Homocysteine-induced nitric oxide production in vascular smooth-muscle cells by NF-kappa B-dependent transcriptional activation of Nos2

Abstract

Increased plasma levels of homocysteine are an independent risk factor for atherothrombosis. While the endothelial cytotoxicity of homocysteine has been attributed to oxidative stress associated with the reactivity of the thiol group, the oxidative effect of homocysteine on vascular smooth-muscle cells has not been investigated. Recent evidence suggests that expression of inducible nitric oxide synthase (iNOS), or Nos2 gene product, in vascular smooth-muscle cells may, in part, promote atherosclerosis by increasing local oxidative stress. We therefore hypothesized that homocysteine contributes to atherosclerosis by affecting cytokine-induced production of nitric oxide (NO) by vascular smooth-muscle cells. Confluent rat aortic smooth-muscle cells were exposed to a range of concentrations of homocysteine for 4 hr, then were treated with interferon-gamma, interleukin-1 beta, and lipopolysaccharide to induce iNOS. Media NOx content (nitrite plus S-nitrosothiol) was measured over 24 hr using the Saville reaction. As compared to controls, 5, 50, and 500 microM homocysteine produced a dose-dependent increase in media NOx content, an effect that was primarily a consequence of increased S-nitrosothiol production. iNOS enzyme activity and iNOS protein levels were increased significantly in the homocysteine-treated cells as compared with controls. Northern analysis showed that homocysteine treatment increased steady-state Nos2 mRNA levels by 61% at 6 hr as compared with controls, an effect that was not caused by changes in message stability. By electrophoretic mobility shift assay, homocysteine activated NF-kappa B and also potentiated cytokine activation of NF-kappa B. These data demonstrate that exposure of vascular smooth-muscle cells to pathophysiologically relevant concentrations of homocysteine prior to cytokine stimulation leads both to an increase in NO production and to an NF-kappa B-mediated increase in Nos2 transcription. Upregulation of Nos2 may contribute to the inflammatory response that characterizes early atherogenesis and may, in part, account for the adverse vascular effects of hyperhomocysteinemia.