Secretome of Human Endothelial Cells under Shear Stress - Journal...
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Secretome of Human Endothelial Cells under Shear Stress Sandra Burghoff * and J€urgen Schrader Institute for Cardiovascular Physiology, Heinrich Heine University, Duesseldorf, Germany
bS Supporting Information ABSTRACT: Endothelial cells are exposed to different types of shear stress which triggers the secretion of subsets of proteins. In this study, we analyzed the secretome of endothelial cells under static, laminar, and oscillatory flow. To differentiate between endogenously expressed and added proteins, isolated human umbilical vein endothelial cells were labeled with L-Lysine-13 C6,15N2 and L-Arginine-13C6,15N4. Shear stress was applied for 24 h using a cone-and-plate viscometer. Proteins from the supernatants were isolated, trypsinized, and finally analyzed using LC-MS/MS (LTQ). Under static control condition 395 proteins could be identified, of which 78 proteins were assigned to the secretome according to Swiss-Prot database. Under laminar shear stress conditions, 327 proteins (83 secreted) and under oscillatory shear stress 507 proteins (79 secreted) were measured. We were able to identify 6 proteins specific for control conditions, 8 proteins specific for laminar shear stress, and 5 proteins specific for oscillatory shear stress. In addition, we identified flow-specific secretion patterns like the increased secretion of cell adhesion proteins and of proteins involved in protein binding. In conclusion, the identification of shear stress specific secreted proteins (101 under different flow conditions) emphasizes the role of endothelial cells in modulating the plasma composition according to the physiological requirements. KEYWORDS: endothelial cells, shear stress, LC-MS/MS, secretome, laminar flow, oscillatory flow
’ INTRODUCTION The vascular endothelium forms a multifunctional, dynamic interface that is constantly exposed to wall shear stress generated by flowing blood. Blood flow, in addition, regulates the internal diameter of arterial vessels both acutely, by relaxation and contraction of smooth muscle cells, and chronically, by vascular remodeling of cellular and extracellular components. This regulation involves modulation of membrane proteins and ion channels, activation of transcription factors, cellular reorganization and change of cell shape. These responses are accomplished within seconds to hours and may be mechanistically important in the pathogenesis of vascular diseases such as atherogenesis.1,2 Several proteins have been described to be differentially responsive to fluid shear stress. Topper et al. described mRNA up-regulation for manganese superoxide dismutase (Mn SOD), cyclooxygenase (COX)-2 and endothelial nitric oxidase (NO) synthase (eNOS) by steady laminar shear stress.2 In further studies, two members of the MAD family, namely Smad6 and Smad7, and the bumetanide-sensitive cotransporter BSC2, one of the two major isoforms of Na-K-Cl cotransporters present in mammalian cells, were identified to be up-regulated upon laminar shear stress.3,4 Whether expression of endothelin-1 is altered upon shear stress is an ongoing discussion.5 The identification of not just a few but of all secreted proteins (secretome) from cells is still a major challenge, and advanced approaches are inevitable. Using mass spectrometry analysis, r 2010 American Chemical Society
Dupont et al. succeeded in the identification of 18 different proteins that were secreted from human arterial smooth muscle cells.6 The identified proteins are involved in a broad range of biological functions like regulation of fibrinolysis (Plasminogen activator inhibitor-1), proteolysis (collagenase), ion transport (serotransferrin) and others. In vascular smooth muscle cells, the heat shock protein 90-R and cyclophilin B were identified as secreted factors after oxidative stress induction.7 Again, Dupont et al. established the first secretome of human macrophages comprising 38 proteins.8 Among the identified proteins, known secreted proteins (e.g., serotransferrin, vimentin) but also proteins not known to be secreted (e.g., Glyceraldehyde 3-phosphate deshydrogenase (GAPDH), transaldolase) were detected. Using an in vitro “foam cell” model of atherosclerosis, 59 proteins in the supernatant could be identified whose secretion was increased upon oxidized LDL treatment in comparison to treatment with LDL.9 In the search for potential biomarkers using atherosclerotic plaque secretome, Duran et al. were able to identify 14 proteins in the supernatant of noncomplicated plaques.10 Again, some of the identified proteins were expected (e.g., apolipoproteins) whereas the identification of others cannot be easily understood (e.g., ubiquitin carboxy-terminal hydrolase). Still, in another study, more than 80 proteins Received: September 15, 2010 Published: December 26, 2010 1160
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Journal of Proteome Research differentially expressed in complicated atherosclerotic plaques versus adjacent fibrous plaques could be identified.11 Again, the identified proteins cover a wide range of biological functions like signal transduction, transcription factors, cell communication and protein transporters. In addition, Tunica et al. identified 182 human proteins in the supernatant of human umbilical vein endothelial cells under static conditions of which 70 are known to be secreted.12 Again, in this study, the identified proteins are widespread in function. The proteomic analysis of endothelial cells in response to shear stress is still in its infancies. Wang et al. revealed that 142, 213, and 186 candidate proteins in vascular endothelial cells were up- or down-regulated after 10 min, 3 h, and 6 h of laminar flow.13 These intracellular proteins encompass many signaling pathways like integrins, PI3K/AKT, apoptosis, Notch and cAMP-mediated pathways. Whereas this study investigated the intracellular proteom so far no study has been undertaken in which the secretome of endothelial cells in response to shear stress was investigated. In the present study, we used LC-MS/MS to qualitatively profile the secretome of human umbilical vein endothelial cells (HUVECs) in response to physiological levels of laminar and oscillatory shear stress. To avoid bias with serum proteins during mass spectrometry analysis, all proteins expressed by HUVECs were labeled with heavy isotope amino acids. We identified 395 proteins after no flow conditions, 327 proteins after 24 h of steady laminar flow, and 507 proteins after 24 h of oscillatory flow. These results provide the first comprehensive analysis of secreted proteins that are flow specific, and this will open the way to study the role these proteins in shear stress mediated development of vascular disease such as atherosclerosis.
’ MATERIALS AND METHODS HUVEC Isolation and Labeling
HUVECs were harvested by collagenase (Biochrom AG, Berlin, Germany) and cultured to subconfluency in 4 mL Basal Medium supplemented with endothelial single quots (PromoCell, Heidelberg, Germany) on gelatin-precoated 60-mm culture dishes. For cell labeling Basal Medium and single quots without arginine and lysine were used (PromoCell, Heidelberg, Germany) supplemented with 0.3 mmol/L L-Arginine-13C6,15N4 hydrochloride and 1 mmol/L L-Lysine-13C6,15N2 hydrochloride (Sigma, Taufkirchen, Germany). Freshly isolated HUVECs were grown for 5 cell doublings using this medium. Application of Shear Stress
HUVECs (with 4 mL medium) were subjected to laminar shear stress at 15 dyn/cm2 and oscillatory shear stress (8 dyn/ cm2) in a cone-and-plate viscometer for 24 h one day after reaching subconfluency as described previously.14,15 The viscometer consists of a cone with an angle of 0.5° rotating on top of a cell culture dish. A control dish (no shear stress) accompanied each cell culture dish from the same HUVEC preparation. Each experiment was carried out in duplicate. Analysis of Proteins
Supernatant (4 mL) after shear stress application was depleted of cell debris by ultracentrifugation (150 000 g; 2 h, 4 °C). Soluble proteins were bound to StrataClean Resin (Stratagene, La Jolla, CA) for 1 h at 8 °C. Resins with bound proteins were transferred into SDS-loading buffer (156.25 mM Tris-HCl pH 6.8,
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2.3% (w/v) SDS, 4% (v/v) glycerol, 0.5% (v/v) 2-mercaptoethanol), boiled (5 min, 95 °C) and then subjected to gradient SDS-PAGE (6-18%). Each lane was divided into 20 fragments. The proteins within the gel fragments were in-geltrypsinized at 37 °C overnight. In order to extract the peptides, 80 μL 50 mM (NH4)CO3, and 2 100 μL 50% (v/v) acetonitril, 5% (v/v) formic acid were added successively to the gel pieces. The volume of the extraction solution was reduced to 0.1. For positive identification of a protein at least 2 peptides per protein had to meet these restrictions. Expasy web server (http://www.expasy.org/sprot) was used to identify the currently known subcellular localization and gene ontology. Cell Viability Tests
After shear stress application cells were stained with trypan blue (1:1 in PBS) for 5 min. For quantification, three different sites of each culture dish were photographed and the number of viable and nonviable cells were counted from these images. In addition, the LDH-content within the supernatant was determined, which is a marker for cytotoxicity. To do so, we used the LDH-Cytotoxicity Assay Kit II (Biovision, Mountain View, CA) according to the manufacturers instructions. Briefly, supernatant of the cell culture was centrifuged to remove cell debris. Then, 10 μL of the cleared supernatant was mixed with 100 μL LDH reaction mix, incubated for 30 min and the absorbance was 1161
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Figure 1. Cellular component distribution of identified proteins under static (control) conditions. HUVECs were kept at 37 °C and 5% CO2 for 24 h without shear stress. Supernatant was collected, heavy-amino acid containing proteins were identified and organized according to their subcellular annotation based on Swiss-Prot prediction. Shown is the name of the subcellular component, the number of identified proteins and the percentage referred to all proteins.
measured at 450 nm. The percentage of cytotoxicity was calculated using the absorbance after lysis of all cells in a control dish set to 100%.
’ RESULTS To identify proteins that are secreted under different shear stress conditions we used primary endothelial cells (HUVECs) at passage 2. For these experiments HUVECs were grown in the presence of L-Lysine-13C6,15N2 hydrochloride and L-Arginine-13 C6,15N4 hydrochloride, whereas the culture medium was deprived of the amino acids lysine and arginine. Within 5 cell doublings proteins of HUVECs can be assumed to be homogenously labeled.16 On the other hand, proteins contained in the culture medium (e.g., growth factors, proteins within FCS) did not carry the label. Thus, using this method, proteins released by HUVECs can be selectively identified by mass spectrometry. For the identification of the proteins a mass shift of þ8.0 for lysine modification and þ10.0 for arginine modification was considered. When cultured under static (control) conditions 395 proteins with stabile isotope incorporations were identified in the supernatant of the cell culture (Figure 1). Most of them can be attributed to the cytoplasm (195), the membrane (43) and the nucleus (70) (Figure 1, Supplemental Table 1, Supporting Information). From all identified proteins, only 78 (17%) were secreted according to Swiss-Prot database (Figure 1, Table 1). Identified proteins include MMP1 (interstitial collagenase), MIF (macrophage migration inhibitory factor), PAI1 (plasminogen activator inhibitor 1), vWF (von Willebrand factor), ANG2 (angiopoietin-2), IL8 (interleukin 8) and PDGFB (plateletderived growth factor subunit B). To obtain an estimate on the validity of our measurements, we calculated the ratio of the number of assigned peptides per identified protein, which describes how many peptides on average were identified per protein. The ratio of assigned peptides per secreted protein was 9.6 ( 17.24 (n = 78), whereas
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the ratio for all nonsecreted proteins was 4.35 ( 4.34 (n = 317) (p < 0.001). This indicates that for the identification of one secreted protein about twice as many peptides were found compared to nonsecreted proteins. In a next set of experiments, we applied either laminar (15 dyn/cm2) or oscillatory (8 dyn/cm2) shear stress to HUVECs for 24 h. Qualitative identification of proteins released into the supernatants revealed that 327 proteins could be identified from the laminar sample (Figure 2A), whereas 507 proteins were identified from the oscillatory sample (Figure 2B). There were no major differences in the cellular component distribution. Again, most proteins can be attributed to the cytoplasm (169 and 234, respectively), the membrane (26 and 58, respectively) and the nucleus (62 and 91, respectively). The fraction of secreted proteins due to laminar flow was 22.6% (83 proteins) and 13.8% (79 proteins) after oscillatory flow (Figure 2, Table 2). Again, the ratio of assigned peptides per identified protein was significantly higher in secreted proteins (10.36 ( 17.67 for laminar shear stress, 11.22 ( 21.20 for oscillatory shear stress) compared to all other proteins (4.77 ( 4.31 for laminar shear stress, 5.43 ( 6.49 for oscillatory shear stress) (p < 0.001 for both groups). In Table 2, data are summarized for all 101 proteins secreted by HUVEC under control, laminar, and oscillatory conditions. As can be seen, of these 101 proteins 6 proteins were identified that were expressed only under no flow conditions, such as latent transforming growth factor beta binding protein 3 (LTBP3) and platelet derived growth factor subunit B (PDGFB); 8 proteins being expressed under laminar flow conditions, like LTBP4 and PDGFA; and 5 proteins specific for oscillatory flow, like aminopeptidase N (CD13, ANPEP), calreticulin (CALR) and endothelin-1 (EDN1). Furthermore, 8 proteins were identified after laminar shear stress and after control conditions, 10 proteins after any flow condition but not under static control conditions and 7 proteins after no flow and oscillatory flow conditions. Finally, 57 proteins could be identified after any treatment. To further analyze the identified secreted proteins we grouped all 101 proteins according to their Gene Ontology molecular function (Supplemental Figure 1, Supporting Information; shown are all GO entries where at least 3 proteins were assigned to) and Gene Ontology biological function (Supplemental Figure 2 Supporting Information; shown are all GO entries where at least 3 proteins were assigned to). Supplemental Figure 1 (GO molecular function, Supporting Information) shows that most proteins are associated with binding to proteins, calcium ions, heparin, cytokine and/or are structural constituents of extracellular matrix. To visualize dynamic changes dependent on the flow situation, Figure 3 shows differences in the GO molecular function between all 3 experimental groups. The number of proteins assigned to a specific GO molecular function is set to 0 for the control condition, and the number of additional or lacking proteins under the two flow conditions is illustrated. Compared to static conditions most differences under laminar and oscillatory flow occur by the secretion of hydrolyzing proteins exhibiting peptidase activity, especially serine-type peptidase activity and serine-type endopeptidase activity, and proteins with hydrolase activity (Figure 3). In particular the expression of serine protease HTRA1 (Swiss-Prot entry Q92743) and tissue type plasminogen activator (Swiss-Prot entry P00750), which both cleave many proteins in the blood plasma, and of aminopeptidase N (Swiss-Prot entry P15144), which cleaves amino acids from oligopeptides, account for these differences. 1162
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Table 1. Proteins Secreted by HUVECs under Control Conditions (No Shear Stress) Swiss-Prot
Table 1. Continued Swiss-Prot
name
P08253
72 kDa type IV collagenase
MMP2
Q13443 Q9UHI8
ADAM 9 A disintegrin and metalloproteinase
ADAM9 ADAMTS1
with thrombospondin motifs 1 O75173
ADAMTS-4
ADAMTS4
O15123
Angiopoietin-2
ANGP2, ANGPT2
P07355
Annexin A2 isoform 1
ANXA2
P08758
Annexin A5
ANXA5
P98160
Basement Membrane-specific Heparan
PGBM
P61769
Sulfate Proteoglycan core protein Beta-2-microglobulin
B2M
P22004
Bone morphogenetic protein 6
BMP6
P13987
CD59 glycoprotein
CD59
P10909
Clusterin
CLUS, CLU
Collagen alpha-1(V) chain
COL5A1, CO5A1
Q9BXJ0
Complement C1q tumor
C1QTNF5
P08603
necrosis factor-related protein 5 Complement factor H
CFH
P29279
Connective tissue growth factor
CTGF
P01034
Cystatin-C
CST3, CYTC
O94907
Dickkopf-related protein 1
DKK1
Q12805
EGF-containing fibulin-like
FBLN3
extracellular matrix protein 1 P35555
Fibrillin-1
FBN1
P35556 P02751
Fibrillin-2 Fibronectin, Isoform 1
FBN2 FN1
Q12841
Follistatin-related protein 1
FSTL1
P09382
Galectin-1
LGALS1, LEG1
P06396
Gelsolin
GELS, GSN
Q99988
Growth/differentiation factor 15
GDF15
P09341
Growth-regulated alpha protein
CXCL1
Q96QV1
Hedgehog-interacting protein
HHIP
P18065
Insulin-like growth factor-binding protein 2
IGFBP2, IBP2
P22692
Insulin-like growth
IGFBP4, IBP4
factor-binding protein 4 Q16270
Insulin-like growth
IGFBP-7
factor-binding protein 7 O00622
Insulin-like growth
CYR61
factor-binding protein 10 P10145 P03956
Interleukin 8 Interstitial collagenase
IL8 MMP1
Q14767
latent transforming growth
LTBP-2
factor beta-binding protein 2 Q9NS15
latent transforming growth
LTBP3
factor beta-binding protein 3 Q16363
Laminin subunit alpha-4
LAMA4
P07942
Laminin subunit beta-1
LAMB1
P55268 P11047
Laminin subunit beta-2 Laminin subunit gamma-1
LAMB2 LAMC1
Q9Y4K0
Lysyl oxidase homologue 2
LOXL2
P14174
Macrophage migration inhibitory factor
MIF
P08493 P01033
Matrix Gla protein Metalloproteinase Inhibitor 1
MGP TIMP1
P16035
Metalloproteinase Inhibitor 2
TIMP2
P55001
Microfibrillar-associated protein 2
MFAP2
P21741
Midkine
MDK, MK
Q13201
Multimerin-1
MMRN1
Q9H8L6
Multimerin-2
MMRN2
P26022
Pentraxin-related protein PTX3
PTX3
P05121
Peroxidasin Plasminogen activator inhibitor 1
PXDN PAI1, SERPINE1
P01127
Platelet-derived growth factor subunit B
PDGFB
P55145
Protein ARMET
ARMET
P07237
Protein disulfide-isomerase
P4HB, PDI
P21980
Protein-glutamine
TGM2
P09486
Secreted protein acidic
SPARC
O95084
and rich in cysteine Serine protease 23
PRSS23, PRS23
P52823
Stanniocalcin-1
STC1
O00391
Sulfhydryl oxidase 1
QSOX1
Q08629
Testican-1, SPOCK
TICN1, SPOCK
P05452
Tetranectin
TETN, CLEC3B
P07996
Thrombospondin-1
TSP1
P10646
Tissue factor pathway inhibitor
TFPI
P48307 P20062
Tissue factor pathway inhibitor 2 Transcobalamin-2
TFPI2 TCN2
Q9GZM7
Tubulointerstitial nephritis antigen-like
TINAGL1
P04275
von Willebrand factor
vWF
gamma-glutamyltransferase 2
Coiled-coil domain-containing protein 80 CCDC80 P20908
name
When all 101 secreted proteins are organized according to their biological function (Supplemental Figure 2, Supporting Information, shown are all GO entries with at least 3 proteins assigned to), cell adhesion is the predominant function of the proteins. In addition, signal transduction, multicellular organismal development and proteolysis are among the most cited ones. Also for this group there are dynamic changes between the 3 groups. Again, the number of proteins assigned to specific GO biological function is set to 0 for control condition. Compared to control conditions secretion of proteins involved in cell adhesion is increased (Figure 4). Here, collagen alpha-1(VI) (COL6A1, Swiss-Prot entry P12109), laminin subunit alpha-5 (LAMA5, Swiss-Prot entry O15230), and laminin subunit gamma-2 (LAMC2, Swiss-Prot entry Q13753) were found, as well as carboxypeptidase-like protein X2 (CPXM2, Swiss-Prot entry Q8N436) and thrombospondin-2 (TSP2, Swiss-Prot entry P35442). Besides cell adhesion, the secretion of proteins performing proteolysis is also increased. Here, we identified proteins like serine protease HTRA1 (Swiss-Prot entry Q92743), tissue type plasminogen activator (Swiss-Prot entry P00750) and aminopeptidase N (Swiss-Prot entry P15144). Interestingly, the number of proteins with the GO annotation multicellular organismal development was decreased after laminar shear stress (-2) but increased after oscillatory fluid flow (þ2). In an additional set of experiments, we checked the viability of the cultured HUVECs. The fraction of cells without shear stress that could be stained with trypan blue solution was 0.093 ( 0.108% 1163
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Figure 2. Cellular component distribution of identified proteins under laminar and turbulent shear stress conditions. HUVECs were kept at 37 °C and 5% CO2 for 24 h with (A) 15 dyn/cm2 laminar shear stress or (B) 8 dyn/cm2 oscillatory shear stress. Supernatants were collected; heavyamino acid containing proteins were identified and organized according to their subcellular annotation based on Swiss-Prot prediction. Shown is the name of the subcellular component, the number of identified proteins, and the percentage referred to all proteins.
of all cells (n = 4). In addition, lactate dehydrogenate (LDH) measurement in the supernatant revealed that 1.93% (n = 3) of all cells died within this period of time. After 24 h of shear stress, trypan blue staining revealed that 0.153 ( 0.041% (n = 3) of all cells were nonviable, whereas LDH measurements show that 3.74% of all cells died within the same period of time. Supporting this finding, LDH was also detected with MS/MS in all samples.
’ DISCUSSION This study investigated the influence of shear stress on the secretome of HUVECs. To this end, we applied laminar or oscillatory flow to HUVECs and analyzed the supernatant with sensitive mass spectrometric techniques. We have identified a total of 395 proteins liberated into the supernatant under static
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conditions, and this fraction remained unchanged under laminar (327 proteins) and oscillatory (507 proteins) flow conditions. The fraction of proteins assigned to be secreted amounted to only 78 proteins under static conditions (17%) and was similar with laminar flow (83 proteins) and oscillatory flow (79 proteins). Among the 101 proteins identified to be secreted, 57 were found to be secreted independently of the flow condition. This study is the second to explore the secretome of HUVECs in detail. In 2009, Tunica et al.12 reported 70 secreted proteins of which 43 were identified by us as well. In addition, we have identified additional 58 proteins that are known to be secreted proteins. While the former study by Tunica et al. restricted the analysis to static flow conditions, the present study explored changes in the secretome under laminar and turbulent flow conditions. The dynamic changes of the endothelial secretome found in this study in response to shear stress may be functionally relevant, because shear stress is the physiological stimulus for endothelial cells and changes in shear stress are known to be of importance for the development of vascular diseases such as atherogenesis. Aside from the secreted proteins, the majority of proteins identified could be attributed to specific cellular compartments, such as cytoplasm, membrane, and nucleus. On the one hand, this shows the sensitivity of the MS-based method that permits the detection of proteins 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 between extracellular and intracellular proteins. To discriminate between endogenously expressed proteins and proteins that were required for optimal cell culture conditions, we have labeled HUVEC with heavy isotope amino acids during our experiments. As to extracellular contaminating proteins, we measured cell integrity by trypan blue staining and found 0.093% of nonsheared cells and 1.93% of all cells after shear stress to be nonviable. Similarly, the corresponding values from LDH cytotoxicity assays were 0.153 and 3.74%, respectively. These data show that the fraction of dead/dying cells is rather low and comparable to data in the literature.17 However, mass spectrometry is so sensitive that this small fraction can considerably “contaminate” the measurement of secreted proteins. Many proteins identified by us were also found under similar experimental conditions by others such as HSP27,11 nucleoside diphosphate kinase B,11,12 cathepsin D,6,6,8,11,12 protein disulfide isomerase,8,11,12 vimentin,6,8,9,11,12 filamin,11,12 60 kDa heat shock protein,8 R-enolase,6,8,9 fructose-bisphosphate aldolase A8 and B,12 annexin A1,8 F-actin capping protein βunit,8 tropomyosin R 3 chain,8,12 glutathione S-transferase P8 and glyceraldehyde 3-phosphate dehydrogenase.6,12 As to the accuracy of protein identification by MS it should be noted that the false positive rate has been reported to be only about 1% (e.g., Huttlin et al.,18 Xie et al.,19 Lu et al.20). Identified proteins were assigned to their cellular compartment according to Swiss-Prot database. In this database, proteins are assigned as being secreted when they have been positively identified as secreted proteins by other biochemical experiments. This implies that it is quite possible that some of the proteins identified by us and assigned to cytoplasmic or membrane origin might also have been secreted. This, however, cannot be decided on the basis of our experiments, since it will not be possible to fully exclude cell death/lysis even when using inhibitors of cell apoptosis. Regarding the reproducibility of our data, it should be 1164
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Table 2. Proteins Secreted by HUVECs under Control Conditions and Laminar and Oscillatory Shear Stress Swiss-Prot
Name detected only under control conditions
P22004
Bone morphogenetic protein 6
Q9UGM3
deleted in malignant brain tumor 1
BMP6 DMBT1
Q9NS15
latent transforming growth factor beta-binding protein 3
LTBP3
P55001
Microfibrillar-associated protein 2
MFAP2
P01127
Platelet-derived growth factor subunit B
PDGFB
P07998
Ribonuclease pancreatic
RNASE1, RNAS1
O00468 Q9NQ79
Agrin Cartilage acidic protein 1
detected only under laminar shear stress
Q99715
AGRN CRTAC1
Collagen alpha-1(XII) chain
COL12A1
LAMA5
LAMA5
latent transforming growth factor beta-binding protein 4
LTBP4
Meteorin-like protein
METRNL
P12272
Parathyroid hormone-related protein
PTHR, PTHLH
P04085
Platelet-derived growth factor subunit A
PDGFA
Q16352 P15144
Alpha-internexin Aminopeptidase Na
INA, AINX ANPEP
P27797
Calreticulin
CALR
P05305
Endothelin-1
EDN1
P01344
Insulin-like growth factor II
IGF2
Q16363
Laminin subunit alpha-4
P55268
Laminin subunit beta-2
LAMB2
Q96RW7 P09486
Hemicentin-1, Fibulin 6 Secreted protein acidic and rich in cysteinea
HMCN1, FIBL-6 SPARC
P52823
Stanniocalcin-1
STC1
P05452
Tetranectin
TETN, CLEC3B
P20062
Transcobalamin-2
TCN2
Q9H8L6
Multimerin-2a
MMRN2
O15123
Angiopoietin-2
ANGP2, ANGPT2
P10145
Interleukin 8
IL8
Q969H8 P00387
Interleukin 25a NADH-cytochrome b5 reductase 3
IL25, C19orf10 CYB5R3
Q15063
Periostin
POSTN
P07237
Protein disulfide-isomerasea
P4HB, PDI
Q9BRX8
Uncharacterized protein C19orf58
P21810
Biglycan
BGN
Q8N436
Carboxypeptidase-like protein X2
CPXM2
P12109
Collagen alpha-1(VI) chain
COL6A1
Q9Y287 O15230
Integral membrane protein 2B Laminin subunit alpha-5
ITM2B LAMA5
Q13753
Laminin subunit gamma-2
LAMC2
Q92743
Serine protease HTRA1
HTRA1
P00750
Tissue-type plasminogen activator
TPA, PLAT
P35442
Thrombospondin-2
TSP2
P49767
Vascular endothelial growth factor C
VEGFC
detected only under oscillatory shear stress
detected under control conditions and laminar shear stress LAMA4
detected under control conditions and oscillatory shear stress
detected under laminar and oscillatory shear stress
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Table 2. Continued Swiss-Prot
Name detected under control conditions, laminar and oscillatory shear stress
P08253
72 kDa type IV collagenasea
Q13443
ADAM 9
ADAM9
Q9UHI8
A disintegrin and metalloproteinase with thrombospondin motifs 1
ADAMTS1
O75173
ADAMTS-4
ADAMTS4
P07355
Annexin A2 isoform 1a
ANXA2
P08758
Annexin A5a
ANXA5
P98160
Basement Membrane-specific Heparan Sulfate Proteoglycan core proteina
PGBM
P61769 Q16627
Beta-2-microglobulina C-C motif chemokine 14
B2M CCL14
P13987
CD59 glycoproteina
CD59
P10909
Clusterin
CLUS, CLU
Coiled-coil domain-containing protein 80
CCDC80
MMP2
P20908
Collagen alpha-1(V) chain
COL5A1, CO5A1
P39060
Collagen alpha-1(XVIII) chain
COL18A1
Q9BXJ0
Complement C1q tumor necrosis factor-related protein 5
C1QTNF5
P08603 P29279
Complement factor H Connective tissue growth factora
CFH CTGF
P01034
Cystatin-Ca
CST3, CYTC
O94907
Dickkopf-related protein 1
DKK1
Q12805
EGF-containing fibulin-like extracellular matrix protein 1a
FBLN3
P35555
Fibrillin-1
FBN1
P35556
Fibrillin-2
FBN2
P02751
Fibronectin, Isoform 1
FN1
Q12841 P09382
Follistatin-related protein 1a Galectin-1a
FSTL1 LGALS1, LEG1
P06396
Gelsolin
GELS, GSN
Q99988
Growth/differentiation factor 15
GDF15
P09341
Growth-regulated alpha proteina
CXCL1
Q96QV1
Hedgehog-interacting protein
HHIP
P18065
Insulin-like growth factor-binding protein 2a
IGFBP2, IBP2
P22692
Insulin-like growth factor-binding protein 4a
IGFBP4, IBP4
Q16270 O00622
Insulin-like growth factor-binding protein 7a Insulin-like growth factor-binding protein 10a
IGFBP-7 CYR61
P03956
Interstitial collagenasea
MMP1
Q14767
latent transforming growth factor beta-binding protein 2
LTBP-2
P07942
Laminin subunit beta-1
LAMB1
P11047
Laminin subunit gamma-1
LAMC1
Q9Y4K0
Lysyl oxidase homologue 2a
LOXL2
P14174
Macrophage migration inhibitory factor
MIF
P08493 P01033
Matrix Gla protein Metalloproteinase Inhibitor 1a
MGP TIMP1
P16035
Metalloproteinase Inhibitor 2
TIMP2
P21741
Midkine
MDK, MK
Q13201
Multimerin-1a
MMRN1
P26022
Pentraxin-related protein PTX3a
PTX3
Peroxidasin
PXDN
P05121
Plasminogen activator inhibitor 1a
PAI1, SERPINE1
P55145 P21980
Protein ARMETa Protein-glutamine gamma-glutamyltransferase 2a
ARMET TGM2
O95084
Serine protease 23a
PRSS23, PRS23
O00391
Sulfhydryl oxidase 1
QSOX1
Q08629
Testican-1, SPOCKa
TICN1, SPOCK
P07996
Thrombospondin-1a
TSP1 1166
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Table 2. Continued Swiss-Prot
a
Name a
P10646 P48307
Tissue factor pathway inhibitor Tissue factor pathway inhibitor 2
TFPI TFPI2
Q9GZM7
Tubulointerstitial nephritis antigen-like
TINAGL1
P04275
von Willebrand factora
vWF
Previously identified in the secretome of HUVECs.12
Figure 3. Differences in the number of identified proteins per Gene Ontology molecular function; comparison between all 3 study groups (control, laminar, oscillatory). Static control is set to zero, differences from there are shown for laminar and oscillatory flow. Shown are all comparisons where the number of assigned proteins differ for 2 at least.
noted that out of the 101 proteins known to be secreted 76 were identified in each of the two flow-identical experiments. The reproducibility is in the same range as was reported recently by others.21 Our study identified several proteins the expression/secretion of which has been reported to be dependent on shear stress. Examples are MMP2,22 insulin-like growth factor-binding proteins, 23 metalloproteinase inhibitor 1 and 2 24,25 and von Willebrand factor.26 The influence of shear stress on the expression of endothelin-1 is dependent both on the level of shear stress and the duration of application. Wang et al. showed that an intermediate level of shear stress leads to a maximum secretion of endothelin-1.27 Kuchan et al. reported, that the secretion of endothelin-1 increases initially, but decreases after 6 h of laminar flow28 while Dancu et al. found an increased endothelin-1 secretion during asynchronous hemodynamics.29 Similar results were obtained by Walshe et al. who reported an increased expression of endothelin-1 after oscillatory flow.30 Collectively these data suggest very low levels of secreted endothelin-1 in noflow samples and after long-time laminar shear stress, but elevated levels after oscillatory flow. Consistent with these
studies we identified endothelin-1 only after oscillatory shear flow, but not under laminar flow and static conditions. Although the mixture of secreted proteins is quite complex, several proteins could be clearly attributed to the flow condition: LTBP3 to static no-flow condition, BMP6 and PDGFB to flow, LAMA5, PDGFA and LTBP4 to laminar flow and aminopeptidase N and endothelin-1 to oscillatory flow. Apparently, the number of proteins with peptidase activity is increased under flow conditions. We found fluid shear stress to stimulate the secretion of tissue plasminogen activator by endothelial cells whereas the secretion of plasminogen activator inhibitor type-1 remained unaltered which is consistent with data in the literature.31 Whereas plasminogen activator inhibitor type-1 was identified under all experimental conditions, we detected tissue plasminogen activator only with laminar and oscillatory flow. This suggests that the fibrinolytic potential of endothelial cells increases in response to hemodynamic forces. We also identified the serine protease HTRA1 to be secreted both after laminar and oscillatory flow. HTRA1 is part of the insulin-growth-factor signaling pathway and cleaves IGF binding proteins. Upregulation of its mRNA in osteoblasts in response to fluid shear has been reported32 which 1167
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Figure 4. Differences in the number of identified proteins per Gene Ontology biological function; comparison between all 3 study groups (control, laminar, oscillatory). Static control is set to zero, differences from there are shown for laminar and oscillatory flow. Shown are all comparisons where the number of assigned proteins differ for 2 at least.
takes place downstream of the early mechano-responsive genes Igtb1 and Cox-2. Another peptidase with broad specificity identified by us to be secreted only after oscillatory flow is aminopeptidase N (CD13). CD13 was found to be secreted by HL60 cells when agitated and the expression of CD13 was proportional to the agitation intensity.33 In summary, this is to our knowledge the first comprehensive study of secretome profiling in human endothelial cells as influenced by shear stress. We identified 587 different proteins of which several are secreted in a flow specific manner. In addition, we have identified several previously unreported proteins and show that the secretome is modulated by the type of shear stress applied. Further studies on the secretome of endothelial cells will have to answer the question whether proteins secreted by endothelial cells can alter the composition of plasma proteins and may be involved in the development of flowdependent diseases such as atherogenesis.
’ ASSOCIATED CONTENT
bS
Supporting Information Supplementary figures and tables. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Dr. Sandra Burghoff, Heinrich Heine University Duesseldorf, Institute for Cardiovascular Physiology Universitaetsstr. 1, 40225 Duesseldorf. Tel.: þ49-211/8112671. Fax: þ49-211/8112672. E-mail: sandra.burghoff@uni-duesseldorf.de.
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