Mechanisms of H sub(2)O sub(2)-induced oxidative stress in endothelial cells

• Published in: Free radical biology & medicine 2006-06, Vol.40 (12), p.2206-2213
• Main Authors: Coyle, Christian H, Martinez, Luis J, Coleman, Mitchell C, Spitz, Douglas R, Weintraub, Neal L, Kader, Khalid N
• Format: Article
• Language: English
• ISSN: 0891-5849
• DOI: 10.1016/j.freeradbiomed.2006.02.017

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H sub(2)O sub(2) induces production of O sub(2) super(-) by activating NADPH oxidase. However, the mechanisms whereby H sub(2)O sub(2) induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H sub(2)O sub(2) on O sub(2) super(-) levels on porcine aortic endothelial cells (PAEC). Treatment with 60 mu mol/L H sub(2)O sub(2) markedly increased intracellular O sub(2) super(-) 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 sub(2) super(-) levels and attenuated cytotoxicity resulting from treatment with H sub(2)O sub(2). L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O sub(2) super(-) levels in PAEC treated with H sub(2)O sub(2), suggesting that both NOS and NADPH oxidase contribute to H sub(2)O sub(2)-induced O sub(2) super(-) in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O sub(2) super(-) levels in PAEC treated with H sub(2)O sub(2) to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H sub(2)O sub(2) produces oxidative stress in endothelial cells by increasing intracellular O sub(2) super(-) levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H sub(2)O sub(2) and oxidant- generating enzymes that may contribute to endothelial dysfunction.