HOMOCYSTEINE-INDUCED NITRIC-OXIDE PRODUCTION IN VASCULAR SMOOTH-MUSCLE CELLS BY NF-KAPPA-B-DEPENDENT TRANSCRIPTIONAL ACTIVATION OF NOS2

Increased plasma levels of homocysteine are an independent risk factor for atherothrombosis. While the endothelial cytotoxicity of homocyste ine has been attributed to oxidative stress associated with the reacti vity of the thiol group, the oxidative effect of homocysteine on vascu lar smooth-muscle cells has not been investigated. Recent evidence sug gests that expression of inducible nitric oxide synthase (iNOS), or No s2 gene product, in vascular smooth-muscle cells may, in part, promote atherosclerosis by increasing local oxidative stress. We therefore hy pothesized that homocysteine contributes to atherosclerosis by affecti ng cytokine-induced production of nitric oxide (NO) by vascular smooth -muscle cells. Confluent rat aortic smooth-muscle cells were exposed t o a range of concentrations of homocysteine for 4 hr, then were treate d with interferon-gamma, interleukin-1 beta, and lipopolysaccharide to induce iNOS. Media NOx content (nitrite plus S-nitrosothiol) was meas ured over 24 hr using the Saville reaction. As compared to controls, 5 , 50, and 500 mu M homocysteine produced a dose-dependent increase in media NOx content, an effect that was primarily a consequence of incre ased 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 co mpared with controls, an effect that was not caused by changes in mess age stability. By electrophoretic mobility shift assay, homocysteine a ctivated NF-kappa B and also potentiated cytokine activation of NF-kap pa B. These data demonstrate that exposure of vascular smooth-muscle c ells to pathophysiologically relevant concentrations of homocysteine p rior to cytokine stimulation leads both to an increase in NO productio n and to an NF-kappa B-mediated increase in Nos2 transcription. Upregu lation of Nos2 may contribute to the inflammatory response that charac terizes early atherogenesis and may, in part, account for the adverse vascular effects of hyperhomocysteinemia.