Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H2O2 induces production of O-2(center dot-) by activating NADPH oxidase. However, the mechanisms whereby H2O2 induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H2O2 on O center dot- levels on porcine aortic endothelial cells (PAEC). Treatment with 60 mu mot/L H2O2 markedly increased intracellular O-2(center dot-) levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O-2(center dot-) levels and attenuated cytotoxicity resulting from treatment with H2O2. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O-2(center dot-) levels in PAEC treated with H2O2, suggesting that both NOS and NADPH oxidase contribute to H2O2-induced O-2(center dot-) in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with Lsepiapterin together reduced O-2(center dot-) levels in PAEC treated with H2O2 to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H2O2 Produces oxidative stress in endothelial cells by increasing intracellular O-2(center dot-) levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H2O2 and oxidant-generating enzymes that may contribute to endothelial dysfunction. (c) 2006 Elsevier Inc. All rights reserved.