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 O₂{radical 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 O₂{radical dot}^- levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂{radical 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 O₂{radical 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 O₂{radical dot}^- levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂{radical dot}^- in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂{radical 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 O₂{radical 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.