Increased superoxide production in coronary arteries in hyperhomocysteinemia: role of tumor necrosis factor-alpha, NAD(P)H oxidase, and inducible nitric oxide synthase.

In coronary arteries, hyperhomocysteinemia (HHcy, a known risk factor for coronary heart disease) impairs flow-induced dilations, which can be reversed by superoxide dismutase (SOD). To evidence increased O2*- generation and elucidate its source, we characterized changes in activity (lucigenin chemiluminescence, hydroethidine staining) and expression of arterial pro- and antioxidant systems (Western blotting, immunohistochemistry, cDNA microarray, reverse-transcription polymerase chain reaction) in the coronary arteries of rats by using methionine diet-induced HHcy. The increased generation of O2*- by HHcy coronary arteries was inhibited by SOD, diphenyleneiodonium, apocynin, and apocynin plus amino guanidine but was unaffected by allopurinol and rotenone. Also, diphenyleneiodonium-sensitive NADPH-driven O2*- generation was increased in HHcy vessels. In HHcy arteries expression of the smooth muscle-confined NAD(P)H oxidase subunit nox1 and that of iNOS was increased. Expression of p67phox, p22phox, and p47phox subunits and that of endothelial nitric oxide synthase, Cu,Zn-SOD, Mn-SOD, extracellular SOD (mRNA), and xanthine oxidase was unchanged. Microarray analysis showed increased expression of tumor necrosis factor (TNF)-alpha (confirmed by reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry) that was localized in smooth muscle. In vitro incubation (18 hours) of HHcy arteries with anti-TNF-alpha antibody decreased O2*- production, whereas incubation of control vessels with TNF-alpha increased O2*- generation and nox1 expression. In coronary arteries, HHcy increases TNF-alpha expression, which enhances oxidative stress through upregulating a nox1-based NAD(P)H oxidase and inducible nitric oxide synthase. Thus, TNF-alpha induces a proinflammatory vascular phenotype in HHcy that potentially contributes to the development of coronary atherosclerosis.