This study investigated whether endothelin-1 (ET-1), a potent vasoconstrictor, which also stimulates cell proliferation, contributes to endothelial dysfunction and atherosclerosis. Apolipoprotein E (apoE)-deficient mice and C57BL͞6 control mice were treated with a Western-type diet to accelerate atherosclerosis with or without ET A receptor antagonist LU135252 (50 mg͞kg͞d) for 30 wk. Systolic blood pressure, plasma lipid profile, and plasma nitrate levels were determined. In the aorta, NO-mediated endotheliumdependent relaxation, atheroma formation, ET receptorbinding capacity, and vascular ET-1 protein content were assessed. In apoE-deficient but not C57BL͞6 mice, severe atherosclerosis developed within 30 wk. Aortic ET-1 protein content (P < 0.0001) and binding capacity for ET A receptors was increased as compared with C57BL͞6 mice. In contrast, NO-mediated, endothelium-dependent relaxation to acetylcholine (56 ؎ 3 vs. 99 ؎ 2%, P < 0.0001) and plasma nitrate were reduced (57.9 ؎ 4 vs. 93 ؎ 10 mol͞liter, P < 0.01). Treatment with the ET A receptor antagonist LU135252 for 30 wk had no effect on the lipid profile or systolic blood pressure in apoE-deficient mice, but increased NO-mediated endothelium-dependent relaxation (from 56 ؎ 3 to 93 ؎ 2%, P < 0.0001 vs. untreated) as well as circulating nitrate levels (from 57.9 ؎ 4 to 80 ؎ 8.3 mol͞liter, P < 0.05). Chronic ET A receptor blockade reduced elevated tissue ET-1 levels comparable with those found in C57BL͞6 mice and inhibited atherosclerosis in the aorta by 31% without affecting plaque morphology or ET receptor-binding capacity. Thus, chronic ET A receptor blockade normalizes NO-mediated endothelial dysfunction and reduces atheroma formation independent of plasma cholesterol and blood pressure in a mouse model of human atherosclerosis. ET A receptor blockade may have therapeutic potential in patients with atherosclerosis.Diseases related to atherosclerosis such as myocardial infarction and stroke account for the majority of deaths in industrialized countries (1). In patients with cardiovascular risk factors such as hypercholesterolemia, hypertension, or aging (2, 3), endothelial dysfunction precedes the development of atherosclerosis and predisposes to the development of structural vascular changes (1, 4). The endothelium releases vasoactive mediators such as NO and endothelin (ET-1), both of which are importantly involved in the regulation of vascular tone (5, 6) and structure (7,8). Endothelial NO synthase (9-11) converts L-arginine into NO and L-citrulline (12) and its expression (13), and the release of NO (14) is reduced in atherosclerosis. In experimental atherosclerosis, inhibition of the L-arginine͞NO pathway accelerates lesion progression in hypercholesterolemic rabbits (15-17) and low density lipoprotein (LDL) receptor-deficient mice (18). Furthermore, superoxide release in atherosclerosis inactivates NO resulting in formation of peroxynitrite (19,20), the production of which is further enhanced by cholesterol (21).In patients with coronary a...

Constitutive nitric oxide synthase (cNOS) with insufficient cofactor (6R)-5,6,7,8-tetrahydrobiopterin (H4B) may generate damaging superoxide (O2-). This study was designed to determine whether cNOS-dependent generation of O2- occurs in spontaneously hypertensive rats (SHR) before the onset of hypertension. Aortas from 4-wk-old SHR and Wistar-Kyoto rats were used. cNOS was stimulated by calcium ionophore A23187. In situ measurements of nitric oxide and hydrogen peroxide by electrochemical sensors and O2- production by chemiluminescence method were performed. Isometric tension was continuously recorded. H4B by high performance liquid chromatography and [3H]citrulline assay were determined in homogenized tissue. The A23187-stimulated production of O2- and its superoxide dismutase product hydrogen peroxide were significantly higher, whereas nitric oxide release was reduced in SHR aortas, with opposite results in the presence of exogenous H4B. Furthermore, NG-monomethyl-L-arginine inhibited the generation of cNOS-dependent O2- by approximately 70%. Natural H4B levels were similar in both strains; however, equivalent cNOS activity required additional H4B in SHR. The endothelium-dependent relaxations to A23187 were significantly inhibited by catalase, and enhanced by superoxide dismutase, only in SHR; however, these enzymes had no effect in the presence of H4B. Thus, dysfunctional cNOS may be a source of O2- in prehypertensive SHR and contribute to the development of hypertension and its vascular complications.

Our results therefore demonstrate that angiotensin II increases the production of endothelin in the blood vessel wall that, via ET(A) receptors, mediates changes in vascular structure of the cerebral and mesenteric circulation. Endothelin antagonists may therefore be of value to reduce blood pressure and to prevent vascular structural changes in conditions of increased activity of the renin-angiotensin system.

Abstract-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. Key Words: tetrahydrobiopterin Ⅲ nitric oxide synthase Ⅲ nitric oxide Ⅲ antioxidants Ⅲ superoxide anion N itric oxide (NO) is a potent vasodilator and plays a key role in control of the cardiovascular system. 1 NO is mainly formed in endothelial cells from L-arginine by oxidation of its terminal guanidino-nitrogen, 2 requiring the cofactors NADPH, (6R)-5,6,7,8-tetrahydrobiopterin (BH 4 ), FAD, FMN, heme, and Zn 2ϩ . 3,4 The formation of NO occurs via endothelial NO-synthase (eNOS) which is expressed constitutively. 5,6 Relaxations in response to the abluminal release of endothelium-derived NO are associated with stimulation of soluble guanylyl cyclase (sGC) and in turn formation of cyclic guanosine 3Ј,5Ј-monophosphate (cGMP) in vascular smooth muscle cells. 7 Inducible NOS (iNOS) enzyme can be expressed in vascular smooth muscle cells, endothelium, and macrophages. This enzyme activity is Ca 2ϩ -independent and produces large amounts of NO; it is induced by cytokines such as interleukin 1␤ and tumor necrosis factor-␣ and hence is activated in atherosclerosis and inflammatory processes. 8 -11 BH 4 is an essential cofactor required for activity of all NOS isoforms. 4,12 During activation of NOS, BH 4 is needed for allosteric and redox activation of its enzymatic activity. 4,13 Accumulating evidence suggests that alterations in the NO pathway, such as increased NO decomposition by superoxide anion (O 2 Ϫ ) or altered NOS...