Proteomic profiling of human bone marrow mesenchymal stem cells ...

Mol Cell Biochem (2010) 341:9–16 DOI 10.1007/s11010-010-0432-7

Proteomic profiling of human bone marrow mesenchymal stem cells under shear stress Wei Yi • Yang Sun • Xufeng Wei • Chunhu Gu • Xiaochao Dong • Xiaojun Kang • Shuzhong Guo Kefeng Dou

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Received: 8 December 2009 / Accepted: 26 February 2010 / Published online: 21 April 2010 Ó Springer Science+Business Media, LLC. 2010

Abstract Mesenchymal stem cells (MSCs) are promising seed cells for tissue engineering of blood vessels. As seed cells, MSCs must endure blood fluid shear stress after transplantation. It has been shown that fluid shear stress can regulate the proliferation and differentiation of MSCs. However, the effects of fluid shear stress on MSCs including the types of proteins modulated are still not well understood. In this study, we exposed human mesenchymal stem cells (HMSCs) to 3 dyn/cm2 shear stress for 6 h and compared them to a control group using proteomic analysis. Thirteen specific proteins were affected by shear stress, 10 of which were up-regulated. Shear stress especially induced sustained increases in the expression of Annexin A2 and GAPDH, which have been specifically shown to affect HMSCs function. We present here the first

Wei Yi and Yang Sun contribute equally to this study.

Electronic supplementary material The online version of this article (doi:10.1007/s11010-010-0432-7) contains supplementary material, which is available to authorized users. W. Yi K. Dou (&) Department of Hepatobiliary Surgery, Xijing Hospital, 4th Military Medical University, 127 Changle West Road, Xi’an 710032, Shaanxi, People’s Republic of China e-mail: [email protected] Y. Sun S. Guo (&) Department of Plastic Surgery, Xijing Hospital, 4th Military Medical University, 127 Changle West Road, Xi’an 710032, Shaanxi, People’s Republic of China e-mail: [email protected] X. Wei C. Gu X. Dong X. Kang Department of Cardiovascular Surgery, Xijing Hospital, 4th Military Medical University, 127 Changle West Road, Xi’an 710032, Shaanxi, People’s Republic of China

comparative proteome analysis of effect of shear stress on HMSCs. Keywords Fluid shear stress Mesenchymal stem cells Proteome Tissue-engineered blood vessel

Introduction Nowadays, the pathology that affects the small- and medium-sized blood vessels has posed great danger to many people, and a large number of vascular grafts are needed [1]. Autologous veins and autologous arteries are widely used in cardiac and peripheral bypass surgery. Unfortunately, because of the deficiency or sometimes the pathology of autologous vessels, many patients do not have appropriate blood vessels for replacements. Synthetic vascular grafts such as ePTFE and Dacron have been widely used in clinic to replace the large arteries ([6 mm internal diameter), but they fail to replace the smaller diameter vessels due to thrombus formation [1]. Therefore, an alternative supply of vessels to replace smaller diameter diseased arteries is urgently needed. Many studies have shown that tissue engineering offers the potential of providing vessels that can be used to replace diseased native blood vessels. Recent studies have focused on applying bone marrow mesenchymal stem cells (MSCs) as seed cells to tissueengineered blood vessels. MSCs are nonhematopoietic and multipotent stromal cells derived from bone marrow. They have the capacity to develop both in vitro and in vivo into terminally differentiated mesenchymal phenotypes such as osteoblasts, chondrocytes, adipocytes, and smooth muscle cells in response to different microenvironmental cues [2, 3]. Due to their self-renewal and differentiation potential,

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MSCs are considered to be a suitable candidate for use in regenerative medicine and cell-based therapy. It has already been shown that MSCs-derived vascular smooth muscle cells (SMCs) and endothelial cells (ECs) can be used to construct tissue-engineered vascular grafts for blood vessel replacement [4, 5]. Tissue-engineered vessels will, however, be challenged by blood shear after transplantation. Recent studies have shown that blood shear stress can regulate the proliferation and differentiation of MSCs through a variety of signaling pathways [6–8]. However, the effects of fluid shear stress on MSCs including the types of proteins modulated are not well understood. Proteomics may provide a powerful approach to evaluate quantitative and qualitative effects of shear stress on protein expression of MSCs, in addition to giving additional insight into cell structure and function [9]. In this study, human mesenchymal stem cells (HMSCs) were exposed to 3 dyn/cm2 shear stress for 6 h and compared to unexposed cells by proteomic analysis. 32 specific protein spots were identified. Thirteen proteins showed differential expression, 10 of which were up-regulated. These findings indicate that shear stress affects HMSCs, while the physiological implications for tissue engineering of blood vessels will require further study.

Materials and methods Cell isolation and cultivation Human bone marrow was obtained with consent from the iliac crest of volunteers undergoing hip replacement operations at Xijing Hospital, Xi’an, China. The experiments were performed in adherence with Chinese National Institutes of Health Guidelines and were proved by 4th Military Medical University committee on Ethnics. HMSCs were isolated and cultured as described previously [10]. HMSCs identification To confirm that they maintained their phenotype after expansion in culture, HMSCs were subjected to flow cytometric analysis [11]. After detachment with trypsin, cells were centrifuged and washed with PBS. Cells were resuspended and nonspecific binding sites were blocked by incubation with 1% bovine serum albumin (Sigma St. Louis, MO, USA) for 30 min. For primary antibodies conjugated with FITC CD14, CD34, CD29, CD45 (all from Santa Cruz, CA), and CD105 (Chemicon, Temecula, CA), samples were incubated with individual primary antibodies for 30 min, washed with PBS containing 3% fetal bovine

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Mol Cell Biochem (2010) 341:9–16

serum, and incubated with fluorescent-conjugated secondary antibody for 30 min. Samples were placed in a FACSCalibur cytometer (Becton Dickinson), and data were analyzed using CELLQUEST software (Becton Dickinson). FITC-conjugated monoclonal antibodies (mAbs) were used (Pharmingen International). To further demonstrate that expanded HMSCs maintained their pluripotent differentiation potential, HMSCs at passage 6 were tested for differentiation into adipogenic and osteogenic cells using established protocols [3, 12–14]. Fluid shear stress experiments A parallel plate flow chamber was used to impose fluid shear stress on HMSCs [15, 16]. HMSCs were cultured on the bottom of the flow chamber (0.05 cm in height, 2.5 cm in width, and 10 cm in length) for 1 day before exposure to fluid shear stress. Fluid was perfused through an inlet and outlet in the chamber. Initiation of fluid flow by a cardiopulmonary bypass unit (Cobe) generated a laminar shear stress where the specific shear stress level could be obtained by adjusting the flow rate. The shear stress level was calculated using the following equation: s = 6Qg/wh2, where Q is the flow rate across the chamber (0.24 ml/s), g is the viscosity of the medium (0.013 Pa s 37°C), w is the chamber width (2.5 cm), and h is the gasket height (0.05 cm). Shear stress at 3 dyn/cm2 was applied to HMSCs, with a static control provided by a no flow condition. The flow chamber was placed in an incubator maintained at 37°C, and DMEM with 10% FCS was circulated through the flow system for 6 h. Cell lysis Total HMSCs proteins of three donors were prepared as reported [17]. The protein content of the supernatant was measured using the Bradford assay [18]. Two-dimensional gel electrophoresis (2-DE) 2-DE was performed using a commercial kit as the manufacturer’s instructions (Amersham Biosciences). Silver staining was then performed as described [19]. Three pairs of gels were prepared for each cell line to minimize the effect of experimental variation. Image analysis and spot identification Stained 2-DE gels were scanned with a ScanMaker 8700 scanister (MICROTEK, China). The images were saved in TIFF format and then exported to ImageMaster 2D platinum Software 5.0 (Amersham-Pharmacia, Sweden). Aiming protein spots were selected based on differential expression


(greater than 2.0- or less than 0.5-fold) between two groups or abundant expression in both groups as determined by automatic analyses.

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intensity of Annexin A2 and GAPDH was measured and normalized via the expression of actin. Immunofluorescence analysis

Protein identification Specific protein spots were extracted from the gels by punch and moved to 1.5-ml tubes; a protein-free gel slab was used as a negative control. Gel-spots were washed with double-distilled water three times. After drying in a vacuum centrifuge, gel pieces were incubated in 10 ll digestion solution (40 mM NH4HCO3 in 9% ACN solution and 20 lg/ml proteomics grade trypsin) for 10–12 h at 37°C. The tryptic peptide mixture was further mixed with a-cyano-4-hydroxycinnamic acid matrix solution and vortexed gently, followed by purification with a Millipore ZipTip C18 column. A 1 ll aliquot of the mixture was loaded on a stainless steel plate and air-dried, and then analyzed with peptide mass fingerprinting using matrixassisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) after loading onto a MALDI plate (Applied Biosystems, USA). The peptide mass profiles produced by MALDI-MS were submitted to Aldente or Profound software. UniProtKB/Swiss-PROT and NCBI nonredundant protein databases were used to match the peptide mass fingerprints (PMF). All searches used a mass window of 14 and 120 kDa with the Homo sapiens taxon, and allowed for modifications such as carboxyamidomethyl, oxidation Met, phosphorylation, and phosphorylation. The thresholds for positive identification of proteins were set as follows: shift max 0.2, slope max 200, internal error max 50, minimum number of hits 4, P value max less than 5. Other parameters were unchanged from the default. Western blot analysis Three HMSCs samples before and after 3 dyn/cm2 shear stress for 6 h were validated by the protein expression. Total protein lysate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The blots were blocked with Trisbuffered saline containing 0.1% Tween-20 and 5% nonfat milk, followed by incubation with primary antibodies against Annexin A2 (mouse antihuman, Chemicon, Temecula, CA), GAPDH (mouse antihuman, ProSci, Poway, CA) and actin (mouse anti-human, ProSci, Poway, CA) at 1:1,000 dilution. The bound primary antibodies were detected by using a goat anti-mouse IgGhorseradish peroxidase conjugated antibody for Annexin A2, GAPDH, and actin (1:1,000) and bands were visualized by electrogenerated chemiluminescence (ECL). Band

After culturing HMSCs before and after 3 dyn/cm2 shear stress for 6 h on slides, cells were washed with PBS and fixed in 95% ethanol for 10 min at room temperature, then washed again with PBS. Nonspecific binding was blocked with 3% BSA in PBS for 1 h, and then slides incubated for 1 h at room temperature with a monoclonal mouse antihuman primary antibodies to Annexin A2 (1:200 dilution) and GAPDH (1:100 dilution). After washing with PBS, rabbit anti-mouse FITC-conjugated secondary antibody (1:200 dilution) was incubated for 1 h at room temperature, then slides washed and coverslips mounted using vectashield mounting medium with DAPI (Vector Laboratories, Peterborough, UK). Samples were analyzed by immunofluorescence microscopy using a Leica microscope with a 209 objective. The complete area of each slide was carefully examined and at least 20 representative images of each sample taken. Statistical analysis All values in the text and figures are presented as mean ± SEM/SD of n independent experiments. ANOVA across all investigated groups was conducted first, and post hoc pairwise tests for certain group pairs with assessment of statistical significance were then performed after Bonferroni correction of the overall significance level. Western blot densities were analyzed with the Kruskal–Wallis test followed by the Dunn post hoc test. Probabilities of B0.05 were considered statistically significant. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Results Characterization of HMSCs HMSCs up to passage 6 were used in our experiments. HMSCs were stained for a set of cell surface markers to confirm their characteristics after in vitro expansion. As shown in Fig. 1, HMSCs at passage 6 expressed CD29 and CD105 (SH2) in the absence of the hematopoietic lineage markers CD14, CD34, and CD45, suggesting that the expanded HMSCs maintained their phenotype. To further confirm their pluripotent differentiation potential, HMSCs at passage 6 were induced into adipogenic and osteogenic cells. After culture in adipogenic medium, several clusters

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Fig. 1 Identification of HMSCs. a Flow cytometric analysis of cell surface markers in HMSCs. As described in ‘‘Materials and Methods’’, HMSCs at passage 6 were subjected to flow cytometry after staining with FITCconjugated CD14, CD29, CD34, CD45, or CD105 antibodies. The chart shows that HMSCs were CD29 and CD105 positive while CD14, CD34 and CD45 were negative. Error bars reflect the mean (±SEM) across 3 HMSCs. b Photomicrograph of HMSCs after induction to adipogenic cells. Cells were stained with Oil red O solution and several clusters of adipocytes were present containing intracellular lipid vacuoles. c Photomicrograph of HMSCs after induction to osteogenic cells. Calcium deposition was assayed by immunohistochemistry against osteocalcin. The umber particles deposited in the endochylema show positive staining

of cells appeared that contained intracellular lipid vacuoles staining positive for Oil Red O. Osteogenic differentiation of HMSCs was confirmed by expression of the osteogenic differentiation marker osteocalcin. These findings indicate that the HMSCs used in this study maintained their pluripotent differentiation potential, as they were capable of differentiating along osteogenic and adipogenic lineages as previously described by numerous studies [13, 20, 21]. Shear stress induced proteome changes in HMSCs A pair of representative two-dimensional gel images of protein lysates from no treatment controls (a) or cells treated with 3 dyn/cm2 shear stress for 6 h (b) is shown in Fig. 2. At pH 3–10, 2-DE maps of a and b displayed about 750 spots each. Overall, 32 protein spots were identified with high confidence. The location of each spot is labeled with a number. 13 proteins were found to be consistently up- or down-regulated by over twofold after 3 dyn/cm2 shear stress treatment for 6 h, and 10 proteins were upregulated and 3 down-regulated. Based on information from UniProtKB/Swiss-PROT and NCBI nonredundant

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protein databases, the 13 identified up- or down-regulated proteins were listed in Table 1. There were 19 other protein spots that remained unchanged after exposed to 3 dyn/cm2 shear stress for 6 h (Table 2). The shear stress regulated proteins are involved in various cellular processes and include heat shock cell membrane proteins (e.g. Annexin A2), proteins involved in protein synthesis, degradative proteins (e.g. T complex protein 1), chaperones (e.g. 60 kDa heat shock protein), metabolic enzymes (e.g. pyruvate kinase isozymes M1/M2), and nuclear and chromatin proteins (e.g. poly(rC)-binding protein 1). Spot 21 showed a great increase (5.1-fold) after shear stress treatment. This protein was identified as Annexin A2. Both spot 5 and Spot 55 were identified as GAPDH, and they both showed significant increase after shear stress treatment, 2.3-fold and 4.2-fold, respectively. Confirmation studies Proteomics findings were evaluated in HMSCs samples before and after 3 dyn/cm2 shear stress derived from original sample, as well as two additional patient samples.


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Fig. 2 Two-dimensional reference maps for HMSCs before (a) and after (b) shear stress at 3 dyn/cm2 for 6 h showing up- and downregulated protein spots. The protein samples were prepared and separated as described in ‘‘Materials and methods,’’ and the gels were subjected to silver staining and image analysis. IEF (pH 3–10 nonlinear gradient) is in the horizontal direction. PAGE (10% gel) is in the vertical direction. Spots of interest were excised from the gels and digested with trypsin, and the proteins were identified. 13 proteins were obviously affected by shear stress (listed in Table 1). c The chat shows 10 up-regulated proteins under shear stress. d The chart shows 3 down-regulated proteins under shear stress. Error bars reflect the mean (±SEM) across 3 HMSCs under 3 dyn/cm2 shear stress compared with untreated control

Table 1 Different proteins identified in two-dimensional electrophoretic gels ID no. from Fig. 2

Shear stress induced change folds

Protein identification

Accession number

Protein molecular weight (kDa)

pI

5

2.3

Glyceraldehyde-3-phosphate dehydrogenase

P04406

36

8.6

11

2.2

Pyruvate kinase isozymes M1/M2

P14618

58

8.0

12

3

Pyruvate kinase isozymes M1/M2

P14618

58

8.0

18

2.1

Isocitrate dehydrogenase [NADP] cytoplasmic

075874

47

6.5

20

2.3

Poly(rC)-binding protein 1

Q15365

37

6.7

21

5.1

Annexin A2

P07355

38

7.5

46

3.3

T-complex protein 1 subunit beta

P78371

57

6.0

55 60

4.2 2.1

Glyceraldehyde-3-phosphate dehydrogenase 60 kDa heat shock protein

P04406 P10809

36 58

8.6 5.2

65

2.7

Voltage-dependent anion-selective 1 channel protein

P21796

31

8.6

34

-3.4

Putative glycerophosphodiester phosphodiesterase 5

Q9NPB8

76

5.3

62

-1.9

Elongation factor 2

P13639

95

6.4

63

-1.8

Elongation factor 2

P13639

95

6.4

2

HMSCs were either kept as a no treatment control or treated with 3 dyn/cm shear stress for 6 h. Proteins affected are denoted as follows: positive number indicates up-regulated, and negative number indicates down-regulated

The significantly changed proteins Annexin A2 and GAPDH expression were validated by western blot analysis and immunofluorescence cell staining. The protein results were analogous to the protein presentation observed in proteomic study. Western blot analysis results showed

that Annexin A2 was up-regulated in HMSCs under 3 dyn/ cm2 shear stress compared to HMSCs before shear stress (Mean folds: 3.994, SD: 0.849, n = 8, P \ 0.001). GAPDH expression was also up-regulated after shear stress (Mean folds: 2.139, SD: 0.538, n = 8, P \ 0.001) (Fig. 3).

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Table 2 Proteins unchanged and identified in two-dimensional electrophoresis gels ID no. from Fig. 2

Shear stress induced change folds

Protein identification

Accession number

Protein molecular weight (kDa)

pI

4

1.3

Fructose-bisphosphate aldolase A

P04075

39

8.4

26

1.5

Synaptic vesicle membrane protein VAT-1 homolog

Q99536

42

5.9

33 35

1.2 1.3

Endoplasmin Annexin A6

P14625 P08133

90 76

4.7 5.4

36

1.1

Protein disulfide-isomerase

P07237

55

4.7

37

1.1

Vimentin

P08670

54

5.1

38

1.1

Tubulin beta chain

P07437

50

4.8

40

1.3

Vimentin

P08670

54

5.1

41

1.2

Vimentin

P08670

54

5.1

42

1.0

Vimentin

P08670

54

5.1

44

1.0

Bifunctional purine biosynthesis protein

P31939

65

6.3

45

1.0

Stress-induced-phosphoprotein 1

P31948

63

6.4

47

1.2

Protein disulfide-isomerase A3

P30101

54

5.6

49

1.3

Beta-crystallin A3

P05813

25

5.8

50

1.2

Major vault protein

Q14764

99

5.3

57

1.2

Heat shock cognate 71 kDa protein

P11142

71

5.4

61

1.5

HERV-K_5q13.3 provirus ancestral Env polyprotein

Q9UKH7

21

5.7

68

1.2

Alpha-enolase

P06733

47

7.0

72

1.1

Actin, cytoplasmic 1

P60709

42

5.3

Immunofluorescence staining of Annexin A2 and GAPDH further demonstrated the up-regulated expression in HMSCs after 3 dyn/cm2 shear stress (Annexin A2 Mean folds: 3.907, SD: 0.912, n = 8, P \ 0.001; GAPDH Mean folds: 2.264, SD: 0.514, n = 8, P \ 0.001) (Fig. 4).

Discussion In the clinic, the possibility of tissue-engineered blood vessels has gained great attention due to the inadequacy of autogenic vessel grafts. In previous studies, SMCs and ECs were applied as seed cells but only with limited success. Repopulation grafts using MSCs show excellent long-term patency and exhibit well-organized layers of ECs and SMCs similar to native arteries. When used as seed cells in tissue-engineered blood vessels, MSCs are bound to undergo fluid shear stress after implantation to grafts. The effects of shear stress on a variety of cell types have been widely studied, but the effect of shear stress on HMSCs has not been investigated comprehensively. The range of shear stress in arteries has been reported to be approximately 10–25 dyn/cm2 compared with approximately 1.5–3 dyn/ cm2 in veins [22]. Shear stress can induce detachment of seed cells followed by anoikis, especially in ECs, just in an early period of time after implanted, which is one of the

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major difficulties for application of tissue-engineered blood vessels in clinic. Currently, some studies have demonstrated that precondition of low shear stress applied ex vivo improves cell retention and tissue morphological integrity for tissue-engineered blood vessels after implantation [23]. However, the effects of low fluid shear stress on HMSCs, including the types of proteins affected, are not well understood. Proteomics provides tools to globally analyze cellular activity at protein level. Besides, this proteomic profiling will allow the elucidation of connections between broad cellular pathways and molecules that were previously impossible to predict using only traditional biochemical analysis. By supplementing this analysis with traditional biophysical and biochemical methods, we have gained certain insights into the mechanisms of low shear stress regulation of HMSCs. A significant finding of this study is that low shear stress induced a sustained increase of Annexin A2 expression in HMSCs. Annexin A2 is a calcium-dependent phospholipidbinding protein found in many cell types. Based on analysis of Ca2? concentration, Annexin A2 has been reported to participate in a variety of membrane-related events such as cell proliferation, cell motility, linkage of membraneassociated protein complexes to the actin cytoskeleton, endocytosis, fibrinolysis, ion channel formation, cellmatrix and cell-cell interactions, angiogenesis and


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Fig. 3 Western blot analysis of Annexin A2 and GAPDH expression in HMSCs before and after shear stress at 3 dyn/cm2 for 6 h. a Annexin A2 and GAPDH in HMSCs were both highly expressed under 3 dyn/cm2 shear stress compared with untreated control. The housekeeping gene actin was uniformly expressed in two groups. b The chart shows respectively up-regulated Annexin A2 and GAPDH expression in HMSCs under shear stress (Annexin A2 Mean folds: 3.994, SD: 0.849, n = 8, P \ 0.001; GAPDH Mean folds: 2.139, SD: 0.538, n = 8, P \ 0.001)

maintaining the plasticity of the dynamic membraneassociated actin cytoskeleton as well as interactions with plasmin that result in stimulation of ERK1/2 and p38MAPK, cyclooxygenase-2, and PGE(2) that in turn lead to increased MMP-1 production [18, 28]. To our knowledge, there have been no previous reports of Annexin A2 regulation expression by shear stress. Additionally, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression increased significantly under the action of low shear stress. GAPDH has been considered a classical metabolic protein for its pivotal role in energy production. It is also utilized as a model protein for analysis of protein structure and enzyme mechanisms. Besides, GAPDH was reported to be a multifunctional protein with defined functions in numerous subcellular processes. As a membrane protein, GAPDH is involved in endocytosis; in cytoplasm, it acts in the translational control of gene expression; in nucleus, it functions in nuclear tRNA export, DNA repair, and DNA replication [29–31]. Recently, it has been reported that GAPDH also functions in cell apoptosis. GAPDH is over-expressed and accumulates in the nucleus during apoptosis induced by kinds of insults in diverse cell types. It is demonstrated that a variety of apoptotic stimuli can activate NO formation, which S-nitrosylates GAPDH, and the S-nitrosylation confers upon GAPDH the ability to bind to Siah, an E3-ubiquitin-ligase, which translocates GAPDH to the nucleus. In the nucleus, GAPDH stabilizes the rapidly turning over Siah, enabling it to degrade selected target proteins and affect apoptosis. There is also evidence suggesting that GAPDH may be an intracellular sensor of oxidative stress during early apoptosis [32, 33]. Additional investigations relate a substantial role for nuclear GAPDH in hyperglycemic stress and the development of metabolic syndrome [31, 34].

Fig. 4 Immunofluorescence analysis of Annexin A2 and GAPDH expression before and after 3 dyn/cm2 shear stress for 6 h. HMSCs were kept as an untreated control (a, c) or treated with 3 dyn/cm2 shear stress for 6 h (b, d). Cells were immunostained for Annexin A2 (green, a and b) and GAPDH (green, c and d), nucleostained with DAPI (blue), and then visualized with immunofluorescence

microscopy. Comparison of them suggests that Annexin A2 expression in the treated group is obviously higher than in the untreated control (Mean folds: 3.907, SD: 0.912, n = 8, P \ 0.001). GAPDH is also highly expressed after shear stress compared to untreated control (Mean folds: 2.264, SD: 0.514, n = 8, P \ 0.001) (Color figure online)

metastasis of cancer [24, 25], and the bridging of membranes. Additionally, Annexin A2 may be an important player in cellular differentiation and related disorders [26, 27]. It has also been reported to have essential roles in

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To our knowledge, we present here the first proteomic analysis of HMSCs after exposure to shear stress. Proteomic profiling has provided a variety of novel molecular bullets that can form the basis of more in-depth investigations into the effects of shear stress on in vitro HMSCs proliferation, differentiation, and apoptosis, which may in turn significantly influence applications in stem cell therapy and tissue regeneration. This study identified that HMSCs respond to low shear stress. The exact mechanism of how shear stress affects HMSCs should be made clear for further progress in tissue engineering of blood vessels using multipotent HMSCs as seed cells. Acknowledgments This study was supported by the National Natural Science Foundation of China (30672086) and was funded in part by 863 Project (2006AA02A138). We thank Yusong Ruan, Ph.D., Department of Research Center of Bioinformatics, Life and Information Engineering College, Xi’an Jiao Tong University, for the supply of IMAGE clones and their sequence verification.

References 1. Ratcliffe A (2000) Tissue engineering of vascular grafts. Matrix Biol 19:353–357 2. Uccelli A, Pistoia V, Moretta L (2007) Mesenchymal stem cells: a new strategy for immunosuppression? Trends Immunol 28:219– 226 3. Wang D, Park JS, Chu JS et al (2004) Proteomic profiling of bone marrow mesenchymal stem cells upon transforming growth factor beta1 stimulation. J Biol Chem 279:43725–43734 4. Schuleri KH, Boyle AJ, Hare JM (2007) Mesenchymal stem cells for cardiac regenerative therapy. Handb Exp Pharmacol 180:195– 218 5. Kurpinski K, Park J, Thakar RG et al (2006) Regulation of vascular smooth muscle cells and mesenchymal stem cells by mechanical strain. Mol Cell Biomech 3:21–34 6. Park JS, Huang NF, Kurpinski KT et al (2007) Mechanobiology of mesenchymal stem cells and their use in cardiovascular repair. Front Biosci 12:5098–5116 7. Hsu S, Thakar R, Liepmann D et al (2005) Effects of shear stress on endothelial cell haptotaxis on micropatterned surfaces. Biochem Biophys Res Commun 337:401–409 8. Bakker A, Klein-Nulend J, Burger E (2004) Shear stress inhibits while disuse promotes osteocyte apoptosis. Biochem Biophys Res Commun 320:1163–1168 9. Blackstock WP, Weir MP (1999) Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol 17:121– 127 10. Pittenger MF, Mackay AM, Beck SC et al (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147 11. Short B, Brouard N, Occhiodoro-Scott T et al (2003) Mesenchymal stem cells. Arch Med Res 34:565–571 12. Choi YS, Park SN, Suh H (2005) Adipose tissue engineering using mesenchymal stem cells attached to injectable PLGA spheres. Biomaterials 26:5855–5863 13. Potier E, Ferreira E, Andriamanalijaona R et al (2007) Hypoxia affects l stromal cell osteogenic differentiation and angiogenic factor expression. Bone 40:1078–1087

123

Mol Cell Biochem (2010) 341:9–16 14. Dennis JE, Carbillet JP, Caplan AI et al (2002) The STRO-1? marrow cell population is multipotential. Cells Tissues Organs 170:73–82 15. Jacobs CR, Yellowley CE, Davis BR et al (1998) Differential effect of steady versus oscillating flow on bone cells. J Biomech 31:969–976 16. Li YJ, Batra NN, You L et al (2004) Oscillatory fluid flow affects human marrow stromal cell proliferation and differentiation. J Orthop Res 22:1283–1289 17. Wagner W, Feldmann RE Jr, Seckinger A et al (2006) The heterogeneity of human mesenchymal stem cell preparations–evidence from simultaneous analysis of proteomes and transcriptomes. Exp Hematol 34:536–548 18. Ramagli LS (1999) Quantifying protein in 2-D PAGE solubilization buffers. Methods Mol Biol 112:99–103 19. Barry FP, Murphy JM (2004) Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 36:568–584 20. Krampera M, Pizzolo G, Aprili G et al (2006) Mesenchymal stem cells for bone, cartilage, tendon and skeletal muscle repair. Bone 39:678–683 21. Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276:71–74 22. Kamiya A, Bukhari R, Togawa T (1984) Adaptive regulation of wall shear stress optimizing vascular tree function. Bull Math Biol 46:127–137 23. Andrews KD, Feugier P, Black RA et al (2008) Vascular prostheses: performance related to cell-shear responses. J Surg Res 149:39–46 24. Camors E, Monceau V, Charlemagne D (2005) Annexins and Ca2? handling in the heart. Cardiovasc Res 65:793–802 25. Sharma MR, Koltowski L, Ownbey RT et al (2006) Angiogenesis-associated protein annexin II in breast cancer: selective expression in invasive breast cancer and contribution to tumor invasion and progression. Exp Mol Pathol 81:146–156 26. Gilmore WS, Olwill S, McGlynn H et al (2004) Annexin A2 expression during cellular differentiation in myeloid cell lines. Biochem Soc Trans 32:1122–1123 27. Chiang Y, Rizzino A, Sibenaller ZA et al (1999) Specific downregulation of annexin II expression in human cells interferes with cell proliferation. Mol Cell Biochem 199:139–147 28. Zhang Y, Zhou ZH, Bugge TH et al (2007) Urokinase-type plasminogen activator stimulation of monocyte matrix metalloproteinase-1 production is mediated by plasmin-dependent signaling through annexin A2 and inhibited by inactive plasmin. J Immunol 179:3297–3304 29. Sirover MA (1997) Role of the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase, in normal cell function and in cell pathology. J Cell Biochem 66:133–140 30. Sirover MA (1999) New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase. Biochim Biophys Acta 1432:159–184 31. Sirover MA (2005) New nuclear functions of the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase, in mammalian cells. J Cell Biochem 95:45–52 32. Chuang DM, Hough C, Senatorov VV (2005) Glyceraldehyde-3phosphate dehydrogenase, apoptosis, and neurodegenerative diseases. Annu Rev Pharmacol Toxicol 45:269–290 33. Hara MR, Snyder SH (2006) Nitric oxide-GAPDH-Siah: a novel cell death cascade. Cell Mol Neurobiol 26:527–538 34. Nishikawa T, Araki E (2007) Impact of mitochondrial ROS production in the pathogenesis of diabetes mellitus and its complications. Antioxid Redox Signal 9:343–353