d’Uscio, Livius V.

Background and Purpose— Hypercholesterolemia is associated with endothelial dysfunction. Inducible nitric oxide synthase (iNOS) is upregulated in atherosclerotic vessels. However, its role in the regulation of vascular function is not completely understood. We examined the effect of adenovirus-mediated gene transfer of iNOS to the hypercholesterolemic rabbit carotid artery in vivo. Methods— Rabbits were fed a high-cholesterol or chow diet for 10 weeks. Two doses (1×10 8 and 1×10 9 plaque-forming units [pfu]/mL) of adenoviral vectors encoding iNOS (AdiNOS) or β-galactosidase (Adβgal) were luminally delivered to the carotid arteries from chow- and cholesterol-fed animals. Vascular reactivity and superoxide levels were assessed in Adβgal- and AdiNOS-transduced vessels from chow- and cholesterol-fed animals after 3 days. Results— Endothelium-dependent vasorelaxation was impaired in the carotid artery from cholesterol-fed animals. In AdiNOS-transduced arteries, transgene expression was demonstrated by positive immunostaining in the endothelium. Transduction with low-dose (1×10 8 pfu/mL) AdiNOS did not affect vascular reactivity in arteries from chow- and cholesterol-fed animals. In contrast, high-dose (1×10 9 pfu/mL) AdiNOS significantly reduced endothelium-dependent relaxation in vessels from cholesterol- but not chow-fed rabbits. After both low- and high-dose iNOS gene transfer, levels of O 2 · − were significantly ( P <0.05) elevated in carotid arteries from cholesterol-fed animals. Incubation with an O 2 · − scavenger did not reverse vascular dysfunction in these arteries. Conclusions— Adenoviral-mediated overexpression of iNOS results in increased production of O 2 · − in carotid arteries from cholesterol- but not chow-fed animals. High-dose AdiNOS gene transfer is associated with reduced endothelium-dependent and -independent relaxation in vessels from cholesterol-fed animals.

In cultured endothelial cells, the antioxidant, l -ascorbic acid (vitamin C), increases nitric oxide synthase (NOS) enzyme activity via chemical stabilization of tetrahydrobiopterin. Our objective was to determine the effect of vitamin C on NOS function and tetrahydrobiopterin metabolism in vivo. Twenty-six to twenty-eight weeks of diet supplementation with vitamin C (1%/kg chow) significantly increased circulating levels of vitamin C in wild-type (C57BL/6J) and apolipoprotein E (apoE)–deficient mice. Measurements of NOS enzymatic activity in aortas of apoE-deficient mice indicated a significant increase in total NOS activity. However, this increase was mainly due to high activity of inducible NOS, whereas eNOS activity was reduced. Significantly higher tetrahydrobiopterin levels were detected in aortas of apoE-deficient mice. Long-term treatment with vitamin C restored endothelial NOS activity in aortas of apoE-deficient mice, but did not affect activity of inducible NOS. In addition, 7,8-dihydrobiopterin levels, an oxidized form of tetrahydrobiopterin, were decreased and vascular endothelial function of aortas was significantly improved in apoE-deficient mice. Interestingly, vitamin C also increased tetrahydrobiopterin and NOS activity in aortas of C57BL/6J mice. In contrast, long-term treatment with vitamin E (2000 U/kg chow) did not affect vascular NOS activity or metabolism of tetrahydrobiopterin. In vivo, beneficial effect of vitamin C on vascular endothelial function appears to be mediated in part by protection of tetrahydrobiopterin and restoration of eNOS enzymatic activity.