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OBJECTIVE@#To investigate the effects of endothelial progenitor cells (EPCs) under shear stress on the biological function such as proliferation, adhesion, migration, apoptosis and expression of α-smooth muscle actin (α-SMA), collagen-I and collagen-Ⅲ of hepatic stellate cells (HSCs).@*METHODS@#HSCs and EPCs were inoculated into the upper and lower layers of the co-culture chamber respectively and co-incubated for 24 hours. Then, 12 dyne/cm shear stress was applied to EPCs cells for another 24 hours. After that, proliferation, adhesion, migration and apoptosis of HSCs were detected by cell counting kit-8 (CCK-8) kit, cell adherent assay, Boyden cell migration assay and flow cytometry respectively. Fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expression of alpha -SMA, collagen I and collagen-Ⅲ in HSCs.@*RESULTS@#Under shear stress, EPCs ecological niche could obviously inhibit the proliferation, adhesion and migration of HSCs, promote the apoptosis of HSCs, and down-regulate the mRNA and protein expression of collagen-I, collagen-Ⅲ in HSC cells.@*CONCLUSIONS@#Under shear stress, EPCs ecological niche could inhibit the fibrosis development of HSCs to a certain extent.
Subject(s)
Actins , Apoptosis , Cell Proliferation , Cells, Cultured , Collagen Type I , Endothelial Progenitor Cells , Hepatic Stellate CellsABSTRACT
Objective To investigate the effects of shear stress on late endothelial progenitor cells (EPCs) functions in vitro and in vivo. Methods Density gradient centrifugation-isolated rat bone marrow mononuclear cells were cultured in EGM-2MV and induced into EPCs. The 3rd~4th generation of EPCs, namely late EPCs, were treated with shear stress (1.2 Pa). Then cell biological functions, such as proliferation, adhesion, migration and ability of tube formation, were assayed with EdU incorporation assay, adhesion testing, Boyden chamber assay and Matrigel, respectively. The gene expression of VEFG was analyzed by real time RT-PCR. The apoptosis and aging situation of late EPCs were assayed by FACS and senescence-associated β-galactosidase (SA-β-gal) staining. The reendothelialization capacity of late EPCs treated by shear stress was evaluated by establishing models of freshly balloon-injured carotid arteries of rats and cell transplantation in situ. Results Shear stress could increase proliferation, adhesion, migration and tube formation of late EPCs (P＜0.05), upregulate the gene expression of VEGF, inhibit EPC apoptosis and delayed EPC aging (P＜0.05). Transplantation of late EPCs treated by shear stress facilitated in vivo reendothelialization in the injured arterial segment and inhibited neointima formation. Conclusions Shear stress within the physiological range can improve the functions of late EPCs and enhance their therapeutic ability of repairing vascular endothelial injury, which provides experimental basis for the clinic application of EPCs and shear stress-mediated cell therapy.
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Objective To investigate effects of F-actin cytoskeleton on differentiation of endothelial progenitor cells (EPCs) under laminar shear stress. MethodsEPCs isolated from rat bone marrow were treated with laminar shear stress (1.2 Pa). Then the gene and protein expressions of the endothelial cell differentiation markers, such as vWF and CD31, were assayed with real time RT-PCR and Flow Cytometry. The effects of laminar shear stress on F-actin cytoskeleton and Ras activity were investigated by immunofluorescence technique and Pull-down assay. Results Compared with the untreated group, the expressions of vWF and CD31 were obviously increased in the group treated with laminar shear stress (P<0.05). Moreover, exposure of EPCs to laminar shear stress led to the reorganization of cytoskeleton and enhanced the activity of Ras in EPCs. The treatment to EPCs with either F-actin stabilizer jasplakinolide or depolymerizers cytochalasin D inhibited the cytoskeleton reorganization induced with laminar shear stress, the activity of Ras and the up-regulation of the vWF and CD31 genes. However, over-expression of Ras augmented the up-regulation of the vWF and CD31 genes induced by laminar shear stress in EPCs.Conclusions The mechanism that laminar shear stress accelerates the differentiation of EPCs may be related with the laminar shear stress-induced cytoskeleton rearrangement and Ras activation. This study is of significance in revealing the mechanism of vascular endothelial repair which could be useful for the prevention and treatment of atherosclerosis.