Protective Effects Of Fluvastatin On The Cultured Endothelial Progenitor Cells Following Oxidative Injury By Hydrogen Peroxide In Vitro

Objective: To determine the biological traits and optimal condition for the induction and differentiation of endothelial progenitor cells from peripheral blood in healthy adults. Then investigate the protective effects of fluvastatin on endothelial progenitor cells (EPCs) which were damaged by hydrogen peroxide (H2O2).Methods: Total hPBMNCs were isolate from peripheral blood of human volunteers by Histopaque-1077 density-gradient centrifugation. The cells were suspended in endothelial basal medium (EBM-2) supplemented with EGM-2-MV-SingleQuots and plated on fibronectin coated culture dishes. After 7 days culture, the adherent cells were subjected to immunohistochemistry and immunofluorescence to analyze the expression of CD31, CD34, VEGFR-2 and vWF. Flow cytometry(FCM) was performed to count the number of CD34⁺/AC133⁺ and VEGFR-2⁺ cells. MTT assay were performed to detect the proliferate effect of H₂O₂ and fluvastatin on EPCs and to determine the concentration of them to further study. The cycle distributions of EPCs were determined by FCM after being exposed to certain concentration of H₂O₂, fluvastatin and both of them.Results: The adherent cells exhibited clonal morphology and cobblestone morphology after 7 days and 4 weeks culture respectively. They were positive stain for CD31, CD34, VEGF-2 and vWF. The number of CD34+/AC133+ cells increased significantly after 2 weeks culture. In MTT assay, H2O2 inhibited proliferation of EPCs in a dose-dependent manner (P<0.01). We observed morphological changes of EPCs after injured by hydrogen peroxide, which just like apoptosis. Meanwhile ljUmol/1 fluvastatin promoted EPCs proliferation in the same test. When EPCs were exposed to both of them after 24h, the concentration of H2O2 and fluvastatin were 10Qwmol/l and lfimolA respectively, cell-cycle analysis indicated that the percentage of EPCs in stage Gl was decreased markedly and there was a down regulation of the percentage of EPCs in stage Sub-Gl, as compared with only exposed to H2O2 and control (P<0.05).Conclusion: EPCs can be obtained from hPBMNCs and be expanded enough to harvesting in vitro under certain condition. It is suggested that fluvastatin may promote EPCs proliferation and protect EPCs from H2O2 induced oxidative injury.