The Effects Of Shear Stress On Bilological Characteristics Of Late Endothelial Progenitor Cells On Differernt ECM

Objective:To investigate the effects of various extracellular matrix(ECM) proteins on the biological characteristics of late endothelial progenitor cells and the shear stress responsiveness of EPCs seeded on different ECM.Methods:1. Density gradient centrifugation-isolated rat bone marrow mononuclear cells were cultured in complete M199 medium(including 15% fetal bovine serum, 10 μg/L VEGF and 5 μg/L b FGF). EPCs were seeded on substrates containing various extracellular matrix(ECM) proteins, such as Fibronectin, laminin and collagen. The cells on 3 dishes coated with different ECM were defined as Fn group, Ln group and Col group. The 3r d generation EPCs, namely late EPCs, was harvested and cell biological functions, such as proliferation, migration, adhesion and the ability of forming tubes were assayed with CCK-8, wound healing assay, adhesion test and Matrigel respectively. The gene expression of endothelial cell differentiation markers, such as v WF and CD31, was analyzed by real time RT-PCR.2. EPCs seeded on different ECM were treated with shear stress(12 dyne/cm2). The effects of shear stress on F-actin cytoskeleton was investigated by immunofluorescence technique. Then proliferation, adhesion, migration, the ability of forming tubes and, the gene expression of v WF and CD31 were assayed by CCK-8, adhesion test, boyden chamber Matrigel and real time RT-PCR, respectively.Results:1. Cell proliferation ability of Fn group and Col group are higher than the Ln group(P<0.05); the adhesion, migration and tube formation in Fn group were significantly increased compared to other groups(P< 0.01); the gene expression of v WF and CD31 in those cells were no difference.2. Exposure of EPCs to shear stress led to cytoskeleton reorganization, especially, the cells from Ln group. After shear stress treatment, EPC proliferation, migration and tube forming abilities of Ln group were higher than Fn group or Col group(P<0.05).Conclusion:These results suggest the ECM affects the biological functions of late EPCs. Moreover, the shear stress responsivenes of EPCs seeded on Fn, Col and Ln are different.