelium-intact (E) and denuded (E-) rings were mounted in myographs and data
were recorded in a PowerLab system. Cyclic GMP was measured using EIA kits. Real-time
PCR and western blot were used to assess gene and protein expression of sGC subunits.
RESULTS: In E rings, BAY 412272 (0.0011 M) induced relaxations in a
concentration-dependent manner, with pEC50 values of 7.53 0.03 (WT), 7.76 0.06
(eNOS -/-) and 7.56 0.04 (nNOS -/-). In E- rings, the curve for BAY 412272 was shifted
to the right by approximately 3-fold in WT (7.08 0.05) and nNOS -/- (7.15 0.03).
However, BAY 412272 relaxation in E- rings from eNOS -/- was similar to E (7.71
0.06). The sGC inhibitor ODQ (10 M) markedly displaced the curve for BAY 412272 to
the right in both E and E- rings of WT (30- and 10-fold), eNOS -/- (80- and 20-fold) and
nNOS -/- (30- and 6-fold). The NO synthesis inhibitor L-NAME (100 M) caused significant
inhibition in the relaxations evoked by BAY 412272 in rings from WT and nNOS -/-, but
not eNOS -/-. Incubation of E- rings with BAY 412272 (0.0011 M) or sodium
nitroprusside (SNP, 0.0011 M) caused significant rightward shifts in the contractile
responses to phenylephrine (PE, 0.00110 M). Co-incubation of BAY 412272 and SNP
caused a synergistic rightward shift in the curves to PE (5.6-fold; p0.01) along with
marked increase in cGMP levels in an ODQ-sensitive manner. Increase in cGMP in
response to BAY 412272 was significantly higher (p0.01) in eNOS -/- aorta, compared
to WT and nNOS -/-. Real-time PCR and Western blot analysis revealed a decreased
expression of both subunits of sGC at the mRNA and protein level, respectively.
CONCLUSION: BAY 412272 potently relaxes the mouse aorta in a synergistic fashion with
NO. Despite the decreased expression of sGC, this study demonstrates that higher enzyme
activity accounts for the increased sensitivity to NO-independent sGC stimulators in eNOS
-/- aorta. Support: HL-74167
 e94 Hypertension Vol 48, No 4 October 2006
P256
-Secretase is New Therapeutic Target for Anti-angiogenic Therapy
Hiroki Hayashi, Hironori Nakagami, Yukihiro Saito, Yoichi Takami, Tomoyuki Nishikawa,
Naoyuki Sato, Ryuichi Morishita, Yasufumi Kaneda; Osaka Univ, Suita, Japan
It has been well-known that -secretase cleavaged the transmembrane domains of several
integral membrane proteins (i.e. amyloid precursor protein, Notch, deleted in colorectal cancer).
The involvement of -secretase in Notch signaling during vasculogenesis is well-documented
but poorly understood in adult angiogenesis. Thus, we examined the effect of potent
-secretase inhibitor (GSI), L-685,458, in endothelial cells (ECs) and smooth muscle cells
(SMCs). We examined the expression of Notch receptors (Notch1/2/3/4), ligands (Delta-like1/
4(Dll1/4), jagged1), and downstream targets of Notch (cardiovascular-related bHLH transcrip-
tion factor1/2, hairy/enhancer split gene (Hes) 1/ 5) in ECs and SMCs by reverse transcription-
PCR (RT-PCR). We also confirmed that the inhibitory effect of GSI in ECs by the down-regulation
of Dll4 and Hes-1 (82% and 98% inhibition, respectively) after the treatment of growth factors
(human recombinant vascular endothelial growth factor (hrVEGF) 25ng/ml, fibroblast growth
factor-2 (hrFGF-2) 25ng/ml, epidermal growth factor (hrEGF) 25ng/ml), P0.0005, respec-
tively) by realtime quantitative RT-PCR (qRT-PCR). The treatment of GSI (1 - 10 M)
significantly decreased ECs proliferation by MTS assay and migration by modified Boyden
chamber method in response to growth factors in a dose dependent manner. Notch signaling
during vascular development is postulated as the downstream of VEGF. However, we also found
that the treatment of GSI (110 M) down-regulated mRNA expression of VEGF and FGF-2 in
SMCs by realtime qRT-PCR (76% and 87% inhibition, P0.05, respectively), accompanied with
the suppression of VEGF promoter activity (66% inhibition, P0.005). The treatment with not
only GSI (10M) but also another -secretase inhibitor DAPT (10M) attenuated hrVEGF
induced-tube formation of ECs (P0.05). Moreover, we observed that the addition of GSI into
matrigel decreased hrFGF-2 induced-migration of ECs in mice (hrFGF-2 : 163.77.0,
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
hrFGF-2GSI 20M : 13612.0, P0.05) quantified by FITC-Lectin stain of ECs. These
results suggest that GSI would have anti-growth effect on ECs and down-regulated angiogenic
growth factors. Therefore, -secretase can be a new therapeutic target for anti-angiogenic
therapy.
P257
Cross-Talk between Vascular Endothelial Cells and Smooth Muscle Cells
Negatively Modulates NADPH Oxidase-Derived Reactive Oxygen Species
Shaoping Xu, Rhian M Touyz; Univ of Ottawa, Ottawa, Canada
Communication between the endothelium and vascular smooth muscle is fundamental for the
maintenance of vascular function and structure. In vivo, endothelial cells (EC) and vascular
smooth muscle cells (VSMC) act as a coupled system for signalling between cell types. In vitro
studies usually involve single cell cultures, which exclude biologically important interactions
between different cell types. Here we tested the hypothesis that EC influence VSMC generation
of reactive oxygen species (ROS) by modulating expression/activation of VSMC NADPH oxidase
and MAP kinases. Modulatory effects of VSMC on EC function were also tested. Human VSMC,
isolated from resistance arteries, were co-cultured with human arterial EC for 4 days. EC and
VSMC monocultures were also examined. Intracellular superoxide (O2-), hydrogen peroxide
(H2O2) and nitric oxide (NO) were measured by FACScan and specific fluorescent dyes, DHE,
DCFDA and DAF respectively. Compared to monoculture, VSMC co-cultured with EC exhibited
decreased O2- (201.5 vs 323 arbitrary fluorescence units (AFU)) and H2O2 (350.5 vs
7919 AFU) levels, whereas generation of NO in EC co-cultured with VSMC was significantly
increased (266 vs 221 AFU, p0.05). Generation of H2O2 in ECs co-cultured with VSMCs
was significantly lower than in EC monoculture (787 vs 11127, p0.05). Expression of
VSMC NADPH oxidase subunits Nox4 and gp91phox, determined by immunoblotting, was
markedly reduced (2-fold) by EC co-culture compared with monoculture. Furthermore, EC
differentially regulated MAP kinase phosphorylation by increasing ERK1/2 (2-fold) and
decreasing p38 MAPK (1.5-fold) in VSMC. Our data demonstrate that EC negatively influence
ROS formation in VSMC and vice versa. Under physiological conditions such processes may
prevent against vascular oxidative damage by blunting O2- and H2O2 production and by
increasing NO formation. These findings provide novel insights into how interactions between
EC and SMC influence redox-dependent processes in human vascular biology.

Late Breaking Presentations
LB1
Increased Expression of AT1 Receptor and Angiotensinogen mRNA and Ang
II in Human Placental Chorionic Villi of Preeclamptic Pregnant Subjects
Lauren Anton, David C Merrill, Liomar A Neves, Patricia E Gallagher, Cheryl Moorefield,
Courtney Gruver, Carlos M Ferrario, K. B Brosnihan; Wake Forest Univ Sch of Medicine,
Winston-Salem, NC
The chorionic villi (CV) are an essential component of the placenta involved in maternal-fetal
oxygen and nutrient transport. Although the renin angiotensin system (RAS) is activated during
normal pregnancy, its presence and regulation in the CV of normal and preeclamptic placentas
have not yet been examined. These studies assessed the regulation of the placental CV RAS
during normal and preeclamptic pregnancies. Placental chorionic villous tissue was collected
from nulliparous third-trimester normotensive pregnant subjects (n25) or preeclamptic
subjects (n21). Preeclamptic subjects had no previous history of chronic hypertension or
renal, connective-tissue, or metabolic disease, but at the time of delivery had significant
hypertension (1733 mmHg systolic, 1052 diastolic mmHg) and 1 proteinuria. The
tissue was analyzed by radioimmunoassays (RIA) for angiotensin peptides or by reverse
transcriptase, real-time polymerase chain reaction for angiotensinogen (Aogen), renin, ACE,
ACE2, and the AT1 and the AT2 receptors. CV angiotensin II (Ang II) levels were significantly
increased in preeclamptic subjects (14.61.5 vs 34.38.3 fmol/mg protein, p0.05).
However, there was no change in Ang I between normal and preeclamptic CV (1.00.1 vs
0.930.1 fmol/mg protein). Aogen (1.020.1 vs 2.430.4 U, p0.005) and AT1 receptor
(1.00.1 vs 3.0 0.7 U, p0.01) mRNAs were significantly increased in preeclamptic CV
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
compared to normal. A trend was seen for an increase in renin mRNA (1.010.1 vs 1.780.8
U); however it did not reach statistical significance. No change was observed for ACE or ACE2
mRNA in normal versus preeclamptic CV. AT2 receptor mRNA was not detectable. These results
provide evidence for enhanced placental chorionic villous tissue expression of select RAS
components, namely Aogen and AT1 receptor mRNA, and Ang II levels, in preeclamptic CV.
These results indicate that the increased Ang II, resulting from increased Aogen, acting through
an up-regulated AT1 receptor may favor vasoconstriction in the chorionic villous tissue of the
placenta leading ultimately to the abnormal regulation of maternal-fetal blood flow and thus
development of preeclampsia.
LB2
17-beta Estradiol Reverses the Decline in Renal Function Associated with
Diabetic Nephropathy
Alexis Dixon, Christine Maric; Georgetown Univ Med Cntr, Washington, DC
Unlike most progressive diseases, in which the female gender is a protective factor, the
female advantage appears to be lost in diabetes; the incidence of diabetes and its
complications, such as diabetic nephropathy is equal or exceeds that in men. We have
previously shown that supplementation with 17-estradiol (E2) from the onset of diabetes
attenuates the decline in renal function associated with prolonged diabetes. The aim of this
study was to examine whether in addition to preventing diabetic nephropathy from developing,
E2 can also reverse the disease process once it has already developed. The study was
performed in Sprague-Dawley non-diabetic (ND), streptozotocin (STZ)-induced diabetic (D) and
STZ-induced diabeticE2 (DE2) rats (n6/group). In both diabetic groups, diabetes was
developed for 8 weeks before the E2 treatment started for additional 8 weeks. Following
treatment, D was associated with increased albumin extraction (UAE; ND, 0.50.1; D,
14.30.1), glomerulosclerosis (GSI; ND, 0.90.1; D, 1.90.3 AU), tubulointerstitial fibrosis
(TIFI, 0.40.1; D, 2.00.3 AU) and increased TGF-1 protein expression (ND, 0.10.09; D,
0.50.02 RLU). Supplementation with E2 attenuated these processes (UAE, 0.90.1 mg/day;
GSI, 1.10.2 AU; TIFI, 0.50.09; TGF-1, 0.30.1 RLU). We conclude that in addition to being
able to attenuate the development of diabetic nephropathy when administered from the onset
of diabetes, supplementation with E2 also reverses the decline in renal function and associated
with diabetic nephropathy. The results suggest that E2 has potential therapeutic benefits in the
prevention and treatment of diabetic renal and possibly other end-organ complications
associated with diabetes.
LB3
Counter-Regulation of ACE and ACE2 Gene Expression in the Kidney of
Normal Pregnant Rats
JaNae Joyner, Liomar Neves, Patricia Gallagher, David Merrill, Carlos Ferrario, K. Bridget
Brosnihan; Wake Forest Univ Sch of Medicine, Winston Salem, NC
Pivotal processing within the renin-angiotensin system occurs by ACE and ACE2. Understanding
their regulation may determine the predominate peptide of the system, i.e., either Ang II or
Ang-(17). In normal pregnancy we have previously demonstrated that kidney Ang I and
Ang-(17) are increased with no change in Ang II. In order to understand the regulation of the
Ang II forming enzyme, ACE, and the Ang-(17) forming enzyme, ACE2, during normal
pregnancy (5, 15, 19 days gestation), we examined gene expression of these enzymes in
kidney cortex and medulla by reverse transcriptase, real-time polymerase chain reaction.
Results were expressed as relative gene expression (ratio of target/18S rRNA control). During
normal pregnancy ACE mRNA decreased in the medulla at 5d (0.750.06 U, p0.01) as
compared to virgins (1.010.06 U) with further decreases at 15d (0.460.06 U, p0.001) and
19d (0.430.05 U, p0.001). In the cortex ACE mRNA decreased during early gestation
(0.630.05 U, p0.05) as compared to virgins (1.020.09 U) with no further change
throughout the remainder of pregnancy. ACE2 mRNA in the medulla increased at days 15
(1.710.23 U, p0.05) and 19 (1.700.14 U, p0.01) of pregnancy as compared to virgin
CHBPR ConferenceLate Breaking Presentations e95
(1.010.07 U) with no change in the cortex. In conclusion normal pregnancy induces a
counterbalancing downregulation of ACE and upregulation of ACE2 in the renal medulla, a
pattern that favors the production of Ang-(17) over Ang II as the predominate peptide. These
studies are consistent with estrogens influence over the regulation of ACE and ACE2 but other
hormonal influences found during pregnancy need to be considered.
LB4
Sex Differences in Subpressor Angiotensin II-induced Hypertension
Julio C Sartori-Valinotti, Wanda A Dorsett-Martin, Radu Iliescu, Licy L Yanes, Huimin Zhang,
Jane F Reckelhoff; Univ of Mississippi Med Cntr, Jackson, MS
Subpressor angiotensin (Ang) II is capable of producing hypertension when given chronically,
and there are sex differences in the pressor responses. However, most of the previous studies
have been conducted without blocking the endogenous renin angiotensin system (RAS). To
determine whether there are sex differences in Ang-II induced hypertension in the presence of
endogenous RAS blockade, male and female Sprague-Dawley rats, 12 weeks old, n3/group,
were assigned to receive either Ang II (150 ng/kg/min SC by osmotic minipump) or vehicle for
3 weeks. To block the endogenous RAS, enalapril was given in the drinking water (250 mg/L)
to all animals starting 4 days prior to the administration of Ang II until the end of the protocol.
To test the effect of salt intake on Ang II pressor response, rats were kept on regular diet (1%
NaCl) during the first week of Ang II infusion, and thereafter, were challenged to 4% NaCl diet
for the remaining 2 weeks. BP was continuously recorded by telemetry. Baseline BP
measurements were similar between males and females (1062 and 1023 mmHg,
respectively). Enalapril reduced BP to a lower level in control females than in males (774 vs.
901 mmHg, p 0.05). At the end of the first week of Ang II, the increment in BP was greater
in females than in males (358 vs. 251 mmHg, respectively); this difference persisted with
4% NaCl diet at the end of the 2nd week (4312 vs 376 mmHg, females vs males) and was
further exacerbated at the end of the 3rd week (496 vs. 325 mmHg, females vs males). Ang
II infusion caused a 35% increase in proteinuria in males and females on 1% NaCl diet.
However, females exhibited significantly more proteinuria during weeks 1 and 2 on high salt
diet than did males (wk1: increased 129% in females, 73% in males; wk2: increased 200% in
females and 115% in males). These data suggest that in females the RAS plays a greater role
in sustaining BP than in males, and when the RAS is blocked, females have a greater pressor
response to Ang II than males. Females also exhibit a higher level of salt sensitivity to Ang II
than males. The mechanisms responsible for these sex differences will require further study.
Supported by HL 51971, HL 66072 and HL 69194.
LB5
Effects of Estrogen on the Renal and Cardiovascular Actions of
Aldosterone in Ovariectomized Rats
Min Shi, Virginia Shalkey, Kathryn Sandberg, Carolyn A Ecelbarger, Darren M Roesch;
Georgetown Univ, Washington, DC
We have recently reported that male rats fed a standard 1% NaCl diet develop hypertension in
response to aldosterone infusion (200 g/day) while weight-matched females do not. The
distal convoluted tubule (DCT) of the nephron is an important site for aldosterone action and
the regulation of sodium balance and blood pressure. Sodium reabsorption in the DCT is
mediated by Na,K ATPase on the basolateral surface and the thiazide sensitive sodium-chloride
cotransporter (NCC) on the apical surface. We have found that female rats have higher basal
circulating levels of aldosterone (ALDO) and immunodensities of Na,K ATPase and NCC in
whole kidney extracts compared to males. Interestingly, the abundance of these proteins
increased in males but not in females after ALDO infusion. In this study, we tested the
hypothesis that estrogens derived from functional ovaries protect female rats from ALDO-
induced hypertension. Four groups of animals (n6 for each group) fed a standard 1% NaCl
diet were studied over a four week period: (1) OVX: ovariectomized rats; (2) OVX E2: OVX rats
treated with estradiol (E2, 10 g/day); (3) OVX ALDO: OVX rats treated with ALDO (200
g/day); and (4) OVXE2ALDO: OVX rats treated with E2 and ALDO infusion. ALDO treatment
increased mean arterial blood pressure (MAP) in OVX rats while E2 treatment decreased MAP
in both OVX and ALDO-infused OVX rats [MAP (mm Hg): OVX, 102 1; OVXE2, 94 1;
OVXALDO, 116 1; OVXE2ALDO, 105 1]. ALDO treatment increased the renal protein
abundance of Na,K ATPase in OVX rats by approximately 2-fold as did E2 treatment; however,
E2 treatment did not add to the effect of ALDO. On the other hand, ALDO but not E2 increased
the renal protein abundance of NCC in OVX rats by approximately 2-fold; and E2 had no effect
on the increase observed with ALDO. These results indicate that functional ovaries protect
females from aldosterone-induced hypertension. The finding that E2 has mineralocorticoid-like
effects on the abundance of Na,K ATPase and no effect on NCC abundance suggests E2 and
perhaps other ovary-derived endocrine factors contribute to the protection of females from
aldosterone-induced hypertension through mechanisms that are independent of renal NCC and
Na,K ATPase.
LB6
Role of Adaptive Immune Response in the Pathogenesis of Angiotensin
II-Dependent Hypertension And Vascular Dysfunction
Tomasz J Guzik, Nyssa Hoch, Kathryn A Brown, Louise McCann, Sergey Dikalov, Jorg
Goronzy, Cornelia Weyand, David G Harrison; Emory Univ Schof Medicine, Atlanta, GA
Angiotensin II (Ang II) activates the NADPH oxidase in the vessel, brain and kidney. The
interaction of these systems in the genesis of hypertension remains unclear. A circulating cell
 e96 Hypertension Vol 48, No 4 October 2006

might interact with these organs and mediate hypertension. Lymphocytes possess an NADPH
oxidase and Ang II causes their proliferation. Accordingly, we investigated the role of
lymphocytes in Ang II dependent hypertension. The hypertension caused by Ang II (500
ng/kg/min) was markedly reduced in RAG-1-/- mice, which lack T and B lymphocytes,
compared to wild-type animals as measured by telemetry (1244 vs. 1424; n12;
p0.01). The increase in aortic O2- caused by Ang II was blunted in RAG-1-/- mice (25 5%
vs. 217 30%; p0.01). Endothelium-dependent vasodilation was preserved in RAG-1-/-
compared to WT treated with Ang II (AChmax: 68 5% vs 49 7%). Adoptive transfer of T
cells, but not B cells restored the hypertensive response to Ang II (MAP 1575 mmHg; n5)
as well as the increase in vascular O2- (21050%). Adoptive transfer of p47phox-/- T cells only
partially restored the hypertensive response to Ang II (MAP: 1394 vs. 1575 mmHg;
p0.01). Ang II infusion increased the number of CD4 cells with the activation markers
CD69 (6.01.3% vs 9.91.5% of CD4; p0.01; n10) and CD25 (7.60.5% vs
11.11.5% of CD4; p0.01) in the peripheral blood and spleen. Ang II also increased T cell
CD44 and CCR5 in peripheral blood T cells and marked aortic T cell infiltration. Ang II caused
similar significant co-stimulatory effects on T cells in vitro. Ang II-induced hypertension also
increased Th1 type cytokine production (TNF-alpha 18520%, IFN-gamma: 25050% and
IL-2: 16515% p0.05 n6). These changes were accompanied by increased T cell O2- as
estimated by electron spin resonance (11.73 vs 30.18 pmol/mln), which was absent in
p47phox-/- T cells. Thus, T cells play a critical role in Ang II mediated hypertension. We
hypothesize that Ang II activates T cells, which infiltrate the kidney and vessels and release
cytokines which in turn stimulate oxidant production in these tissues, leading to vasoconstric-
tion, sodium retention and hypertension. T cell activation likely contributes to the inflammation
associated with hypertension and provides a link between atherosclerosis and hypertension.
LB7
Increasing Macula Densa Intracellular pH Enhances Superoxide Production
Via NAD(P)H Oxidase During Tubuloglomerular Feedback
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0,04 vs 0,62 0,07 mg). Our data indicate that the Ang(17) anti-thrombotic effect is due to
Mas-mediated NO release. Furthermore, this study suggests that the Ang(17)-Mas axis should
be considered as a putative target for development of a new class of drugs for treating
thrombotic diseases.
LB9
Angiotensin II-Induced Hypertension and Endothelial Dysfunction are
Attenuated in Transgenic Mice Expressing the Omega-3 Desaturase Gene
Kathryn A Brown, Mushtaq Ahmad, R. W Alexander, David G Harrison; Emory Univ Sch of
Medicine, Atlanta, GA
It has become widely accepted that omega-3 (n-3) fatty acids have beneficial effects on the
cardiovascular system. Mammals are unable to endogenously produce n-3 fatty acids from the
more widely consumed omega-6 (n-6) fatty acids. Transgenic mice have recently been created
that overexpress the C. elegans fat-1 gene, which encodes a desaturase enzyme capable of
converting n-6 into n-3 fatty acids. These mice have significant decreases in tissue ratios of
n-6 to n-3 fatty acids when compared to wild-type mice. We hypothesized that fat-1 mice
would be protected against angiotensin II-induced hypertension due to the increase in tissue
levels of n-3 fatty acids. Wild-type C57BL/6 and transgenic fat-1 mice were implanted with
osmotic mini-pumps that allowed subcutaneous infusion of angiotensin II (Ang II, 500
ng/min/kg) over a 2-week period. Sham-operated animals underwent an identical surgical
procedure with mini-pumps releasing buffer instead of angiotensin II. Systolic blood pressures
were measured via a computerized tail-cuff system prior to surgery, and then daily for 2 weeks
after mini-pump implantation. At baseline fat-1 mice had lower systolic blood pressure than
wild-type mice (105 2 versus 119 3, p 0.05). Moreover, the hypertension caused by
two weeks of Ang II was markedly diminished in fat-1 compared to wild-type mice (e.g. 129
4 mmHg versus 155 5 mmHg, p 0.001). The alteration of aortic endothelium-dependent

Ruisheng Liu, Jeffrey L Garvin, Yilin Ren, Patrick J Pagano, Oscar A Carretero; Henry Ford
Hosp, Detroit, MI
Increases luminal NaCl enhance superoxide (O2-) production by the macula densa (MD), which
in turn, enhances tubuloglomerular feedback (TGF). The mechanism of O2- generation by the
MD is not clear. We hypothesized that O2- production by NAD(P)H oxidase is enhanced during
TGF due to an increase in pHi at the MD. The thick ascending limb with intact MD plaque
adherent to the glomerulus were microdissected and perfused. The MD perfusate was changed
from 10 mM to 80 mM NaCl to mimic the conditions that induce TGF. We used the O2--sensitive
fluorescent dye dihydroethidium (DHE) to measure intracellular O2- production. In control TGF,
O2- production increased from 0.41 0.07 to 2.75 0.1 units/min (p 0.01). Tempol (0.1
mM) blocked this increase. To study the source of O2-, we used the NAD(P)H oxidase inhibitor
apocynin. In control TGF, intracellular O2- increased from 0.47 0.21 to 2.72 0.31
units/min. Then luminal NaCl was switched back to 10 mM and apocynin (10-5 M) was added
to the lumen and bath for 60 min. When luminal NaCl was again switched from 10 to 80 mM,
O2- increased from 0.53 0.25 to 0.92 0.40 units/min (p 0.01 vs control). To study the
role of pHi in O2- generation, we used dimethylamiloride (DMA) to inhibit Na/H exchange and
block increases in pHi during TGF. In control TGF, intracellular O2- increased by 0.32 0.06
to 2.5 0.30 units/min (p 0.01). DMA reduced the increase in O2- by 40% (p 0.01). To
study whether the effect of DMA is due to changes in MD pHi, we clamped pHi with a K/H
ionophore, nigericin. Nigericin (10-5 M) was added to 10 and 80 mM NaCl solutions, with pH
adjusted to 7.0. In control TGF, intracellular O2- increased from 0.31 0.06 to 1.54 0.25
units/min (p 0.01). Then we raised the pHi of 80 mM NaCl to 7.8. Intracellular O2- increased
from 1.54 0.25 to 2.11 0.45 units/min (p 0.01). We conclude that: 1) increasing luminal
NaCl induces O2- production by the MD; 2) NAD(P)H oxidase is the main source of O2- production
during TGF; and 3) increases in pHi caused by increasing luminal NaCl enhance but do not
initiate O2- production by the MD.
LB8
The Anti-thrombotic Effect of Angiotensin (17) is Abolished in
Mas-knockout Mice
Rodrigo A Fraga-Silva, Sr., Andrey C Goncalves, Federal Univ of Minas Gerais, Belo
Horizonte, Brazil; Michael Bader, Nathalia Alenina, Max-Delbruck Cntr for Molecular
Medicine, Berlin, Germany; Robson A Santos; Federal Univ of Minas Gerais, Belo Horizonte,
Brazil
It has been described that Angiotensin (17) [Ang(17)] has anti-thrombotic activity in rats.
However, the mechanism of this action is not known. In this study we tested for the presence
of the Ang(17) receptor Mas in rat platelets, the effect of Ang (17) on NO release in these
cells and whether the Ang(17) antithrombotic effect is dependent of Mas. The presence of
receptor Mas in rat platelets was evaluated by Western Blot using a polyclonal anti-Mas
antibody. To test if Ang(17) is able to induce NO release from rat platelets, we used the NO
indicator 4-amino-5 methylamino-2,7-difluorofluorescein diacetate (DAF-FM - 2.5 mol/L) in
rat platelet suspended in Krebs-HEPES-buffer and subsequently incubated with Ang(17)(10-9
and 10-8 mol/L). The NO release was evaluated by confocal microscopy. Western Blot revealed
a single band corresponding to the Mas protein. Ang(17) at 10-9 and 10-8 mol/L released NO
from rat platelets. To examine the role of Mas on the Ang(17) antitrombotic effect, thrombus
formation was induced in male C57/Bl-6, mas-KO and wild type mice. After anesthesia, the
vena cava was exposed and carefully ligated with a cotton thread just below the left renal vein.
A filter paper steeped in a 35 % ferric chloride solution was applied below the ligature. The
mice were submitted to intravenous infusion of Ang(17) (1, 10, 102, 103 pmol/Kg/min in
C57/Bl-6 and 102 pmol/Kg/min in wild type and mas-KO) or vehicle through the jugular vein
starting 15 minutes before the thrombus induction and lasting for the entire experimental
period. After 30 minutes the thrombus formed was carefully removed and dried. The thrombotic
response was measured by the thrombus weight. Ang(17) produced a dose-dependent
inhibition of thrombus formation in C57/Bl-6 mice with a maximal effect at 10 pmol/Kg/min
(0,34 0,03 vs 0,76 0,12 mg). Strikingly, the anti-thrombotic effect of Ang(17) was
abolished in Mas-knockout mice (Mas-KO: 0,84 0,09 vs 0,76 0,15 mg; wild type: 0,24
relaxation caused by Ang II infusion was more pronounced in the wild-type mice compared to
fat-1 mice (maximal percent relaxation to acetylcholine: 59 2 vs. 71 4, p 0.01). These
data indicate that increasing the aortic composition of omega-3 fatty acids has a protective
effect in preventing hypertension and endothelial dysfunction and may help explain the
beneficial effects of fish oils in cardiovascular health.
LB10
Adiponectin Mediates the PPARgamma Ligand Effect to Decrease Egr-1
Expression and Inhibit Angiotensin II-accelerated Atherosclerosis in LDLR-/-
Mice
Alan R Collins, Shu Wakino, Joey Z Liu, Yasunori Takata, Fen Yin, Christopher J Lyon,
Florian Blaschke, Ronald E Law, Rajendra K Tangirala, Willa A Hsueh; UCLA, Los Angeles,
CA
Peroxisomal proliferator activated receptor- (PPAR) is expressed in vascular cells, has
anti-inflammatory activity and when activated, inhibits atherosclerosis in LDLR-/- mice fed
diets that primarily produce fatty streaks. We hypothesized that PPAR ligands would attenuate
more advanced atherosclerosis by inhibiting expression of early growth response gene-1
(Egr-1), an orchestrator of inflammatory events in vascular smooth muscle cells (VSMCs) in a
model accelerated by angiotensin II (AngII) which has profound proinflammatory activity.
Minipump infusion of AngII elevated blood pressure by 60 mmHg, dramatically accelerating
lesion formation (44.46.3% aorta covered with lesions) and progression (fibrous caps)
compared to normotensive controls only receiving high fat diet (0.40.1%). The area of lesion
coverage was prominently attenuated by treatment with rosiglitazone 17.61.8% (60.4%
inhibition), pioglitazone 15.01.2% (64.7% inhibition) or a non-TZD PPAR ligand 22.92.4%
(48.4% inhibition). These changes occurred in the absence of improved cholesterol, plasma
triglycerides, insulin, glucose or blood pressure. Attenuation of aortic lesions by PPAR ligands
correlated with an early (2 weeks post-AngII infusion) and pronounced downregulation of
vascular expression of Egr-1 as well as Egr-1-regulated proinflammatory genes. Addition of
PPAR ligands to VSMCs did not inhibit while adenoviral overexpression of adiponectin did
inhibit AngII-induced Egr-1 expression. In addition, Egr-1 siRNA inhibited AngII stimulated
expression of intracellular adhesion molecule-1, macrophage chemoattractant protein and
tumor necrosis factor-, while control siRNA had no effect, suggesting Egr-1 mediates the
proinflammatory effects of AngII in VSMC. These data suggest that PPAR ligands exert their
antiatherogenic activity in VSMCs in large part through anti-inflammatory effects of adiponectin,
which may be a key vascular protective therapeutic target in accelerated atherosclerosis. .
LB11
Hypertension and Endothelial Dysfunction is Associated with Increased
Vascular Cyp 4a8 Expression and 20-hete Synthesis in DHT-treated Rats
Harpreet singh, Huan Deng, Rowena Kemp, Tsuneo Ishizuka, Alberto Nasjletti, Michal L
Schwartzman; New york Med college, valhalla, NY
We have shown that rats injected with adenoviral vectors carrying CYP4A constructs, feature
increased vascular production of 20-HETE associated with elevation of blood pressure,
impaired expression of the NO-dependent component of the vasorelaxing action of acetylcho-
line and elevated vascular production of superoxide anion. It is also known that androgen
induces expression of CYP4A enzymes. In this study, we investigated the consequences of
5-dihydrotestosterone (DHT) treatment on renal vascular CYP 4A expression, 20-HETE
synthesis, vasorelaxing response to acetylcholine and blood pressure in male Sprague Dawley
rats. DHT (70 mg/Kg bw/day for 2 weeks) increased blood pressure from 1272.5 to 1483.5
mmHg (p 0.005). DHT treatment increased (p0.05) renal interlobar arterial levels of CYP
4A-immunoreactive protein and CYP 4A8 mRNA (measured by real time PCR) by 2.160.36
and 3.02 0.72 fold, respectively, compared to vehicle-treated rats. No changes in the CYP
4A1, CYP 4A2 and CYP4A3 mRNAs as well as CYP4F1, CYP4F5 and CYP4F6 mRNA were
detected. Relative to data in vehicle-treated rats, interlobar arteries from DHT-treated rats

produced increased levels of 20-HETE (12.261.18 vs 16.81.34 ng/mg protein, p0.05)
and displayed higher levels of oxidative stress as measured by increased generation of
superoxide anion (217.741.3 vs 83.388.17 chemiluminescence cpm/g protein) and
increased levels of 3-nitrosylated proteins. Moreover, maximal relaxing response to acetylcho-
line in phenylephrine-preconstricted renal interlobar arteries from DHT-treated rats significantly
decreased (54.17.98 vs 88.92.2 % in vehicle-treated rats), which was offset by treatment
with an inhibitor of CYP4A (86.28.4%). Collectively, these findings demonstrate that DHT is
associated with upregulation of CYP 4A expression, specifically CYP4A8, and increased
production of vascular 20-HETE, which may impair endothelial function, promote constrictor
mechanisms and contribute to the development of androgen-dependent hypertension.
LB12
ACE2 is Preferentially Localized in the Tunica Media Layer in Renal
Vasculature and its Expression Increases after Administration of a Type 1
Receptor Antagonist
Maria J Soler, Jan Wysocki, Josette William, Minghao Ye, Ivan Cokic, Daniel Batlle; Div of
Nephrology & Hypertension. Northwestern Univ Feinberg Sch of Medicine., Chicago, IL
Angiotensin converting enzyme (ACE)2 is a carboxypeptidase involved in the degradation of
angiotensin II and other peptides. We have shown that ACE2 is localized in epithelial cells of
renal tubule and glomeruli (podocytes). This study was aimed to characterize ACE2 expression
within the renal vasculature and to investigate whether telmisartan, a specific angiotensin II
type 1 receptor blocker, is capable of altering its expression. Methods: Female C57BLKS/J mice
(8 weeks of age) were given either vehicle or telmisartan (2mg/kg/day) for 2 weeks. The renal
vascular pattern of ACE and ACE2 expression was characterized by confocal microscopy using
specific cellular markers. ACE2 expression in renal vasculature was also investigated by
immunohistochemistry. Results: The systolic blood pressure, measured by tail-cuff, in
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telmisartan-treated mice was lower than in the untreated mice (108.84.72 vs.
97.83.87mmHg, respectively p0.05). Both ACE and ACE2 were expressed in renal
arterioles of control and telmisartan-treated animals. While ACE colocalized with PECAM-1
(endothelial cell marker), ACE2 mainly colocalized with -smooth muscle actin (a marker of
smooth muscle cell in the tunica media). Semiquantitative analysis revealed a significant
increase in ACE2 expression in renal vessels from telmisartan-treated mice (n5) in
comparison to vehicle-treated mice (70.2%6.4% vs. 17.3%9.2%, respectively, p0.05). In
conclusion, in renal vessels ACE2 is mainly present in the tunica media layer, whereas ACE is
expressed in endothelial and adventitia layers. Our finding that the angiotensin-II type 1
receptor blocker, telmisartan, amplifies ACE2 in the renal vasculature suggests that its
renoprotective effect may involve enhanced angiotensin II peptide degradation in addition to its
well known direct blockade of the angiotensin II type 1 receptor.
LB13
Menkes protein as a Novel Modulator for Extracellular SOD: Role in
Angiotensin II-Induced Hypertension
Zhenyu Qin, Maria C Gongora, Shinichi Itoh, Ha Won Kim, Masuko Ushio-Fukai, David G
Harrison, Tohru Fukai; Emory Univ, Atlanta, GA
The extracellular superoxide dismutase (ecSOD), a secretory copper enzyme, regulates blood
pressure and endothelial function by modulating levels of extracellular superoxide anion (O2-)
and bioavailability of nitric oxide. We have previously demonstrated that the copper transporter
protein Menkes (MNK) is required for full activity of ecSOD via delivering copper through a
copper-dependent direct MNK-ecSOD interaction in the trans-Golgi network. We sought to
understand the role of MNK in modulating ecSOD activity in hypertension using MNK mutant
(MNKmut) mice. At baseline, the activity of ecSOD was decreased in MNKmut compared to
wild-type (WT) aortas (0.7 0.1 vs.1.5 0.1 U/mg total protein, p0.01), while Cu/ZnSOD
activity was normal. Two weeks of angiotensin II (Ang II) infusion (0.5 mg/kg/day) increased
ecSOD, but not Cu/ZnSOD activity in aortas from WT mice, while having little effect on ecSOD
activity in MNKmut mice. Interestingly, co-immunoprecipitation assays showed that Ang II
increased aortic association of MNK with ecSOD which might increase copper delivery to
ecSOD by MNK, thereby increasing ecSOD activity. Ang II increased systolic blood pressure
more in MNKmut mice (1334 mmHg vs 1204 mmHg, p0.01). Vascular O2- production
as assessed lucigenin chemiluminescence (4.00.2 vs. 2.50.2 fold, p0.01) and in situ
dihydroethidium (DHE) staining was also increased in MNKmut compared to WT aortas.
Consistent with this, Ang II impaired acetylcholine-evoked vasodilatation to a greater extent in
aortas of MNKmut than WT mice (max relaxation 60.60.1 vs. 77.50.1 %, p0.001). These
alterations of blood pressure, O2- production and endothelial function caused by Ang II in
MNKmut mice were rescued by co-infusion of the SOD mimetic tempol. In contrast,
norepinephrine (NE) infusion in MNKmut and WT mice caused a similar degree of hypertension
and no increase in vascular O2- production. Taken together, these results suggest that MNK
plays an important role as a modulator of vascular O2- production and blood pressure induced
by Ang II and might become a target of anti-oxidant therapy.
LB14
Lack of -Calcitonin Gene-Related Peptide Enhances DOC-Salt Induced
Renal Damage
Khurshed A Katki, Scott &White Hosp Rsch Cntr, Temple, TX; Valorie Chiasson, Texas A&M
Univ System Health Science Cntr, Temple, TX; Parag Patel, Arundhati Rao, Sheldon Chaffer,
Mae Lopez, Scott &White Hosp Rsch Cntr, Temple, TX; Donald J DiPette, Scott C Supowit;
Texas A&M Univ System Health Science Cntr, Temple, TX
We have reported that DOC-salt hypertension (HTN, 21days), results in enhanced inflammation,
renal damage and reduced renal function in -CGRP KO compared to WT mice. Due to a higher
baseline MAP (1520 mmHg), the KO mice exhibit a consistently higher BP than the WT mice
for the duration of the DOC-salt protocol. Therefore, the aim of this study was to determine the
CHBPR ConferenceLate Breaking Presentations e97
role of the elevated BP and/or the absence of -CGRP in DOC-salt HTN induced renal
dysfunction. DOC-salt HTN was induced in telemetry probe implanted 8 10 week old -CGRP
KO and WT mice. To equalize the BP to that of the DOC-salt WT mice, a separate group of
DOC-salt KO mice were given 0.025% hydralazine (HYD) p.o. Basal MAP was significantly
elevated in KO (1164 mmHg) compared to WT (1023 mmHg) mice. At the end of the 21
day study period, the DOC-salt protocol increased the MAP in the KO (to 1412 mmHg) and
WT (to 1282 mmHg) mice. HYD treatment equalized the MAP (1262 mmHg) in the DOC-salt
KO mice to that of the WT-DOC salt mice. Renal sections from DOC-salt KO mice exhibited
marked histopathologic damage, which was absent in DOC-salt WT mice. HYD treatment in the
DOC-salt KO mice reduced the renal damage by only 25 % despite BP reduction. Plasma
C-reactive protein (CRP), a marker for inflammation, was significantly elevated in DOC-salt KO
mice (80.7 g/ml) compared DOC-salt WT mice (6.70.3 g/ml). HYD treatment in the
DOC-salt KO mice did not change plasma CRP levels (9.20.5 g/ml). On day 21, creatinine
clearance was markedly reduced, but to an equal extent, in the DOC-salt KO (60%) and
KO-HYD mice (50%) compared to WT-DOC mice. At the end of the protocol, 24 hr urinary
microalbumin excretion was significantly higher in the DOC-salt KO mice (3-fold) and was even
further elevated (4-fold) in the KO-HYD mice, when compared to WT-DOC mice. Twenty four
hour urinary isoprostane, a marker of oxidative stress, was elevated 4-fold in DOC-salt KO mice
(27885 ng/ml/24hr) and 2-fold in KO-HYD mice (13834 ng/ml/24hr) on day 21, when
compared to WT-DOC mice (7712 ng/ml/24hr). In conclusion, in DOC-salt HTN, the lack of
-CGRP rather than the elevated BP appears to be the predominant mechanism contributing
to the inflammation, renal damage and decrease in renal function. This suggests that -CGRP
is reno-protective in this setting.
LB15
Renal Injury and Impaired Myogenic Response in Obese Zucker Rats
Radu Iliescu, Michael J Ryan, Univ of Mississippi Med Cntr, Jackson, MS; Radu Cazan,
Georgia Institute of Technology, Atlanta, GA; Julio C Sartori-Valinotti, Licy L Yanes, Gerald R
McLemore, Jr., Jane F Reckelhoff; Univ of Mississippi Med Cntr, Jackson, MS
Obesity is a risk factor for the development of hypertension and kidney disease. Renal
hemodynamic alterations in obesity may enhance transmission of systemic arterial pressure to
the glomerulus, leading to injury. We tested the hypothesis that impaired myogenic and/or
tubuloglomerular feedback (TGF) mechanisms of renal blood flow (RBF) control may contribute
to renal injury in a rat model of obesity. Obese Zucker rats (OZR, 16 weeks of age) had
significantly higher body weight, plasma triglyceride and total cholesterol concentrations as
compared to their lean counterparts (LZR). Blood pressure, as determined by telemetry
monitoring, was similar in obese and lean rats (1062 vs 1012 mmHg). OZR had renal
injury, as indicated by 75% higher excretion of urinary protein and albuminuria as compared
the LZR. Dynamic characteristics of RBF autoregulation in response to spontaneous fluctuations
in systemic blood pressure (BP) were determined in chronically instrumented, conscious rats.
Steady state levels of renal blood flow were not different between OZR and LZR (13.81.1 vs
14.41.4 mL/min). Transfer function analysis revealed that the strength of the myogenic
response, determined as the magnitude of the fractional gain in admittance at the characteristic
myogenic resonance peak, was significantly attenuated in OZR (1.70.1 vs 2.60.2, p0.05),
while its natural frequency was not altered (0.23 and 0.26 Hz, respectively). The TGF resonance
peak was similar both in magnitude and operating frequency in OZR and LZR. These data
indicate that an impairment of the renal myogenic response in OZR might contribute to the
development of renal injury even in the absence of overt hypertension.
LB16
Pivotal Role of Smooth Muscle-Specific PPAR in the Regulation of
Vascular Function
Carmen M Halabi, Andreas M Beyer, Henry L Keen, Willem J de Lange, Frank M Faraci,
Curt D Sigmund; Univ of Iowa, Iowa City, IA
Activation of peroxisome proliferator activated receptor gamma (PPAR), a member of the
nuclear receptor superfamily of ligand activated transcription factors, has been shown to
improve blood pressure and vascular function in animal models of acute or chronic
hypertension as well as in humans. Furthermore, patients heterozygous for dominant negative
mutations in the coding region of PPAR (V290M or P467L) were reported to exhibit severe
insulin resistance leading to type II diabetes mellitus, and early onset hypertension. To explore
the role of PPAR in vascular smooth muscle (VSM), we generated transgenic mice expressing
a dominant negative form of PPAR (P467L) specifically targeted to VSM cells using the smooth
muscle myosin heavy chain promoter. Transgene expression in tissues containing SMC,
including the aorta, was confirmed and differentiated from endogenous PPAR gene expression
via RNase protection. Using transgenic (Tg) animals and their non-transgenic (NT) littermates,
we performed functional studies of the SMC-P467L mice using aorta, and observed differential
responses to several vasoconstrictors. Remarkably, aorta of SMC-P467L mice contracted
considerably more (30229 mg at 100 nM, n8, P0.001) to increasing doses of
endothelin-1 than did aorta of NT littermates (446 mg at 10 nM, n9). In contrast, the
contractile response of the aorta to dopamine was significantly decreased in Tg animals
(5515 mg at 10 M, n8, P0.001) than NT littermates (23134 mg at 10 M, n9).
Whereas, no difference was observed in the response of aorta from male animals to serotonin
(950100 mg NT n5 vs. 953144 mg Tg at 10 M n4), aorta from female Tg mice
contracted more to serotonin (1,18391 mg at 10 M, n4) than did those of female NT
littermates (58476 mg, n4). These data suggest that normal expression of PPAR in
vascular smooth muscle is an important determinant in the regulation of vascular tone. Studies
are currently underway to determine the molecular mechanisms by which PPAR exerts its
effects in vascular muscle.
 e98 Hypertension Vol 48, No 4 October 2006
LB17
Kidney Specific Induction of HO-1 Prevents Angiotensin-II Dependent
Hypertension in Mice
Trinity Vera, David E Stec; Univ of Mississippi Med Cntr, Jackson, MS
Several studies have demonstrated that systemic induction of Heme Oxygenase-1 (HO-1) can
attenuate the development of hypertension in several models including Angiotensin II (AngII)
dependent hypertension. However, the mechanism behind the blood pressure lowering actions
of HO-1 induction is not known. In this study, we tested the hypothesis that induction of HO-1
specifically in the kidney can prevent the development of Ang-II dependent hypertension.
Experiments were performed in uninephrectomized mice in which a renal medullary interstitial
catheter was implanted into the remaining left kidney. Infusion of cobalt protoporphyrin (CoPP;
250 g/ml; 50 l/hr), a known inducer of HO-1, through the renal medullary interstitial
catheter for 48 hours resulted in a significant induction of HO-1 mainly in the renal medulla
when examined 2 weeks following infusion. No induction of HO-1 was observed in any other
organs such as the brain, heart, and liver. Next, the effect of renal medullary interstitial infusion
of CoPP on the development of Ang II dependent hypertension was examined. CoPP or Vehicle
(0.1 M NaOH, pH 8.3) was infused as indicated above 2 days prior to implantation of an osmotic
minipump which delivered Ang II at a rate of 1 g/kg/min subcutaneously. Mean arterial
pressure (MAP) was measured in conscious, unrestrained mice for 3 consecutive days starting
on day 7 following the implantation of the Ang II minipumps. MAP averaged 98 4, 160
4 and 128 11 mmHg in Vehicle Vehicle, Vehicle Ang II and CoPP Ang II treated mice
(n4). These results indicate that renal medullary induction of HO-1 is sufficient to prevent the
development of Ang II dependent hypertension in mice. Induction of HO-1 in the renal medulla
may be the mechanism by which systemic delivery of CoPP lowers blood pressure in Ang-II
dependent hypertension.
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LB18
Lack of Glutathione Peroxidase-1 Accelerates Cardiac-Specific Hypertrophy
and Dysfunction in Angiotensin II-Induced Hypertension
Noelia Ardanaz, Kyle W Jackson, Xiao-Ping Yang, Oscar A Carretero, Patrick J Pagano;
Henry Ford Hosp, Detroit, MI
Glutathione peroxidase-1 (Gpx1) is a selenium-dependent enzyme that plays an important role
in cellular antioxidant defense by reducing hydrogen peroxide and organic hydroperoxides.
However, the effect of Gpx1 on various target organs in hypertension has not been elucidated.
We tested the hypothesis that in mice lacking Gpx1 (Gpx1-/-) the development of hypertension,
cardiovascular hypertrophy, and dysfunction after AngII infusion for a short period of time (7
days) would be more severe compared with wild-type controls (WT; C57BL/6J). Mice were
infused with AngII (521 ng/kg*min, s.c.) or vehicle. Mean blood pressure (BP) was measured
by telemetry, and cardiac remodeling and function were evaluated by echocardiography (see
table for data). AngII infusion produced a similar increase in BP in WT and Gpx1-/-mice. AngII
increased total heart weight/body weight (THW/BW) in Gpx1-/- but not in the WT group.
Interventricular septum thickness (IVST) and posterior wall thickness (PWT) were increased to
a greater degree in Gpx1-/- than in WT. Left ventricular shortening fraction (SF) was decreased
in Gpx1-/- treated with AngII but not in WT. Vascular hypertrophy was increased similarly in WT
and Gpx1-/- treated with AngII. Thus, although AngII was infused for only 7 days, the lack of
Gpx1-/- led to a specific increase in cardiac hypertrophy and dysfunction without affecting
vascular hypertrophy and blood pressure. This study highlights a major role of peroxides in the
development of cardiac hypertrophy and dysfunction and potentially suggests a more important
role for Gpx1 in the heart.
WT Gpx1-/-
Vehicle AngII Vehicle AngII
BP (mm Hg)(n37) 1193 1352* 1173 1365*
THW/BW (mg/10g)(n 910) 53.61.3 56.61.8 55.01.6 66.42.7*#
IVST (% change)(n 910) 9.03.5 18.86.1 3.72.6 36.25.4*#
PWT (% change)(n 910) 11.47.3 24.24.9 1.32.8 41.13.9*#
SF (% change)(n 910) 0.72.6 1.32.4 -1.13.5 -12.84.1*#
*p0.05 vs. vehicle within strains, # p0.05 WT AngII vs. Gpx1-/- AngII. Echocardiography data expressed
as % change from day 0.
LB19
Familial Atherosclerotic Disease and Hypertension Localised To
Chromosome 7p in the British Genetics of Hypertension Study
Sandosh Padmanabhan, Claire E Hastie, BHF Glasgow Cardiovascular Rsch Cntr, Univ of
Glasgow, Glasgow, United Kingdom; Chris Wallace, Patricia B Munroe, Richard Dobson,
William Harvey Rsch Institute, Barts and the London Sch of Medicine, London, United
Kingdom; Morris Brown, Clinical Pharmacology and the Cambridge Institute of Med Rsch,
Univ of Cambridge, Cambridge, United Kingdom; Nilesh J Samani, Dept of Cardiology, Univ
of Leicester, Leicester, United Kingdom; David Clayton, Cambridge Institute of Med Rsch,
Univ of Cambridge, Cambridge, United Kingdom; Martin Farrall, Wellcome Trust Cntr for
Human Genetics, Univ of Oxford, Oxford, United Kingdom; John Webster, Clinical
Pharmacology Unit, Aberdeen Royal Infirmary, Aberdeen, United Kingdom; Mark Lathrop,
Cntr National de Genotypage, Evry, France; Mark Caulfield, William Harvey Rsch Institute,
Barts and the London Sch of Medicine, London, United Kingdom; John M Connell, Anna F
Dominiczak; BHF Glasgow Cardiovascular Rsch Cntr, Univ of Glasgow, Glasgow, United
Kingdom
Most conventional risk factors (blood pressure, diabetes, lipids, obesity) for atherosclerosis
show polygenic inheritance and many of them cluster in the metabolic syndrome predicting
increased cardiovascular risk. The small but significant residual familial relative risk after
regression on these conventional risk factors suggests the presence of unmeasured genetic
risk factors. The MRC BRIGHT study comprises 2010 non-diabetic non-obese Caucasian
hypertensive affected sibling pairs, of whom 2785 individuals (80.3%) had complete family and
phenotypic information allowing detailed classification by cardiovascular risk. Thus, 570
sib-pairs were identified with a history of parental cardiovascular mortality, where both sibs
also had CVD (defined by at least one of the following: MI, stroke, CABG, angiogram,
angioplasty, angina). Of these, 311 pairs were also negative for metabolic syndrome by
NCEP/ATPIII criteria. Nonparametric linkage analysis carried out using 450 markers with
MERLIN showed significant linkage on chromosome 7p (LOD 6.67, 39.3 cM, Figure 1) which
persisted after excluding those with the metabolic syndrome phenotype (LOD 5.18). This region
overlaps two mouse atherosclerotic loci Ath18 and Ath21 and multiple rat and mouse QTLs for
blood pressure, lipids, diabetes and weight on conserved syntenic analysis, and may explain
the clustering of metabolic traits commonly associated with increased atherosclerotic
cardiovascular risk. Our results indicate that this novel locus on chromosome 7p may contain
genes influencing the stability and progression of atherosclerotic lesions by mechanisms
different from or additive to conventional risk factors.
LB20
The Role of the NADPH Oxidase in Activation of T Cells By Angiotensin II
Nyssa E Hoch, Tomasz J Guzik, Cornelia M Weyand, Jorg J Goronzy, David G Harrison;
Emory Univ Sch Of Medicine, Atlanta, GA
NADPH oxidase produced reactive oxygen species (ROS) have been implicated in the genesis
of angiotensin II (Ang II)-dependent hypertension. AT-1 receptors and the NADPH oxidase have
been previously identified in T cells. Hypertension is associated with perturbations of the
immune system, which might contribute to inflammation and elevated blood pressure. We
hypothesize that Ang II acts as an accessory stimulus on the T cell AT-1 receptor and via
activation of the NADPH oxidase modulates T cell function. Culture of murine T cells with Ang
II and anti-CD3 for 3 hours significantly increased the percent of CD4 T cells expressing CD69
(an early indicator of T cell activation, 68.3% vs. 56.6%, Ang II vs. no Ang II, n11, p0.03)
as detected by FACS. Likewise, the percent of CD4 cells expressing CD25, a late indicator
T cell activation, was also increased significantly following 6-hour exposure to Ang II (67.4%
vs. 61.4%, Ang II vs. no Ang II, n11, p0.03). These increases in CD69 and CD25 were
inhibited with losartan (1uM), as well as the NADPH oxidase inhibitor apocynin (300 nM),
suggesting a role of the AT-1 receptor and NADPH oxidase, respectively. These effects were
also not observed in T cells from p47phox-/- mice. Infusion of Ang II (500 ng/kg/min) for 2 weeks
markedly increased T cell expression of p47phox. Exposure of T cells to Ang II for 48 hours
increased production of the Th1-related cytokine TNF-alpha by 5-fold (p0.04). CD4 cells
expressing CCR5, a chemokine receptor involved in homing of T cells to tissues, were
increased 92% by Ang II and anti-CD3 (12.86% vs. 6.67%, Ang II vs. no Ang II, p0.02). In
the absence of T cell receptor activation with anti-CD3, Ang II had no effect. Together, these
findings suggest that Ang II acts directly on T cells as an accessory stimulus which primes cells
to exhibit an augmented response to T cell receptor engagement. These effects of Ang II could
increase production of Th1 cytokines and homing of CD4 cells to tissues including the
vascular wall. These effects are at least in part mediated by the NADPH oxidase and might
explain a mechanism whereby T cells contribute to hypertension.
LB21
Angiotensin I and Angiotensin II Metabolism in Glomerular Epithelial Cells
is ACE2 but not ACE Dependent
Jan Wysocki, Maria J Soler, Minghao Ye, Daniel Batlle; Div of Nephrology & Hypertension,
Northwestern Univ Feinberg Sch of Medicine, Chicago, IL
We have recently shown that angiotensin converting enzyme (ACE) 2, a homologue of ACE
involved in the degradation of angiotensin peptides, is present in mouse glomerular epithelial
cells (podocytes). Moreover, we found ACE2 to be reduced in podocytes from diabetic mice,
which could lead to decreased degradation of angiotensin peptides within the glomerulus. To
examine the role of ACE2 activity on angiotensin (ANG) I and ANG II metabolism more directly
we used growth-restricted, conditionally immortalized mouse podocytes. By western blotting,
cultured podocytes expressed ACE2 abundantly, whereas ACE was not detectable. ACE mRNA
was also not detectable by real time RT-PCR. To establish whether ACE2 is involved in ANG I
and ANG II degradation, podocyte cell lysates were spiked with exogenous angiotensin peptides
(10-8M of ANG I or ANG II) and incubated with or without the addition of a specific ACE2
inhibitor, MLN-4760. ACE2 inhibition caused a marked decrease in ANG I degradation, as

shown by an increase in levels of the remaining, not degraded ANG I (1106149% of the
control without the inhibitor, n8, p0.001). The presence of the ACE2 inhibitor resulted in
more than a two-fold accumulation of ANG II when compared to podocytes incubated without
ACE2 inhibitor (27833% of the control, n8, p0.001). Strikingly, podocytes did not show
any measurable formation of ANG II from exogenous ANG I, unless when transfected with ACE
(-0.240.24 vs. 5.250.34 pg ANG II/g protein/hr, respectively, p0.001). The conversion
of ANG II from ANG I was completely abolished by the addition of captopril (-0.170.13 pg ANG
II/g protein/hr). By contrast, this ACE inhibitor had no effect on ANG II formation in
non-transfected podocytes. We conclude that in the podocyte ACE2 is important for angiotensin
peptide metabolism by degrading both ANG II and ANG I. The podocyte, a cell that lacks ACE
as a mechanism of angiotensin formation, has an effective mechanism of angiotensin peptide
degradation via ACE2, which may protect this cell from ANG II overactivity, a cause of increased
glomerular permeability and albuminuria.
LB22
Cardiovascular Profile of Nitric Oxide-releasing Enalapril in Aged
Spontaneously Hypertensive Rats
Magdalena Alonso-Galicia, Chrissa Dooling, Merck Rsch Laboratories, Rahway, NJ; Hillary K
Cresswell, Theodore J Detwiler, James C Hershey, Christopher Gibson, Charles Lin, Cheri M
Faust, Merck Rsch Laboratories, West Point, PA; Paola Tocchetti, Nicoletta Almirante,
Cristina Presotto, Ennio Ongini; NicOx Rsch Institute, Milan, Italy
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Nitric oxide (NO) plays an important role in vascular tone regulation. Recently, a variety of well
known drugs have been modified to incorporate a NO-donating moiety in order to enhance their
pharmacological properties. One such drug is NCX 899, a NO-releasing derivative of the
angiotensin-converting enzyme (ACE) inhibitor enalapril. The aim of this study was to compare
the anti-hypertensive efficacy and potency of NCX 899 vs. enalapril. In isolated rat aortic rings
pre-contracted with methoxamine, NCX 899 produced a dose-dependent vasodilation (EC503
uM) while enalapril had no effect. IV bolus injections of enalapril given to conscious aged male
SHRs (9 10 mo) at 0.26, 0.8, 2.6 and 5.2 umol/kg lowered systolic blood pressure (SBP) 4h
post-dosing by -93 (n11), -142 (n9), -144 (n12), and -155 (n11) mmHg,
respectively, while NCX 899 at equimolar doses lowered SBP by -92 (n12), -243 (n11),
-274 (n12) and -308 (n8) mmHg. In this acute study, enhanced BP lowering efficacy
of NCX 899 was achieved at equivalent plasma enalaprilat levels or degree of ACE inhibition,
and it was associated with a 25 fold increase of basal plasma nitrate/nitrate (NOx) levels. The
chronic anti-hypertensive effects of NCX 899 vs. enalapril were compared in aged male SHR
with telemetry devices dosed orally for 7 days (8 umoL/kg QD, n13 rats per group). Daily
early and late SBP responses were obtained by averaging values from hours 2 6 and hours
20 23 after dosing. After 7 days, NCX 899 treated rats had statistically significant lower SBP
(342 vs. 253 mmHg) 2 6 h post dosing. NCX 899 superior BP lowering effect after chronic
treatment was achieved at a similar degree of ACE inhibition (408 vs. 577 % inhibition,
NCX 899 vs. enalapril) and equivalent plasma enalaprilat levels, and it was associated with a
statistically significant 2-fold increase in plasma NOx. These results suggest that sustained
superior efficacy of NCX 899 is most likely due to NO release and that intermittent exposure
to NO donation from this compound does not lead to tolerance or diminished BP lowering
efficacy. NO-releasing derivatives of ACE inhibitors could provide better blood pressure control
and additional benefits such as enhanced vascular, renal and cardiac protection.
LB23
Race-dependent Cardiovascular Characteristics in Youth with
Prehypertension
Haidong Zhu, Weili Yan, Dongliang Ge, Frank A Treiber, Gregory A Harshfield, Gaston
Kapuku, Harold Snieder, Yanbin Dong; Med College of Georgia, Augusta, GA
Background: The cardiovascular structure and function in youth with prehypertension are
incompletely understood. In particular, whether the cardiovascular phenotypes of prehyper-
tension may vary dependent upon race remains unknown. Methods and Results: We examined
arterial stiffness by pulse wave velocity (PWV) measurement, hemodynamic profiles by
noninvasive impedance cardiography, and left ventricular structure and function by echocar-
diography in a cohort of black and white youth (n970, mean age17.63.3). The cohort
consisted of monozygotic and dizygotic (DZ) twins (blacks, 44.5%; DZ, 52.2%). Blacks
compared to whites had a higher incidence of both prehypertension (13.0% vs. 10.7%) and
hypertension (5.0% vs. 1.1%). In whites, prehypertensives (PHTs) compared with normoten-
sives (NTs) showed increased radial (6.800.14 vs. 6.240.06 m/s, p0.001) and foot PWV
(7.410.11 vs. 7.010.05 m/s, p0.001). In whites, total peripheral resistance index (TPX)
was greater in PHTs than NTs (28.811.03 vs. 25.710.35 mmHg/L/min/m2, p0.005). In
contrast, in blacks, cardiac index (CI) was higher in PHTs than NTs (3.280.12 vs. 2.960.05,
p0.004). Furthermore, in whites, radial and foot PWV, TPX, and relative wall thickness of left
ventricle showed a significantly increasing trend across NTs, PHTs and hypertensives (HTs),
respectively. In blacks, CI, and left ventricular mass index were sequentially increased from
NTs, PHTs to HTs. All the p values were adjusted for age, sex and BMI. Conclusion:
Prehypertension seems to be a critical phase during the development of hypertension.
Prehypertension in youth is associated with increased cardiovascular risks. Cardiovascular
characteristics of prehypertension appear to be race-dependent, indicating heterogeneity of its
etiology.
CHBPR ConferenceLate Breaking Presentations e99
LB24
Evidence for Impaired NOx Release in Collecting Duct-Specific ET-1
Knockout Mice
Markus P Schneider, Jennifer S Pollock, Med College of Georgia, Augusta, GA; Donald E
Kohan, Univ of Utah, Salt Lake City, UT; David M Pollock; Med College of Georgia, Augusta,
GA
Pressure diuresis is mediated by increased renal nitric oxide (NO) production, but the signaling
pathways have not been clarified. Mice with deletion of endothelin-1 in the collecting duct (CD
ET-1 KO) develop salt-sensitive hypertension. We hypothesized that pressure diuresis and renal
NO production are impaired in CD ET-1 KO mice. Blood pressure (BP) was measured via an
intracarotid catheter in 7 wild type (WT) and 6 CD ET-1 KO mice under 1.5% isoflurane
anesthesia. Bovine serum albumin (1%) was infused intravenously at a rate of 12.5 l/min.
After a 30 min baseline period, BP was increased stepwise by tying off the celiac and
mesenteric arteries, then further, 30 min later, by tying off the abdominal aorta distal to the
renal arteries. During each period, urine was collected via bladder catheter and urinary
nitrate/nitrite (NOx) concentration was measured by chemiluminescence. Mean BP was similar
in both groups at baseline (meanSE: 72.85.3 mmHg in CD ET-1 KO vs 77.14.0 in WT)
and during the first and second pressure increase (85.25.1 vs 87.04.4 vs and 93.73.3
vs 91.04.7 mmHg, respectively, n.s.). In response to increasing BP, urine flow rate increased
less in CD ET-1 KO (from 2.30.8 to 5.20.9 and then to 10.72.4l/min/g kidney)
compared to WT (from 2.90.4 to 6.81.4 and then to 13.23.3l/min/g kidney, p0.04 by
2-way ANOVA). Urinary NOx excretion rate increased in both groups, but was also affected by
genotype: 26979 to 878307 and 1886695 pmol/min/g kidney in CD ET-1, and from
26365 to 2027782 and 32751396 in WT (p0.02 interaction by 2-way ANOVA). These
results suggest production of ET-1 from the collecting duct is an upstream mediator of renal
NO production during pressure induced changes in urine flow. Furthermore, dysfunction of the
inner medullary ET-1 system may lead to hypertension by impaired pressure diuresis.
LB25
Roles of NAD(P)H Oxidase and NO Synthase in Superoxide Production by
the Renal Medullary Thick Ascending Limb from Normal and Diabetic Rats
Pamela K Carmines, Jing Yang, Univ of Nebraska College of Medicine, Omaha, NE; Jennifer
S Pollock; Med College of Georgia, Augusta, GA
Net Na reabsorption by the medullary thick ascending limb (mTAL) is modulated by superoxide
anion (O2-), which is produced in excess by many tissues during type 1 diabetes (T1D). The
purpose of the present study was to determine the impact of T1D on O2- production by the
mTAL and to test the hypothesis that both NAD(P)H oxidase and NO synthase (NOS) contribute
to O2- production in hyperglycemic environments. O2- production (lucigenin chemilumines-
cence) by mTAL suspensions prepared from normal rats (n8) averaged 38868 RLU/sec/mg
protein in 5.5 mM glucose media and was significantly accelerated after 30 min incubation in
20 mM glucose media (987193 RLU/sec/mg). Pre-incubation in either 100 M apocynin
(NAD(P)H oxidase inhibitor) or 250 M L-NAME (NOS inhibitor) prevented the stimulatory
impact of 20 mM glucose on O2- production. In mTAL suspensions prepared from rats with
streptozotocin-induced T1D (STZ rats, 3 4 wk after onset; n8) studied in 20 mM glucose to
maintain the chronic in vivo condition of the donor rats, O2- production averaged 2568814
RLU/sec/mg (P0.05 vs mTALs from normal rats studied in either 5.5 or 20 mM glucose). O2-
production by mTALs from STZ rats was not altered by 30 min exposure to L-NAME; however,
apocynin restored O2- production to 544218 RLU/sec/mg (not significantly different from
values obtained in untreated mTALs from normal rats). We conclude that both NAD(P)H oxidase
activity and NOS uncoupling contribute to acute glucose-stimulated O2- production by the
mTAL, while the chronic hyperglycemic condition of T1D promotes O2- production primarily via
an NAD(P)H oxidase-dependent mechanism. We speculate that the chronic pro-oxidant state of
T1D triggers compensatory events that minimize glucose-induced NOS uncoupling in the
mTAL. The accelerated NAD(P)H oxidase-dependent O2- production by the mTAL may
contribute to the Na retention and increased exchangeable Na that accompany the early
stage of moderately hyperglycemic T1D in animals and in humans.
LB26
Angiotensin-(17) Receptor Mas Modulates Cardiovascular Reflex
Reponses
Marina M Moura, Robson A Santos, Federal Univ of Minas Gerais, Belo Horizonte, Brazil;
Michael Bader, Natalia Alenina, Max Delbruck Cntr, Berlin, Germany; Andrea S Haibara;
Federal Univ of Minas Gerais, Belo Horizonte, Brazil
Mas was recently described as an endogenous receptor for Angiotensin-(17) [Ang-(17)].
Several studies have suggested an important role of this peptide in the modulation of
cardiovascular reflex responses. The aim of this study was to evaluate the influence of the
Ang-(17) receptor Mas deletion on the cardiovascular reflex responses in conscious mice.
Methods: Wild Type FVB/N mice (WT), Mas-knockout mice on FVB/N background (Mas-KO)
were implanted with polyethylene catheters into the femoral artery and vein for direct
measurement of cardiovascular parameters and drug administration, respectively. The surgery
procedure was performed under ketamine (4.5mg/kg) xylazine (0.2mg/kg) anesthesia, 24 h
before the experimental protocol for conscious mice. Baroreflex sensitivity was evaluated by an
index relating the changes in pulse interval (PI) and in mean arterial pressure (MAP) induced
by intravenous bolus injection of phenylephrine or sodium nitroprusside (0.25 0.5.0g/5l,
i.v.). Bezold-Jarisch reflex was evaluated by phenylbiguanide (1.0g/5l i.v.) injections and
chemoreflex was induced by potassium cyanide (2.5/5l i.v.) injections. Results: The basal
mean arterial pressure was higher in Mas-KO (n22) when compared with WT mice (n21)
(1181 vs 1082 mmHg, p0.001). However, the basal heart rate (HR) (65114 vs 63423
bpm) was not different from WT mice. The baroreflex bradycardic response was smaller in
Mas-KO mice (1.780.2 vs 2.440.3 ms/mmHg, p0.05) whereas the baroreflex tachycardic
response was higher in Mas-KO mice (1.210.25 vs 0.530.09 ms/mmHg, p0.05). The
 e100 Hypertension Vol 48, No 4 October 2006
depressor (-195 vs -388 mmHg, p0.05) and bradycardic (-18826 vs -35341 bpm,
p0.01) components of the Bezold-Jarisch reflex were smaller in Mas-KO mice. In addition,
the chemoreflex pressor response (203 vs 184 mmHg) was not different but the
bradycardic response (-33834 vs -20975 bpm, 2.5g/5l i.v., p0.01) was significantly
greater in Mas-KO compared with WT mice. Conclusion: These results show that Ang-(17)
receptor Mas is importantly involved in the modulation of cardiovascular reflex responses.
LB27
Axl Contributes to Vascular Impairment in DOCA-Salt Induced Hypertension
Matthew Daul, Vyacheslav A Korshunov, Michael P Massett, Bradford C Berk; Univ of
Rochester, Rochester, NY
Axl, a tyrosine kinase receptor, has recently been identified as a candidate gene in the
development of salt induced hypertension via an integrated genomic-transcriptomic approach.
Previously our group found that Axl is important in vascular remodeling. Here we investigate
the role of Axl in the pathogenesis of hypertension in a deoxycorticosterone-acetate (DOCA) salt
model. Hypertension was induced in Axl wild-type (Axl/) mice and Axl deficient (Axl-/-)
mice employing uninephrectomy and subcutaneous implantation of a DOCA pellet (75mg, 60
days release). Mice were subsequently given 1% NaCl solution to drink in addition to normal
chow for 6 weeks. Controls were uninephrectomized and received tap water and regular chow
ad libitum. Systolic Blood Pressure (SBP) was measured using a non-invasive tail-cuff method
and recorded 3 days before surgery and weekly thereafter. SBP of Axl/ DOCA mice was 30
mmHg greater than Axl/ controls beginning at week 1 and persisting for 5 weeks. In
contrast Axl-/- DOCA mice SBP levels were the same as Axl-/- controls at 5 weeks. Axl-/- mice
gained weight faster following surgery compared to Axl/ counterparts. DOCA-salt increased
relative kidney weight by 40% compared to controls similarly in both genotypes. Consistent
with DOCA-salt induced hypertension Axl/ mice exhibited increased maximal vasocon-
striction responses to KCl and phenylephrine (23 fold) as well as increased sensitivity (ED50).
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017
In contrast, vasoconstriction in Axl-/- DOCA mice was attenuated compared to wild-types.
Endothelium-dependent vasorelaxation (Acetylcholine, log[IC50]) was slightly impaired in
Axl/ DOCA mice (-7.0 M) compared to Axl/ controls (-7.4 M) while in Axl-/- DOCA mice
relaxation responses were augmented (-7.4 M) compared to Axl -/- controls (-7.1 M).
Additionally, endothelium-independent vasorelaxation (Sodium nitroprusside, log[IC50]) was
improved in Axl-/- DOCA mice (-8.5 M) compared to Axl/ DOCA mice (-7.8 M). These data
confirm the role for Axl as a key mediator of vascular impairment in salt-induced hypertension.
LB28
Acute Deletion of Ecsod Has No Effect on Blood Pressure but Causes
Profound Lung Injury
Maria C Gongora, Heinrich Lob, Ulf Landmesser, Zhenyu Qin, Graciela Gamez, Sergey I
Dikalov, Michael B Gravanis, Tohru Fukai, David G Harrison; Emory Univ, Atlanta, GA
The extracellular superoxide dismutase (ecSOD) is highly expressed in vessels, lung and heart.
Mice with traditional deletion of ecSOD have normal hemodynamics at baseline, but develop
augmented hypertension and endothelial dysfunction in resistance vessels in response to
angiotensin II. In addition, when given 100% oxygen, ecSOD-/- mice develop severe lung injury.
Traditional deletion of ecSOD could allow compensatory mechanisms that would mask the true
function of this protein. We sought to determine if acute deletion of ecSOD would alter
hemodynamics in adult animals. Mice were generated with loxP sites flanking the ecSOD
coding region and were backcrossed to the C57Blk/6 background. These mice were crossed
with mice transgenic for Tamoxifen (Tam)-inducible Cre (Tgcre/tam) and then bred to homozy-
gosity for the loxP flanked ecSOD. These ecSODloxP x Tgcre/tam mice were then treated with Tam
(3 mg/20g) IP for 5 days. Western blot confirmed 70% decrease in ecSOD protein in aorta
and lungs of ecSODloxP x Tgcre/tam mice after Tam injection and lung ecSOD enzyme activity was
similarly diminished. Tam injection had no effect on these parameters in C57Blk/6 mice. These
results were confirmed by immunostaining. Strikingly, acute deletion of ecSOD caused death
in 90% of ecSODloxP x Tgcre/tam mice within 15 days, while injection of Tam in C57Blk/6 was
without effect. IP injection of vehicle (corn oil) had no effect in either C57Blk/6 or ecSODloxP x
Tgcre/tam mice. Histology revealed severe lung damage following acute deletion of ecSOD, with
marked thickening of the alveolar septa, obliteration of alveolar spaces and massive
inflammatory infiltrate. Using ESR, we found acute deletion of ecSOD increased lung superoxide
by 6 fold. At baseline blood pressure by telemetry was 1221/951 mmHg, and was
1173/923 mmHg after ecSOD deletion. Cardiac function, as determined by echocardio-
gram was not altered by ecSOD deletion. These results indicate that while acute deletion of
ecSOD has no effects on cardiovascular parameters, ecSOD is essential for survival in the
presence of ambient levels of oxygen. Acute inactivation of ecSOD, as can occur upon exposure
to peroxynitrite or peroxides, could lead to the adult respiratory distress syndrome.
LB29
NOX4 Suppresses Angiotensin II-Induced Vascular Myofibroblast Migration
Mounir J Haurani, Alexander D Shepard, Patrick J Pagano; Henry Ford Hosp, Detroit, MI
The vascular adventitia is increasingly being recognized as an important modulator of vessel
tone and remodeling. Adventitial myofibroblasts were shown to migrate toward the intima after
vessel injury and thus appear to contribute significantly to restenosis. The mechanisms by
which this occurs are not well understood, however evidence from our lab as well as others
implicates angiotensin II (AII) and the NADPH oxidases, including nox2 and nox4, in this
process. We hypothesized that AII and/or TGF-beta1 (TGFb1) would enhance adventitial
myofibroblast migration in vitro and that overexpression of nox4-based NADPH would enhance
myofibroblast migration. Rat adventitial myofibroblasts were cultured to passage 6 9 and
characterized morphologically and by Western blot for smooth muscle -actin. Adventitial
myofibroblasts were plated into an 8-m Transwell system (Costar) and AII (10 nM) or vehicle
was added. Cells that migrated through the pores of the Transwell system were stained with
crystal violet and quantified by spectrophotometry. We found that treatment with 10 nM AII
alone caused a 61 23 % increase in migration vs. vehicle control (p0.029). Intriguingly,
myofibroblasts transfected with nox4 and its essential cytochrome b558 counterpart p22-phox
vs. its empty vector control reduced the increase in AII-induced migration by 89% (41 14
% vs. 6.2 7.3 % increase in migration, p0.038). We also tested whether addition of TGFb1
(10 ng/ml) would enhance AngII-induced migration. TGFb1 alone increased migration by 37
15% whereas AII TGFb1 caused a 131 45% increase in migration. In nox4
p22-phox-transfected cells, AII TGFb1 increased migration 42 11% (a 68% reduction vs.
AII TGFb1 alone, p0.048). Our data suggest that TGFb1 potentiates adventitial myofibro-
blast migration and also imply that nox4-based oxidase suppresses growth factor-induced
myofibroblast migration. These data are in contrast to our previous findings suggesting that
nox2 oxidase promotes migration, and are consistent with an isoform-specific effect of nox2
vs. nox4 oxidase on migration. The unique effects of the 2 noxes may be explained by
differences in compartmentalization and/or reactive oxygen species production, or another yet
undefined mechanism.
LB30
Is Change in Pulse Wave Contour a Better Predictor of Left Ventricular
Mass Reduction than Cuff Pressure?
Junichiro Hashimoto, St. Vincents Clinic/Univ of New South Wales, Darlinghurst, Australia;
Yutaka Imai, Tohoku Univ, Sendai, Japan; Michael F ORourke; St. Vincents Clinic/Univ of
New South Wales, Darlinghurst, Australia
Relatively large numbers of patients have been required to associate regression of left
ventricular mass (LVM) with fall in brachial cuff blood pressure (BP) in treatment of
hypertension. Hence, we prospectively examined potential superiority of pulse wave analysis
over conventional BP measurement in predicting treatment-induced LVM reduction. Forty-six
untreated patients (mean age, 578 years) with hypertension received standard medical
treatment for a 1-year period. LVM was measured by echocardiography (Penn convention) with
identical equipment on two occasions and with operators blinded to the LV load indices.
Antihypertensive treatment significantly reduced left ventricular load, manifest by decrease in
measured brachial BP (BPB, 153 20/9012 to 12215/7310 mmHg, P0.001), estimated
aortic BP (BPA, 14420/9112 to 11415/7410 mmHg, P0.001), carotid-femoral pulse
wave velocity (PWVCF, 9.01.4 to 8.21.3 m/s, P0.001), aortic augmentation index [AIx(A),
339 to 3011 %, P0.047], aortic augmented pressure (AugP, 1910 to 137 mmHg,
P0.001), and radial augmentation index [AIx(R), 9412 to 8915 %, P0.008]. These
changes were accompanied by significant reduction in LVM index (LVMI, 12119 to 11518
g/m2, P0.001). The treatment-induced LVMI change was not correlated with changes in BPB
(r0.09) or PWVCF (r0.00), but was closely correlated with factors influenced by wave
reflection - changes in AIx(A) (r0.51, P0.001), AIx(R) (r0.41, P0.005), AugP (r0.33,
P0.03) and amplification of pulse pressure between the aorta and brachial artery (Amp,
r0.43, P0.003). On multivariate analysis, AIx(A) change was the strongest determinant of
LVMI change, independent of BPB and PWVCF (0.51, P0.001). Estimated subject numbers
required for predicting a significant LVMI reduction were far less when wave reflection-related
factors than conventional BPB were used; for (two-sided) 0.05 and 0.20, numbers were
AIx(A) 28, Amp 41, AIx(R) 45, AugP 70, systolic BPA 270, diastolic BPB 543, and systolic BPB 966.
Reduction in wave reflection is an important therapeutic strategy for reducing LVM, and can be
predicted with modest subject numbers.
LB31
Endogenous Substance P Modulates Human Cardiovascular Regulation
Matthew V Dzurik, Andre M Diedrich, Bonnie Black, Sachin Y Paranjape, Satish R Raj,
Daniel W Byrne, David Robertson; Vanderbilt Univ, Nashville, TN
Background: Substance P (SP) is a peptide neurotransmitter identified in the brain and in many
afferent neural pathways. Its precise role in human physiology has been difficult to elucidate.
We used the selective neurokinin 1 (NK1) antagonist aprepitant as a pharmacologic probe to
determine the role of endogenous SP in human cardiovascular regulation. Methods and
Results: We performed this randomized, double blind, placebo controlled, crossover trial in
healthy subjects in electrolyte balance on a metabolic ward. SP antagonism raised heart rate
with standing by 38%. It reduced resting muscle sympathetic activity (MSNA) by 38%, reduced
systemic vascular resistance by 25%, and increased cardiac index by 47%. This constellation
of changes did not however alter either blood pressure or heart rate in the supine position.
There was a mildly attenuated vagal baroreflex with SP blockade. Conclusions: In human
subjects, Substance P exerts a tonic enhancement of sympathetic outflow to some cardiovas-
cular structures, manifested as an increase in MSNA and peripheral vascular resistance. The
role of Substance P is likely modulatory at sites that, in the aggregate, are relatively
well-compensated at baseline, but its actions emerge with cardiovascular perturbations. This
study thus demonstrates a role for Substance P in supporting peripheral vascular resistance in
human subjects.

LB32
Key Roles of Lipoxygenase and Downstream Inflammatory Genes in
Visceral Adipocytes from Obese Zucker Rat Model
Jiali Gu, Yeshao Wen, Susanna Keller, Hong Pei, Michael Williams, Swarup K Chakrabarti,
Jerry L Nadler; Univ of Virginia, Charlottesville, VA
The obese Zucker rat is a model of the cardiovascular metabolic syndrome and shows marked
increases in visceral fat deposition. In this study, we showed that mRNA expression of
5-lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and 12-LO were markedly
increased, 3.75-fold (p 0.01), 3.42-fold (p 0.017), and 6.93-fold (p 0.001) respectively,
in adipocytes isolated from visceral fat tissues of obese rats over that in visceral adipocytes
from lean rats. Furthermore, 5-LO products, 5-hydroxyeicosatetraenoic acid (5-HETE) (2.20-
fold, p 0.01) and leukotriene B4 (LTB4) (4.64-fold, p 0.001) as well as the 12-LO product,
12-HETE, (2.1-fold, 0 0.01) were also significantly increased. Visceral adipocytes from obese
Zucker rats also showed greater expression of interleukin (IL)-6 (3.11-fold, p 0.02), tumor
necrosis factor (TNF)- (3.0-fold, p 0.01), and monocyte chemoattractant protein (MCP)-1
(3.5-fold, p 0.02). We next tested the effect of (R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine
(lisofylline or LSF), a novel anti-inflammatory drug, which has been shown to protect the vessel
wall in the Zucker rats from injury. Obese Zucker rats were injected twice a day for 10 days
with a dose of 25 mg/kg LSF. We observed that LSF significantly suppressed 5-LO (77%, p
0.01), 5-FLAP (89%, p 0.05), and 12-LO mRNA (83%, p 0.001). LSF also reduced IL-6
(p 0.001) and MCP-1 (p 0.01), but not TNF- mRNA expression in obese Zucker rats
compared with that in saline-treated control obese Zucker rats. The results showed that
visceral adipocytes express major LO genes that produce downstream inflammatory actions.
Novel anti-inflammatory agents such as LSF could provide therapeutic effects in obese-insulin
resistant models or type 2 diabetes by reducing expression of key inflammatory genes in fat
tissue. Support from NIH P01 HL55798
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LB33
Renal Dysfunction in Diabetic SHR is Attenuated by Treatment with
Angiotensin-(17)
Ibrahim F Benter, Mariam H Yousif, Constantin Cojocel, May Al-Maghrebi, Kuwait Univ
Faculty of Medicine, Safat, Kuwait; Debra I Diz; Wake Forest Univ Sch of Medicine,
Winston-Salem, NC
In WKY rats, streptozotocin (STZ)-induced diabetes is assoicated with impaired endothelial
function, enhanced vascular reactivity and proteinuria that is attenuated by chronic adminis-
tration of angiotensin-(17) [Ang-(17)]. We examined the influence of chronic treatment with
Ang-(17) (576 g/kg/day) on mean arterial pressure (MAP) and development of renal
dysfunction in STZ-treated spontaneously hypertensive rats (diabetic SHR). MAP, urine volume
and protein, and vascular responsiveness of isolated renal ring segments to vasoactive agonists
were studied in vehicle- or Ang-(17)-treated diabetic SHR. MAP was attenuated by either
diabetes (163 3 mm Hg; p0.05) or Ang-(17) (172 5 mm Hg; p0.05) versus untreated
SHR (205 11 mm Hg). There was a significant increase in urine protein and urine volume
(266 221 mg/24hrs and 118 5 ml/24hrs, respectively; p0.05) in diabetic SHR as
compared to SHR (112 13 mg/24hrs and 12 2 ml/24hrs). Treatment of diabetic SHR with
Ang-(17) resulted in a significant reduction in urine protein (185 23 mg/24hrs; p0.05)
without change in urine volume (123 5 ml/24hrs). Treatment with Ang-(17) also prevented
the abnormal renal vascular responsiveness to endothelin-1 (ET-1), norepinephrine (NE),
angiotensin II (Ang II), carbachol and Ang-(17) in diabetic SHR:
Response to vasoconstrictor agonists Response to vasodilator
(mg/mg tissue wt) ET-1 (10-9M) agonists (%) Carbachol
NE (10-7M) (10-7M) Ang-(17)
Animal Group Ang II (10-7M) (107M)
WKY 8214 13427 3912 564 613
SHR 14915* 22020* 7714* 205* 244*
SHR-Ang-(17) 11914 9820# 6910 5211# 437#
Diabetic SHR 24632*# 30946*# 1636*# 286* 266*
Diabetic 12231 18132 6714 353# 351# not been investigated in this model even though ET-1-dependent vasoconstriction frequently
SHR-Ang-(17)
Data presented as mean SEM (N5 8) * Value significantly different compared to WKY, p0.05. # Value
significantly different compared to SHR, p0.05. Value significantly different compared to diabetic SHR,
p0.05. Ang-(17) did not correct the hyperglycemia in the diabetic groups. These results suggest that
therapeutic strategies involving elevation of Ang-(17) could reduce renal dysfunction in diabetic hypertensives,
despite persistance of hyperglycemia.
LB34
Angiotensin III Induces Natriuresis in Mice Overexpressing AT2 Receptors in
the Renal Proximal Tubule
Brandon A Kemp, Shetal H Padia, Nancy L Howell, Helen E McGrath, Pete D Meliagros,
Robert M Carey; Univ Virginia Sch Med, Charlottesville, VA
Previous studies have indicated that the angiotensin (Ang) AT2 receptor may play a role in
pressure-natriuresis and that des-aspartyl1-Ang II (AngIII) may be the preferred agonist in the
natriuretic response. The present study employed female transgenic mice (Tg) which
specifically overexpress proximal tubule AT2 receptors via a kidney androgen-regulated protein
(KAP) promoter fused to the rat HA-tagged AT2 receptor gene. In the presence of testosterone
(T), the Tg mice (N 3) demonstrated a significant increase in renal cortical AT2 receptor
protein expression (179.4 12.6 density units (DU)) compared to wild type mice with T
(91.5 16.6 DU, P0.01) or without T (105.9 15.3 DU, P0.01). Given these observations,
the Tg mice were utilized to examine the role of the AT2 receptor in Ang III-induced natriuresis.
Tg (N6) and wild-type mice (N6 with T; N6 without T) were anesthetized and infused
intravenously for two hours with vehicle (D5W, 8 l/min) followed by a 1 hour Ang III (14
nmol/kg/min) or vehicle infusion. Blood pressure (BP) was monitored directly via a carotid artery
catheter and urinary Naexcretion rate (UNaV) was determined after each 1- hour collection
CHBPR ConferenceLate Breaking Presentations e101
period via a urethral catheter. In Tg mice with T, UNaV increased from a baseline of 0.03 0.01
to 0.08 0.02 mol/min (P0.01) after Ang III infusion, accompanied by a BP increase from
84.56 5.30 to 133.38 7.92 mm Hg (P0.0001) after Ang III infusion. In the female
wild-type mice with and without T, UNaV also increased, but not significantly (0.03 0.01 to
0.05 0.01 mol/min and 0.03 0.01 to 0.05 0.01 mol/min, respectively; P NS). BP,
however, increased significantly in the wild-type mice with and without T (77.73 1.98 to
121.11 7.43 mm Hg; P0.0001 and 79.68 4.79 to 117.91 6.58 mm Hg; P0.0005),
indicating that pressure-natriuresis only occurred in response to intravenous Ang III infusion in
the Tg mice with T. In conclusion, these data suggest that Ang III may be involved in
pressure-natriuresis via the activation of renal proximal tubule AT2 receptors.
LB35
Partial Prevention of Salt-induced Hypertension by Icv Infusion of the
Aldosterone Synthase Inhibitor Fad286 in Dahl S Rats
Bing S Huang, Univ of Ottawa Heart Institute, Ottawa, Canada; Arco Y Jeng, Novartis
Pharmaceuticals Corp., East Hanover, NJ; Frans H Leenen; Univ of Ottawa Heart Institute,
Ottawa, Canada
Dahl S rats on high salt intake develop hypertension associated with an increase in CSF [Na],
and icv infusion of a MR or ENaC blocker or the steroid synthase inhibitor trilostane prevents
the hypertension. In the present study, we examined whether chronic blockade of CNS
aldosterone production with the specific aldosterone synthase (CPY11B2) inhibitor FAD286
(FAD) can prevent the hypertension and CSF [Na]1 in Dahl S rats on high salt. Six weeks old
Dahl S rats were divided into 5 groups (n57) and started via osmotic minipump an icv
infusion of FAD at 10 or 80 g/kg/day or vehicle, or subcutaneous infusion of FAD at 80
g/kg/day as control for possible peripheral effects of icv dosing. Regular salt diet (RNa) was
started in one of icv vehicle groups and high salt diet (8% Na, HNa) in other groups. After 4
weeks of treatments, resting BP and HR were recorded in free-moving rats via catheter inserted
in carotid artery 18 h earlier. Blood samples were then taken, and CSF withdrawn from the
cisterna magna in anesthetized rats for electrolytes measurement.
[Na], mM
MAP (mmHg) HR (bpm) CSF plasma
RNaicv vehicle 1282* 4206 1481* 1462
HNa icv vehicle 1655 50710* 1542 1472
HNa icv FAD, 10 g/kg/d 1493a 46716b 1542 1462
HNa icv FAD, 80 g/kg/d 1453a 43710 1532 1472
HNa sc FAD, 80 g/kg/d 1564 4638b 1533 1483
Data are meansSE. * p0.05 vs others, a: p0.05 vs HNaveh, b: p0.05 vs RNaveh. Icv infusion
of FAD at both doses partially prevented hypertension, but did not affect increases in CSF [Na] on high salt
intake. Since central MR blockade fully prevents salt-induced hypertension, these results suggest that besides
aldosterone other MR agonists, e.g. corticosterone in the CNS may also be involved in the development of
salt-induced hypertension.
LB36
Altered Coronary Artery Vasoreactivity in Rats Exposed to Intermittent
Hypoxia/Hypercapnia
Tom W Cherng, Univ of New Mexico, Albuquerque, NM; Matthew J Campen, Lovelace
Respiratory Rsch Institute, Albuquerque, NM; Nancy L Kanagy; Univ of New Mexico,
Albuquerque, NM
Sleep apnea is a common problem in the United States and has been associated with many
cardiovascular diseases including hypertension, myocardial infarction and coronary artery
disease (CAD). Rats exposed to intermittent hypoxia and hypercapnia (IH/HC) to mimic sleep
apnea were previously shown to have elevated plasma endothelin 1 (ET-1) levels and
augmented ET-1 constrictor sensitivity in mesenteric arteries. However, coronary reactivity has
contributes to ischemic injury in CAD. We hypothesized IH/HC-exposure increases coronary
artery constrictor sensitivity to ET-1 and decreases acetylcholine (ACh)-induced dilation.
Endothelium-intact intraseptal coronary arteries (inner diameter 192 13 m) from SHAM
and IH/HC rats were isolated, pressurized to 60 mmHg and loaded with the Ca2-binding dye,
Fura-2 AM. Myogenic tone and basal vessel wall [Ca2] was not different between groups of
arteries. ET-1 (10-10 to 10-8 M) constricted IH/HC arteries more than SHAM arteries (p 0.05)
but increased vessel wall [Ca2]i similarly in both groups. ACh-induced dilation of ET-1 (10 nM)
constricted arteries was less in IH/HC than in SHAM arteries (Max 62 6% SHAM, 19
5% IH/HC, p 0.01) and ACh decreased Fura-2 ratio less in IH/HC than in SHAM arteries.
Similar to our previous observations in mesenteric arteries, coronary arteries from IH/HC rats
show augmented ET-1 constrictor sensitivity, increased Ca2-sensitivity and diminished
ACh-induced dilation. These findings suggest IH/HC-induced ET-1 synthesis should lead to
augmented ET-1 dependent coronary vasoconstriction and may contribute to cardiac ischemic
injury in sleep apnea.
LB37
Na,K-ATPase Exhibits Higher Affinity for Sodium and Insensitivity to
Angiotensin II Receptors Action in Proximal Tubule of
Streptozotocin-induced Diabetic Rat
Athar H Siddiqui, Tahir Hussain; Univ of Houston, Houston, TX
Recent reports suggest that the angiotensin II AT2 receptor, via a tubular effect, greatly
contributes to enhanced urinary Na excretion in streptozotocin (STZ)-induced diabetic rats,
while the AT1 receptor does not affect the renal Na excretion. However, the biochemical
mechanism affecting angiotensin II receptors function in diabetic animals is not known. In the
 e102 Hypertension Vol 48, No 4 October 2006
present study, we tested the hypothesis that AT2 receptor activation causes greater
inhibition of the Na,K-ATPase (NKA) activity in the proximal tubules (PTs) of STZ-induced
diabetic compared with control Sprague Dawley rats, and the activation of AT1 receptor
does not affect the enzyme activity in diabetic animals. Western blotting of the PTs
proteins revealed a significant increase in the AT2 receptor and no change in the AT1
receptor levels in diabetic compared with control rats. In control rat PTs, CGP42112, AT2
receptor agonist (0.1100 nM) inhibited NKA activity; the CGP42112-induced inhibition
was attenuated by the AT2 receptor antagonist PD123319 and not by the AT1 receptor
antagonists losartan or candesartan, suggesting the involvement of the AT2 receptor.
Surprisingly, CGP42112 alone or in the presence of AT2 or AT1 receptor antagonists did
not affect the NKA activity in diabetic rats. Angiotensin II (100 pM) via AT1 receptor
stimulated NKA activity in control rats, but had no effect on the NKA activity in diabetic
rats. Determination of the NKA kinetic parameters in the PT membranes revealed that Km
for Na was significantly lower (4.750.47 mM) in diabetic compared with control rats
(11.750.85 mM), while Vmax were similar in both rat groups (cont.-11114 vs diabetic
12614 nmoles/mg protein/min), suggesting higher NKA affinity for Na in diabetic
compared with control rats. We conclude that (i) the higher affinity of NKA for Na in the
proximal tubules of diabetic rats renders the enzyme insensitive to hormone action
especially further stimulation, but provides as a compensatory mechanism of preventing
Na loss due to hyper-filtration in diabetes, and (ii) the inability of CGP42112 to inhibit NKA
activity in the proximal tubule indicates that the AT2 receptor-mediated Na excretion in
diabetic rat as reported earlier, may be a distal tubule mechanism.
LB38
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Urinary C-Reactive Protein Excretion Remains Elevated Independent of
Aldosterone Antagonism in Cyp1a1-Ren2 Transgenic Rats with Inducible
Ang II-Dependent Malignant Hypertension
Rudy M Ortiz, Univ of California, Merced, CA; John J Mullins, Univ of Edinburgh, Edinburgh,
United Kingdom; Kenneth D Mitchell; Tulane Univ, New Orleans, LA
Elevated aldosterone levels contribute to the pathogenesis of various hypertensive states.
Administration of the mineralocorticoid receptor antagonist, sprironolactone, in combination
with either an ACEI or ARB has been shown to reduce the expression of C-Reactive Protein
(CRP) in individuals with dilated cardiomyopathy suggesting that aldosterone may contribute to
inflammatory phenotype characterized by elevated CRP. However, the contribution of aldoste-
rone to this phenotype in malignant hypertension has not been elucidated. The present study
was performed to determine if chronic mineralocorticoid receptor blockade attenuates the
development of inflammatory phenotype during malignant hypertension in transgenic rats with
inducible expression of the Ren2 gene [TGR(Cyp1a1Ren2)]. Systolic blood pressure was
measured by radiotelemetry in conscious male Cyp1a1-Ren2 rats (n8) during control
conditions and during dietary administration of indole-3-carbinol (I3C; 0.3%) for 10 days. Rats
induced with I3C exhibited elevated SBP (1334 vs. 1826 mmHg), increased urinary CRP
(23 7 vs. 287 74 ng/d) and total urinary protein excretion (10.5 1.5 vs. 25 5 mg/d). Systolic/diastolic, mmHg
Spironolactone treatment (3.3 mg/day, sc) in hypertensive rats (n8) alleviated the proteinuria
(13 3 mg/d) but did not alter urinary CRP excretion (424 151 ng/d). These data indicate
that ANG II-dependent malignant hypertension is associated with an inflammatory phenotype
characterized by elevated CRP; however, blockade of mineralocorticoid receptors does not
reduce CRP suggesting that aldosterone does not contribute markedly to the elevated CRP
levels and cardiovascular inflammation induced by malignant hypertension in Cyp1a1-Ren2
transgenic rats.
LB39
Angiotensin II Enhances Intracellular Calcium Responses to ATP in Renal
Vascular Smooth Muscle Cells
Andrew Fuller, L.G. Navar; Tulane Univ, New Orleans, LA
In addition to its direct vasoconstrictor action, Angiotensin II (ANGII) also facilitates or
enhances vasoconstrictor responses to other stimuli. Because ANGII has been shown to
enhance tubuloglomerular feedback responses which have been associated with changes
in renal interstitial fluid ATP concentrations, we evaluated the ability of ANGII to augment
early peak and sustained increases in intracellular calcium (Ca2i) in renal vascular smooth
muscle cell (RVSMC) response to ATP. RVSMC were isolated and exposed to 5M ATP with
and without pretreatment with ANGII (1nM). The average control initial peak Ca2i response
was 66 5nM followed by a decrease to a sustained level of 28 3nM above baseline
(n9). RVSMC pre-incubated with 1nM ANGII for 60 seconds (s) before exposure to 5M
ATP had significantly greater responses with an average peak response of 124 7nM
followed by a decrease to a sustained level of 47 6nM (n11). The augmenting effect
of ANGII was prevented by blockade of the AT1 receptors. Cells pre-incubated with 1M
of the AT1 receptor blocker, candesartan, for 100s and 1nM ANGII for 60s before exposure
to 5M ATP did not show an augmentation and had an average peak response of 69
7nM followed by a sustained response of 26 9nM (n5). The effect of ANGII to augment
Ca2i response to ATP appears to be agonist specific. Cells exposed to 1M Vasopressin,
had an average peak response of 58 11nM followed by a sustained response of 10
8nM (n7). This effect was not enhanced by ANGII, cells pre-incubated with 1nM ANGII
for 60s before exposure to 1M Vasopressin had an average peak response of 63 12nM
followed by a sustained response of 21 10nM (n7). These data indicate that low
nanomolar concentrations of ANGII activate AT1 receptors leading to augmented peak and
sustained Ca2i responses to ATP. The results provide cellular evidence to explain the
mechanism by which increased renal interstitial fluid ANGII concentrations enhance the
sensitivity of the tubuloglomerular feedback mechanism.
LB40
Angiotensin II AT2 Receptor Agonist Produces Natriuresis in Spontaneously
Hypertensive Rats
Tahir Hussain, Dept of Pharmacological and Pharmaceutical Sciences, College of Pharmacy,
Univ of Houston, Houston, TX; Negin N Fouladi; Dept of Pharmacological and
PharmaceuticalSciences, College of Pharmacy, Univ of Houston, Houston, TX
Enhanced renal angiotensin II AT1 receptor function leading to increase in renal Na
reabsorption has been implicated in various forms of hypertension including in the animal
models of obesity and spontaneous hypertension. Recently we have shown that the renal
AT2 receptors are up-regulated and promote urinary Na excretion in obese Zucker rats,
and thereby may have a compensatory role against enhanced AT1 receptor function.
Present study was designed to investigate the renal expression levels and function of AT2
receptor on urinary Na excretion in spontaneously hypertensive rats (SHR) and age
matched Wistar Kyoto rats (WKY). Western blotting revealed that 12 week old SHR express
AT2 receptors (45 kDa), which are 2.5 fold higher in the cortical homogenates compared
to WKY rats but similar in the cortical (basolateral, BLM and brush border, BBM)
membranes. In pre-hypertensive SHR (5 wk), the AT2 receptor levels either in the total
cortical homogenate or in BLM/BBM were similar as compared with 5 wk WKY rats. Renal
function parameters, measured under anesthesia in 12 wk old SHR and WKY, suggest that
infusion of CGP 42112A (i.v. 1 g/kg/min) an AT2 receptor agonist, increased in UF and
UNaV over basal in SHR, but not in WKY. Interestingly, the infusion of the agonist did not
affect FENa either in SHR or in WKY rats. The CGP42112 infusion significantly increased
GFR and decreased blood pressure in SHR and not in WKY rats. *- p 0.05 versus basal
SHR. The data suggest that adult SHR express higher level of the AT2 receptor which upon
activation causes an increase in UF and UNaV via a mechanism related to increase in GFR,
and not due to the tubular effect on Na transport, as shown earlier in diabetic rat models.
The enhanced renal function of the AT2 receptors may be a compensatory mechanism
against increased Na reabsorption and development of hypertension in SHR.
Parameter WKY SHR
Basal CGP Basal CGP
Urine flow, UF l/min 1.890.3 1.90.4 1.670.27 3.30.46*
Urinary Na excretion, UNa 1.10.34 1.070.3 0.530.16 1.90.57*
V mole/min
Fractional Na excretion, FENa % 1.250.5 0.90.24 0.660.35 0.650.15
Glomerular filtration rate, 1.00.15 0.80.12 0.980.14 1.50.11*
GFR ml/min
117/84 111/73 151/124 136*/106*
LB41
Dietary Sodium Enhances the Benzamil Sensitive Component of Myogenic
Constriction in Mesenteric Vessels
Nikki L Jernigan, Univ of New Mexico, Albuquerque, NM; Kathy L Cockrell, Josh Speed,
Joey P Granger, Heather A Drummond; Univ of Mississippi Med Cntr, Jackson, MS
Increases in dietary sodium are known to affect vascular reactivity. Recent work from our
laboratory indicates that Epithelial Na Channel (ENaC) function plays an important role in
modulating myogenic vascular reactivity. While previous studies have demonstrated that
dietary salt intake regulates ENaC expression and activity in epithelial tissue, the importance
of dietary salt on ENaC expression in vascular smooth muscle cells (VSMC) and its role in
myogenic constriction is unknown. Therefore, the goal of the present study was to determine
whether dietary salt modulates ENaC expression and function in vascular myogenic vasocon-
striction. To accomplish this goal, we examined 1) ENaC expression in freshly dispersed VSMC,
and 2) agonist-induced vascular reactivity and 3) pressure-induced vasoconstrictor responses
in isolated mesenteric resistance arteries from normotensive Sprague-Dawley rats fed normal
salt (NS; 0.4% NaCl) or HS diet (8% NaCl; 2 wks). VSMCs from mesenteric arteries of NS fed
animals express , , and ENaC. However, pressure-induced constriction was unaltered by
ENaC inhibition with 1 M benzamil, suggesting ENaC proteins do not contribute to myogenic
constriction in mesenteric arteries under normal salt intake. HS induced a non-coordinate
regulation of , and ENaC expression and distribution, marked by a 48% reduction in
ENaC and 14 % increase in ENaC protein expression. Furthermore, high salt diet induced
a translocation ( 340 nm) of ENaC from intracellular regions towards the VSMC membrane.
Associated with these changes in expression was an increased benzamil sensitive component
of the myogenic constrictor response (Table 1). These data suggest that dietary sodium alters
the quantitative importance of the vascular ENaC signaling pathway contributing to myogenic
constriction.
TABLE 1. % MYOGENIC TONE AT 100 MM HG
Diet Control 1 M Benzamil
NS (n5) 44.63.9 43.42.0
HS (n5) 43.45.6 30.23.6*

LB42
Renoprotective Effects of Raloxifene in Diabetic Nephropathy
Alexis Dixon, Sandhya Singh, Regina Babayan, Christine Maric; Georgetown Univ Med Cntr,
Washington, DC
Our previous studies have shown that supplementation with estrogen from the onset of
diabetes attenuates diabetic nephropathy. But, estrogen may have adverse side effects on
other organs and have feminizing side effects if supplemented in males. The current study
examined the renoprotective effects of a selective estrogen receptor modulator, raloxifene, in
an experimental model of diabetic nephropathy. Raloxifene activates estrogen receptors and
estrogen receptor-mediated cellular events without the side effects of estrogen. The study was
performed in female Sprague-Dawley non-diabetic (ND), streptozotocin (STZ)-induced diabetic
(D) and STZ-induced diabeticraloxifene (DRal) rats (n5/group). After 12 weeks of
treatment, D was associated with increased albumin extraction (UAE; ND, 4.20.4; ND,
41.39.0 mg/day), increased plasma levels of interleukin-6 (IL-6; ND, 14.85.0; D,
51.314.0 pg/ml), glomerulosclerosis (GSI; ND, 0.260.04; D, 1.860.8 AU), tubulointersti-
tial fibrosis (TIFI, ND, 0.370.05; D, 2.120.5 AU) and transforming growth factor beta protein
expression (TGF- ND, 0.260.04; D, 0.460.05 AU). Treatment with raloxifene attenuated
these processes (UAE, 21.05.0 mg/day; IL-6, 31.25.0 pg/ml; GSI, 0.40.06 AU; TIFI,
0.200.04; TGF-, 0.160.02 AU). Our data indicate that raloxifene, when administered from
the onset of diabetes prevents the decline in renal function commonly observed in both humans
and animal models after longer-term diabetes. Further studies are needed to determine if in
addition to being able to attenuate the decline in renal function and structure associated with
diabetes, raloxifene can also reverse these processes and thus be used as an effective
therapeutic agent in the treatment of diabetic renal complications.
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CHBPR ConferenceLate Breaking Presentations e103
LB43
Genetic Deletion of the Angiotensin-(17) Receptor Mas in FVBN Mice
Produces a Metabolic Syndrome-like State
Sergio H Santos, Sr., Luciana R Fernandes, Jaqueline I Alvarez-Leite, Federal Univ of Minas
Gerais, Belo Horizonte, Brazil; Michael Bader, Sr., Natalia Alenina, Max-Delbruck Cntr for
Molecular Medicine, Berlin, Germany; Robson A Santos, Sr.; Federal Univ of Minas Gerais,
Belo Horizonte, Brazil
The metabolic syndrome, also known as insulin resistance syndrome, is characterized by the
variable coexistence of obesity, insulin resistance, dislipidemy and hypertension. It has been
shown that the G protein-coupled receptor, Mas, mediates many actions of angiotensin-(17).
We have recently observed that Mas-knockout male mice (Mas-/-) in the pure, FVBN genetic
background, presents elevated blood pressure levels and endothelial dysfunction, alterations
present in the metabolic syndrome. The aim of this study was to ascertain whether genetic
deletion of Mas also changes lipidic and glycemic profile. Ten weeks old Mas-/- and WT mice
were used. Curves of plasma glycemia versus time were built after intraperitoneal application
of insulin (0.75U/Kg BW) or glucose (2g/Kg BW). Despite of having normal body weight (24.7
0.35 vs 24.8 0.24 g in WT), young Mas-/- mice presented a state of insulin resistance and
glucose intolerance as well as an increase in the fasting glycemia levels (86.6 6.43 vs
56.40 4.98 mg/dl in WT). Furthermore a significant increases in total cholesterol (92.2
3.65 vs 74.6 5.67 mg/dl in WT) and triglycerides (70.6 13.3 vs 41.4 4.07 mg/dl in WT)
levels were observed. Altogether these findings indicate an important role of receptor Mas in
the cardiovascular and metabolic function in FVBN mice and suggest the angiotensin-(17)/
Mas axis as a potential target for treatment of the metabolic syndrome and its complications.
 Conference Abstracts

Hypertension. 2006;48:E25-E103
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright 2006 American Heart Association, Inc. All rights reserved.
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