Atorvastatin antagonizes homocysteine-induced functional injury of endothelial progenitor cells

AIM: To investigate the oxidative mechanism of homocysteine (Hcy)-induced functional injury in endothelial progenitor cells (EPCs) and the protective effects of atorvastatin. METHODS: Total mononuclear cells were isolated from the mouse bone marrow by Ficoll density gradient centrifugation and cultured in vitro. After being cultured for 7 days，the cells were identified by fluorescence microscopy. Cells were divided into six groups. Cells were treated with Hcy (0 μmol/L, 50 μmol/L, 500 μmol/L) for 24 h or pretreated with atorvastatin (0.1 μmol/L, 1 μmol/L, 10 μmol/L) for 0.5 h, then cultured with 500 μmol/L Hcy for 24 h. EPC proliferation, migration and in vitro vasculogenesis activity were assayed with MTT assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPC adhesion assay was performed by replating those on fibronectin-coated dishes, and then adherent cells were counted. Reactive oxygen species (ROS) levels in cells were measured using H2DCF-DA as a fluorescence probe. Activities of NADPH oxidases were evaluated with lucigenin-enhanced chemiluminescence. NO in the supernatant was detected by nitrate reductase assay. RT-PCR was performed to determine the eNOS expression. RESULTS: Hcy impaired the proliferation, adhesion, migration activity and in vitro vasculogenesis capacity of EPCs, increased ROS accumulation, NADPH oxidase activation, and decreased the secretion of NO, and eNOS mRNA expression, compared with the control group (P<0.05 or P<0.01). Atorvastatin inhibited the effects of Hcy in a dose-dependent manner, compared with the 500 μmol/L Hcy group (P<0.05 or P<0.01). CONCLUSION: Hcy induces ROS via activating NADPH oxidase, decreases NO secretion and eNOS mRNA expression leading to EPC functional injury. Atorvastatin partially antagonizes these effects.