Proteomic Analysis of Differentially Expressed Proteins in Endothelial Cells Under Low Shear Stress

Background: Shear stress (SS) affects the morphology, proliferation, differentiation and migration of endothelial cells, and regulates protein expression. Methods: This study aims to conduct a proteomic analysis of human umbilical vein endothelial cells (HUVECs) under low SS. HUVECs were cultured on glass slides (test group n=30; control group n=30) and transferred to parallel-plate flow chamber. The test group was subjected to low SS at 4.58 dyne/cm 2 for 2 hours, and the control group was maintained under static conditions. Approximately 2  10 7 cells were collected from each group and analyzed by two-dimensional electrophoresis. The protein from each spot were identified by mass spectrometry. The biological functions and processes associated with the identified proteins were evaluated using the NCBInr, SwissProt, and DAVID databases. The protein expression of 14-3-3 proteins, annexin I, annexin II, zyxin, filamin A, filamin B, Ras-related nuclear protein (Ran), and talin were assessed by Western blotting. Results: A total of 234 proteins were analyzed; 14 proteins were upregulated and 78 were downregulated in the test group compared with the control group. The expression of 14-3-3 proteins, annexin I, annexin II, zyxin, filamin A, and filamin B was significantly decreased, whereas the expression of Ran and talin was significantly increased in the test group. Conclusion: The current results provide evidence that SS changes the protein profile of HUVECs. Shear stress; HUVECs: human umbilical vein endothelial cells; Ras-related nuclear protein; TNF: tumor necrosis factor; KEGG ： Kyoto Encyclopedia of Genes and Genomes; ： hypertrophic cardiomyopathy ； ： Visualization, and Integrated


Background
Vascular endothelial cells (VECs) are located in the inner lining of blood vessels and serve as an interface between blood flow and the vascular wall. VECs are sensitive to changes in blood shear stress, blood pressure, inflammatory signals, and hormone levels in the circulation. Furthermore, VECs help maintain homeostasis and play a critical role in pathophysiological processes [1]. The expression of several genes is regulated by laminar shear stress in endothelial cells; these genes are involved in cell proliferation and differentiation, cell-cell adhesion, maintenance of vascular tone, as well as physiological and pathological processes, including inflammation and immune regulation [2]. High shear stress (>15 dyne/cm 2 ) upregulates the expression of vasodilators, growth factors, fibrinolytic components, and antioxidants and downregulates the expression of vasoconstrictors, growth factors, inflammatory mediators, and adhesion molecules to protect endothelial cells (ECs) from damage and prevent atherosclerosis [3,4]. Low shear stress (±4 dyne/cm 2 ) increases the response of ECs to chemical substances, including oxidized low-density lipoprotein and tumor necrosis factor (TNF), which promote cell proliferation and platelet adhesion, and ultimately results in the dysfunction of ECs [4,5] and atherosclerosis [2]. Atheromas typically occur in vascular bends and bifurcations, where shear stress is low and uneven. Low and oscillatory shear have been shown to promote atherosclerosis in animal models. The alteration in hemodynamics and its effects on cell rheology may play a significant role in the pathogenesis of atherosclerosis; however, the regulation of protein expression following shear stress is not fully understood.
Proteomics has advanced rapidly in recent years. In this study, we investigated the proteomic profile of HUVECs that were subjected to either shear stress or static conditions. The proteomic analysis identified differentially expressed proteins associated with mechanotransduction of low shear stress. The current results provide evidence that shear stress changes the protein profile of HUVECs.

Endothelial cell culture and treatment
HUVEC EA. hy926 cell line was purchased from Shanghai Cell Bank and were routinely sub-cultured and cultured in an incubator (37 °C and 5% CO 2 ) in M199 complete medium containing 10% fetal bovine serum and 1% double antibody according to supplier instructions. Third-passage cells were used for shear stress treatment. A slide (75 mm × 25 mm) was treated with 1 mL fibronectin (20%) at 37 °C for 30 min. Trypsinized cells were then seeded on the treated slides with the cell concentration of 1×10 6 /mL. After cell confluence, the slides were transferred to a parallel-plate flow chamber system (Fig. 1), and treated with (4.58 dyne/cm 2 ) shear stress for 2 hr., then slides were removed from the chamber, and the adhered cells were collected and counted for the downstream experiment.

Two-dimensional electrophoresis
The cell suspensions were centrifuged and washed three times with PBS (room temperature, 1000g, 5 min for each wash). The cells were transferred to 1.

5-mL
Eppendorf tubes, and the residual PBS was collected. Then 500 μL of bidirectional electrophoretic lysis buffer was added to the cell suspension. The cells were lysed by sonication with fifteen 15-s pulses at 30-s intervals at a temperature of 4 °C and power output of 15%. The lysates were centrifuged at 40,000g for 1 h, the supernatant was collected, and protein was quantified using the Bradford method. The samples were aliquoted and stored at -80 ℃ until further use. Samples were rehydrated into 24-cm pH 3-10 IPG strips (Bio-Rad) at -20 °C for 18 hr. The hydrated strips were transferred to the isoelectric focusing tray; the cathodic and anodic ends of the strip were placed on the respective cathodic and anodic ends of the tray. Then 3 mL of mineral oil was added to each strip, and isoelectric focusing was performed ( Table 1).
The strip was removed from the tray and sequentially treated with dithiothreitol and iodoacetamide each for 15 min. The strips were subjected to 12% SDS-PAGE. After electrophoresis, the strips were removed, stained with Coomassie brilliant blue, destained, and washed. Images were subsequently acquired using the PDQuest software. Ten distinct protein spots were subjected to enzymatic hydrolysis and analyzed using Q Exactive mass spectrometer.

Image processing and data analysis
Ten different spots were selected for mass spectrometry analysis. The structure, function, and biological processes of DEPs were evaluated using the databases NCBInr; SwissProt; Database for Annotation, Visualization, and Integrated Discovery (DAVID); and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING).

Western blotting
After shear treatment the slides were removed from the parallel-plate flow chamber, washed twice with cold PBS, and dried. The adhered cells were detached in PBS using a scraper. The cell suspension was transferred to 1.5-mL sterile Eppendorf tubes and centrifuged at high speed for 3-5 min. PBS was then removed. The protein lysate was added to the test tubes and incubated on ice for 30 min with agitation at 10-min intervals. The test tubes were centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatant was transferred to a new sterile test tube. The protein concentration was measured using the Price BCA Protein Assay kit according to the manufacturer's instructions. In brief, 30 ug of protein was isolated on a 12% SDS-PAGE gel and transferred onto a polyvinyl difluoride membrane overnight at 4 °C. The membrane was blocked with 5% skim milk powder dissolved in TBST buffer for 6 hr. The membrane was then incubated with a primary antibody (1:1000) overnight at 4 °C, washed three times (10 min for each wash) with TBST at room temperature, incubated with a secondary antibody (1:1000) at room temperature in the dark for 2 hr under agitation, and then washed three times with TBST (10 min for each wash) and once with distilled water for 5 min each. After this step, the membrane was incubated with a color development solution (1:1) for 3 min. Images were acquired using a chemiluminescence imager (Bio-Rad). The detected proteins were 14-3-3 (

Statistical analysis
The results are expressed as mean ± SD (n=3). Comparisons were made using a one-way analysis of variance. All statistical analyses were performed with SPSS software version 17.0. P-values of less than 0.05 were considered significant.

Determination of total protein concentration
The protein concentration of the cell lysates was determined by the Bradford method using 1 mg/mL bovine serum albumin as the standard. The concentration in the test and control group was 4.935 and 4.89 mg/mL, respectively.

Two-dimensional electrophoresis
Two-dimensional electrophoresis was performed on the cell lysates. This method detected 79 protein spots in the low shear stress group ( Fig. 2A) and 189 specific protein spots in the control group (Fig. 2B). Ten protein spots were selected, subjected to enzymatic digestion, and analyzed by mass spectrometry (Figs. 2C-D). The analysis indicated that the test and control group presented 234 and 92 DEPs, respectively, with a fold-change 2.

Functional analysis of DEPs using the DAVID database and interaction network analysis
The main biological processes identified in the DAVID database were metabolic and catabolic processes, apoptosis, programmed cell death, protein transport, assembly and transport of macromolecular complexes, cytoskeletal composition, RNA processing and splicing, mRNA processing and metabolism, protein folding, and cellular stress response (Fig. 3). The main pathways identified in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were cell cycle, neurotrophin signaling, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy, cardiac muscle contraction, Hippo signaling, and PI3K-Akt signaling (Fig. 4).

Analysis of DEPs under low shear stress
Eight DEPs identified by mass spectrometry were selected for Western blot analysis.

Discussion
Molecular and cellular studies have demonstrated that ECs regulate vascular tone and maturation by secreting vasoactive substances with multiple functions, including the prevention of thrombosis. Other studies have shown that endothelial dysfunction is a critical event in early cardiovascular disease (CVD) and is closely related to the occurrence and development of atherosclerosis, hypertension, and heart failure. [6] In cardiovascular system, mechanical factors play an important role in regulating vascular pathophysiology. ECs are subjected to different types of shear stress caused by the tangential frictional force of blood flow, such as laminar shear stress and disturbed flow, which can affect endothelial function [7,8]. Under normal conditions, high laminar shear stress can increase the expression of genes with hypoplastic, anti-inflammatory, and anti-thrombotic effects that can consequently aid in preventing atherosclerosis [3]. In contrast, low shear stress or disturbed flow (and other blood flow changes) induces the expression of pro-inflammatory genes and promotes cell proliferation and apoptosis [4,9]. Studies have shown that shear stress achieves the pathophysiological regulation of angiogenesis by modulating endothelial genes [2]. The present results confirmed that the expression of fractalkine on ECs was highest at low shear stress (4.58 dyne/cm 2 ) for 2 hr. Fractalkine can promote the formation and development of atherosclerotic plaques [10].
Therefore, we intended to use 4.58 dyne/cm 2 as a low shear stress value, and proteomics to analyze DEPs in VECs under low laminar shear stress.
The proteomics analysis identified 78 downregulated proteins with a fold-change  2 in the low shear group compared with the control. The DEPs identified using DAVID were involved in several biological processes, including metabolic processes, protein localization, apoptosis, programmed cell death, protein transport, assembly and transport of macromolecular complexes, energy metabolism, cytoskeletal composition, RNA processing and splicing, mRNA processing and metabolism, protein folding, cellular stress response, and translation. The most enriched pathways in the KEGG analysis were cell cycle, neurotrophin signaling, HCM, dilated cardiomyopathy, myocardial contraction, Hippo signaling, and PI3K-Akt signaling. The identification of CVD-related proteins in these pathways provides evidence that the development of CVD is induced by low shear stress.
The results of Western blotting showed that the expression of 14-3-3 proteins was significantly reduced after of 2-hr low shear stress. These proteins are highly conserved in eukaryotic organisms and play different roles in regulatory processes, including cell growth, apoptosis, signal transduction, protein transport, and stress response. The subtype 14-3-3σ is an epithelial marker found in the epithelium. In recent years, the study of 14-3-3 proteins and tumors has become a popular topic.
The current results demonstrate that low shear stress decreases the expression of annexins I and II. Annexin I is a calcium-and phospholipid-binding protein involved in various biological processes, including cellular differentiation, proliferation, apoptosis, and migration [12] and suppresses and resolves inflammation predominantly by targeting neutrophils [13]. Accumulating evidences suggest that neutrophils play fundamental roles in atherogenesis and contribute to the initiation, progression, and destabilization of atherosclerotic plaques [14][15][16][17]. Moreover, annexin I have been shown to modulate apoptosis, efferocytosis, and macrophage polarization, which are dysregulated in atherosclerosis [18]. Annexin II is expressed in several cell types, including tumor cells, ECs, macrophages, and mononuclear cells. Annexin II binds to the p50 subunit of NF-κB in a calcium-independent manner, and the annexin II-p50 complex is translocated into the nucleus. Furthermore, annexin II increases the transcriptional activity of NF-κB in both resting and activated state, and upregulates the transcription of several target genes downstream of NF-κB, including interleukin-6 [19] and NF-κB genes, which play an essential role in inflammatory response. However, the biological function of intracellular annexin II and the mechanism underlying the production thereof are not fully elucidated.
Zyxin is predominantly localized to cell adhesion sites. Mechanisms by which zyxin affects gene expression have been proposed [20] and this protein is a potential therapeutic target to impact the onset of endothelial cell phenotype. Zyxin is usually associated with focal adhesions and stress fibers, and plays major role in stretch-induced endothelial gene expression [21,22]. Mechanical forces mobilize zyxin from focal adhesions to actin filaments and regulate cytoskeletal reinforcement [23]. Furthermore, zyxin regulates cAMP agonist-induced Weibel-Palade body exocytosis in cultured human primary ECs, and zyxin deletion impairs vascular hemostasis and thrombosis [24]. The current results demonstrate that zyxin protein expression was significantly downregulated, suggesting that low shear stress affect zyxin production.
Filamins are involved in cytoskeletal formation and play a crucial role in cell adhesion, proliferation, migration, signal transduction, and gene therapy [25]. FLNA is a actin-binding protein that helps maintain cell integrity and is critical for cell migration [26]. FLNB is widely distributed across species and is the second most abundant actin filament protein in humans, being highly expressed in normal endothelial cells. Studies have shown that the endothelial cells of FLNA-deficient mice displayed structural disorder, and the migration ability of endothelial cells in mice lacking FLNB is altered. FLNA and FLNB are expressed in endothelial cells and play an essential role in vascular development [27]. In this study, mass spectrometry analysis demonstrated that FLNB expression was downregulated under low shear stress conditions, indicating that the stimulation of the physical condition can change the migration ability of endothelial cells.
Ran family is unique in the Ras-GTPase superfamily and helps regulate nuclear import and export [27,28]. Ran is activated in the cytoplasm and nucleus and acts as a molecular switch that cycles between the GDP-and GTP-bound state [29]. The deregulation of Ran-dependent processes has been implicated in the development of severe diseases such as cancer and neurodegenerative disorders [30]. Our results show that the expression of Ran was substantially increased by low shear stress. However, the function of Ran in vascular disease and Ran regulation were unclear until recently.
Talin is a large focal adhesion protein that links intracellular networks with the extracellular matrix (ECM) via the cytoskeleton and integrins. Talin binds and recruits cytoskeletal proteins involved in mechanotransduction [31,32]. The dysregulation of talin activators may lead to disease states in which aberrant integrin activation and mechanotransduction promote changes in cell spreading, migration, and survival [33].
The present results indicate that talin expression was upregulated in ECs under mechanotransduction.
In summary, the proteomic analysis demonstrated that the expression of VEC proteins changed under low shear stress conditions. These results may help elucidate the mechanism by which shear stress affects VECs.              Analysis of differentially-expressed proteins in vascular endothelial cells under shear stress or static conditions. Electrophoretogram of two-dimensional electrophoresis of differentially expressed proteins under shear stress and static conditions. A total of 79 protein spots were identi ed in the shear stress group (A) and 189 spots were identi ed in the static control group (B). Of these, ten protein spots were excised and analyzed by mass spectrometry (C, D).

Figure 2
Analysis of differentially-expressed proteins in vascular endothelial cells under shear stress or static conditions. Electrophoretogram of two-dimensional electrophoresis of differentially expressed proteins under shear stress and static conditions. A total of 79 protein spots were identi ed in the shear stress group (A) and 189 spots were identi ed in the static control group (B). Of these, ten protein spots were excised and analyzed by mass spectrometry (C, D).

Figure 3
Enriched biological processes.

Figure 3
Enriched biological processes.