Unexpected role of anticoagulant protein C in
controlling epithelial barrier integrity and
intestinal inflammation
Stefania Vetranoa, Victoria A. Ploplisb, Emanuela Salaa, Mayra Sandoval-Cooperb, Deborah L. Donahueb,
Carmen Correalea, Vincenzo Arenac, Antonino Spinellid, Alessandro Repicia, Alberto Malescia, Francis J. Castellinob,1,2,
and Silvio Danesea,1,2
a
IBD Unit, Division of Gastroenterology, Istituto Clinico Humanitas IRCCS, Rozzano 20089 Milan, Italy; bW. M. Keck Center for Transgene Research, University
of Notre Dame, Notre Dame, IN 46556; cDepartment of Pathology, Catholic University of Rome, 00168 Rome, Italy; and dDivision of General Surgery III, Istituto
Clinico Humanitas, 20089 Rozzano Milan, Italy
Edited* by Charles A. Dinarello, University of Colorado Denver, Aurora, CO, and approved October 25, 2011 (received for review May 12, 2011)
The protein C (PC) pathway is a well-characterized coagulation
system. Endothelial PC receptors and thrombomodulin mediate
the conversion of PC to its activated form, a potent anticoagulant
and anti-inflammatory molecule. Here we show that the PC path-
way is expressed on intestinal epithelial cells. The epithelial ex-
pression of PC and endothelial PC receptor is down-regulated In
patients with inflammatory bowel disease. PC−/−/PC(Tg) mice,
expressing only 3% of WT PC, developed spontaneous intestinal
inflammation and were prone to severe experimental colitis.
These mice also demonstrated spontaneous elevated production
of inflammatory cytokines and increased intestinal permeability.
Structural analysis of epithelial tight junction molecules revealed
that lack of PC leads to decreased JAM-A and claudin-3 expression
and an altered pattern of ZO-1 expression. In vitro, treatment of
epithelial cells with activated PC led to protection of tight junction
disruption induced by TNF-α, and in vivo, topical treatment with
activated PC led to mucosal healing and amelioration of colitis. Taken
together, these findings demonstrate that the PC pathway is a
unique system involved in controlling intestinal homeostasis and in-
flammation by regulating epithelial barrier function.
intestinal barrier integrity | DSS-induced colitis | intestinal permeability and
tight junction proteins
I nflammation and coagulation are closely linked, interdepen-
dent processes (1, 2). Under physiological conditions, the
molecules within the microcirculation of tissues act in an anti-
coagulant and anti-inflammatory manner. However, when in-
flammation occurs, coagulation is also triggered and participates
in the spreading of inflammation. Unexpected roles of hemo-
stasis in the humoral and cellular mechanisms of innate immu-
nity have been reported recently (2). One of the major systems
that forms a bridge between inflammation and coagulation is
the protein C (PC) pathway (3). This pathway is composed of
thrombomodulin (TM), endothelial cell PC receptor (EPCR),
PC, and protease-activated receptor-1 (PAR-1). TM and EPCR
are expressed mainly by vascular endothelial cells and form a
complex on the surface of these cells that converts circulating PC
into its active form (3).
Although the function of the PC pathway is classically con-
sidered anticoagulative, mounting evidence indicates that this
pathway also plays a dominant role in inflammation, with each
component of the pathway displaying remarkably potent anti-
inflammatory activity (2–4). Indeed, PC is now emerging as a
participant in the pathogenesis of acute and chronic inflam-
matory diseases, such as sepsis, asthma, inflammatory bowel
disease (IBD), atherosclerosis, and lung and heart inflammation,
and might represent a previously unexpected therapeutic target
for intervention (3, 5).
Crohn’s disease (CD) and ulcerative colitis (UC) are the two
major forms of IBD. Although these are immune-mediated
diseases, we and others have shown that nonimmune cells [e.g.,
19830–19835 | PNAS | December 6, 2011 | vol. 108 | no. 49
epithelial cells (ECs), vascular endothelium, platelets] and co-
agulation are deeply involved in the pathogenesis of IBD (6–10).
In particular, we recently reported that the PC pathway controls
microvascular inflammation by down-regulating endothelial
EPCR and TM expression, thereby impairing activation of PC by
the inflamed mucosal microvasculature (5, 10). Whether PC also
could be involved in intestinal barrier function or in cytopro-
tection is unclear, however (5, 10).
In this paper we report the surprising observation that, along
with its known expression in the microcirculation of the gut
mucosa, the epithelial layer of the intestine expresses proteins of
the PC pathway, which play a unique role in regulating intestinal
permeability and controlling the integrity of tight junctions. Ex-
pression of epithelial PC is altered in patients with CD or UC,
and its deletion in mice leads to spontaneous colitis. Restoring
epithelial PC is therapeutically effective in mice and could rep-
resent a novel treatment approach in humans with IBD.
Results
Expression of EPCR, PAR-1, PC, and TM by ECs from Healthy Individuals
and Patients with IBD. To investigate whether intestinal epithelium
expresses components of the PC system, we performed confocal
microscopy using specific antibodies to PC, EPCR, PAR-1, and
TM, and an antibody for pan-cytokeratin, a specific epithelial
marker, on sections of colon obtained from 16 healthy individ-
uals (Fig. 1A) (11). PC, EPCR, and PAR-1, but not TM, were
expressed by ECs in the intestine, as indicated by colocalization
with pan-cytokeratin (Fig. 1A). Staining for the same proteins in
specimens from patients with active CD (n = 12) and active UC
(n = 13) revealed a significant decrease in the epithelial ex-
pression of EPCR and PC compared with healthy individuals
(Fig. 1A). No significant difference was found in PAR-1 ex-
pression on ECs between NL subjects and patients with CD,
whereas PAR-1 was significantly down-regulated in patients with
UC compared with NL subjects and patients with CD. We
quantified protein expression in the tissue by a semiquantitative
score (10, 12). We found a significant decrease in the expression
of epithelial EPCR in tissue samples from patients with UC
(0.8 ± 0.2) and those with CD (0.8 ± 0.2) compared with mucosa
from normal individuals (2.7 ± 0.1) (P < 0.001). Similar results
were obtained for PC expression, which was significantly
Author contributions: F.J.C. and S.D. designed research; S.V., E.S., M.S.-C., D.L.D., C.C.,
V.A., A.S., A.M., and A.R. performed research; F.J.C. contributed new reagents/analytic
tools; S.V., V.A.P., and F.J.C. analyzed data; and S.V. and S.D. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1
F.J.C. and S.D. contributed equally to this work.
2
To whom correspondence may be addressed: E-mail: fcastell@nd.edu or sdanese@
hotmail.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1107140108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1107140108
A intestine, we evaluated the susceptibility to experimental colitis in
PC-deficient mice. Given that the genetic deletion of PC leads to
death soon after birth, we used PC−/−/PC(Tg) (“low-PC”) mice,
which express only 3% of the amount of PC expressed by WT
mice (13, 14). When mice (6–8 wk old) underwent colonoscopy,
low-PC mice had an endoscopic colitis score indicating the
presence of spontaneous colitis characterized by mucosal hyper-
emia and friability (P < 0.05) (Fig. 2 A and B). The degree of
colitis increased after administration of dextran sodium sulfate
(DSS), with more significant colon damage seen in low-PC mice
(P < 0.05). Bleeding and the presence of mucosal erosions that
were absent in WT mice characterized this damage.
In addition, minimal doses of DSS were sufficient to induce
significant clinical colitis in low-PC mice, characterized by a sig-
nificant loss in body weight and a progressive increase in the
overall disease activity index (Fig. 2 C and D). Indeed, admin-
istration of 3% DSS led to 100% mortality in low-PC mice after
3–4 d of administration.
After mice were killed, the low-PC mice exhibited spontane-
ous intestinal inflammation characterized by the presence of
B
A B

Fig. 1. Decreased expression of components of the PC pathway in intestinal

epithelial cells of patients with IBD. (A) Immunofluorescent staining shows
the expression and colocalization of EPCR, PC, and PAR-1, but not of TM (in
green) with the EC marker pan-cytokeratin (in red) in normal subjects (NL).
Marked reductions in EPCR, PC, and PAR-1 were detected in the colon of
C D
patients with active Crohn’s disease (CD) and patients with active ulcerative
colitis (UC) with DAPI nuclear stain (blue). The images were acquired with an
oil immersion objective (60×, 1.4 NA Plan-Apochromat; Olympus). (B) Rela-
tive changes in mRNA expression of EPCR, PC, PAR-1, and TM relative to
GAPDH in NL subjects (n = 10), patients with CD (n = 6), and patients with UC
(n = 6). *P < 0.05.

decreased in the epithelium of patients with UC (0.5 ± 0.1) and

those with CD (0.6 ± 0.2) compared with mucosa from control
individuals (2.4 ± 0.1) (P < 0.001). Furthermore, TM was not
expressed by the intestinal epithelium. No decrease in PC and E F
EPCR staining was found in specimens from patients in an in-
active disease state.
We next analyzed the expression of the PC pathway in primary
ECs by FACS analysis. ECs in healthy subjects expressed PC,
EPCR, and PAR-1. In patients with IBD, PC and EPCR expres-
sion was decreased by 47%, and PAR-1 expression was decreased
by 30% (Fig. S1). These results suggest that inflammation down-
regulates the expression of EPCR and PC on ECs within
the intestine.
We also investigated the expression of EPCR, PAR-1, PC, and
TM by RT-PCR. Results from primary ECs isolated from in-
testinal tissue of healthy subjects and patients with active IBD Fig. 2. Spontaneous inflammation and increased susceptibility to DSS-in-
confirmed down-regulation of mRNA for EPCR, PC, and PAR-1 duced colitis in low-PC mice. (A) Endoscopic images of mucosal damage in
in patients with active CD or UC. The expression of mRNA for the colon of WT and low-PC mice before (day 0) and after 7 d (day 7) of 2%
DSS administration demonstrates the spontaneous mucosal hyperemia and
TM was undetectable (Fig. 1B). No decrease in PC or EPCR
friability in low-PC mice and the presence of mucosal erosions at day 7, as
mRNA was found in ECs from patients with quiescent IBD. indicated by the white arrows. (B) Quantification of mucosal injury by en-
Taken together, these results demonstrate that the intestinal
SYSTEMS BIOLOGY

epithelium of healthy subjects expresses PC and EPCR, and that
this expression is significantly reduced in the inflamed intestines
of patients with active IBD.
Spontaneous Colitis in PC-Deficient Mice. To study the consequences
of the decreased expression of epithelial PC in the inflamed
doscopic score in WT and low-PC mice before and after treatment. (C and D)
Trends toward greater body weight loss (C) and disease activity index (DAI)
(D) in low-PC mice compared with their littermates over the course of DSS
treatment. (E) Red arrows indicate the presence of small lymphoid follicles at
day 0 and increased leukocyte infiltrate at day 7 in the mucosa of low PC
mice. (F) Histological damage scoring at day 0 and day 7 in low-PC and WT
mice. Histological images were taken using a 20× objective.

Vetrano et al. PNAS | December 6, 2011 | vol. 108 | no. 49 | 19831

small lymphoid follicles in the mucosa and submucosa mainly in
the colon but not in the small bowel (Fig. 2 E and F). Moreover,
after DSS challenge, the mucosa of low-PC mice displayed a
significantly higher degree of histological inflammation (P <
0.05), characterized by total crypt loss and diffuse leukocyte in-
filtration that was not observed in WT mice (Fig. 2 E and F). The
finding that deletion of PC leads to spontaneous colitis unveils
a role for PC in intestinal inflammation.
Increased Mucosal Cytokine Production and Intestinal Permeability in
PC-Deficient Mice. We next measured the mucosal production of
various inflammatory mediators, including IL-6, IL-8 (KC), and
macrophage inflammatory protein 2. Consistent with our histo-
logical findings indicating the presence of spontaneous in-
flammation, low-PC mice displayed a significant increase in the
spontaneous production of all inflammatory mediators compared
with WT mice (all mediators; P < 0.05), even before DSS ad-
ministration. These differences were maintained over 7 d of DSS
administration (Fig. 3A).
Given that PC is expressed by ECs, which function to maintain
an intact intestinal barrier, we hypothesized that PC could play
a key role in this process. Thus, we investigated paracellular
epithelial permeability in low-PC and WT mice before and after
DSS administration by measuring transepithelial electrical re-
sistance (TEER) using a Ussing chamber and the Evans blue dye
test. TEER was monitored as an indicator of epithelial barrier
integrity. Low-PC mice demonstrated a spontaneous increase in
intestinal permeability, with significantly lower values of TEER
(28.66 ± 2 Ω/cm2) compared with WT mice (88 ± 2 Ω/cm2)
before DSS challenge (P < 0.0001) (Fig. 3B). After 3 d of DSS
administration, epithelial permeability was still significantly
higher in WT mice (45.9 ± 3.74 Ω/cm2) compared with low-PC
mice (29.85 ± 3.66 Ω/cm2) (P = 0.0005). After 7 d of DSS ad-
ministration, TEER values were markedly diminished in both
groups of mice, with no significant differences (Fig. 3B). Taken
together, these data indicate a leaky intestinal barrier in low-
PC mice.
Epithelial permeability is governed by tight junctions, and
alterations in the expression of the proteins forming these
junctions might lead to a leaky intestinal barrier (15). Thus, we
investigated the expression levels of the tight junction proteins
claudin-3, ZO-1, JAM-A, and claudin-10 in low-PC and WT
mice. Consistent with the observed intestinal permeability, low-
PC mice showed a significant decrease in the expression of
claudin-3 and JAM-A compared with WT mice (P < 0.05) (Fig.
A B
C D
19832 | www.pnas.org/cgi/doi/10.1073/pnas.1107140108
3C), a finding that could explain the leaky intestinal barrier in
low-PC mice. In addition, the expression pattern of ZO-1 was
significantly altered and irregular in low-PC mice compared with
WT mice (Fig. 3C). There was no between-group difference in
claudin-10 expression (Fig. 3C). In addition, there was no be-
tween-group difference in the overall expression of tight junc-
tions after 7 d of DSS-induced colitis.
PC Controls Epithelial Tight Junction Integrity, Wound Healing, and EC
Proliferation. Increased leakiness of the intestinal barrier is con-
sidered a crucial event in the pathogenesis of IBD, and TNF-α
actively contributes to the disruption of barrier function by
down-regulating epithelial tight junctions. To investigate the role
of PC in epithelial barrier function, we first analyzed the ex-
pression of the PC pathway in a monolayer of the colonic EC line
(Caco-2) by immunofluorescence. Similar to primary ECs, Caco-
2 cells expressed PC, PAR-1 and EPCR, but not TM (Fig. S2).
We then cultured Caco-2 cells with and without stimulation by
TNF-α in the absence and presence of exogenous activated PC
(aPC). Changes in TEER were measured over 48 h. As expected,
a decrease in TEER was observed after 48 h of stimulation with
TNF-α (t = 0: 304 ± 23.31 Ω/cm2; + TNF, t = 48: 78.75 ± 6.57
Ω*cm2) (Fig. 4A). However, the addition of exogenous aPC to-
tally abrogated the effects of TNF-α on TEER (TNF + aPC, t =
48: 381.8 ± 26.20 Ω*cm2; P < 0.05). Notably, when the cells were
treated only with aPC an increase of TEER was observed com-
pared with nontreated cells (+ aPC, t = 48: 552 ± 34.5 Ω*cm2;
control, t = 48: 304.8 ± 23.31 Ω*cm2) (Fig. 4A). To test whether
the protective effect observed with aPC was dependent on EPCR
signaling, we performed the same experiments in the presence
and absence of anti-EPCR antibodies. The protective effects of
aPC disappeared in the presence of anti-EPCR antibodies,
suggesting that aPC exerts a direct effect on paracellular epi-
thelial permeability in an EPCR-dependent manner. No effects
on TEER values were seen in the presence of anti-EPCR alone.
Because the aPC–EPCR complex cross-activates sphingosine 1
phosphate (S1P), a signaling sphingolipid that promotes in-
creased endothelial barrier protection, we investigated whether
aPC induces the potent epithelial barrier protective effects via
S1P activation. To assess this hypothesis, we evaluated the
changes in TEER values in Caco-2 cells after 0, 6, and 12 h of
stimulation with S1P (1 μM). Interestingly, S1P not only en-
hanced TEER values similarly to APC (1 μg/mL), but also ab-
rogated TNF-dependent TEER reduction (Fig. S3A). These data
support the evidence suggesting the involvement of S1P in the
Fig. 3. Changes in mucosal cytokine
production and the integrity of intestinal
barrier function in low-PC mice. (A and B)
Secretion of IL-6, KC, and macrophage
inflammatory protein 2 by the mucosa of
the colon (A) and TEER measured from
the colon (B) of low-PC and WT mice on
days 0, 3, and 7 of DSS administration. (C
and D) Immunofluorescent staining and
bar graph showing the expression of
JAM-A, CL-3, CL-10, and ZO-1 tight junc-
tion proteins (green) in the colon of low-
PC and WT mice before DSS administra-
tion (day 0); nuclei were stained with
DAPI (blue). (Scale bar: 30 μm.) Values
represent mean ± SEM; n = 13 for WT
mice and n = 20 for low-PC mice. These
data are representative of three indepen-
dent experiments. *P < 0.05; **P < 0.01.
Vetrano et al.
A regulation of tight junctions (Fig. 4 B and C), whereas the stim-
B a scratch in the monolayers. Significant wound closure was seen
C D
Fig. 4. Protective effects of aPC on epithelial barrier function are mediated
by EPCR. Confluent Caco-2 cells were treated without (control) and with
TNF-α (25 ng/mL) in the presence (TNF aPC) or absence of aPC (1 μg/mL), and
with (TNF aPC EPCR) or without (aPC EPCR) a 1-h pretreatment with the
antibody inhibiting EPCR activity (5 μg/mL). (A) Values of TEER were mea-
sured over 48 h of stimulation. (B–D) After 48 h of stimulation, immuno-
fluorescence analysis of JAM-A and ZO-1 (in green) was performed on Caco-2
cells grown on Transwell filters under different conditions: control, TNF,
TNF + aPC, aPC, TNF + aPC + EPCR, and aPC + EPCR; DAPI nuclear stain
(blue). (Scale bar: 60 μm.) Values are mean ± SEM; the data are represen-
tative of three independent experiments. θP < 0.05 (aPC vs. control); ζP <
0.05 (control vs. TNF and TNF aPC EPCR); φP < 0.01(aPC vs. TNF and TNF aPC
EPCR); *P < 0.01.
epithelial barrier protection. It has been reported that once
formed after the phosphorylation of sphingosine by enzyme
sphingosine kinase (SphK1 and SphK 2), S1P regulates the en-
dothelial barrier function mediating the S1P1 receptor. Because
the Caco-2 cell line expresses S1P1, SphK1, and SphK2 mRNA
(Fig. S3 B and C), we verified whether the effects of aPC on
TEER were S1P1-dependent. After 5 h of pretreatment with
FTY720 (1 μM), which is known to inhibit the S1P1 receptor, the
Caco-2 cells were stimulated with and without TNF-α (25 ng/mL)
in the presence or absence of aPC (1 μg/mL) for 0, 6, and 12 h.
FTY720 treatment displayed no inhibitory effects on aPC ac-
tivity. Indeed, aPC in the presence of FTY720 significantly ab-
rogated (P < 0.01) TNF-dependent TEER values (Fig. S3D). No
differences were observed between control and FTY720 alone.
These results suggest that aPC-enhanced epithelial barrier pro-
tection is S1P1-independent.
To further explore the mechanism by which aPC reinforces
epithelial barrier function, we investigated the expression levels
of the major tight junction proteins in EC lines in the presence
and absence of aPC and before and after stimulation with TNF-α.
Consistent with the in vivo findings in low-PC mice, we found that
aPC inhibits the altered expression of JAM-A and ZO-1 induced
by TNF-α (Fig. 4 B and C). Furthermore, the use of anti-EPCR
antibodies totally inhibited such modification of tight junctions,
suggesting a functional role for EPCR in aPC-mediated
Vetrano et al.
ulation with α-EPCR alone did not alter the expression of JAM-
A or ZO-1. Along with their crucial role in formation of tight
junctions to maintain the epithelial barrier, ECs are able to ac-
tively respond to injury by migrating when wounds occur, and to
heal the disrupted barrier. Thus, we tested whether aPC can in-
duce intestinal wound closure and cell proliferation. To in-
vestigate wound closure, we seeded Caco-2 cells onto plates in
designated areas in the absence or presence of exogenous aPC (1
μg/mL) and monitored cell migration for 48 h after creation of
in the presence of a PC compared with the control, at a level
comparable to that induced by epithelial growth factor (EGF; 50
ng/mL) (Fig. S4 A and B). Pretreatment with α-EPCR antibody
(5 μg/mL) 1 h before the stimulation decreased epithelial wound
closure, supporting the suggestion of a functional role for EPCR
in aPC-mediated epithelial migration (Fig. S4 A and B). Finally,
a [3H]-thymidine incorporation assay revealed that a low con-
centration of aPC (1 μg/mL) induced a high rate of Caco-2 cell
proliferation, comparable to that induced by EGF (50 ng/mL).
The cells pretreated with α-EPCR and stimulated with aPC dis-
played significantly reduced cellular proliferation. Importantly,
pretreatment with α-EPCR alone did not induce any effects on
ECs, reinforcing the hypothesis that aPC signals through EPCR
(Fig. S4C).
Topical aPC Treatment Leads to Improvement of Colitis. Mucosal
healing is believed to be an important target for the treatment of
IBD (16). To explore the therapeutic potential of our in vitro
findings, we investigated whether the intrarectal administration
of aPC would exert a therapeutic effect in mice with experi-
mental colitis. We began by investigating the expression of epi-
thelial PC. Similar to human samples, the ECs of healthy WT
mice expressed high but physiological levels of PC. In contrast, in
mice developing colitis induced by DSS administration, a signif-
icant decrease in PC expression was detected by day 3 and per-
sisted to the end of the experiment (Fig. 5A). Because intestinal
inflammation leads to down-regulation of epithelial PC expres-
sion, we investigated whether topical intrarectal administration
of aPC could improve colitis by favoring epithelial healing. Mice
receiving aPC recovered more rapidly from colitis, as indicated
by a significant increase in body weight compared with saline-
treated mice (P < 0.05) (Fig. 5 A and B). Endoscopically, ad-
ministration of aPC significantly ameliorated mucosal damage
(Fig. 5 C and D), and the mice that received intrarectal aPC
demonstrated significantly less colon shortening compared with
saline-treated mice (P < 0.05) (Fig. S5). Finally, quantification of
mucosal inflammation revealed that aPC-treated mice had a
significantly less severe histological score, characterized by a de-
creased number of ulcers (Fig. 5E). This finding suggests that
aPC has a beneficial and therapeutic effect. Taken together,
these data support the potential therapeutic efficacy of topical
aPC in intestinal inflammation.
Discussion
Inflammation is the result of an imbalance between proinflam-
matory and anti-inflammatory pathways. In the last several years,
it has become clear that inflammation involves unique pathways
that are not classically acknowledged as inflammatory systems,
for example, pathways involved in hemostasis (1, 2). Evidence
supporting this hypothesis has pointed to the involvement of the
coagulation cascade in inflammatory processes, and it has been
firmly established that coagulation and inflammation are in-
terrelated processes, which is particularly true in IBD (8).
The specific hemostasis-related molecules involved in inflam-
SYSTEMS BIOLOGY
mation are only now beginning to be elucidated. The PC pathway
is a major pathway bridging inflammation and coagulation (3).
Mounting evidence also indicates that the PC pathway plays a
dominant role in inflammation, with each component of the
pathway displaying remarkably potent anti-inflammatory activity
(3, 17, 18). Indeed, TM, EPCR, PAR-1, and aPC, individually
PNAS | December 6, 2011 | vol. 108 | no. 49 | 19833

and together, have emerged as crucial controllers of inflammation
in multiple organs, suggesting the potential for development of
novel therapeutic approaches for a wide range of inflammatory
disorders (3, 17, 18). Recently, the PC pathway was suggested to
be involved in endothelial barrier integrity and in cytoprotection
(18). However, up to now, expression of the proteins of the PC
pathway and their roles in controlling intestinal barrier integrity
and cytoprotection, let alone the role of PC in the pathogenesis
of the two major forms of IBD, have not been investigated. In
patients with CD and UC, alterations of the coagulative system
and of the cells involved in coagulation play important roles in
the intestinal immune response (7, 8). In particular, the PC
pathway has been implicated in the control of microvascular
inflammation of the intestine (5, 10).
In this paper we report the surprising finding that beyond its
classical role in vascular biology, PC also acts as an unexpected
mediator of intestinal epithelial barrier function. We demon-
strate that the intestinal epithelial layer expresses the PC path-
way, and that the expression of PC system is lost in patients with
active IBD, whereas no changes in the PC pathway were found in
ECs from patients in an inactive disease state. These findings
suggest that inflammation leads to dysregulation of PC system
components in intestinal ECs, similar to what is seen in intestinal
endothelial cells (10). However, consistent with these human
results, we also found a down-regulation of epithelial PC in mice
during DSS-induced colitis. The altered expression of EPCR and
TM impairs the conversion of PC into its active form, aPC, which
has been widely demonstrated to exert anti-inflammatory and
cytoprotective properties (3, 10). Interestingly, we found that
intestinal ECs are positive for all PC pathway components except
TM, a protein crucial for aPC conversion. Therefore, these data
suggest that although the epithelium expresses EPCR, PC, and
PAR-1, it does not provide an appropriate microenvironment for
aPC production. It is plausible to hypothesize that ECs
expressing EPCR and PAR-1, even if they synthesize PC, must
obtain aPC from other types of cells. Previous reports have de-
scribed the interactions between immune mucosal cells and ECs
that contribute to intestinal homeostasis and intestinal epithelial
permeability (19), indicating the likely presence of cross-talk be-
tween ECs and other cells expressing TM. However, a TM-soluble
form, derived from the endothelial membrane, is released in
plasma, but whether this form is functional is unknown (20).
Why the ECs express the inactive form of PC but are not able
to covert it remains unclear. Once activated, aPC plays an
19834 | www.pnas.org/cgi/doi/10.1073/pnas.1107140108
Fig. 5. Exogenous administration of aPC
ameliorates recovery of intestinal mucosa in
DSS-induced colitis. (A) Immunohistochemi-
cal staining of PC on sections of colon
from WT mice before and after adminis-
tration of 3% DSS. Arrows indicate the
strong expression of PC on ECs in healthy
colon and its dramatic reduction after DSS
treatment. (B) After 8 d of DSS administra-
tion, WT mice were divided into two
groups: One group received saline, and the
other group received recombinant murine
aPC (100 μg/kg) intrarectally in a volume of
80 μL using a straight gavage needle every
other day after colitis was established. Loss
of body weight was monitored daily during
treatment. (C and D) Endoscopic images and
evaluation of mucosal damage in the colons
of healthy and saline- and aPC-treated mice
on day 16. (E) Evaluation of the number of
ulcers on day 16 after topical aPC or saline
treatment. Values are mean ± SEM; n = 6
mice for all groups. These data are repre-
sentative of two independent experiments.
*P < 0.05; ** P < 0.01. Images were taken
using a 20× objective.
important role in governing intestinal homeostasis by inhibiting
the release of proinflammatory cytokines and limiting leukocyte
adhesion (10). When PC activation is impaired, as in low-PC
mice, the intestinal homeostasis is altered, and the transgenic
mice develop spontaneous intestinal inflammation and a more
pronounced experimental colitis phenotype after challenge with
DSS, reinforcing the evidence that the low PC levels in these
mice induce a proinflammatory state, as also indicated by the
high levels of proinflammatory cytokines at baseline. Low-PC
mice, besides being associated with high mortality and pro-
thrombotic and proinflammatory phenotypes, display altered
intestinal barrier function. Tight junction proteins strictly control
intestinal permeability, and their alteration can lead to sponta-
neous intestinal inflammation. We found that low-PC mice had
altered or reduced expression of ZO-1, JAM-A, and claudin-3,
three tight junction proteins required for intestinal barrier
function. Most notable was the down-regulation of JAM-A, a
molecule that we recently identified as a crucial mediator of
intestinal barrier permeability (11, 21). In addition, consistent
with the in vivo findings, aPC had a strong effect on tight junc-
tions in vitro; its presence was able to inhibit the altered ex-
pression of ZO-1 and JAM-A induced by TNF-α. As expected,
TNF-α stimulation induced an increase in epithelial permeabil-
ity, but notably, this effect was abrogated by recombinant aPC.
This indicates that aPC controls the intestinal barrier function
enhancing the expression of tight junction proteins. It has been
reported that most of the cytoprotective effects of aPC are me-
diated by EPCR. In the present study, we have demonstrated
that the aPC’s beneficial protective effects in controlling in-
testinal barrier function are EPCR-dependent, suggesting that
EPCR expressed by the epithelium is functionally active and is
essential for the regulation of cellular permeability.
The protective effects of aPC on endothelial barrier integrity
are mediated through its interaction with EPCR and the sub-
sequent cleavage and activation of protease-activated receptor-1
(PAR-1) (3). The aPC/EPCR/PAR-1 complex cross-activates
S1P, a signaling sphingolipid. It has been reported that S1P is
involved in regulating the proliferation, survival, differentiation,
and migration of many types of cells, including endothelial cells
(22, 23). Furthermore, a recent study indicated that S1P regu-
lates the function of the intestinal epithelial barrier by altering
the expression of E-cadherin, which is an adherens junction (24).
That study demonstrated that ECs express S1P and that the
signaling pathway of S1P is involved in maintaining the integrity
Vetrano et al.
of the epithelial barrier, but it did not address the mechanism of
these effects. The cell-signaling properties of aPC are known to
enhance vascular endothelial barrier function via the S1P1 re-
ceptor. Although the level of S1P1 mRNA expression was very
low in Caco-2 cells, we tested whether the aPC-dependent in-
testinal barrier protective effects were mediated by S1P1. Both
S1P and aPC stimulation exerted similar effects, completely
abrogating the TNF-dependent decrease in TEER, suggesting
that the cytoprotective effects of aPC that we observed in ECs
can be mediated by S1P signaling; however, the inhibition of
S1P1 using FTY720 did not abolish the effect of aPC. FTY720 is
an immunomodulator drug that has been proven to induce in-
ternalization of S1P1 in endothelial cells after 60 min of treat-
ment (25). Here we pretreated the cells with FTY720 for 5 h
before aPC stimulation, but found no difference in TEER values
in the presence or absence of FTY720. It is plausible to suppose
that aPC in ECs activates S1P signaling, but not via S1P1. S1P1
receptor is not a unique receptor for S1P ligand; both S1P2 and
S1P3 are expressed by ECs at higher levels than S1P1 (22). Thus,
further studies are needed to provide evidence of the involve-
ment of S1P receptors in aPC effects.
To maintain intestinal homeostasis, the epithelium must be
able to proliferate and migrate after injury, especially when in-
flammation occurs. Evidence points to the capacity of aPC to
stimulate proliferation, migration, and wound closure in human
keratinocytes (26). In particular, aPC was found to increase
keratinocyte proliferation in a dose-dependent manner. Thus, we
investigated whether aPC promoted this function in intestinal
ECs as well. We found that at low concentrations, aPC accel-
erated wound closure and induced proliferation of ECs similarly
to EGF, in an EPCR-dependent manner.
Because aPC promotes wound closure and intestinal epi-
thelial proliferation, it is plausible that the prolonged healing
process seen in patients with UC and CD could be at least
partially dependent on the reduced expression of PC and
EPCR by the intestinal epithelium. Thus, topical administra-
tion of aPC could promote mucosal healing. A clinical study of
four patients found that treatment of chronic leg ulcers with
topical aPC stimulated wound healing by activating skin ree-
pithelialization (27). Furthermore, the local aPC treatment was
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the capacity of aPC treatment to favor mucosal healing. Indeed,
mice treated with intrarectal injection of aPC every other day
after colitis was established displayed faster recovery of body
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intrarectal route could provide a safer route of administration.
In conclusion, our study provides evidence of an unexpected
function of the anticoagulative PC pathway in the intestine. The
PC pathway controls the process of mucosal homeostasis that is
mediated by ECs through the maintenance of barrier function
and cytoprotection. Altered PC expression in the epithelium of
the gut leads to spontaneous intestinal inflammation, a pre-
viously unrecognized component of the pathogenesis of IBD.
Restoring the function of the PC pathway in intestinal epithe-
lium by acting on a molecule classically recognized as an anti-
coagulant may help guide the development of new treatments for
IBD and mucosal inflammation.
Materials and Methods
Patients. Intestinal tissues were obtained from surgical specimens taken from
patients with active CD or UC. Healthy areas of intestine taken from patients
admitted for bowel resection because of polyps or diverticulosis were used as
controls. Specimens were formalin-fixed and paraffin-embedded or frozen in
Cryoblock Compound (DiaPath) on dry ice and stored at −80 °C. Human studies
were approved by the Ethical Committee of Istituto Clinico Humanitas.
Detailed information is provided in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Sarah A. De La Rue of Readable Science for
her assistance with the manuscript. This study was supported by grants from
Innovative Medicines Initiative (IMI) (115142), Ministero della Salute (GR-
2007 convenzione 23), Fondazione Cariplo (Grant 2010-0733), and Associa-
zione Italiana Ricerca Cancro (IG-2010 10205) to S.D.; IBD Research Founda-
tion (mini Grant 2010), European Crohn’s and Colitis Organization (ECCO)
(Grant 2009), and Ministero della Salute (GR -2009 convenzione 76) to S.V.;
and National Institutes of Health Grant HL019982 (to F.J.C.).
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PNAS | December 6, 2011 | vol. 108 | no. 49 | 19835