Atorvastatin inhibits homocysteine-induced oxidative stress and apoptosis in endothelial progenitor cells involving Nox4 and p38MAPK

Previous studies showed that homocysteine (Hcy) reduces endothelial progenitor cells (EPCs) numbers and impairs functional activity. Atorvastatin, HMG-CoA inhibition has been showed to have protective effects on EPCs. Recent studies have demonstrated that reduced EPCs numbers and activity are associated with EPCs apoptosis. However, the protective mechanisms of atorvastatin on HHcy-induced EPCs apoptosis remain to be determined. This study was designed to examine the effect of atorvastatin on homocysteine-induced reactive oxygen species (ROS) production and apoptosis in EPCs. EPCs were isolated from peripheral blood and characterized, then challenged with Hcy (50-500 mu mol/L) in the presence or absence of atorvastatin (0.01-1 mu mol/L) or various stress signaling inhibitors, including mevalonate (100 mu mol/L), antioxidants N-acetyl cysteine (NAC, 10 mu mol/L), the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI 10 mu mol/L), the eNOS inhibitor N(G)mono-methyl-L-arginine LNMA (1 mmol/L), and the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 (10 mu mol/L). Apoptosis was evaluated by FACS analysis and cell viability was determined by MTT assay. ROS were detected by 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFH-DA). NADPH oxidases were evaluated with lucigenin-enhanced chemiluminescence. Expression of Nox4 mRNA and p-p38MAPK protein was measured by RT-PCR and Western blot analysis, respectively. Our data revealed that atorvastatin significantly suppressed Hcy-induced ROS accumulation and EPCs apoptosis. Atorvastatin also antagonized homocysteine-induced activation of NADPH oxidase and overexpression of Nox4 mRNA and p-p38MAPK protein. Similar effects occurred with EPCs transfected with Nox4 siRNA. These findings demonstrated that atorvastatin may inhibit Hcy-induced NADPH oxidase activation, ROS accumulation, and EPCs apoptosis through Nox4/p38MAPK dependent mechanisms, all of which may contribute to atorvastatin-induced beneficial effects on EPCs function. (C) 2009 Elsevier Ireland Ltd. All rights reserved.