The role of reactive oxygen species in the vascular pathology asiated with atherosclerosis was examined by testing the hypothesis that impaired vascular reactivity results from the reaction of nitric oxide (NO) with superoxide (O2), yielding the oxidant peroynitrite (ONOO-). Contractility studies were performed on femoral arteries from rabbits fed a cholesterol-supplemented diet Endothelium-dependent relaxation is impaired in vessels from atherosclerotic patients (1, 2) and hypercholesterolemic animal models (3-6), suggesting the functional modification of endothelium-derived relaxing factor (EDRF) in hyperlipidemia. The dynamic role ofthe endothelium in the regulation of vascular tone was established when it was observed that relaxation of isolated blood vessels by vasoactive agents, such as acetylcholine (ACh) and the calcium ionophore A23187, was dependent on an intact endothelium and a diffusible factor (EDRF) that stimulated cGMP-dependent relaxation of vascular smooth muscle cells (VSMCs; ref. 7).Nitric oxide (-NO) and EDRF share similar chemical and pharmacological properties (8) and are derived from the oxidation of a terminal guanidino group of L-arginine (9, 10).Numerous mechanisms have been suggested for the defect in vascular relaxation in atherosclerosis and hypercholesterolemic animal models. They include an increased diffusional barrier for 'NO due to intimal cell proliferation and lipid deposition (11), L-argifine depletion (3,12,13) Vessel Contraction Studies. New Zealand White rabbits (2.5-3.0 kg) were maintained on rabbit chow containing 1% cholesterol (Ralston Purina) for 6 months prior to study [cholesterol-fed (Chol-fed) group]. Age-and weight-matched controls were fed a standard diet. After exsanguination under ketamine/rompun anesthesia, vessels were isolated and changes in tension were measured in femoral artery ring segments as described (28). After maximal contraction with 70 mM KCl and recovery, phenylephrine was added to achieve 30% of maximal tone. Rings were then exposed to increasing doses of ACh; relaxation is reported as the percentage decrease in preexisting tone. After the generation of cumulative ACh dose-response curves, rings were exposed to 30 ,uM papaverine. In some experiments, rings from control and Chol-fed rabbits were incubated with 3 mM L-arginine for 30 min prior to administration of ACh. In other studies, vessels were treated with native bovine SOD (200 units/ml) before measuring ACh-induced relaxation. All studies were performed in the presence of 5 !uM indomethacin.

Reactive oxygen species play a central role in vascular inflammation and atherogenesis, with enhanced superoxide (02-) production contributing significantly to impairment of nitric oxide (NO)-dependent relaxation of vessels from cholesterol-fed rabbits. We investigated potential sources of 02-production, which contribute to this loss of endothelium-dependent vascular responses. The vasorelaxation elicited by acetylcholine (ACh) in phenylephrinecontracted, aortic ring segments was impaired by cholesterol feeding. Pretreatment of aortic vessels with either heparin, which competes with xanthine oxidase (XO) for binding to sulfated glycosaminoglycans, or the XO inhibitor allopurinol resulted in a partial restoration (36-40o at 1 ,uM ACh) of ACh-dependent relaxation. Furthermore, 027-dependent lucigenin chemiluminescence, measured in intact ring segments from hypercholesterolemic rabbits, was decreased by addition of heparin, allopurinol or a chimeric, heparin-binding superoxide dismutase. XO activity was elevated more than two-fold in plasma of hypercholesterolemic rabbits. Incubation of vascular rings from rabbits on a normal diet with purified XO (10 milliunits/ml) also impaired 'NO-dependent relaxation but only in the presence of purine substrate. As with vessels from hypercholesterolemic rabbits, this effect was prevented by heparin and allopurinol treatment. We hypothesize that increases in plasma cholesterol induce the release of XO into the circulation, where it binds to endothelial cell glycosaminoglycans. Only in hypercholesterolemic vessels is sufficient substrate available to sustain the production of 02-and impair NO-dependent vasorelaxation. Chronically, the continued production of peroxynitrite, (ONOO-) which the simultaneous generation of NO and 02 -implies, may irreversibly impair vessel function.In recent years, numerous studies have shown that vascular function is compromised in a number of pathological conditions including atherosclerosis and hypertension (1-3). In atherosclerosis, blood vessels undergo marked changes in both structure and function that may predispose for angina and myocardial infarction. Defects in lipoprotein metabolism and vascular reactivity are fundamental pathological responses to hypercholesterolemia, with reactive oxygen species playing an important role in the initiation and progression of these lesions (4, 5). Isolated blood vessels from atherosclerotic patients (6, 7) and hypercholesterolemic animals (8-11) exhibit impaired, endothelium-dependent vascular relaxation. In these models, superoxide (02-) is generated at greater rates in both intracellular and extracellular compartments, ren.cting with 'NO to inhibit endothelium-dependent relaxation ard yielding peroxynitrite (ONOO-) (12). Since ONOO-is capable of oxidizing lipids and proteins, it may further contribute to foam cell formation in the atherosclerotic lesion (13-15).While oxidative injury plays a pivotal role in these processes, the cellular mechanisms of 0i-production and sites of reaction remain to be...

Peroxynitrite stimulated the synthesis of cyclic GMP by rat aortic smooth muscle in a time-and dose-dependent manner. Peak formation of cyclic GMP occurred at 1 rain with 100 /~M peroxynitrite and was inhibited by oxyhemoglobin. Peroxynitrite was less potent than nitric oxide in stimulating cyclic GMP synthesis. Peroxynitrite also enhanced endothelial-dependent cyclic GMP synthesis, via generation of a long-lived substance, which was prevented by inhibition of glutathione synthesis. These data show that peroxynitrite stimulates cyclic GMP synthesis, inferring production of low yields of nitric oxide or associated derivatives. Additionally, vascular exposure to peroxynitrite potentiates endothelial-dependent activation of guanylate cyclase.

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