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...

This study demonstrates that coronary vasa vasorum neovascularization occurs within the first weeks of experimental hypercholesterolemia and prior to the development of endothelial dysfunction of the host vessel, suggesting a role for vasa vasorum neovascularization in the initial stage of atherosclerotic vascular disease.

Oxidative stress has been implicated as an important mechanism of vascular endothelial dysfunction induced by aging. Previous studies suggested that tetrahydrobiopterin (BH 4), an essential cofactor of endothelial NO synthase, could be a molecular target for oxidation. We tested the hypothesis that oxidative stress, in particular oxidation of BH 4, may contribute to attenuation of endothelium-dependent relaxation in aged mice. Vasomotor function of isolated carotid arteries was studied using a video dimension analyzer. Vascular levels of BH 4 and its oxidation products were measured via HPLC. In aged mice (age, 95 Ϯ 2 wk), endothelium-dependent relaxation to ACh (10 Ϫ5 to 10 Ϫ9 M) as well as endothelium-independent relaxation to the NO donor diethylammo-Ϫ5 to 10 Ϫ9 M) were significantly reduced compared with relaxation detected in young mice (age, 23 Ϯ 0.5 wk). Incubation of aged mouse carotid arteries with the cell-permeable SOD mimetic Mn(III)tetra(4-benzoic acid)porphyrin chloride normalized relaxation to ACh and DEA-NONOate. Furthermore, production of superoxide anion in aorta and serum levels of amyloid P component, which is the murine analog of C-reactive protein, was increased in old mice. In aorta, neither the concentration of BH 4 nor the ratio of reduced BH4 to the oxidation products were different between young and aged mice. Our results demonstrate that in mice, aging impairs relaxation mediated by NO most likely by increased formation of superoxide anion. Oxidation of BH 4 does not appear to be an important mechanism underlying vasomotor dysfunction in aged mouse arteries. endothelial dysfunction; nitric oxide; superoxide anion; reactive oxygen species; C-reactive protein AGING IS A RISK FACTOR for vascular disease; however, the role of aging, either as a process or as the result of longer exposure to other risks, is not well defined (16,17). In murine vascular tissue, the age-dependent changes in vasomotor function have not been characterized. Studies on rats have shown impairment of endothelium-dependent relaxation due to increased production of superoxide anions, but the source of the superoxide anions has also not been characterized (16,30). Reactive oxygen species (ROS) have been implicated in endothelial dysfunction associated with aging, hypertension, hypercholesteremia, diabetes, and cigarette smoking (3). ROS can interfere with endothelium-dependent relaxation particularly by the scavenging of NO by superoxide anion (O 2 Ϫ ⅐; Refs. 1, 3, 34).

Abstract-Erythropoietin (EPO) fosters tissue oxygenation by stimulating erythropoiesis. More recently, EPO has been recognized as a tissue-protective cytokine.In this study, we tested the hypothesis that endothelial NO synthase (eNOS) plays a key role in the vascular protective effect of EPO. A murine model of wire-induced injury of carotid artery was used to examine the effect of EPO on endothelial repair and arterial wall architecture. Recombinant human EPO (1000 U/kg, SC, biweekly) was administered for 2 weeks in wild-type and eNOS-deficient mice after which reactivity of isolated carotid arteries was studied in vitro, and the vasculature was histologically assessed. Key Words: nitric oxide synthase Ⅲ endothelium Ⅲ vasculature Ⅲ erythropoietin Ⅲ mice N itric oxide is a potent vasodilator that is generated in endothelial cells from L-arginine by constitutively expressed endothelial NO synthase (eNOS). 1,2 It has been recognized that reduced NO bioavailability is a major mechanism responsible for initiation and progression of endothelial dysfunction in vascular disease. 3 Furthermore, removal of the endothelium by mechanical vascular injury invariably leads to hyperplasia at the site of injury. 4 -7 This suggests that the endothelium also regulates vascular structure and that its presence assures quiescence of vascular smooth muscle cells.Erythropoietin (EPO) is a hypoxia-inducible hormone that is essential for normal erythropoiesis in bone marrow. Administration of recombinant human EPO is an efficient and safe therapeutic approach to anemia associated with chronic renal failure. 8 However, EPO receptors are also widely distributed in the cardiovascular system, including endothelial, smooth muscle, cardiac, and other cell types, and nonhematopoietic effects of EPO are increasingly recognized. 9 -11 For example, it has been reported that EPO has potentially beneficial effects on cardiovascular function. 11 Furthermore, EPO increases the number of functionally active endothelial progenitor cells, thus enhancing angiogenesis. 12,13 However, little is known about the mechanisms underlying vascular effects of EPO in vivo. The present study was, thus, designed to determine whether EPO prevents pathological repair of injured blood vessel. We hypothesized that eNOS plays a critical role in vascular protective effects of EPO. Methods Experimental Animals of Carotid Artery InjuryMale C57BL/6J (wild-type) and eNOS-deficient mice (C57BL/6J-Nos3 tm1Unc ) were obtained from Jackson Laboratory (Bar Harbor, Maine) and were maintained on standard chow with free access to drinking water. All of the experimental protocols were approved by the institutional animal care and use committee of Mayo Clinic. Wire-induced vascular injury was performed in the left common carotid artery. 7 Twelve-week-old mice were anesthetized with 90 mg/kg of ketamine and 10 mg/kg of xylazine (IP). Using a dissecting microscope, a midline incision (1 to 1.5 cm) was made, and the salivary glands were moved laterally to access the left carotid artery. Tw...

Shear stress, imposed on the vascular endothelium by circulating blood, critically sustains vascular synthesis of nitric oxide (NO). Endothelial NO synthase (eNOS) activity is determined by heat shock protein 90 (HSP90), caveolin-1, and the cofactor tetrahydrobiopterin (BH4). To determine whether increased blood flow concomitantly upregulates eNOS and GTP cyclohydrolase I (GTPCH I, the rate-limiting enzyme in BH4 biosynthesis), an aortocaval fistula model in the rat was employed wherein aortic blood flow is enhanced proximal but decreased distal to the fistula. Eight weeks after the creation of the aortocaval fistula, the proximal and distal aortic segments were harvested; sham-operated rats served as controls. Vasomotor function was assessed by isometric force recording. Expression of eNOS, HSP90, caveolin-1, Akt, phosphorylated eNOS (eNOS-Ser1177), and GTPCH I were determined by Western blot analysis. Biosynthesis of BH4 and GTPCH-I activity was examined by HPLC. In the aortic segments exposed to increased flow, contractions to KCl and phenylephrine were reduced, whereas endothelium-dependent relaxations were not affected compared with sham-operated or aortic segments with reduced blood flow. Expression of eNOS, caveolin-1, phosphorylated Akt, and eNOS-Ser1177 was enhanced in aortas exposed to increased blood flow. High flow augmented levels of cGMP and BH4 and increased expression of GTPCH I. In aggregate, these findings provide the first demonstration in vivo that coordinated vascular upregulation of eNOS, and GTPCH I accompanies increased blood flow. This induction of GTPCH I increases BH4 production, thereby optimizing the generation of NO by eNOS and thus the adaptive, vasorelaxant response required in sustaining increased blood flow.