Cigarette smoke-induced proinflammatory alterations in the endothelial phenotype: role of NAD(P)H oxidase activation

Although the cardiovascular morbidity and mortality induced by cigarette smoking exceed those attributable to lung cancer, the molecular basis of smoking-induced vascular injury remains unclear. To test the link between cigarette smoke, oxidative stress, and vascular inflammation, rats were exposed to the smoke of five cigarettes per day (for 1 wk). Also, isolated arteries were exposed to cigarette smoke extract (CSE; 0 to 40 mu g/ml, for 6 h) in organoid culture. We found that smoking impaired acetylcholine-induced relaxations of carotid arteries, which could be improved by the NAD(P)H oxidase inhibitor apocynin. Lucigenin chemiluminescence measurements showed that both smoking and in vitro CSE exposure significantly increased vascular O-2(center dot-) production. Dihydroethidine staining showed that increased O-2(center dot-) generation was present both in endothelial and smooth muscle cells. CSE also increased vascular H2O2 production (dichlorofluorescein fluorescence). Vascular mRNA expression of the proinflammatory cytokines IL-1 beta, IL-6, and TNF-alpha and that of inducible nitric oxide synthase was significantly increased by both smoking and CSE exposure, which could be prevented by inhibition of NAD(P)H oxidase (diphenyleneiodonium and apocynin) or scavenging of H2O2. In cultured endothelial cells, CSE elicited NF-kappa B activation and increased monocyte adhesiveness, which were prevented by apocynin and catalase. Thus we propose that water-soluble components of cigarette smoke (which are likely to be present in the bloodstream in vivo in smokers) activate the vascular NAD(P)H oxidase. NAD(P)H oxidase-derived H2O2 activates NF-kappa B, leading to proinflammatory alterations in vascular phenotype, which likely promotes development of atherosclerosis, especially if other risk factors are also present.