Increased NADPH Oxidase Activity, gp91phox Expression, and Endothelium-Dependent Vasorelaxation During Neointima Formation in Rabbits

• Published in: Circulation research 2002-07, Vol.91 (1), p.54-61
• Main Authors: Paravicini, Tamara M, Gulluyan, Lerna M, Dusting, Gregory J, Drummond, Grant R
• Format: Article
• Language: English
• Subjects: Acetylcholine - pharmacology; Animals; Arteriosclerosis - etiology; Arteriosclerosis - pathology; Atherosclerosis (general aspects, experimental research); Biological and medical sciences; Blood and lymphatic vessels; Braces - adverse effects; Cardiology. Vascular system; Carotid Arteries - drug effects; Carotid Arteries - metabolism; Carotid Arteries - physiopathology; Carotid Artery Injuries - etiology; Carotid Artery Injuries - metabolism; Carotid Artery Injuries - physiopathology; Ditiocarb - pharmacology; Dose-Response Relationship, Drug; Endothelium, Vascular - physiology; Gene Expression; In Vitro Techniques; Isotonic Solutions - pharmacology; Male; Medical sciences; Membrane Glycoproteins - genetics; Membrane Glycoproteins - metabolism; NAD - pharmacology; NADP - pharmacology; NADPH Oxidase 2; NADPH Oxidases - genetics; NADPH Oxidases - metabolism; Rabbits; RNA, Messenger - genetics; RNA, Messenger - metabolism; Superoxides - metabolism; Tunica Intima - metabolism; Tunica Intima - pathology; Tunica Intima - physiopathology; Vasoconstriction - drug effects; Vasoconstriction - physiology; Vasodilation - drug effects; Vasodilation - physiology; Vasodilator Agents - pharmacology
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/01.RES.0000024106.81401.95

Reactive oxygen species including superoxide and hydrogen peroxide are important mediators in atherogenesis. We investigated the enzymatic source of vascular superoxide and its role in endothelium-dependent vasorelaxation during neointima formation. Silastic collars positioned around carotid arteries of rabbits for 14 days induced neointimal thickening. Using lucigenin-enhanced chemiluminescence, superoxide production was detectable in collared artery sections, but not in controls, only after inactivation of endogenous Cu/Zn-superoxide dismutase (Cu/Zn-SOD) with diethyldithiocarbamate (DETCA). Dihydroethidium staining indicated that endothelium and adventitia were the major sites of superoxide generation. Superoxide production in DETCA-treated collared arteries was enhanced further by NADPH and was inhibited by diphenyleneiodonium, suggesting NADPH oxidase was the source of the radical in collared arteries. Moreover, real-time PCR demonstrated 11-fold higher expression of the gp91phox subunit of NADPH oxidase in collared arteries than in controls. In vascular reactivity studies, endothelium-dependent vasorelaxation to acetylcholine did not differ between collared and control sections. However, treatment with DETCA reduced relaxations to acetylcholine in collared rings, but not in controls. NADPH further reduced relaxations to acetylcholine in DETCA-treated collared sections, but not in controls. In DETCA/NADPH-treated collared rings, sensitivity to nitroprusside, in contrast to acetylcholine, exceeded that of controls. Moreover, further treatment of such rings with exogenous Cu/Zn-SOD restored acetylcholine relaxations without altering nitroprusside responses. Thus, early neointimal lesions induced by periarterial collars are associated with elevated gp91phox expression and increased NAPDH-oxidase-dependent superoxide production in endothelium and adventitia. However, endothelium-dependent vasorelaxation is largely preserved due to the actions of Cu/Zn-SOD and increased smooth muscle sensitivity to nitric oxide.