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Cellular glutathione peroxidase (GPx-1) is the most abundant intracellular isoform of the GPx antioxidant enzyme family. In this study, we hypothesized that GPx-1 deficiency directly induces an increase in vascular oxidant stress, with resulting endothelial dysfunction. We studied vascular function in a murine model of homozygous deficiency of GPx-1 (GPx-1 Ϫ/Ϫ ). Mesenteric arterioles of GPx-1 Ϫ/Ϫ mice demonstrated paradoxical vasoconstriction to ␤-methacholine and bradykinin, whereas wildtype (WT) mice showed dose-dependent vasodilation in response to both agonists. One week of treatment of GPx-1 Ϫ/Ϫ mice with L-2-oxothiazolidine-4-carboxylic acid (OTC), which increases intracellular thiol pools, resulted in restoration of normal vascular reactivity in the mesenteric bed of GPx-1 Ϫ/Ϫ mice. We observed an increase of the isoprostane iPF 2␣-III, a marker of oxidant stress, in the plasma and aortas of GPx-1 Ϫ/Ϫ mice compared with WT mice, which returned toward normal after OTC treatment. Aortic sections from GPx-1 Ϫ/Ϫ mice showed increased binding of an anti-3-nitrotyrosine antibody in the absence of frank vascular lesions. These findings demonstrate that homozygous deficiency of GPx-1 leads to impaired endothelium-dependent vasodilator function presumably due to a decrease in bioavailable nitric oxide and to increased vascular oxidant stress. These vascular abnormalities can be attenuated by increasing bioavailable intracellular thiol pools. nitric oxide; peroxynitrite; oxidant stress NITRIC OXIDE (NO) synthesized by the endothelium contributes to vascular tone (23), preserves endothelial integrity (13), inhibits smooth muscle cell migration and proliferation (16), and acts as an antioxidant (30). An increase in reactive oxygen species (ROS), leading to increased oxidant stress in the vasculature, promotes endothelial dysfunction (28) associated with NO insufficiency (15).The enzyme glutathione peroxidase (GPx) is a selenocysteine-containing protein that serves an important role in the cellular defense against oxidant stress (24) by utilizing reduced glutathione (GSH) to reduce hydrogen peroxide (H 2 O 2 ) and lipid peroxides to their corresponding alcohols (37). GPx exists in several isoforms, and the most abundant intracellular isoform is cellular GPx, or GPx-1. We (7, 36, 39) have previously shown that elevated homocysteine concentrations suppress GPx-1 expression in endothelial cells in vitro and in mildly hyperhomocysteinemic mice in vivo and suggested that this effect may account, in part, for the vascular oxidant stress of hyperhomocysteinemic states. Hydrogen peroxide forms the toxic oxygen species hydroxyl radical (⅐OH), which is highly reactive and causes lipid peroxidation, and hydroxide anion (OHϪ), which promotes alkaline tissue damage, a process that is offset in part by catalase and GPx-1-dependent reduction to H 2 O. Elevated levels of lipid peroxides are accompanied by an increase in peroxyl radicals, which can inactivate NO through the formation of lipid peroxynitrites (19,30), although the prec...

The vascular endothelium compensates for oxidant stress by increasing the activity of antioxidant enzymes such as glucose‐6‐phophate dehydrogenase (G6PD). G6PD provides reducing equivalents of NAPDH to maintain glutathione stores and modulates nitric oxide synthase (eNOS) activity. To determine whether deficient G6PD activity perturbs these responses, we treated bovine aortic endothelial cells with dehydroepiandrosterone or an antisense oligodeoxynucleotide to G6PD mRNA to decrease G6PD activity and expression. When exposed to hydrogen peroxide, reactive oxygen species (ROS) accumulation was increased in G6PD‐deficient cells compared with those with normal activity. To determine the source of increased oxidant stress in G6PD‐deficient cells, we used inhibitors of ROS generation, which suggested that eNOS was contributing to ROS production. Treatment with L‐NMMA, an inhibitor of eNOS mediated‐nitric oxide (NO) but not superoxide, production confirmed this observation; in contrast to L‐NAME, L‐NMMA promoted ROS generation in G6PD‐deficient cells. In addition, deficient G6PD activity was associated with a decrease in endothelium‐derived bioavailable NO in response to the agonists A23187 and bradykinin as demonstrated by decreased endothelial cGMP and nitrate/nitrite levels. Enhanced ROS accumulation and decreased NO bioavailability may represent one mechanism by which G6PD deficiency contributes to vascular oxidant stress and endothelial dysfunction.

Abstract-Previous in vitro experiments have shown that hyperhomocysteinemia leads to oxidative inactivation of nitric oxide, in part by inhibiting the expression of cellular glutathione peroxidase (GPx-1). To elucidate the role of intracellular redox status on homocysteine-induced endothelial dysfunction and oxidant stress, heterozygous cystathionine ␤-synthase-deficient (CBS -/ϩ ) and wild-type (CBS ϩ/ϩ ) mice were treated with the cysteine donor L-2-oxothiazolidine-4-carboxylic acid (OTC). CBS -/ϩ mice had significantly lower GPx-1 activity compared with their CBS ϩ/ϩ littermates, and OTC treatment led to a modest increase in tissue GPx-1 activity and significant increases in total thiols and in reduced glutathione levels in both CBS ϩ/ϩ and CBS -/ϩ mice. Superfusion of the mesentery with ␤-methacholine or bradykinin produced dose-dependent vasodilation of mesenteric arterioles in CBS ϩ/ϩ mice and in CBS ϩ/ϩ mice treated with OTC. In contrast, mesenteric arterioles from CBS -/ϩ mice manifested dose-dependent vasoconstriction in response to both agonists. OTC treatment of CBS -/ϩ mice restored normal microvascular vasodilator reactivity to ␤-methacholine and bradykinin. These findings demonstrate that mild hyperhomocysteinemia leads to endothelial dysfunction in association with decreased bioavailable nitric oxide. Increasing the cellular thiol and reduced glutathione pools and increasing GPx-1 activity restores endothelial function. These findings emphasize the importance of intracellular redox balance for nitric oxide bioactivity and endothelial function. Key Words: homocysteine Ⅲ endothelial function Ⅲ oxidant stress Ⅲ nitric oxide Ⅲ P-selectin M ild hyperhomocysteinemia, ie, an elevation of the plasma levels of homocysteine, homocystine, or homocysteine-mixed disulfides, has been shown to be a risk factor for atherosclerotic vascular disease and its thrombotic complications. Population-based, nested case-control studies demonstrate that a 5 mol/L increase in plasma homocysteine concentrations leads to a 30% increase in cardiovascular risk (reviewed in the first 5 references). [1][2][3][4][5] In addition, several prospective studies indicate that the increased risk is higher during short-term follow-up and declines after 3 to 4 years. Hyperhomocysteinemia is an even stronger predictor of cardiovascular risk in patients with preexisting conditions, such as chronic renal failure, 6 coronary heart disease, 7 diabetes mellitus, 8 peripheral arterial occlusive disease, 9 systemic lupus erythematosus, 10 or venous thromboembolism. 11 In accordance with these observations, elevated homocysteine levels are more strongly associated with recurrent cardiovascular events than with first stroke or myocardial infarction. 12 These data suggest that homocysteine promotes acute thrombotic events in the presence of preexisting vascular lesions rather than induces atherosclerotic lesions de novo.The mechanisms by which elevated homocysteine levels alter the vascular environment to facilitate the development and progression of ...

Background— Oxidant stress has been implicated in the pathogenesis of atherothrombosis and other vascular disorders accompanied by endothelial dysfunction. Glutathione peroxidases (GPx) play an important role in the cellular defense against oxidant stress by utilizing glutathione (GSH) to reduce lipid hydroperoxides and hydrogen peroxide to their corresponding alcohols. Cellular GPx (GPx-1) is the principal intracellular isoform of GPx. We hypothesized that GPx-1 deficiency per se induces endothelial dysfunction and structural vascular abnormalities through increased oxidant stress. Methods and Results— A murine model of heterozygous deficiency of GPx-1 (GPx +/−) was investigated to examine this hypothesis. Mesenteric arterioles in GPx-1 +/− mice demonstrated vasoconstriction to acetylcholine compared with vasodilation in wild-type mice (maximal change in vessel diameter, −13.0±2.8% versus 13.2±2.8%, P <0.0001). We also noted an increase in the plasma and aortic levels of the isoprostane iPF 2α -III, a marker of oxidant stress, in GPx-1 +/− mice compared with wild-type mice (170.4±23 pg/mL plasma versus 98.7±7.1 pg/mL plasma, P <0.03; 11.7±0.87 pg/mg aortic tissue versus 8.2±0.55 pg/mg aortic tissue, P <0.01). Histological sections from the coronary vasculature of GPx-1 +/− mice show increased perivascular matrix deposition, an increase in the number of adventitial fibroblasts, and intimal thickening. These structural abnormalities in the myocardial vasculature were accompanied by diastolic dysfunction after ischemia-reperfusion. Conclusions— These findings demonstrate that heterozygous deficiency of GPx-1 leads to endothelial dysfunction, possibly associated with increased oxidant stress, and to significant structural vascular and cardiac abnormalities. These data illustrate the importance of this key antioxidant enzyme in functional and structural responses of the mammalian cardiovascular system.