Increased Superoxide Production in Coronary Arteries in Hyperhomocysteinemia: Role of Tumor Necrosis Factor-α, NAD(P)H Oxidase, and Inducible Nitric Oxide Synthase

Abstract

Objective—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 O 2.−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. Methods and Results—The increased generation of O 2.−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 O 2.−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 p47phoxsubunits 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)-α (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-α antibody decreased O 2.−production, whereas incubation of control vessels with TNF-α increased O 2.−generation and nox1 expression. Conclusions—In coronary arteries, HHcy increases TNF-α expression, which enhances oxidative stress through upregulating a nox1-based NAD(P)H oxidase and inducible nitric oxide synthase. Thus, TNF-α induces a proinflammatory vascular phenotype in HHcy that potentially contributes to the development of coronary atherosclerosis.