PR 100937 A

Article
The secretome of human endothelial cells under shear stress

Sandra Burghoff, and Jürgen Schrader
J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr100937a • Publication Date (Web): 26 December 2010
Downloaded from http://pubs.acs.org on January 26, 2011
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Page 1 of 35 Journal of Proteome Research
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9 The secretome of human endothelial cells under shear stress
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16 Sandra Burghoff* and Jürgen Schrader
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18 Institute for Cardiovascular Physiology, Heinrich Heine University Duesseldorf, Germany
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Corresponding Author:
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35 Dr. Sandra Burghoff
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37 Heinrich Heine University Duesseldorf
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Institute for Cardiovascular Physiology
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42 Universitaetsstr. 1
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44 40225 Duesseldorf
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Tel.: +49-211/8112671
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49 Fax: +49-211/8112672
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51 E-Mail: sandra.burghoff@uni-duesseldorf.de
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Journal of Proteome Research Page 2 of 35
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3 Abstract
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6 Endothelial cells are exposed to different types of shear stress which triggers the secretion of
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8 subsets of proteins. In this study we analyzed the secretome of endothelial cells under static,
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laminar and oscillatory flow. To differentiate between endogenously expressed and added
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13 proteins, isolated human umbilical vein endothelial cells were labelled with L-Lysine-
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15 C6,15N2 and L-Arginine-13C6,15N4. Shear stress was applied for 24 hours using a cone-and-
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plate viscometer. Proteins from the supernatants were isolated, trypsinized and finally
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20 analyzed using LC-MS/MS (LTQ). Under static control condition 395 proteins could be
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22 identified, of which 78 proteins were assigned to the secretome according to SwissProt
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25 database. Under laminar shear stress conditions 327 proteins (83 secreted) and under
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27 oscillatory shear stress 507 proteins (79 secreted) were measured. We were able to identify 6
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29 proteins specific for control conditions, 8 proteins specific for laminar shear stress and 5
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32 proteins specific for oscillatory shear stress. In addition, we identified flow specific secretion
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34 pattern like the increased secretion of cell adhesion proteins and of proteins involved in
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protein binding. In conclusion, the identification of shear stress specific secreted proteins (101
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39 under different flow conditions) emphasizes the role of endothelial cells in modulating the
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41 plasma composition according to the physiological requirements.
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51 Keywords
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53 Endothelial cells, shear stress, LC-MS/MS, secretome, laminar flow, oscillatory flow
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3 Introduction
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6 The vascular endothelium forms a multifunctional, dynamic interface that is constantly
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8 exposed to wall shear stress generated by flowing blood. Blood flow in addition, regulates the
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internal diameter of arterial vessels both acutely, by relaxation and contraction of smooth
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13 muscle cells, and chronically by vascular remodeling of cellular and extracellular
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15 components. This regulation involves modulation of membrane proteins and ion channels,
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activation of transcription factors, cellular reorganisation and change of cell shape. These
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20 responses are accomplished within seconds to hours and may be mechanistically important in
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22 the pathogenesis of vascular diseases such as atherogenesis1;2.
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25 Several proteins have been described to be differentially responsive to fluid shear stress.
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27 Topper et al. described mRNA up-regulation for manganese superoxide dismutase (Mn SOD),
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29 cyclooxygenase (COX)-2 and endothelial nitric oxidase (NO) synthase (eNOS) by steady
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32 laminar shear stress2. In further studies two members of the MAD family, namely Smad6 and
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34 Smad7, and the bumetanide-sensitive cotransporter BSC2, one of the two major isoforms of
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Na-K-Cl cotransporters present in mammalian cells were identified to be up-regulated upon
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39 laminar shear stress3;4. Whether expression of endothelin-1 is altered upon shear stress is an
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41 ongoing discussion5.
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The identification of not just a few but of all secreted proteins (secretome) from cells is still a
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46 major challenge and advanced approaches are inevitable. Using mass spectrometry analysis
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48 Dupont et al. succeeded in the identification of 18 different proteins that were secreted from
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51 human arterial smooth muscle cells6. The identified proteins are involved in a broad range of
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53 biological functions like regulation of fibrinolysis (Plasminogen activator inhibitor-1),
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55 proteolysis (collagenase), ion transport (serotransferrin) and others. In vascular smooth
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58 muscle cells the heat shock protein 90-α and cyclophilin B were identified as secreted factors
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60 after oxidative stress induction7. Again, Dupont et al. established the first secretome of human
macrophages comprising 38 proteins8. Among the identified proteins known secreted proteins
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3 (e.g. serotransferrin, vimentin) but also proteins not known to be secreted (e.g.
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6 Glyceraldehyde 3-phosphate deshydrogenase (GAPDH), transaldolase) were detected. Using
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8 an in vitro “foam cell” model of atherosclerosis 59 proteins in the supernatant could be
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identified whose secretion was increased upon oxidized LDL treatment in comparison to
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13 treatment with LDL9. In the search for potential biomarkers using atherosclerotic plaque
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15 secretome Duran et al. were able to identify 14 proteins in the supernatant of noncomplicated
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plaques10. Again, some of the identified proteins were expected (e.g. apolipoproteins) whereas
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20 the identification of others cannot be easily understood (e.g. ubiquitin carboxy-terminal
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22 hydrolase). Still in another study more than 80 proteins differentially expressed in
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25 complicated atherosclerotic plaques versus adjacent fibrous plaques could be identified11.
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27 Again, the identified proteins cover a wide range of biological functions like signal
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29 transduction, transcription factors, cell communication and protein transporters. In addition,
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32 Tunica et al. identified 182 human proteins in the supernatant of human umbilical vein
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34 endothelial cells under static conditions of which 70 are known to be secreted12. Again, in this
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study the identified proteins are wide-spread in function.
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39 The proteomic analysis of endothelial cells in response to shear stress is still in its infancies.
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41 Wang et al. revealed that 142, 213, and 186 candidate proteins in vascular endothelial cells
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were up- or down-regulated after 10 min, 3 h, and 6 h of laminar flow13. These intracellular
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46 proteins encompass many signalling pathways like integrins, PI3K/AKT, apoptosis, Notch
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48 and cAMP-mediated pathways. Whereas this study investigated the intracellular proteom so
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51 far no study has been undertaken in which the secretome of endothelial cells in response to
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53 shear stress was investigated.
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55 In the present study, we used LC-MS/MS to qualitatively profile the secretome of human
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58 umbilical vein endothelial cells (HUVECs) in response to physiological levels of laminar and
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60 oscillatory shear stress. To avoid bias with serum proteins during mass spectrometry analysis
all proteins expressed by HUVECs were labelled with heavy isotope amino acids. We
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3 identified 395 proteins after no flow conditions, 327 proteins after 24 hours of steady laminar
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6 flow and 507 proteins after 24 h of oscillatory flow. These results provide the first
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8 comprehensive analysis of secreted proteins which are flow specific and this will open the
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way to study the role these proteins in shear stress mediated development of vascular disease
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13 such as atherosclerosis.
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3 Materials and Methods
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6 HUVEC isolation and labelling
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8 HUVECs were harvested by collagenase (Biochrom AG, Berlin, Germany) and cultured to
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subconfluency in 4 ml Basal Medium supplemented with endothelial single quots (PromoCell,
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13 Heidelberg, Germany) on gelatine-precoated 60-mm culture dishes.
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15 For cell labelling Basal Medium and single quots without arginine and lysine were used
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(PromoCell, Heidelberg, Germany) supplemented with 0.3 mmol/l L-Arginine-13C6,15N4
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20 hydrochloride and 1 mmol/l L-Lysine-13C6,15N2 hydrochloride (Sigma, Taufkirchen,
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22 Germany). Freshly isolated HUVECs were grown for 5 cell doublings using this medium.
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27 Application of shear stress
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29 HUVECs (with 4 ml medium) were subjected to laminar shear stress at 15 dyne/cm2 and
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32 oscillatory shear stress (8 dyne/cm2) in a cone-and-plate viscometer for 24 hours one day after
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34 reaching subconfluency as described previously . The viscometer consists of a cone with
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an angle of 0.5° rotating on top of a cell culture dish. A control dish (no shear stress)
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39 accompanied each cell culture dish from the same HUVEC preparation. Each experiment was
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41 carried out in duplicate.
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46 Analysis of proteins
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48 Supernatant (4 ml) after shear stress application was depleted of cell debris by
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51 ultracentrifugation (150,000×g; 2 hours, 4°C). Soluble proteins were bound to StrataClean™
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53 Resin (Stratagene, La Jolla, USA) for 1 hour at 8°C. Resins with bound proteins were
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55 transfered into SDS-loading buffer (156.25 mM Tris-HCl pH 6.8, 2.3% (w/v) SDS, 4% (v/v)
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58 glycerol, 0.5% (v/v) 2-mercaptoethanol), boiled (5 min, 95°C) and then subjected to gradient
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60 SDS-PAGE (6-18%). Each lane was divided into 20 fragments. The proteins within the gel
fragments were in-gel-trypsinized at 37°C overnight. In order to extract the peptides, 80 µl
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3 50 mM (NH4)CO3, and 2× 100 µl 50% (v/v) acetonitril, 5% (v/v) formic acid were added
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6 successively to the gel pieces. The volume of the extraction solution was reduced to <20 µl
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8 using a SpeedVac.
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13 Chromatographic separation of peptides and MS/MS analysis
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15 Peptides were separated by C18 reversed phase hydrophobic interaction chromatography
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using a C18 PepMap100 column (3 µm, 100 Å; Dionex, Idstein, Germany) on an Ettan
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20 microLC system (Amersham Biosciences, Freiburg im Breisgau, Germany). The two buffer
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22 solutions used were 4% (v/v) acetonitrile, 0.012% (v/v) heptafluorobutyric acid (HFBA),
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25 0.5% (v/v) acetic acid (solution A) and 80% (v/v) acetonitrile, 0.012% (v/v) HFBA, 0.5%
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27 (v/v) acetic acid (solution B). Peptides were directly eluted into the mass spectrometer with a
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29 linear gradient from 5-60% solution B for 140 min, 60-99% solution B for 5 min, and 99%
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32 solution B for 5 min. The flowrate of 100 µl/min at the HPLC was reduced to 200 nl/min by
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34 an integrated flow splitter.
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Mass spectrometric measurements were done with a Finnigan LTQ ion trap mass
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39 spectrometer equipped with a nanospray ionization source (Thermo Finnigan, Langenselbold,
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41 Germany). A spray voltage of 2.0 kV was applied to the column and continous cycles of one
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full scan (m/z 400-1,600) followed by four data-dependent MS/MS measurements at 35%
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46 normalized collision energy were performed. Operation of the Ettan microLC and the mass
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48 spectrometer was fully automated during the entire process using Unicorn (Amersham
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51 Biosciences) and Xcalibur 2.0 (Thermo Finnigan software), respectively.
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53 MS/MS spectra were sequence database searched against the human International Protein
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55 Index (IPI) database (version 3.18) from the European bioinformatics institute using the
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58 SEQUEST algorithm. The search parameters used included trypsin specificity, a peptide mass
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60 tolerance of 2.0, +8.0 for lysine modification and +10.0 for arginine modification. Differential
modifications were set to +79.966331 for serine, threonine and tyrosine, +57.021464 for
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3 cysteine and +15.994920 for methionine. In order to obtain reliable protein identification,
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6 only peptides with a minimal cross-correlation score (Xcorr) of 1.8, 2.5, and 3.5 for charge
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8 states +1, +2, and +3, respectively, were taken into account. The DeltaCN had to be >0.1. For
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positive identification of a protein at least 2 peptides per protein had to meet these restrictions.
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13 Expasy web server (http://www.expasy.org/sprot) was used to identify the currently known
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15 subcellular localization and gene ontology.
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20 Cell viability tests
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22 After shear stress application cells were stained with trypan blue (1:1 in PBS) for 5 minutes.
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25 For quantification 3 different sites of each culture dish were photographed and the number of
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27 viable and nonviable cells were counted from these images. In addition, the LDH-content
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29 within the supernatant was determined, which is a marker for cytotoxicity. To do so, we used
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32 the LDH-Cytotoxicity Assay Kit II (Biovision, Mountain View, USA) according to the
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34 manufacturers instructions. Briefly, supernatant of the cell culture was centrifuged to remove
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cell debris. Then, 10 µl of the cleared supernatant was mixed with 100 µl LDH reaction mix,
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39 incubated for 30 minutes and the absorbance was measured at 450 nm. The percentage of
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41 cytotoxicity was calculated using the absorbance after lysis of all cells in a control dish set to
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100%.
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3 Results
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6 To identify proteins that are secreted under different shear stress conditions we used primary
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8 endothelial cells (HUVECs) at passage 2. For these experiments HUVECs were grown in the
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presence of L-Lysine-13C6,15N2 hydrochloride and L-Arginine-13C6,15N4 hydrochloride,
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13 whereas the culture medium was deprived of the amino acids lysine and arginine. Within 5
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15 cell doublings proteins of HUVECs can be assumed to be homogenously labelled16. On the
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other hand, proteins contained in the culture medium (e.g. growth factors, proteins within
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20 FCS) did not carry the label. Thus, using this method, proteins released by HUVECs can be
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22 selectively identified by mass spectrometry. For the identification of the proteins a mass shift
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25 of +8.0 for lysine modification and +10.0 for arginine modification was considered.
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29 When cultured under static (control) conditions 395 proteins with stabile isotope
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32 incorporations were identified in the supernatant of the cell culture (figure 1). Most of them
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34 can be attributed to the cytoplasm (195), the membrane (43) and the nucleus (70) (figure 1,
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table 1 supplement). From all identified proteins only 78 (17%) were secreted according to
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39 SwissProt database (figure1, table 1). Identified proteins include MMP1 (interstitial
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41 collagenase), MIF (macrophage migration inhibitory factor), PAI1 (plasminogen activator
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inhibitor 1), vWF (von Willebrand factor), ANG2 (angiopoietin-2), IL8 (interleukin 8) and
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46 PDGFB (platelet-derived growth factor subunit B).
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51 In order to obtain an estimate on the validity of our measurements, we calculated the ratio of
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53 the number of assigned peptides per identified protein, which describes how many peptides on
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55 average were identified per protein. The ratio of assigned peptides per secreted protein was
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58 9.6 ± 17.24 (n=78), whereas the ratio for all nonsecreted proteins was 4.35 ± 4.34 (n=317)
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60 (p < 0.001). This indicates that for the identification of one secreted protein about twice as
many peptides were found compared to nonsecreted proteins.
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6 In a next set of experiments we applied either laminar (15 dyne/cm2) or oscillatory (8
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8 dyne/cm2) shear stress to HUVECs for 24 hours. Qualitative identification of proteins released
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into the supernatants revealed that 327 proteins could be identified from the laminar sample
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13 (figure 2A), whereas 507 proteins were identified from the oscillatory sample (figure 2B).
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15 There were no major differences in the cellular component distribution. Again, most proteins
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can be attributed to the cytoplasm (169 and 234, respectively), the membrane (26 and 58,
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20 respectively) and the nucleus (62 and 91, respectively). The fraction of secreted proteins due
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22 to laminar flow was 22.6% (83 proteins) and 13.8% (79 proteins) after oscillatory flow (figure
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25 2, table 2). Again, the ratio of assigned peptides per identified protein was significantly higher
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27 in secreted proteins (10.36 ± 17.67 for laminar shear stress, 11.22 ± 21.20 for oscillatory shear
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29 stress) compared to all other proteins (4.77 ± 4.31 for laminar shear stress, 5.43 ± 6.49 for
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32 oscillatory shear stress) (p < 0.001 for both groups).
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In table 2 data are summarized for all 101 proteins secreted by HUVEC under control,
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39 laminar and oscillatory conditions. As can be seen, of these 101 proteins 6 proteins were
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41 identified that were expressed only under no flow conditions, such as latent transforming
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growth factor beta binding protein 3 (LTBP3) and platelet derived growth factor subunit B
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46 (PDGFB); 8 proteins being expressed under laminar flow conditions, like LTBP4 and
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48 PDGFA; and 5 proteins specific for oscillatory flow, like aminopeptidase N (CD13, ANPEP),
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51 calreticulin (CALR) and endothelin-1 (EDN1). Furthermore, 8 proteins were identified after
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53 laminar shear stress and after control conditions, 10 proteins after any flow condition but not
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55 under static control conditions and 7 proteins after no flow and oscillatory flow conditions.
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58 Finally, 57 proteins could be identified after any treatment.
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3 To further analyze the identified secreted proteins we grouped all 101 proteins according to
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6 their Gene Ontology molecular function (figure 1 supplemental data; shown are all GO entries
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8 where at least 3 proteins were assigned to) and Gene Ontology biological function (figure 2
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supplemental data; shown are all GO entries where at least 3 proteins were assigned to).
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13 Figure 1 supplemental data (GO molecular function) shows that most proteins are associated
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15 with binding to proteins, calcium ions, heparin, cytokine and/or are structural constituents of
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extracellular matrix. In order to visualize dynamic changes dependent on the flow situation,
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20 figure 3 shows differences in the GO molecular function between all 3 experimental groups.
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22 The number of proteins assigned to a specific GO molecular function is set to 0 for the control
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25 condition, and the number of additional or lacking proteins under the two flow conditions is
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27 illustrated. Compared to static conditions most differences under laminar and oscillatory flow
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29 occur by the secretion of hydrolysing proteins exhibiting peptidase activity, especially serine-
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32 type peptidase activity and serine-type endopeptidase activity, and proteins with hydrolase
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34 activity (figure 3). In particular the expression of serine protease HTRA1 (SwissProt entry
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Q92743) and tissue type plasminogen activator (SwissProt entry P00750), which both cleave
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39 many proteins in the blood plasma, and of aminopeptidase N (SwissProt entry P15144),
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41 which cleaves amino acids from oligopeptides, account for these differences.
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46 When all 101 secreted proteins are organized according to their biological function (figure 2
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48 supplemental data, shown are all GO entries with at least 3 proteins assigned to), cell adhesion
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51 is the predominant function of the proteins. In addition, signal transduction, multicellular
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53 organismal development and proteolysis are among the most cited ones. Also for this group
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55 there are dynamic changes between the 3 groups. Again, the number of proteins assigned to
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58 specific GO biological function is set to 0 for control condition. Compared to control
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60 conditions secretion of proteins involved in cell adhesion is increased (figure 4). Here,
collagen alpha-1(VI) (COL6A1, SwissProt entry P12109), laminin subunit alpha-5 (LAMA5,
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3 SwissProt entry O15230), and laminin subunit gamma-2 (LAMC2, SwissProt entry Q13753)
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6 were found, as well as carboxypeptidase-like protein X2 (CPXM2, SwissProt entry Q8N436)
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8 and thrombospondin-2 (TSP2, SwissProt entry P35442). Besides cell adhesion, the secretion
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of proteins performing proteolysis is also increased. Here, we identified proteins like serine
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13 protease HTRA1 (SwissProt entry Q92743), tissue type plasminogen activator (SwissProt
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15 entry P00750) and aminopeptidase N (SwissProt entry P15144). Interestingly, the number of
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proteins with the GO annotation multicellular organismal development was decreased after
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20 laminar shear stress (-2) but increased after oscillatory fluid flow (+2).
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25 In an additional set of experiments we checked the viability of the cultured HUVECs. The
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27 fraction of cells without shear stress that could be stained with trypan blue solution was 0.093
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29 ± 0.108% of all cells (n=4). In addition, lactate dehydrogenate (LDH) measurement in the
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32 supernatant revealed that 1.93% (n=3) of all cells died within this period of time. After 24
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34 hours of shear stress trypan blue staining revealed that 0.153 ± 0.041% (n=3) of all cells were
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nonviable, whereas LDH measurements show that 3.74% of all cells died within the same
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39 period of time. Supporting this finding, LDH was also detected with MS/MS in all samples.
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3 Discussion
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6 This study investigated the influence of shear stress on the secretome of HUVECs. To this
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8 end, we applied laminar or oscillatory flow to HUVECs and analysed the supernatant with
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sensitive mass spectrometric techniques. We have identified a total of 395 proteins liberated
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13 into the supernatant under static conditions, and this fraction remained unchanged under
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15 laminar (327 proteins) and oscillatory (507 proteins) flow conditions. The fraction of proteins
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assigned to be secreted amounted to only 78 proteins under static conditions (17%), and was
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20 similar with laminar flow (83 proteins) and oscillatory flow (79 proteins). Among the 101
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22 proteins identified to be secreted, 57 were found to be secreted independently of the flow
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25 condition.
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29 This study is the second to explore the secretome of HUVECs in detail. In 2009 Tunica et al.12
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32 reported 70 secreted proteins of which 43 were identified by us as well. In addition, we have
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34 identified additional 58 proteins which are known to be secreted proteins. While the former
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study by Tunica et al. restricted the analysis to static flow conditions, the present study
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39 explored changes in the secretome under laminar and turbulent flow conditions. The dynamic
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41 changes of the endothelial secretome found in this study in response to shear stress may be
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functionally relevant, because shear stress is the physiological stimulus for endothelial cells
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46 and changes in shear stress are known to be of importance for the development of vascular
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48 diseases such as atherogenesis.
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53 Aside from the secreted proteins the majority of proteins identified could be attributed to
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55 specific cellular compartments, such as cytoplasm, membrane and nucleus. On the one hand
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58 this shows the sensitivity of the MS-based method which permits the detection of proteins
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60 derived from even a small fraction of dying cells (see below); on the other hand it addresses
the important issue of cellular integrity of cultured cells and the ability to differentiate
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3 between extracellular and intracellular proteins. In order to discriminate between
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6 endogenously expressed proteins and proteins which were required for optimal cell culture
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8 conditions we have labelled HUVEC with heavy isotope amino acids during our experiments.
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As to extracellular contaminating proteins we measured cell integrity by trypan blue staining
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13 and found 0.093% of non-sheared cells and 1.93% of all cells after shear stress to be
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15 nonviable. Similarly, the corresponding values from LDH cytotoxicity assays were 0.153%
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and 3.74%, respectively. These data show that the fraction of dead/dying cells is rather low
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20 and comparable to data in the literature17. However, mass spectrometry is so sensitive that this
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22 small fraction can considerably “contaminate” the measurement of secreted proteins. Many
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25 proteins identified by us were also found under similar experimental conditions by others such
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27 as HSP2711, nucleoside diphosphate kinase B11;12, cathepsin D6;6;8;11;12, protein disulfide
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isomerase8;11;12, vimentin6;8;9;11;12, filamin11;12, 60 kDa heat shock protein8, α-enolase6;8;9,
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32 fructose-bisphosphate aldolase A8 and B12, annexin A18, F-actin capping protein β-unit8,
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tropomyosin α 3 chain8;12, glutathione S-transferase P8 and glyceraldehyde 3-phosphate
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37 dehydrogenase6;12. As to the accuracy of protein identification by MS it should be noted that
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39 the false positive rate has been reported to be only about 1% (e.g. Huttlin et al.18, Xie et al.19,
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42 Lu et al.20).
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46 Identified proteins were assigned to their cellular compartment according to SwissProt
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48
49 database. In this database proteins are assigned as being secreted when they have been
50
51 positively identified as secreted proteins by other biochemical experiments. This implies, that
52
53
54
it is quite possible that some of the proteins identified by us and assigned to cytoplasmic or
55
56 membrane origin might also have been secreted. This, however, cannot be decided on the
57
58 basis of our experiments, since it will not be possible to fully exclude cell death/lysis even
59
60
when using inhibitors of cell apoptosis. Regarding the reproducibility of our data it should be
noted that out of the 101 proteins known to be secreted 76 were identified in each of the two
14
1
2
3 flow-identical experiments. The reproducibility is in the same range as was reported recently
4
5
6 by others21.
7
8
9
10
Our study identified several proteins the expression/secretion of which has been reported to be
11
12
13 dependent on shear stress. Examples are MMP222, insulin-like growth factor-binding
14
15 proteins23, metalloproteinase inhibitor 1 and 224;25 and von Willebrand factor26. The influence
16
17
18
of shear stress on the expression of endothelin-1 is dependent both on the level of shear stress
19
20 and the duration of application. Wang et al. showed that an intermediate level of shear stress
21
22 leads to a maximum secretion of endothelin-127. Kuchan et al. reported, that the secretion of
23
24
25 endothelin-1 increases initially, but decreases after 6 h of laminar flow28 while Dancu et al.
26
27 found an increased endothelin-1 secretion during asynchronous hemodynamics29. Similar
28
29 results were obtained by Walshe et al. who reported an increased expression of endothelin-1
30
31
32 after oscillatory flow30. Collectively these data suggest very low levels of secreted endothelin-
33
34 1 in no-flow samples and after long-time laminar shear stress, but elevated levels after
35
36
oscillatory flow. Consistent with these studies we identified endothelin-1 only after oscillatory
37
38
39 shear flow, but not under laminar flow and static conditions.
40
41
42
43
44
Although the mixture of secreted proteins is quite complex, several proteins could be clearly
45
46 attributed to the flow condition: LTBP3 to static no-flow condition, BMP6 and PDGFB to
47
48 flow, LAMA5, PDGFA and LTBP4 to laminar flow and aminopeptidase N and endothelin-1
49
50
51 to oscillatory flow. Apparently, the number of proteins with peptidase activity is increased
52
53 under flow conditions. We found fluid shear stress to stimulate the secretion of tissue
54
55 plasminogen activator by endothelial cells whereas the secretion of plasminogen activator
56
57
58 inhibitor type-1 remained unaltered which is consistent with data in the literature31. Whereas
59
60 plasminogen activator inhibitor type-1 was identified under all experimental conditions, we
detected tissue plasminogen activator only with laminar and oscillatory flow. This suggests
15
1
2
3 that the fibrinolytic potential of endothelial cells increases in response to hemodynamic
4
5
6 forces. We also identified the serine protease HTRA1 to be secreted both after laminar and
7
8 oscillatory flow. HTRA1 is part of the insulin-growth-factor signaling pathway and cleaves
9
10
IGF binding proteins. Upregulation of its mRNA in osteoblasts in response to fluid shear has
11
12
13 been reported32 which takes place downstream of the early mechano-responsive genes Igtb1
14
15 and Cox-2. Another peptidase with broad specificity identified by us to be secreted only after
16
17
18
oscillatory flow is aminopeptidase N (CD13). CD13 was found to be secreted by HL60 cells
19
20 when agitated and the expression of CD13 was proportional to the agitation intensity33.
21
22
23
24
25 In summary, this is to our knowledge the first comprehensive study of secretome profiling in
26
27 human endothelial cells as influenced by shear stress. We identified 587 different proteins of
28
29 which several are secreted in a flow specific manner. In addition, we have identified several
30
31
32 previously unreported proteins and show that the secretome is modulated by the type of shear
33
34 stress applied. Further studies on the secretome of endothelial cells will have to answer the
35
36
question whether proteins secreted by endothelial cells can alter the composition of plasma
37
38
39 proteins and may be involved in the development of flow-dependent diseases such as
40
41 atherogenesis.
42
43
44
45
46 Supporting Information
47
48 Supporting Information Available: This material is available free of charge via the Internet at
49
50
51 http://pubs.acs.org.
52
53
54
55
56
57
58
59
60
16
1
2
3 Reference List
4
5
6 1. Gimbrone, M. A., Jr. Atherogenesis: current concepts. Monogr Pathol. 1995, 37 1-11
7
8 2. Topper, J. N.; Cai, J.; Falb, D.; Gimbrone, M. A., Jr. Identification of vascular
9 endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-
10 2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are
11
12
selectively up-regulated by steady laminar shear stress. Proc. Natl. Acad. Sci. U. S. A
13 1996, 93 (19), 10417-22
14
15 3. Topper, J. N.; Cai, J.; Qiu, Y.; Anderson, K. R.; Xu, Y. Y.; Deeds, J. D.; Feeley, R.;
16 Gimeno, C. J.; Woolf, E. A.; Tayber, O.; Mays, G. G.; Sampson, B. A.; Schoen, F. J.;
17 Gimbrone, M. A., Jr.; Falb, D. Vascular MADs: two novel MAD-related genes
18
selectively inducible by flow in human vascular endothelium. Proc. Natl. Acad. Sci.
19
20 U. S. A 1997, 94 (17), 9314-9
21
22 4. Topper, J. N.; Wasserman, S. M.; Anderson, K. R.; Cai, J.; Falb, D.; Gimbrone, M. A.,
23 Jr. Expression of the bumetanide-sensitive Na-K-Cl cotransporter BSC2 is differentially
24 regulated by fluid mechanical and inflammatory cytokine stimuli in vascular
25 endothelium. J. Clin. Invest 1997, 99 (12), 2941-9
26
27
28 5. Groenendijk, B. C.; Van der, H. K.; Hierck, B. P.; Poelmann, R. E. The role of shear
29 stress on ET-1, KLF2, and NOS-3 expression in the developing cardiovascular system
30 of chicken embryos in a venous ligation model. Physiology (Bethesda. ) 2007, 22
31 380-9
32
33 6. Dupont, A.; Corseaux, D.; Dekeyzer, O.; Drobecq, H.; Guihot, A. L.; Susen, S.;
34
35
Vincentelli, A.; Amouyel, P.; Jude, B.; Pinet, F. The proteome and secretome of human
36 arterial smooth muscle cells. Proteomics. 2005, 5 (2), 585-96
37
38 7. Liao, D. F.; Jin, Z. G.; Baas, A. S.; Daum, G.; Gygi, S. P.; Aebersold, R.; Berk, B. C.
39 Purification and identification of secreted oxidative stress-induced factors from vascular
40 smooth muscle cells. J. Biol. Chem. 2000, 275 (1), 189-96
41
42
43
8. Dupont, A.; Tokarski, C.; Dekeyzer, O.; Guihot, A. L.; Amouyel, P.; Rolando, C.; Pinet,
44 F. Two-dimensional maps and databases of the human macrophage proteome and
45 secretome. Proteomics. 2004, 4 (6), 1761-78
46
47 9. Fach, E. M.; Garulacan, L. A.; Gao, J.; Xiao, Q.; Storm, S. M.; Dubaquie, Y. P.; Hefta,
48 S. A.; Opiteck, G. J. In vitro biomarker discovery for atherosclerosis by proteomics.
49
Mol. Cell Proteomics. 2004, 3 (12), 1200-10
50
51
52 10. Duran, M. C.; Mas, S.; Martin-Ventura, J. L.; Meilhac, O.; Michel, J. B.; Gallego-
53 Delgado, J.; Lazaro, A.; Tunon, J.; Egido, J.; Vivanco, F. Proteomic analysis of human
54 vessels: application to atherosclerotic plaques. Proteomics. 2003, 3 (6), 973-8
55
56 11. Vivanco, F.; Martin-Ventura, J. L.; Duran, M. C.; Barderas, M. G.; Blanco-Colio, L.;
57
Darde, V. M.; Mas, S.; Meilhac, O.; Michel, J. B.; Tunon, J.; Egido, J. Quest for novel
58
59 cardiovascular biomarkers by proteomic analysis. J. Proteome. Res. 2005, 4 (4),
60 1181-91
12. Tunica, D. G.; Yin, X.; Sidibe, A.; Stegemann, C.; Nissum, M.; Zeng, L.; Brunet, M.;
Mayr, M. Proteomic analysis of the secretome of human umbilical vein endothelial cells
17
1
2
3 using a combination of free-flow electrophoresis and nanoflow LC-MS/MS.
4
5
Proteomics. 2009, 9 (21), 4991-6
6
7 13. Wang, X. L.; Fu, A.; Raghavakaimal, S.; Lee, H. C. Proteomic analysis of vascular
8 endothelial cells in response to laminar shear stress. Proteomics. 2007, 7 (4), 588-96
9
10 14. Morawietz, H.; Talanow, R.; Szibor, M.; Rueckschloss, U.; Schubert, A.; Bartling, B.;
11 Darmer, D.; Holtz, J. Regulation of the endothelin system by shear stress in human
12
13
endothelial cells. J. Physiol 2000, 525 Pt 3 761-70
14
15 15. Schubert, A.; Cattaruzza, M.; Hecker, M.; Darmer, D.; Holtz, J.; Morawietz, H. Shear
16 stress-dependent regulation of the human beta-tubulin folding cofactor D gene. Circ.
17 Res. 2000, 87 (12), 1188-94
18
19 16. Mann, M. Functional and quantitative proteomics using SILAC. Nat. Rev. Mol. Cell
20
21
Biol. 2006, 7 (12), 952-8
22
23 17. Koo, D. D.; Welsh, K. I.; West, N. E.; Channon, K. M.; Penington, A. J.; Roake, J. A.;
24 Morris, P. J.; Fuggle, S. V. Endothelial cell protection against ischemia/reperfusion
25 injury by lecithinized superoxide dismutase. Kidney Int. 2001, 60 (2), 786-96
26
27 18. Huttlin, E. L.; Hegeman, A. D.; Harms, A. C.; Sussman, M. R. Prediction of error
28
29
associated with false-positive rate determination for peptide identification in large-scale
30 proteomics experiments using a combined reverse and forward peptide sequence
31 database strategy. J. Proteome. Res. 2007, 6 (1), 392-8
32
33 19. Xie, H.; Rhodus, N. L.; Griffin, R. J.; Carlis, J. V.; Griffin, T. J. A catalogue of human
34 saliva proteins identified by free flow electrophoresis-based peptide separation and
35
tandem mass spectrometry. Mol. Cell Proteomics. 2005, 4 (11), 1826-30
36
37
38 20. Lu, B.; Motoyama, A.; Ruse, C.; Venable, J.; Yates, J. R., III Improving protein
39 identification sensitivity by combining MS and MS/MS information for shotgun
40 proteomics using LTQ-Orbitrap high mass accuracy data. Anal. Chem. 2008, 80 (6),
41 2018-25
42
43 21. Piersma, S. R.; Fiedler, U.; Span, S.; Lingnau, A.; Pham, T. V.; Hoffmann, S.; Kubbutat,
44
45 M. H.; Jimenez, C. R. Workflow comparison for label-free, quantitative secretome
46 proteomics for cancer biomarker discovery: method evaluation, differential analysis, and
47 verification in serum. J. Proteome. Res. 2010, 9 (4), 1913-22
48
49 22. Sultan, S.; Gosling, M.; Nagase, H.; Powell, J. T. Shear stress-induced shedding of
50 soluble intercellular adhesion molecule-1 from saphenous vein endothelium. FEBS
51
52
Lett. 2004, 564 (1-2), 161-5
53
54 23. Elhadj, S.; Akers, R. M.; Forsten-Williams, K. Chronic pulsatile shear stress alters
55 insulin-like growth factor-I (IGF-I) binding protein release in vitro. Ann. Biomed. Eng
56 2003, 31 (2), 163-70
57
58 24. Nanjo, H.; Sho, E.; Komatsu, M.; Sho, M.; Zarins, C. K.; Masuda, H. Intermittent short-
59
60
duration exposure to low wall shear stress induces intimal thickening in arteries exposed
to chronic high shear stress. Exp. Mol. Pathol. 2006, 80 (1), 38-45
18
1
2
3 25. Uchida, C.; Milkiewicz, M.; Fudalewski, T.; Gee, E.; Hankenson, K. D. TIMP-1 is
4
5
increased by shear stress in the skeletal muscle microvasculature. FASEB J. 2008, 22
6 925-8
7
8 26. Galbusera, M.; Zoja, C.; Donadelli, R.; Paris, S.; Morigi, M.; Benigni, A.; Figliuzzi, M.;
9 Remuzzi, G.; Remuzzi, A. Fluid shear stress modulates von Willebrand factor release
10 from human vascular endothelium. Blood 1997, 90 (4), 1558-64
11
12
13
27. Wang, G. X.; Cai, S. X.; Wang, P. Q.; Ouyang, K. Q.; Wang, Y. L.; Xu, S. R. Shear-
14 induced changes in endothelin-1 secretion of microvascular endothelial cells.
15 Microvasc. Res. 2002, 63 (2), 209-17
16
17 28. Kuchan, M. J.; Frangos, J. A. Shear stress regulates endothelin-1 release via protein
18 kinase C and cGMP in cultured endothelial cells. Am. J. Physiol 1993, 264 (1 Pt 2),
19
H150-H156
20
21
22 29. Dancu, M. B.; Berardi, D. E.; Vanden Heuvel, J. P.; Tarbell, J. M. Asynchronous shear
23 stress and circumferential strain reduces endothelial NO synthase and cyclooxygenase-2
24 but induces endothelin-1 gene expression in endothelial cells. Arterioscler. Thromb.
25 Vasc. Biol. 2004, 24 (11), 2088-94
26
27
30. Walshe, T. E.; Ferguson, G.; Connell, P.; O'Brien, C.; Cahill, P. A. Pulsatile flow
28
29 increases the expression of eNOS, ET-1, and prostacyclin in a novel in vitro coculture
30 model of the retinal vasculature. Invest Ophthalmol. Vis. Sci. 2005, 46 (1), 375-82
31
32 31. Diamond, S. L.; Sharefkin, J. B.; Dieffenbach, C.; Frasier-Scott, K.; McIntire, L. V.;
33 Eskin, S. G. Tissue plasminogen activator messenger RNA levels increase in cultured
34 human endothelial cells exposed to laminar shear stress. J. Cell Physiol 1990, 143 (2),
35
36 364-71
37
38 32. Lau, K. H.; Kapur, S.; Kesavan, C.; Baylink, D. J. Up-regulation of the Wnt, estrogen
39 receptor, insulin-like growth factor-I, and bone morphogenetic protein pathways in
40 C57BL/6J osteoblasts as opposed to C3H/HeJ osteoblasts in part contributes to the
41 differential anabolic response to fluid shear. J. Biol. Chem. 2006, 281 (14), 9576-88
42
43
44
33. McDowell, C. L.; Papoutsakis, E. T. Increased agitation intensity increases CD13
45 receptor surface content and mRNA levels, and alters the metabolism of HL60 cells
46 cultured in stirred tank bioreactors. Biotechnol. Bioeng. 1998, 60 (2), 239-50
47
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19
1
2
3 Figure legend
4
5
6
7
8 Figure 1
9
10
Cellular component distribution of identified proteins under static (control) conditions.
11
12
13 HUVECs were kept at 37°C and 5% CO2 for 24 hours without shear stress. Supernatant was
14
15 collected, heavy-amino acid containing proteins were identified and organized according to
16
17
18
their subcellular annotation based on SwissProt prediction. Shown is the name of the
19
20 subcellular component, the number of identified proteins and the percentage referred to all
21
22 proteins.
23
24
25
26
27 Figure 2
28
29 Cellular component distribution of identified proteins under laminar and turbulent shear stress
30
31
32 conditions. HUVECs were kept at 37°C and 5% CO2 for 24 hours with 15 dyne/cm2 laminar
33
34 shear stress (A) or 8 dyne/cm2 oscillatory shear stress (B). Supernatants were collected,
35
36
heavy-amino acid containing proteins were identified and organized according to their
37
38
39 subcellular annotation based on SwissProt prediction. Shown is the name of the subcellular
40
41 component, the number of identified proteins and the percentage referred to all proteins.
42
43
44
45
46 Figure 3
47
48 Differences in the number of identified proteins per Gene Ontology molecular function -
49
50
51 comparison between all 3 study groups (control, laminar, oscillatory). Static control is set to
52
53 zero, differences from there are shown for laminar and oscillatory flow. Shown are all
54
55 comparisons where the number of assigned proteins differ for 2 at least.
56
57
58
59
60 Figure 4
20
1
2
3 Differences in the number of identified proteins per Gene Ontology biological function -
4
5
6 comparison between all 3 study groups (control, laminar, oscillatory). Static control is set to
7
8 zero, differences from there are shown for laminar and oscillatory flow. Shown are all
9
10
comparisons where the number of assigned proteins differ for 2 at least.
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
21
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18 Static (control) condition: 395 proteins
19
20
21
22
23
24 lysosome: 3 (1%)
25
26
mitochondrium: 28
27 (6%) endosome: 2 (0%)
28 unknow n: 4 (1%)
29
30
31
secreted: 78
32 Golgi: 8 (2%) (17%)
33
34 ER: 17 (4%)
35
36
37
38
39
40
41
42 nucleus: 70 (16%)
43
44
45 membrane: 43
46 (10%)
47 cytoplasm: 195
48
49 (43%)
50
51
52
53
54
55
56
57
58
59
60
Fig. 1
22
1
2
3
4
5
6
7
8
9 A Laminar condition: 327 proteins
10
11
mitochondrium: 7 lysosome: 2 (1%)
12 endosome: 1 (0%)
13 (2%)
14
15 unknow n: 4 (1%)
16 Golgi: 4 (1%)
17 ER: 10 (3%) secreted: 83
18 (23%)
19
20
21
22 nucleus: 62 (17%)
23
24
25 membrane: 26
26 (7%)
27
28
29
30
31
32
cytoplasm: 169
33
(45%)
34
35
36
37 B Oscillatory condition: 507 proteins
38
39 mitochondrium: 52
40 (9%)
lysosome: 6 (1%) endosome: 2 (0%)
41
unknow n: 4 (1%)
42
43
44 secreted: 79 (14%)
Golgi: 13 (2%)
45
46
47 ER: 32 (6%)
48
49
50
51
52
53
54 nucleus: 91 (16%)
55
56 cytoplasm: 234
57 (41%)
58
59 membrane: 58
60 (10%)
Fig. 2
23
9
8
7
6
5
4
3
2
1
60
59
58
57
56
55
54
53
52
51
50
49
48
47
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30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
number of differences GO molecular function
st
ru
ct
u ra
lm
ol
ec
-2
-1
0
1
2
3
4
5
ul
e
ac
tiv
se i ty
r in pr
e- ot
ty e in
pe
en bi
nd
do in
pe g
p tid
se as
rin e
e- ac
ty ti v
pl pe
at ity
el pe
et pt
-d id
er as
iv e
ed ac
gr tiv
ow ity
th
fa
ct
or
bi
nd
in
g
he
pa
r in
bi
nd
in
pe g
pt
id
as
e
ac
ti v
ity
zi
nc
io
n
bi
nd
in
hy g
Journal of Proteome Research

dr
ol
as
e
ac
ti v
ity
Differences in comparison to control - GO molecular function
bi
gr nd
in
ow g
th
fa
ct
or
a ct
iv
ity
in cy
s ul to
in ki
- lik ne
e ac
gr tiv
ow ity
th
24
fa
ct
o rb
in
di
ng
D laminar
Fig. 3
D oscillatory
Page 24 of 35
9
8
7
6
5
4
3
2
1
60
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49
48
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30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
number of differences GO biological function
ce
-3
-2
-1
0
1
2
3
4
5
6
ll a
dh
po es
io
Page 25 of 35
si
m
ti ve n
or re pr
ph gu ot
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of ll
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br
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or
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ce
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Differences in comparision to control - GO biological function
lo
pm
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25
on
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iff
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D laminar
Fig. 4
D oscillatory
1
2
3 Tab. 1 Proteins secreted by HUVECs under control conditions (no shear stress).
4
5 SwissProt Name
6 P08253 72 kDa type IV collagenase MMP2
7 Q13443 ADAM 9 ADAM9
8 Q9UHI8 A disintegrin and metalloproteinase with thrombospondin motifs 1 ADAMTS1
9 O75173 ADAMTS-4 ADAMTS4
10 O15123 Angiopoietin-2 ANGP2, ANGPT2
11 P07355 Annexin A2 isoform 1 ANXA2
12 P08758 Annexin A5 ANXA5
13 Basement Membrane-specific Heparan Sulfate Proteoglycan core
14 P98160 protein PGBM
15
P61769 Beta-2-microglobulin B2M
16
P22004 Bone morphogenetic protein 6 BMP6
17
18 P13987 CD59 glycoprotein CD59
19 P10909 Clusterin CLUS, CLU
20 Coiled-coil domain-containing protein 80 CCDC80
21 P20908 Collagen alpha-1(V) chain COL5A1, CO5A1
22 Q9BXJ0 Complement C1q tumor necrosis factor-related protein 5 C1QTNF5
23 P08603 Complement factor H CFH
24 P29279 Connective tissue growth factor CTGF
25 P01034 Cystatin-C CST3, CYTC
26 O94907 Dickkopf-related protein 1 DKK1
27
Q12805 EGF-containing fibulin-like extracellular matrix protein 1 FBLN3
28
P35555 Fibrillin-1 FBN1
29
30 P35556 Fibrillin-2 FBN2
31 P02751 Fibronectin, Isoform 1 FN1
32 Q12841 Follistatin-related protein 1 FSTL1
33 P09382 Galectin-1 LGALS1, LEG1
34 P06396 Gelsolin GELS, GSN
35 Q99988 Growth/differentiation factor 15 GDF15
36 P09341 Growth-regulated alpha protein CXCL1
37 Q96QV1 Hedgehog-interacting protein HHIP
38 P18065 Insulin-like growth factor-binding protein 2 IGFBP2, IBP2
39
P22692 Insulin-like growth factor-binding protein 4 IGFBP4, IBP4
40
41 Q16270 Insulin-like growth factor-binding protein 7 IGFBP-7
42 O00622 Insulin-like growth factor-binding protein 10 CYR61
43 P10145 Interleukin 8 IL8
44 P03956 Interstitial collagenase MMP1
45 Q14767 latent transforming growth factor beta-binding protein 2 LTBP-2
46 Q9NS15 latent transforming growth factor beta-binding protein 3 LTBP3
47 Q16363 Laminin subunit alpha-4 LAMA4
48 P07942 Laminin subunit beta-1 LAMB1
50 P11047 Laminin subunit gamma-1 LAMC1
51
Q9Y4K0 Lysyl oxidase homolog 2 LOXL2
52
53 P14174 Macrophage migration inhibitory factor MIF
54 P08493 Matrix Gla protein MGP
55 P01033 Metalloproteinase Inhibitor 1 TIMP1
57 P55001 Microfibrillar-associated protein 2 MFAP2
58 P21741 Midkine MDK, MK
59 Q13201 Multimerin-1 MMRN1
60 Q9H8L6 Multimerin-2 MMRN2
P26022 Pentraxin-related protein PTX3 PTX3
Peroxidasin PXDN
26
1
2
3 P05121 Plasminogen activator inhibitor 1 PAI1, SERPINE1
4 P01127 Platelet-derived growth factor subunit B PDGFB
5
P55145 Protein ARMET ARMET
6
7 P07237 Protein disulfide-isomerase P4HB, PDI
8 P21980 Protein-glutamine gamma-glutamyltransferase 2 TGM2
9 P09486 Secreted protein acidic and rich in cysteine SPARC
10 O95084 Serine protease 23 PRSS23, PRS23
11 P52823 Stanniocalcin-1 STC1
12 O00391 Sulfhydryl oxidase 1 QSOX1
13 Q08629 Testican-1, SPOCK TICN1, SPOCK
14 P05452 Tetranectin TETN, CLEC3B
15 P07996 Thrombospondin-1 TSP1
16
P10646 Tissue factor pathway inhibitor TFPI
17
P48307 Tissue factor pathway inhibitor 2 TFPI2
18
19 P20062 Transcobalamin-2 TCN2
20 Q9GZM7 Tubulointerstitial nephritis antigen-like TINAGL1
21 P04275 von Willebrand factor vWF
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
27
1
2
3 Tab. 2 Proteins secreted by HUVECs under control conditions, laminar and oscillatory shear
4
5
stress.
6 detected only under control conditions
7 P22004 Bone morphogenetic protein 6 BMP6
8 Q9UGM3 deleted in malignant brain tumor 1 DMBT1
9 Q9NS15 latent transforming growth factor beta-binding protein 3 LTBP3
10 P55001 Microfibrillar-associated protein 2 MFAP2
11 P01127 Platelet-derived growth factor subunit B PDGFB
12 P07998 Ribonuclease pancreatic RNASE1, RNAS1
13
14 detected only under laminar shear stress
15 O00468 Agrin AGRN
16 Q9NQ79 Cartilage acidic protein 1 CRTAC1
17 Q99715 Collagen alpha-1(XII) chain COL12A1
18 LAMA5 LAMA5
19 latent transforming growth factor beta-binding protein 4 LTBP4
20 Meteorin-like protein METRNL
21 P12272 Parathyroid hormone-related protein PTHR, PTHLH
22
P04085 Platelet-derived growth factor subunit A PDGFA
23
24 detected only under oscillatory shear stress
25 Q16352 Alpha-internexin INA, AINX
a
26 P15144 Aminopeptidase N ANPEP
27 P27797 Calreticulin CALR
28 P05305 Endothelin-1 EDN1
29 P01344 Insulin-like growth factor II IGF2
30
31 detected under control conditions and laminar shear stress
32 Q16363 Laminin subunit alpha-4 LAMA4
34 Q96RW7 Hemicentin-1, Fibulin 6 HMCN1, FIBL-6
a
35 P09486 Secreted protein acidic and rich in cysteine SPARC
36 P52823 Stanniocalcin-1 STC1
37 P05452 Tetranectin TETN, CLEC3B
38 P20062 Transcobalamin-2 TCN2
39 a
Q9H8L6 Multimerin-2 MMRN2
40
41 detected under control conditions and oscillatory shear stress
42 O15123 Angiopoietin-2 ANGP2, ANGPT2
43 P10145 Interleukin 8 IL8
a
44 Q969H8 Interleukin 25 IL25, C19orf10
45 P00387 NADH-cytochrome b5 reductase 3 CYB5R3
46 Q15063 Periostin POSTN
47 P07237 Protein disulfide-isomerase
a
P4HB, PDI
48
Q9BRX8 Uncharacterized protein C19orf58
49
50 detected under laminar and oscillatory shear stress
51 P21810 Biglycan BGN
52 Q8N436 Carboxypeptidase-like protein X2 CPXM2
53 P12109 Collagen alpha-1(VI) chain COL6A1
54 Q9Y287 Integral membrane protein 2B ITM2B
55 O15230 Laminin subunit alpha-5 LAMA5
56 Q13753 Laminin subunit gamma-2 LAMC2
57
Q92743 Serine protease HTRA1 HTRA1
58
59 P00750 Tissue-type plasminogen activator TPA, PLAT
P49767 Vascular endothelial growth factor C VEGFC
detected under control conditions, laminar and oscillatory shear stress
a
P08253 72 kDa type IV collagenase MMP2
28
1
2
3 Q13443 ADAM 9 ADAM9
4 Q9UHI8 A disintegrin and metalloproteinase with thrombospondin motifs 1 ADAMTS1
5
O75173 ADAMTS-4 ADAMTS4
6 a
7 P07355 Annexin A2 isoform 1 ANXA2
a
8 P08758 Annexin A5 ANXA5
9 Basement Membrane-specific Heparan Sulfate Proteoglycan core
a
10 P98160 protein PGBM
a
11 P61769 Beta-2-microglobulin B2M
12 Q16627 C-C motif chemokine 14 CCL14
a
13 P13987 CD59 glycoprotein CD59
14 P10909 Clusterin CLUS, CLU
15 Coiled-coil domain-containing protein 80 CCDC80
16 P20908 Collagen alpha-1(V) chain COL5A1, CO5A1
17 P39060 Collagen alpha-1(XVIII) chain COL18A1
18 Q9BXJ0 Complement C1q tumor necrosis factor-related protein 5 C1QTNF5
19
P08603 Complement factor H CFH
20 a
21 P29279 Connective tissue growth factor CTGF
a
22 P01034 Cystatin-C CST3, CYTC
23 O94907 Dickkopf-related protein 1 DKK1
a
24 Q12805 EGF-containing fibulin-like extracellular matrix protein 1 FBLN3
27 P02751 Fibronectin, Isoform 1 FN1
28 Q12841 Follistatin-related protein 1
a
FSTL1
29 P09382 Galectin-1
a
LGALS1, LEG1
30
P06396 Gelsolin GELS, GSN
31
Q99988 Growth/differentiation factor 15 GDF15
32 a
33 P09341 Growth-regulated alpha protein CXCL1
34 Q96QV1 Hedgehog-interacting protein HHIP
a
a
a
37 Q16270 Insulin-like growth factor-binding protein 7 IGFBP-7
a
38 O00622 Insulin-like growth factor-binding protein 10 CYR61
39 a
P03956 Interstitial collagenase MMP1
40 Q14767 latent transforming growth factor beta-binding protein 2 LTBP-2
42
P11047 Laminin subunit gamma-1 LAMC1
43 a
Q9Y4K0 Lysyl oxidase homolog 2 LOXL2
44
45 P14174 Macrophage migration inhibitory factor MIF
46 P08493 Matrix Gla protein MGP
a
49 P21741 Midkine MDK, MK
a
50 Q13201 Multimerin-1 MMRN1
51 a
P26022 Pentraxin-related protein PTX3 PTX3
52 Peroxidasin PXDN
53 P05121 Plasminogen activator inhibitor 1
a
PAI1, SERPINE1
54 a
P55145 Protein ARMET ARMET
55 a
P21980 Protein-glutamine gamma-glutamyltransferase 2 TGM2
56 a
57 O95084 Serine protease 23 PRSS23, PRS23
58 O00391 Sulfhydryl oxidase 1 QSOX1
a
59 Q08629 Testican-1, SPOCK TICN1, SPOCK
a
a
P10646 Tissue factor pathway inhibitor TFPI
P48307 Tissue factor pathway inhibitor 2 TFPI2
Q9GZM7 Tubulointerstitial nephritis antigen-like TINAGL1
29
1
2
3 P04275 von Willebrand factor
a
vWF
4 12
5 a) Previously identified in the secretome of HUVECs
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
30
1
2
3 Table of Contents (TOC) Synopsis
4
5
6 In this paper we report on the shear stress dependent secretome of human endothelial cells
7
8 (HUVEC). We identified 587 different proteins of which several are secreted in a flow
9
10
specific manner (no-flow; laminar and turbulent flow). In addition, several previously
11
12
13 unreported proteins were identified and show that the secretome is modulated by the type of
14
15 shear stress applied. Future studies will have to answer the role these secreted proteins in
16
17
18
flow-dependent diseases such as endothelial dysfunction and atherogenesis.
19
20
21
22
23
24
25 Procedure Number of identified Proteins
26 isolation of HUVEC control laminar
27 condition shear stress
labelling of HUVEC with L-Lysine-13C6 15N2 (78) (83)
28 and L-Arginine-13C6 15N4
29
application of shear stress
30
31 ultracentrifugation of supernatant
32
1D SDS-PAGE of soluble proteins
33
34 in-gel trypsinisation of proteins oscillatory
35 analysis of peptides with LC-MS/MS
shear stress
(79)
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
31
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16 Static (control) condition: 395 proteins
17
18
19
20
21
22
23
lysosome: 3 (1%)
25 (6%) endosome: 2 (0%)
26 unknow n: 4 (1%)
27
28 secreted: 78
29
30 Golgi: 8 (2%) (17%)
31
32 ER: 17 (4%)
33
34
35
36
37
38
39 nucleus: 70 (16%)
40
41
42 membrane: 43
43
(10%)
44
cytoplasm: 195
45
46 (43%)
47
48
49
50
51
52
53
54
55
56
57 Fig. 1
58
59
60

1
2
3
4
5
6
7
8 A Laminar condition: 327 proteins
9
10 mitochondrium: 7 lysosome: 2 (1%)
11 (2%) endosome: 1 (0%)
12
13
unknow n: 4 (1%)
14 Golgi: 4 (1%)
15
ER: 10 (3%) secreted: 83
16
(23%)
17
18
19
20
nucleus: 62 (17%)
21
22
23 membrane: 26
24 (7%)
25
26
27
28
29
30 cytoplasm: 169
31 (45%)
32
33
34
B Oscillatory condition: 507 proteins
35
36
lysosome: 6 (1%) endosome: 2 (0%)
38 (9%)
39 unknow n: 4 (1%)
40
41 secreted: 79 (14%)
Golgi: 13 (2%)
42
43
44 ER: 32 (6%)
45
46
47
48
49
50
51 nucleus: 91 (16%)
52
53 cytoplasm: 234
54 (41%)
55
membrane: 58
56
(10%)
57 Fig. 2
58
59
60

9
8
7
6
5
4
3
2
1
60
59
58
57
56
55
54
53
52
51
50
49
48
47
46
45
44
43
42
41
40
39
38
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
number of differences GO molecular function
st
ru
ct
ur
al
m
ol
ec
-2
-1
0
1
2
3
4
5
ul
e
ac
tiv
se i ty
r in pr
e- ot
ty ei
p e n
en bi
nd
do in
pe g
p tid
se
r in
as
e- e
ty ac
pl pe t iv
at pe it y
el
et pt
-d id
er as
iv e
ed ac
gr tiv
ow ity
th
fa
ct
or
bi
nd
in
g
he
pa
r in
bi
nd
in
pe g
pt
id
as
e
ac
ti v
ity
zi
n c
io
n
bi
nd
in
hy g
dr

ol
as
e
ac
ti v
ity
Differences in comparison to control - GO molecular function
bi
gr nd
in
ow g
th
fa
ct
or
a ct
iv
it y
in cy
s ul to
in ki
n
- li
ke e
ac
gr t iv
ow it y
th
fa
ct
or
bi
nd
in
g
D laminar
Fig. 3
D oscillatory
Page 34 of 35
9
8
7
6
5
4
3
2
1
60
59
58
57
56
55
54
53
52
51
50
49
48
47
46
45
44
43
42
41
40
39
38
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
number of differences GO biological function
ce
-3
-2
-1
0
1
2
3
4
5
6
ll a
Page 35 of 35
dh
po es
si io
m
ti ve n
or re pr
ph gu ot
o ge l at eo
n io l ys
es n is
is of
of ce
e ll
m di
vi
br s
y io
on n
ic
ep
or i th
ga el
n iu
m
m
or
ph
ep o ge
id
er ne
m sis
is
de
ve
lo
p m
ce en
ll-
c t
el
po ls
si ig
tiv na
e lin
re g
gu an
l at gi
io og
n e ne
of
ce si
ne
ll s
rv
o us
pr
ol
sy ife
s ra
te t io
po m n
si de
tiv ve
e lo
re pm
tra gu
ns sk l en
fo e
at
io t
r m le
ta n
in l of
g sy m
gr st it o
ow em si
th s
de
fa ve
ct lo
or pm
be
ta en
t
re

ce os
p to sif
r si ica
gn tio
re a lin n
gu
l at g
io pa
n th
of w
ce ay
l lp
m ro
ul lif
t ic er
el at
lu io
l ar n
or
ga
n ism
tra
ns
al po
de rt
ve
Differences in comparision to control - GO biological function
l op
m
im en
m t
un
e
re
sp
on
ce se
ll d
iff
er
en
t ia
tio
n
D laminar
Fig. 4
D oscillatory

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Article

The secretome of human endothelial cells under shear stress

Journal of Proteome Research is published by the American Chemical Society.

fragments were in-gel-trypsinized at 37°C overnight. In order to extract the peptides, 80 µl

many peptides were found compared to nonsecreted proteins.

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