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iTRAQ quantitative proteomics-based identification of cell adhesion as a dominant phenotypic modulation in thrombin-stimulated human aortic endothelial cells
TR-05873; No of Pages 7 Thrombosis Research xxx (2015) xxx–xxx
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Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Regular Article
iTRAQ quantitative proteomics-based identification of cell adhesion as a dominant phenotypic modulation in thrombin-stimulated human aortic endothelial cells Huang-Joe Wang a,b, Sung-Fang Chen c,1, Wan-Yu Lo d,⁎,1 a
School of Medicine, China Medical University, Taichung, Taiwan Division of Cardiology, Department of Medicine, China Medical University Hospital, Taichung, Taiwan c Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan d Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan b
a r t i c l e
i n f o
Article history: Received 20 July 2014 Received in revised form 16 February 2015 Accepted 24 February 2015 Available online xxxx Keywords: Thrombin quantitative proteomics human aortic endothelial cells Isobaric tags for relative and absolute quantitation (iTRAQ)
a b s t r a c t Introduction: The phenotypic changes in thrombin-stimulated endothelial cells include alterations in permeability, cell shape, vasomotor tone, leukocyte trafficking, migration, proliferation, and angiogenesis. Previous studies regarding the pleotropic effects of thrombin on the endothelium used human umbilical vein endothelial cells (HUVECs)—cells derived from fetal tissue that does not exist in adults. Only a few groups have used screening approaches such as microarrays to profile the global effects of thrombin on endothelial cells. Moreover, the proteomic changes of thrombin-stimulated human aortic endothelial cells (HAECs) have not been elucidated. Materials and methods: HAECs were stimulated with 2 units/mL thrombin for 5 h and their proteome was investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and the MetaCoreTM software. Results: A total of 627 (experiment A) and 622 proteins (experiment B) were quantified in the duplicated iTRAQ analyses. MetaCoreTM pathway analysis identified cell adhesion as a dominant phenotype in thrombinstimulated HAECs. Replicated iTRAQ data revealed that “Cell adhesion_Chemokines and adhesion,” “Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity,” and “Cell adhesion_Integrin-mediated cell adhesion and migration” were among the top 10 statistically significant pathways. The cell adhesion phenotype was verified by increased THP-1 adhesion to thrombin-stimulated HAECs. In addition, the expression of ICAM-1, VCAM-1, and SELE was significantly upregulated in thrombin-stimulated HAECs. Conclusions: Several regulatory pathways are altered in thrombin-stimulated HAECs, with cell adhesion being the dominant altered phenotype. Our findings show the feasibility of the iTRAQ technique for evaluating cellular responses to acute stimulation. © 2015 Elsevier Ltd. All rights reserved.
Introduction One of the most important functions of normal endothelium is to maintain blood in the fluid state. The non-thrombogenic properties of endothelium can be damaged by several inflammatory insults, Abbreviations: CAM, Cell adhesion molecule; SELE, E-selectin; FDR, false discovery rate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HAEC,human aorticendothelialcell; HUVECs, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule 1; iTRAQ, isobaric tags for the relative and absolute quantification; LC-ESI-MS/MS, liquid chromatography-electrospray ionization-tandem mass spectrometry; LDL, low-density lipoprotein; PCR, polymerase chain reaction; SCX, strong cation exchange chromatography; VCAM-1, vascular cell adhesion protein 1 ⁎ Corresponding author at: Graduate Integration of Chinese and Western Medicine, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan. Tel.: +886 4 22053366x3513; fax: +886 4 22056139. E-mail address: [email protected] (W.-Y. Lo). 1 These authors contributed equally to this work.
including hypoxia, fluid shear stress, oxidants, interleukin-1, tumor necrosis factor, γ-interferon, endotoxin, and thrombin [1]. Thrombin is a serine protease that plays a key role in vascular diseases. In atherothrombosis, thrombin is generated in the site of a ruptured plaque, which in turn triggers the coagulation process. Besides its role in coagulation, thrombin also plays a significant role in the pro-inflammatory response [2]. Thrombin-stimulated endothelial cells undergo multiple phenotypic changes, including alterations in permeability, cell shape, vasomotor tone, leukocyte trafficking, migration, proliferation, and angiogenesis [3,4]. Most studies investigating the pleotropic effects of thrombin on the endothelium have used tissue obtained from the human umbilical vein endothelial cell (HUVECs), which is a fetal tissue that does not exist in adults. In addition, major studies on thrombin have focused on a few specific genes. Only a few groups have used screening approaches to profile the global thrombin effects on endothelial cells using cDNA
http://dx.doi.org/10.1016/j.thromres.2015.02.031 0049-3848/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: Wang H-J, et al, iTRAQ Quantitative Proteomics-Based Identification of Cell Adhesion as a Dominant Phenotypic Modulation in Thrombin-Stimulated Human Aortic Endothelial Cells, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.02.031
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microarray techniques [3,5–7]. These experiments are invaluable for identification of novel target genes. However, such approaches failed to reveal the important post-transcriptional control mechanisms in the endothelium [8]; the real proteomic changes in the thrombinstimulated endothelial cells are not yet fully elucidated. Proteomic approaches offer a large potential for obtaining the comprehensive protein expression data of endothelial cells. The progress of proteomics and bioinformatics further help scientists to identify important signal transduction pathways and functional cellular information. Isobaric tags for the relative and absolute quantification (iTRAQ) is a shotgun approach that enable high-throughput quantitative proteomics analysis [9]. Briefly, the iTRAQ technique labels samples with four independent isobaric tags that, upon fragmentation in MS/MS, produce unique reporter ions that provide quantitative data based on the integration of the peak areas of the different samples [10]. MetaCoreTM is a data-mining and bioinformatics pathway analysis software developed by GeneGo (a Thomson Reuters business). MetaCoreTM software allows for functional analysis of many data forms, including Next Generation Sequencing, microarray, proteomics, siRNA, and microRNA. Our laboratory previously applied the iTRAQ technique and identified miR-27b as a useful biomarker for oral cancer [11]. Since the previous profiling efforts on thrombin-stimulated endothelial effects focused on fetal tissue (i.e., HUVECs), the present study selected human aortic endothelial cells (HAECs) as an adult endothelial in vitro model. The purpose of the present study is to investigate the global protein expression profiles and related pathway analysis in thrombin-stimulated HAECs by the iTRAQ proteomics and MetaCoreTM bioinformatics techniques. Materials and methods Endothelial cell culture and sample treatments for iTRAQ HAECs were purchased from Cell Applications, Inc. (San Diego, CA) and cultured in endothelial cell growth medium (Cell Applications, Inc.) according to the manufacturer’s recommendations. HAECs were cultured and seeded in 6-well plates with 106 cells/well and grown overnight under serum-starved conditions in M-199 medium supplemented with 1% fetal bovine serum (HyClone, Logan, UT). Cells were treated with and without thrombin (2 units/mL) for 5 h (Sigma–Aldrich, St. Louis, MO) and then harvested by centrifugation at 300 × g for 10 min. The obtained pellets were resuspended in RIPA buffer (SigmaAldrich, St. Louis, MO) to extract total protein. The supernatants were enriched and desalted using a 3-kDa centrifugal filter (Millipore, Merck KGaA, Germany). The amount of protein in each concentrated/ desalted sample was determined by Bradford protein assay (Bio-Rad, Hercules, CA), and stored at -80 °C for subsequent processing. The study protocol was granted and approved by the institutional committee. Chemicals and reagents for iTRAQ Modified porcine trypsin (sequencing grade) was obtained from Promega (Madison, WI). iTRAQ 4-plex reagent kits were purchased from Applied Biosystems (Framingham, MA). All other chemicals and reagents were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise stated. Reduction, alkylation, digestion, and labeling with iTRAQ Seventy μg of each protein samples were reduced, alkylated, digested, and labeled with iTRAQ reagents according to the method described in our previous study [11]. The digested samples were labeled with the iTRAQ reagents, and the samples were labeled as follows: 114 designated as controls (without thrombin) and 117 thrombintreated samples. Using two iTRAQ kits, we performed two biological
replicates of iTRAQ quantitative analysis to monitor the consistency of the results. Strong cation exchange chromatography (SCX) and liquid chromatographyelectrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis Peptide separation was performed by Polysulfoethyl A Column (200 mm L × 2.1 mm i.d., 5 μm, 300 A, PolyLC, Columbia, MD) on an Agilent 1100 binary HPLC (Agilent Technologies, Wilmington, DE). The iTRAQ-labeled peptides (280 μg) were eluted at a flow rate of 200 μL/min. Eighty fractions were collected after SCX fractionation and then pooled into 24 fractions, which were subsequently purified by C-18 spin columns (Thermo Scientific, San Jose, CA) for nano-LCESI-MS/MS analysis [11]. Database search and iTRAQ quantification Protein identification of the iTRAQ samples was performed using the Mascot search algorithm (version 2.3.2). The search was performed against the SwissProt v.2011_08 database (531,473 sequences) using the following search parameters: taxonomy: Homo sapiens; enzyme: trypsin; max. miss cleavages: 1; fixed modifications: methylthiolation, N-terminal iTRAQ 4plex, lysine iTRAQ 4plex; variable modifications: methionine oxidation, tyrosine iTRAQ 4plex; MS peptide tolerance: 1.5 Da; MS/MS tolerance: 0.6 Da. Protein identifications were only accepted when ≥ 2 spectra had an ion score above 35 (95% confidence) [12]. The relative protein expression was determined based on the ratio of the experiment and control reporter ions for the peptides (117:114). A decoy search (based on automatically generated random sequences of the same length) was employed to determine the rate of false-positive identifications. The false discovery rate (FDR) b 3% was set as the criteria according to Käll et al. [13]. Data analysis by bioinformatics In the two independent experiments, a total of 648 and 653 proteins were identified from the first and second iTRAQ replicates, respectively (Supplementary data 1A and B). Exclusion of those proteins without 117/114 values, a total of 627 and 622 proteins were well quantified proteins. These quantified proteins were selected for the automatic processing of “protein network construction and analysis” by the MetaCoreTM software (2014_01 database/ Server: portal.genego.com) without any modification. Monocyte adhesion assay The human monocytic cell line THP-1 (derived from acute monocytic leukemia) was obtained from the American Type Culture Collection (Rockville, MD) and maintained according to the manufacturer’s recommendations. In adhesion experiments, THP-1 cells were labeled with calcein acetoxymethyl ester (Calcein-AM; Molecular Probes, Oregon) dye at a concentration of 7.5 μM for 30 min immediately preceding the adhesion assay. HAECs were maintained in 12 well dishes until 90% confluent. HAECs (105 cells/well) were then treated with/without thrombin for 5 h, and then incubated with the culture medium containing the labeled THP-1 cells (THP-1/HAECs = 7) for 10 min. After incubation, non-adherent THP-1 cells were removed by two gentle washes with PBS for 30 s. The adherent THP-1 cells on the activated HEACs were quantified from 10 randomly selected view fields. Real-time polymerase chain reaction (PCR) mRNA expression levels in the HAECs was analyzed by real-time PCR, as previously described [14]. The primer sequences used for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), vascular
Please cite this article as: Wang H-J, et al, iTRAQ Quantitative Proteomics-Based Identification of Cell Adhesion as a Dominant Phenotypic Modulation in Thrombin-Stimulated Human Aortic Endothelial Cells, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.02.031
H.-J. Wang et al. / Thrombosis Research xxx (2015) xxx–xxx Table 1 Top 10 “statistically significant pathway maps” from the duplicate iTRAQ analysis by the MetaCoreTM software. Sorts of Experiment A pathway
Experiment B
1
Cytoskeleton remodeling_ Cytoskeleton remodeling LRRK2 in neurons in Parkinson's disease Cell adhesion_Chemokines and adhesion
4
Cytoskeleton remodeling_ Cytoskeleton remodeling Cell adhesion_Chemokines and adhesion Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity CFTR folding and maturation
5
Glycolysis and gluconeogenesis
6
Cytoskeleton remodeling_TGF, WNT and cytoskeletal remodeling
7
Cell adhesion_Integrinmediated cell adhesion and migration Regulation of CFTR activity
2 3
8
9
10
3
respectively. These quantified proteins were examined from the two replicates. A total of 378 proteins with same ID were identified in two replicates. The percentage overlap of ID proteins was 58%. Cell adhesion was dominant phenotype in thrombin-stimulated HAECs
Cell adhesion_Integrinmediated cell adhesion and migration Cytoskeleton remodeling_TGF, WNT and cytoskeletal remodeling Cytoskeleton remodeling_ Regulation of actin cytoskeleton by Rho GTPases Cytoskeleton remodeling_ Integrin outside-in signaling
Cell adhesion_Role of tetraspanins in the integrin-mediated cell adhesion wtCFTR and deltaF508 traffic Cytoskeleton remodeling_ Membrane expression Fibronectin-binding integrins in cell motility Cell adhesion_Histamine H1 Cytoskeleton remodeling_ Regulation of actin cytoskeleton by receptor signaling in the interruption of cell barrier Rho GTPases integrity
Based on the independent analysis of each replicate, the top-10 regulated pathways were analyzed by the MetaCoreTM software and listed in Table 1. Interestingly, the cell adhesion phenotype appeared in both iTRAQ replicates. In the first iTRAQ (Experiment A) data, the “Cell adhesion_Chemokines and adhesion” (rank 2, Fig. 1), “Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity” (Rank 3, Fig. 2), and “Cell adhesion_Integrin-mediated cell adhesion and migration” (Rank 7, Fig. 3) were identified as statistically significant maps. In the second iTRAQ replicate (Experiment B), four phenotypes were associated with the cell adhesion phenotype, including the three pathways identified in Experiment A (“Cell adhesion_Chemokines and adhesion” (rank 3), “Cell adhesion_Integrinmediated cell adhesion and migration” (rank 4), “Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity” (Rank 10)). Another cell adhesion phenotype, “Cell-adhesion_Role of tetraspanins in the integrin-mediated cell adhesion” (rank 8), that was not represented in the top-10 enriched pathways in Experiment A, was also identified in Experiment B as one of the top-10 enriched pathways. These findings
cell adhesion protein 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin (SELE) were as follows: GAPDH: Forward primer: 5'-CTCTGCTCCTCCTGTTCGAC-3' Reverse primer: 5'-ACGACCAAATCCGTTGACTC-3' VCAM-1: Forward primer: 5’-TGCACAGTGACTTGTGGACAT-3’ Reverse primer: 5’- CCACTCATCTCGATTTCTGGA-3’ ICAM-1: Forward primer: 5’- CCTTCCTCACCGTGTACTGG-3’ Reverse primer: 5’- AGCGTAGGGTAAGGTTCTTGC-3’ SELE: Forward primer: 5’-ACCAGCCCAGGTTGAATG-3’ Reverse primer: 5’- GGTTGGACAAGGCTGTGC-3’ Statistical analysis Significant differences were determined using the unpaired t-test. Significantly different results were defined as p b 0.05. Results iTRAQ analysis of the HAECs treated with and without thrombin Two replicate iTRAQ experiments were performed as independent biological experiments to gather quantitative information. In each replicate, there were 9765 and 6100 MS/MS spectra identified, which led to the identification of 2111 and 1668 unique peptides with ion scores ≥ 35. The FDR was b 3%, and a total of 648 and 653 proteins were identified from the first and second iTRAQ replicates, respectively. Exclusion of the 21 and 31 proteins without 117/114 values (yellow color in Supplementary data 1A and B), the two independent experiments quantified 627 and 622 proteins in the two replicates,
Fig. 1. The simplified “Cell adhesion_Chemokines and adhesion” pathway sorted from the iTRAQ data of Experiment A by MetaCoreTM analysis.
Please cite this article as: Wang H-J, et al, iTRAQ Quantitative Proteomics-Based Identification of Cell Adhesion as a Dominant Phenotypic Modulation in Thrombin-Stimulated Human Aortic Endothelial Cells, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.02.031
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suggested that cell adhesion-related pathways are a dominant endothelial phenotype upon thrombin stimulation. Therefore, we selected the “cell adhesion” phenotype to examine iTRAQ’s ability to identify functional cellular behavior. Thrombin increased monocytic adhesion to HAECs Upon atherosclerotic plaque rupture, the generated thrombin can damage the overlying endothelial cells. Circulating monocytes adhere to the injured endothelial cells, which is an important initiating event in promoting vascular inflammation and atherosclerosis development [15]. Based on the iTRAQ analysis, we examined the adhesion of THP-1 monocytes to thrombin-stimulated HAECs. As shown in Fig. 4, the adhesion of THP-1 cells was significantly increased in thrombin-stimulated HAECs, suggesting that the MetacoreTM software can identify important cellular phenotypes using iTRAQ proteomic data. Thrombin increased the gene expression of endothelial adhesion molecules Cell adhesion molecules (CAMs) mediate cell adhesion and play a key role in mediating several inflammatory processes [16,17]. As cell adhesion was a dominant phenotype in the MetacoreTM pathway analysis, we further tested whether CAMs were required for monocyte adhesion to endothelial cells. As shown in Fig. 5, gene expression of ICAM-1, VCAM-1, and SELE were significantly upregulated in thrombinstimulated HAECs. Discussion In this study, we used the iTRAQ proteomics technique to quantitatively profile the expression of proteins in HAECs in response to treatment with 2 units/mL thrombin for 5 h. We used iTRAQ labeling
quantitative proteomic analysis to identify how thrombin treatment affects protein levels. We identified several HAEC regulatory pathways that were modulated at this thrombin concentration. Among the top 10 statistically significant maps, the cell adhesion phenotype appeared to be a dominant pathway, suggesting that the inflammatory response is a very early response in thrombin-stimulated HAECs [17]. Profiling the protein changes in thrombin may help advance our understanding of the role of endothelial cells in vascular homeostasis. Only a few proteomics studies of endothelial cells have been reported. Bruneel et al. did a pioneer study to begin to address the endothelial proteome by using two-dimensional gel electrophoresis. They identified 53 proteins that mediated the functions of cellular motility/plasticity, apoptosis, senescence, coagulation, antigen presentation, and enzymatic capabilities in quiescent HUVECs [18]. More recently, Chen et al. also utilized 2-dimensional gel electrophoresis to examine the role of different modified low-density lipoproteins (LDL) in HUVECs [19]. The authors concluded that different forms of oxidized-LDL can regulate HUVEC protein expression in different patterns. The major limitation in both studies is that HUVECs were used as the endothelial cell model. As mentioned above, HUVECs are obtained from the umbilical cord and represent a cell type that does not exist in adults. Therefore, the HUVEC model may not represent a valid model for studying the endothelial function in many human diseases (e.g., atherosclerosis, thrombosis, cancer, or other inflammatory diseases) related to the coagulation and inflammatory process. Additional studies have utilized bovine aortic endothelial cells and proteomics techniques to study the effect of different flow patterns [20]. This research group showed that endothelial Gap G (a member of the gelsolin family) mediated a protective role against unidirectional shear stress. The bovine origin of these endothelial cells is another major concern when applying their findings to human pathophysiology. In contrast, we utilized
Fig. 2. The simplified “Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity” pathway sorted from the iTRAQ data of Experiment A by MetaCoreTM analysis.
Please cite this article as: Wang H-J, et al, iTRAQ Quantitative Proteomics-Based Identification of Cell Adhesion as a Dominant Phenotypic Modulation in Thrombin-Stimulated Human Aortic Endothelial Cells, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.02.031
H.-J. Wang et al. / Thrombosis Research xxx (2015) xxx–xxx
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Fig. 3. The simplified “Cell adhesion_Integrin-mediated cell adhesion and migration” pathway sorted from the iTRAQ data of Experiment A by MetaCoreTM analysis.
HAECs as the endothelial cell model system, which may provide more relevant information for the mechanistic study in thrombinstimulated endothelial cells. Previous cDNA microarray studies had documented that many genes can be modulated within one to two hours in thrombin-stimulated HUVECs [6,21]. In addition, Minamia et al. reported that thrombin induced an early transcriptional cascade within one hour. Their data found that 74 genes (out of 8794 genes on the microarray chip) were induced by thrombin, and 34 of the 74 induced genes peaked at one hour post-treatment [3]. The above experiments suggested that thrombin could induce a very early biological effect on the endothelial cells.
A
Further, we and other groups have shown that optimal tissue factor activity and protein expression can be induced within 4-6 hours of thrombin stimulation [22–25]. Therefore, a 5 hour stimulation period was selected in our study design to examine the acute stress response in thrombin-stimulated HAECs. In addition to the cell adhesion phenotype, MetacoreTM pathway analysis also identified cytoskeleton remodeling as an important phenotypic modulation induced by thrombin. There are several reasons why we chose to verify the cell adhesion phenotype. First, cytoskeleton remodeling is a nonspecific phenotype: a basic cellular response that is required for any further phenotypic modulations, including changes in
B ** 200
C
D
THP-1 cell numbers/field
180 160 140 120 100 80 60 40 20 0 control
thrombin
Fig. 4. THP-1 monocytic adhesion to HAECs was increased in thrombin-stimulated HAECs. (A) control, (B) control (bright field), (C) thrombin, (D) thrombin (bright field). The bars represent the mean ± SEM from 3 experiments. **P b 0.01, as compared with control.
Please cite this article as: Wang H-J, et al, iTRAQ Quantitative Proteomics-Based Identification of Cell Adhesion as a Dominant Phenotypic Modulation in Thrombin-Stimulated Human Aortic Endothelial Cells, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.02.031
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80
Relative gene expression
70 60
*
Control
scientific input. The authors thank the editing company (Editage) for providing language help.
Thrombin
Appendix A. Supplementary data
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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.thromres.2015.02.031.
40 30
*
20
References
*
10 0
VCAM-1
ICAM-1
SELE
Fig. 5. Significant increase in VCAM-1 (6.5-fold), ICAM-1 (10.9-fold), and SELE (56.1-fold) expression in HAECs following simulation with thrombin for 5 h. Bars represent the mean ± SEM from 3 experiments. *P b 0.05, as compared with control.
permeability, migration, proliferation, and angiogenesis [3,26,27]. Second, cell adhesion is also dependent on cytoskeleton remodeling, but is a more specific phenotype [28]. Third, cell adhesion is an important initiating step for vascular inflammation [17]. Therefore, we selected cell adhesion to verify the accuracy of the iTRAQ quantitative proteomics. Our biologically replicated iTRAQ data identified that the same canonical pathway maps of the cell adhesion phenotype were altered in thrombin-stimulated HAECs. These pathways included “Cell adhesion_Chemokines and adhesion”, “Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity,” and “Cell adhesion_Integrin-mediated cell adhesion and migration”. Cell adhesion represents an important cellular communication between different cell types. In the past, using HUVECs as the endothelial in vitro model, several groups reported that the adhesiveness of neutrophils and lymphocytes to endothelial cells was increased in thrombin-stimulated endothelial cells in a NF-κB dependent manner [29–31]. In a monocytic adhesion model using U937 cell lines (derived from a human histiocytic lymphoma), Shankar et al. reported that thrombin increased monocytic adhesion and E-selectin expression in HUVECs [32]. Similarly, Kaplanski et al. showed that thrombin induced functional ICAM-1 and VCAM-1 expression, which were responsible for the increased adhesiveness of THP-1 cells, peripheral blood mononuclear cells, or purified monocytes on HUVECs [33]. The recruitment of the monocytes to the dysfunctional endothelium represents a hallmark of the atherosclerotic lesion. Monocytes and macrophages are key players in the progression of the atherosclerotic plaque [15]. Several CAMs (i.e, ICAM-1, VCAM-1, E-selectin) are known to be important for monocyte/macrophage recruitment to the developing atherosclerotic plaques [16]. In our study, we found that thrombin enhanced expression of these important CAMs and THP-1 adhesiveness to HAECs, similar to the above-mentioned HUVEC studies. In conclusion, using iTRAQ methods, we identified that thrombin stimulation can induce several regulatory pathways including cell adhesion. This study emphasized the feasibility of a non-gel based proteomics study in approaching the cellular response to acute stimulation. Conflict of Interest None. Acknowledgements This study was supported by the Research Laboratory of Pediatrics, Children’s Hospital, China Medical University and by a grant from the China Medical University Hospital (DMR-102-006). H.-J.W. and W.Y.L. designed and performed experiments, analyzed results, and wrote the paper. S.-F.C. performed experiments, analyzed results and provided
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Please cite this article as: Wang H-J, et al, iTRAQ Quantitative Proteomics-Based Identification of Cell Adhesion as a Dominant Phenotypic Modulation in Thrombin-Stimulated Human Aortic Endothelial Cells, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.02.031
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Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Regular Article
iTRAQ quantitative proteomics-based identification of cell adhesion as a dominant phenotypic modulation in thrombin-stimulated human aortic endothelial cells Huang-Joe Wang a,b, Sung-Fang Chen c,1, Wan-Yu Lo d,⁎,1 a
School of Medicine, China Medical University, Taichung, Taiwan Division of Cardiology, Department of Medicine, China Medical University Hospital, Taichung, Taiwan c Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan d Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan b
a r t i c l e
i n f o
Article history: Received 20 July 2014 Received in revised form 16 February 2015 Accepted 24 February 2015 Available online xxxx Keywords: Thrombin quantitative proteomics human aortic endothelial cells Isobaric tags for relative and absolute quantitation (iTRAQ)
a b s t r a c t Introduction: The phenotypic changes in thrombin-stimulated endothelial cells include alterations in permeability, cell shape, vasomotor tone, leukocyte trafficking, migration, proliferation, and angiogenesis. Previous studies regarding the pleotropic effects of thrombin on the endothelium used human umbilical vein endothelial cells (HUVECs)—cells derived from fetal tissue that does not exist in adults. Only a few groups have used screening approaches such as microarrays to profile the global effects of thrombin on endothelial cells. Moreover, the proteomic changes of thrombin-stimulated human aortic endothelial cells (HAECs) have not been elucidated. Materials and methods: HAECs were stimulated with 2 units/mL thrombin for 5 h and their proteome was investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and the MetaCoreTM software. Results: A total of 627 (experiment A) and 622 proteins (experiment B) were quantified in the duplicated iTRAQ analyses. MetaCoreTM pathway analysis identified cell adhesion as a dominant phenotype in thrombinstimulated HAECs. Replicated iTRAQ data revealed that “Cell adhesion_Chemokines and adhesion,” “Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity,” and “Cell adhesion_Integrin-mediated cell adhesion and migration” were among the top 10 statistically significant pathways. The cell adhesion phenotype was verified by increased THP-1 adhesion to thrombin-stimulated HAECs. In addition, the expression of ICAM-1, VCAM-1, and SELE was significantly upregulated in thrombin-stimulated HAECs. Conclusions: Several regulatory pathways are altered in thrombin-stimulated HAECs, with cell adhesion being the dominant altered phenotype. Our findings show the feasibility of the iTRAQ technique for evaluating cellular responses to acute stimulation. © 2015 Elsevier Ltd. All rights reserved.
Introduction One of the most important functions of normal endothelium is to maintain blood in the fluid state. The non-thrombogenic properties of endothelium can be damaged by several inflammatory insults, Abbreviations: CAM, Cell adhesion molecule; SELE, E-selectin; FDR, false discovery rate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HAEC,human aorticendothelialcell; HUVECs, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule 1; iTRAQ, isobaric tags for the relative and absolute quantification; LC-ESI-MS/MS, liquid chromatography-electrospray ionization-tandem mass spectrometry; LDL, low-density lipoprotein; PCR, polymerase chain reaction; SCX, strong cation exchange chromatography; VCAM-1, vascular cell adhesion protein 1 ⁎ Corresponding author at: Graduate Integration of Chinese and Western Medicine, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan. Tel.: +886 4 22053366x3513; fax: +886 4 22056139. E-mail address: [email protected] (W.-Y. Lo). 1 These authors contributed equally to this work.
including hypoxia, fluid shear stress, oxidants, interleukin-1, tumor necrosis factor, γ-interferon, endotoxin, and thrombin [1]. Thrombin is a serine protease that plays a key role in vascular diseases. In atherothrombosis, thrombin is generated in the site of a ruptured plaque, which in turn triggers the coagulation process. Besides its role in coagulation, thrombin also plays a significant role in the pro-inflammatory response [2]. Thrombin-stimulated endothelial cells undergo multiple phenotypic changes, including alterations in permeability, cell shape, vasomotor tone, leukocyte trafficking, migration, proliferation, and angiogenesis [3,4]. Most studies investigating the pleotropic effects of thrombin on the endothelium have used tissue obtained from the human umbilical vein endothelial cell (HUVECs), which is a fetal tissue that does not exist in adults. In addition, major studies on thrombin have focused on a few specific genes. Only a few groups have used screening approaches to profile the global thrombin effects on endothelial cells using cDNA
http://dx.doi.org/10.1016/j.thromres.2015.02.031 0049-3848/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: Wang H-J, et al, iTRAQ Quantitative Proteomics-Based Identification of Cell Adhesion as a Dominant Phenotypic Modulation in Thrombin-Stimulated Human Aortic Endothelial Cells, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.02.031
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microarray techniques [3,5–7]. These experiments are invaluable for identification of novel target genes. However, such approaches failed to reveal the important post-transcriptional control mechanisms in the endothelium [8]; the real proteomic changes in the thrombinstimulated endothelial cells are not yet fully elucidated. Proteomic approaches offer a large potential for obtaining the comprehensive protein expression data of endothelial cells. The progress of proteomics and bioinformatics further help scientists to identify important signal transduction pathways and functional cellular information. Isobaric tags for the relative and absolute quantification (iTRAQ) is a shotgun approach that enable high-throughput quantitative proteomics analysis [9]. Briefly, the iTRAQ technique labels samples with four independent isobaric tags that, upon fragmentation in MS/MS, produce unique reporter ions that provide quantitative data based on the integration of the peak areas of the different samples [10]. MetaCoreTM is a data-mining and bioinformatics pathway analysis software developed by GeneGo (a Thomson Reuters business). MetaCoreTM software allows for functional analysis of many data forms, including Next Generation Sequencing, microarray, proteomics, siRNA, and microRNA. Our laboratory previously applied the iTRAQ technique and identified miR-27b as a useful biomarker for oral cancer [11]. Since the previous profiling efforts on thrombin-stimulated endothelial effects focused on fetal tissue (i.e., HUVECs), the present study selected human aortic endothelial cells (HAECs) as an adult endothelial in vitro model. The purpose of the present study is to investigate the global protein expression profiles and related pathway analysis in thrombin-stimulated HAECs by the iTRAQ proteomics and MetaCoreTM bioinformatics techniques. Materials and methods Endothelial cell culture and sample treatments for iTRAQ HAECs were purchased from Cell Applications, Inc. (San Diego, CA) and cultured in endothelial cell growth medium (Cell Applications, Inc.) according to the manufacturer’s recommendations. HAECs were cultured and seeded in 6-well plates with 106 cells/well and grown overnight under serum-starved conditions in M-199 medium supplemented with 1% fetal bovine serum (HyClone, Logan, UT). Cells were treated with and without thrombin (2 units/mL) for 5 h (Sigma–Aldrich, St. Louis, MO) and then harvested by centrifugation at 300 × g for 10 min. The obtained pellets were resuspended in RIPA buffer (SigmaAldrich, St. Louis, MO) to extract total protein. The supernatants were enriched and desalted using a 3-kDa centrifugal filter (Millipore, Merck KGaA, Germany). The amount of protein in each concentrated/ desalted sample was determined by Bradford protein assay (Bio-Rad, Hercules, CA), and stored at -80 °C for subsequent processing. The study protocol was granted and approved by the institutional committee. Chemicals and reagents for iTRAQ Modified porcine trypsin (sequencing grade) was obtained from Promega (Madison, WI). iTRAQ 4-plex reagent kits were purchased from Applied Biosystems (Framingham, MA). All other chemicals and reagents were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise stated. Reduction, alkylation, digestion, and labeling with iTRAQ Seventy μg of each protein samples were reduced, alkylated, digested, and labeled with iTRAQ reagents according to the method described in our previous study [11]. The digested samples were labeled with the iTRAQ reagents, and the samples were labeled as follows: 114 designated as controls (without thrombin) and 117 thrombintreated samples. Using two iTRAQ kits, we performed two biological
replicates of iTRAQ quantitative analysis to monitor the consistency of the results. Strong cation exchange chromatography (SCX) and liquid chromatographyelectrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis Peptide separation was performed by Polysulfoethyl A Column (200 mm L × 2.1 mm i.d., 5 μm, 300 A, PolyLC, Columbia, MD) on an Agilent 1100 binary HPLC (Agilent Technologies, Wilmington, DE). The iTRAQ-labeled peptides (280 μg) were eluted at a flow rate of 200 μL/min. Eighty fractions were collected after SCX fractionation and then pooled into 24 fractions, which were subsequently purified by C-18 spin columns (Thermo Scientific, San Jose, CA) for nano-LCESI-MS/MS analysis [11]. Database search and iTRAQ quantification Protein identification of the iTRAQ samples was performed using the Mascot search algorithm (version 2.3.2). The search was performed against the SwissProt v.2011_08 database (531,473 sequences) using the following search parameters: taxonomy: Homo sapiens; enzyme: trypsin; max. miss cleavages: 1; fixed modifications: methylthiolation, N-terminal iTRAQ 4plex, lysine iTRAQ 4plex; variable modifications: methionine oxidation, tyrosine iTRAQ 4plex; MS peptide tolerance: 1.5 Da; MS/MS tolerance: 0.6 Da. Protein identifications were only accepted when ≥ 2 spectra had an ion score above 35 (95% confidence) [12]. The relative protein expression was determined based on the ratio of the experiment and control reporter ions for the peptides (117:114). A decoy search (based on automatically generated random sequences of the same length) was employed to determine the rate of false-positive identifications. The false discovery rate (FDR) b 3% was set as the criteria according to Käll et al. [13]. Data analysis by bioinformatics In the two independent experiments, a total of 648 and 653 proteins were identified from the first and second iTRAQ replicates, respectively (Supplementary data 1A and B). Exclusion of those proteins without 117/114 values, a total of 627 and 622 proteins were well quantified proteins. These quantified proteins were selected for the automatic processing of “protein network construction and analysis” by the MetaCoreTM software (2014_01 database/ Server: portal.genego.com) without any modification. Monocyte adhesion assay The human monocytic cell line THP-1 (derived from acute monocytic leukemia) was obtained from the American Type Culture Collection (Rockville, MD) and maintained according to the manufacturer’s recommendations. In adhesion experiments, THP-1 cells were labeled with calcein acetoxymethyl ester (Calcein-AM; Molecular Probes, Oregon) dye at a concentration of 7.5 μM for 30 min immediately preceding the adhesion assay. HAECs were maintained in 12 well dishes until 90% confluent. HAECs (105 cells/well) were then treated with/without thrombin for 5 h, and then incubated with the culture medium containing the labeled THP-1 cells (THP-1/HAECs = 7) for 10 min. After incubation, non-adherent THP-1 cells were removed by two gentle washes with PBS for 30 s. The adherent THP-1 cells on the activated HEACs were quantified from 10 randomly selected view fields. Real-time polymerase chain reaction (PCR) mRNA expression levels in the HAECs was analyzed by real-time PCR, as previously described [14]. The primer sequences used for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), vascular
Please cite this article as: Wang H-J, et al, iTRAQ Quantitative Proteomics-Based Identification of Cell Adhesion as a Dominant Phenotypic Modulation in Thrombin-Stimulated Human Aortic Endothelial Cells, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.02.031
H.-J. Wang et al. / Thrombosis Research xxx (2015) xxx–xxx Table 1 Top 10 “statistically significant pathway maps” from the duplicate iTRAQ analysis by the MetaCoreTM software. Sorts of Experiment A pathway
Experiment B
1
Cytoskeleton remodeling_ Cytoskeleton remodeling LRRK2 in neurons in Parkinson's disease Cell adhesion_Chemokines and adhesion
4
Cytoskeleton remodeling_ Cytoskeleton remodeling Cell adhesion_Chemokines and adhesion Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity CFTR folding and maturation
5
Glycolysis and gluconeogenesis
6
Cytoskeleton remodeling_TGF, WNT and cytoskeletal remodeling
7
Cell adhesion_Integrinmediated cell adhesion and migration Regulation of CFTR activity
2 3
8
9
10
3
respectively. These quantified proteins were examined from the two replicates. A total of 378 proteins with same ID were identified in two replicates. The percentage overlap of ID proteins was 58%. Cell adhesion was dominant phenotype in thrombin-stimulated HAECs
Cell adhesion_Integrinmediated cell adhesion and migration Cytoskeleton remodeling_TGF, WNT and cytoskeletal remodeling Cytoskeleton remodeling_ Regulation of actin cytoskeleton by Rho GTPases Cytoskeleton remodeling_ Integrin outside-in signaling
Cell adhesion_Role of tetraspanins in the integrin-mediated cell adhesion wtCFTR and deltaF508 traffic Cytoskeleton remodeling_ Membrane expression Fibronectin-binding integrins in cell motility Cell adhesion_Histamine H1 Cytoskeleton remodeling_ Regulation of actin cytoskeleton by receptor signaling in the interruption of cell barrier Rho GTPases integrity
Based on the independent analysis of each replicate, the top-10 regulated pathways were analyzed by the MetaCoreTM software and listed in Table 1. Interestingly, the cell adhesion phenotype appeared in both iTRAQ replicates. In the first iTRAQ (Experiment A) data, the “Cell adhesion_Chemokines and adhesion” (rank 2, Fig. 1), “Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity” (Rank 3, Fig. 2), and “Cell adhesion_Integrin-mediated cell adhesion and migration” (Rank 7, Fig. 3) were identified as statistically significant maps. In the second iTRAQ replicate (Experiment B), four phenotypes were associated with the cell adhesion phenotype, including the three pathways identified in Experiment A (“Cell adhesion_Chemokines and adhesion” (rank 3), “Cell adhesion_Integrinmediated cell adhesion and migration” (rank 4), “Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity” (Rank 10)). Another cell adhesion phenotype, “Cell-adhesion_Role of tetraspanins in the integrin-mediated cell adhesion” (rank 8), that was not represented in the top-10 enriched pathways in Experiment A, was also identified in Experiment B as one of the top-10 enriched pathways. These findings
cell adhesion protein 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin (SELE) were as follows: GAPDH: Forward primer: 5'-CTCTGCTCCTCCTGTTCGAC-3' Reverse primer: 5'-ACGACCAAATCCGTTGACTC-3' VCAM-1: Forward primer: 5’-TGCACAGTGACTTGTGGACAT-3’ Reverse primer: 5’- CCACTCATCTCGATTTCTGGA-3’ ICAM-1: Forward primer: 5’- CCTTCCTCACCGTGTACTGG-3’ Reverse primer: 5’- AGCGTAGGGTAAGGTTCTTGC-3’ SELE: Forward primer: 5’-ACCAGCCCAGGTTGAATG-3’ Reverse primer: 5’- GGTTGGACAAGGCTGTGC-3’ Statistical analysis Significant differences were determined using the unpaired t-test. Significantly different results were defined as p b 0.05. Results iTRAQ analysis of the HAECs treated with and without thrombin Two replicate iTRAQ experiments were performed as independent biological experiments to gather quantitative information. In each replicate, there were 9765 and 6100 MS/MS spectra identified, which led to the identification of 2111 and 1668 unique peptides with ion scores ≥ 35. The FDR was b 3%, and a total of 648 and 653 proteins were identified from the first and second iTRAQ replicates, respectively. Exclusion of the 21 and 31 proteins without 117/114 values (yellow color in Supplementary data 1A and B), the two independent experiments quantified 627 and 622 proteins in the two replicates,
Fig. 1. The simplified “Cell adhesion_Chemokines and adhesion” pathway sorted from the iTRAQ data of Experiment A by MetaCoreTM analysis.
Please cite this article as: Wang H-J, et al, iTRAQ Quantitative Proteomics-Based Identification of Cell Adhesion as a Dominant Phenotypic Modulation in Thrombin-Stimulated Human Aortic Endothelial Cells, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.02.031
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suggested that cell adhesion-related pathways are a dominant endothelial phenotype upon thrombin stimulation. Therefore, we selected the “cell adhesion” phenotype to examine iTRAQ’s ability to identify functional cellular behavior. Thrombin increased monocytic adhesion to HAECs Upon atherosclerotic plaque rupture, the generated thrombin can damage the overlying endothelial cells. Circulating monocytes adhere to the injured endothelial cells, which is an important initiating event in promoting vascular inflammation and atherosclerosis development [15]. Based on the iTRAQ analysis, we examined the adhesion of THP-1 monocytes to thrombin-stimulated HAECs. As shown in Fig. 4, the adhesion of THP-1 cells was significantly increased in thrombin-stimulated HAECs, suggesting that the MetacoreTM software can identify important cellular phenotypes using iTRAQ proteomic data. Thrombin increased the gene expression of endothelial adhesion molecules Cell adhesion molecules (CAMs) mediate cell adhesion and play a key role in mediating several inflammatory processes [16,17]. As cell adhesion was a dominant phenotype in the MetacoreTM pathway analysis, we further tested whether CAMs were required for monocyte adhesion to endothelial cells. As shown in Fig. 5, gene expression of ICAM-1, VCAM-1, and SELE were significantly upregulated in thrombinstimulated HAECs. Discussion In this study, we used the iTRAQ proteomics technique to quantitatively profile the expression of proteins in HAECs in response to treatment with 2 units/mL thrombin for 5 h. We used iTRAQ labeling
quantitative proteomic analysis to identify how thrombin treatment affects protein levels. We identified several HAEC regulatory pathways that were modulated at this thrombin concentration. Among the top 10 statistically significant maps, the cell adhesion phenotype appeared to be a dominant pathway, suggesting that the inflammatory response is a very early response in thrombin-stimulated HAECs [17]. Profiling the protein changes in thrombin may help advance our understanding of the role of endothelial cells in vascular homeostasis. Only a few proteomics studies of endothelial cells have been reported. Bruneel et al. did a pioneer study to begin to address the endothelial proteome by using two-dimensional gel electrophoresis. They identified 53 proteins that mediated the functions of cellular motility/plasticity, apoptosis, senescence, coagulation, antigen presentation, and enzymatic capabilities in quiescent HUVECs [18]. More recently, Chen et al. also utilized 2-dimensional gel electrophoresis to examine the role of different modified low-density lipoproteins (LDL) in HUVECs [19]. The authors concluded that different forms of oxidized-LDL can regulate HUVEC protein expression in different patterns. The major limitation in both studies is that HUVECs were used as the endothelial cell model. As mentioned above, HUVECs are obtained from the umbilical cord and represent a cell type that does not exist in adults. Therefore, the HUVEC model may not represent a valid model for studying the endothelial function in many human diseases (e.g., atherosclerosis, thrombosis, cancer, or other inflammatory diseases) related to the coagulation and inflammatory process. Additional studies have utilized bovine aortic endothelial cells and proteomics techniques to study the effect of different flow patterns [20]. This research group showed that endothelial Gap G (a member of the gelsolin family) mediated a protective role against unidirectional shear stress. The bovine origin of these endothelial cells is another major concern when applying their findings to human pathophysiology. In contrast, we utilized
Fig. 2. The simplified “Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity” pathway sorted from the iTRAQ data of Experiment A by MetaCoreTM analysis.
Please cite this article as: Wang H-J, et al, iTRAQ Quantitative Proteomics-Based Identification of Cell Adhesion as a Dominant Phenotypic Modulation in Thrombin-Stimulated Human Aortic Endothelial Cells, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.02.031
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Fig. 3. The simplified “Cell adhesion_Integrin-mediated cell adhesion and migration” pathway sorted from the iTRAQ data of Experiment A by MetaCoreTM analysis.
HAECs as the endothelial cell model system, which may provide more relevant information for the mechanistic study in thrombinstimulated endothelial cells. Previous cDNA microarray studies had documented that many genes can be modulated within one to two hours in thrombin-stimulated HUVECs [6,21]. In addition, Minamia et al. reported that thrombin induced an early transcriptional cascade within one hour. Their data found that 74 genes (out of 8794 genes on the microarray chip) were induced by thrombin, and 34 of the 74 induced genes peaked at one hour post-treatment [3]. The above experiments suggested that thrombin could induce a very early biological effect on the endothelial cells.
A
Further, we and other groups have shown that optimal tissue factor activity and protein expression can be induced within 4-6 hours of thrombin stimulation [22–25]. Therefore, a 5 hour stimulation period was selected in our study design to examine the acute stress response in thrombin-stimulated HAECs. In addition to the cell adhesion phenotype, MetacoreTM pathway analysis also identified cytoskeleton remodeling as an important phenotypic modulation induced by thrombin. There are several reasons why we chose to verify the cell adhesion phenotype. First, cytoskeleton remodeling is a nonspecific phenotype: a basic cellular response that is required for any further phenotypic modulations, including changes in
B ** 200
C
D
THP-1 cell numbers/field
180 160 140 120 100 80 60 40 20 0 control
thrombin
Fig. 4. THP-1 monocytic adhesion to HAECs was increased in thrombin-stimulated HAECs. (A) control, (B) control (bright field), (C) thrombin, (D) thrombin (bright field). The bars represent the mean ± SEM from 3 experiments. **P b 0.01, as compared with control.
Please cite this article as: Wang H-J, et al, iTRAQ Quantitative Proteomics-Based Identification of Cell Adhesion as a Dominant Phenotypic Modulation in Thrombin-Stimulated Human Aortic Endothelial Cells, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.02.031
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80
Relative gene expression
70 60
*
Control
scientific input. The authors thank the editing company (Editage) for providing language help.
Thrombin
Appendix A. Supplementary data
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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.thromres.2015.02.031.
40 30
*
20
References
*
10 0
VCAM-1
ICAM-1
SELE
Fig. 5. Significant increase in VCAM-1 (6.5-fold), ICAM-1 (10.9-fold), and SELE (56.1-fold) expression in HAECs following simulation with thrombin for 5 h. Bars represent the mean ± SEM from 3 experiments. *P b 0.05, as compared with control.
permeability, migration, proliferation, and angiogenesis [3,26,27]. Second, cell adhesion is also dependent on cytoskeleton remodeling, but is a more specific phenotype [28]. Third, cell adhesion is an important initiating step for vascular inflammation [17]. Therefore, we selected cell adhesion to verify the accuracy of the iTRAQ quantitative proteomics. Our biologically replicated iTRAQ data identified that the same canonical pathway maps of the cell adhesion phenotype were altered in thrombin-stimulated HAECs. These pathways included “Cell adhesion_Chemokines and adhesion”, “Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity,” and “Cell adhesion_Integrin-mediated cell adhesion and migration”. Cell adhesion represents an important cellular communication between different cell types. In the past, using HUVECs as the endothelial in vitro model, several groups reported that the adhesiveness of neutrophils and lymphocytes to endothelial cells was increased in thrombin-stimulated endothelial cells in a NF-κB dependent manner [29–31]. In a monocytic adhesion model using U937 cell lines (derived from a human histiocytic lymphoma), Shankar et al. reported that thrombin increased monocytic adhesion and E-selectin expression in HUVECs [32]. Similarly, Kaplanski et al. showed that thrombin induced functional ICAM-1 and VCAM-1 expression, which were responsible for the increased adhesiveness of THP-1 cells, peripheral blood mononuclear cells, or purified monocytes on HUVECs [33]. The recruitment of the monocytes to the dysfunctional endothelium represents a hallmark of the atherosclerotic lesion. Monocytes and macrophages are key players in the progression of the atherosclerotic plaque [15]. Several CAMs (i.e, ICAM-1, VCAM-1, E-selectin) are known to be important for monocyte/macrophage recruitment to the developing atherosclerotic plaques [16]. In our study, we found that thrombin enhanced expression of these important CAMs and THP-1 adhesiveness to HAECs, similar to the above-mentioned HUVEC studies. In conclusion, using iTRAQ methods, we identified that thrombin stimulation can induce several regulatory pathways including cell adhesion. This study emphasized the feasibility of a non-gel based proteomics study in approaching the cellular response to acute stimulation. Conflict of Interest None. Acknowledgements This study was supported by the Research Laboratory of Pediatrics, Children’s Hospital, China Medical University and by a grant from the China Medical University Hospital (DMR-102-006). H.-J.W. and W.Y.L. designed and performed experiments, analyzed results, and wrote the paper. S.-F.C. performed experiments, analyzed results and provided
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Please cite this article as: Wang H-J, et al, iTRAQ Quantitative Proteomics-Based Identification of Cell Adhesion as a Dominant Phenotypic Modulation in Thrombin-Stimulated Human Aortic Endothelial Cells, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.02.031
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Please cite this article as: Wang H-J, et al, iTRAQ Quantitative Proteomics-Based Identification of Cell Adhesion as a Dominant Phenotypic Modulation in Thrombin-Stimulated Human Aortic Endothelial Cells, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.02.031