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Reactive oxygen species (ROS) function as intracellular signaling molecules in a diverse range of biological processes. However, it is unclear how freely diffusible ROS dictate specific cellular responses. In this study, we demonstrate that nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (Nox4), a major Nox isoform expressed in nonphagocytic cells, including vascular endothelium, is localized to the endoplasmic reticulum (ER). ER localization of Nox4 is critical for the regulation of protein tyrosine phosphatase (PTP) 1B, also an ER resident, through redox-mediated signaling. Nox4-mediated oxidation and inactivation of PTP1B in the ER serves as a regulatory switch for epidermal growth factor (EGF) receptor trafficking and specifically acts to terminate EGF signaling. Consistent with this notion, PTP1B oxidation could also be modulated by ER targeting of antioxidant enzymes but not their untargeted counterparts. These data indicate that the specificity of intracellular ROS-mediated signal transduction may be modulated by the localization of Nox isoforms within specific subcellular compartments.

Arsenic exposure is associated with an increased risk of atherosclerosis and vascular diseases. Although endothelial cells have long been considered to be the primary targets of arsenic toxicity, the underlying molecular mechanism remains largely unknown. In this study, we sought to explore the signaling pathway triggered by sodium arsenite and its implication for endothelial phenotype. We found that sodium arsenite produced time-and dose-dependent decreases in human umbilical vein endothelial cell viability. This effect correlated with the induction of [p21.sup.Cip1/Waf1] (up to 10-fold), a regulatory protein of cell cycle and apoptosis. We also found that arsenite-stimulated EGF (ErbB1) and ErbB2 receptor transactivation, manifest as receptor tyrosine phosphorylation, appeared to be a proximal signaling event leading to [p21.sup.Cip1/Waf1] induction, because both pharmacological inhibitors and knockdown of receptors by RNA interference blocked arsenite-induced [p21.sup.Cip1/Waf1] upregulation. Arsenite-induced activation of JNK and p38 MAPK was distinct, with only JNK as a downstream target of the EGF receptor. Moreover, inhibition of JNK with SP-600125 or dominant negative MKK7 inhibited only [p21.sup.Cip1/Waf1] induction, whereas the p38 MAPK inhibitor SB-203580 or dominant negative MKK4 inhibited both [p21.sup.Cip1/Waf1] and p53 induction. Functionally, inhibition of [p21.sup.Cip1/Waf1] induction prevented endothelial apoptosis due to arsenite treatment. Insofar as endothelial dysfunction promotes vascular disease, these data provide a mechanism for the increased incidence of cardiovascular disease dub to arsenite exposure. epidermal growth factor; mitogen-activated protein kinases; c-Jun NH2-terminal kinase

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced, in part, from NADPH oxidase in response to host invasion and tissue injury. Defects in NADPH oxidase impair host defense; however, the role of ROS and RNS in the response to tissue injury is not known. We addressed this issue by subjecting leukocyte oxidase (Nox2)-deficient ([Nox2.sup.-/-]) mice to arterial injury. Femoral artery injury was associated with increased Nox2 expression, ROS/RNS production, and oxidative protein and lipid modification in wild-type mice. In [Nox2.sup.-/-] mice, RNS-mediated protein oxidation, as monitored by protein nitrotyrosine content, was significantly diminished. This was accompanied by reduced neointimal proliferation, as monitored by intimal thickness and intimal/medial ratio, in [Nox2.sup.-/-] compared to wild-type mice. In addition, Nox2 deficiency led to reduced cellular proliferation and leukocyte accumulation. These data indicate that Nox2-mediated oxidant production has a requisite role in the response to tissue injury.

Oxidative stress decreases the bioavailability of endothelium-derived nitric oxide in diabetic patients. We investigated whether impaired endothelium-dependent vasodilation (EDV) in diabetes can be improved by long-term administration of oral antioxidants. Forty-nine diabetic subjects [26 Type 1 (T1) and 23 Type 2 (T2)] and 45 matched healthy control subjects were randomized to receive oral vitamin C (1,000 mg) and vitamin E (800 IU) daily or matching placebo for 6 mo. Vascular ultrasonography was used to determine brachial artery EDV and endothelium-independent vasodilation (EIV). EDV was decreased in both T1 (4.9 [+ or -] 0.9%, P = 0.015) and T2/4.1 [+ or -] 1.0%, P < 0.01) subjects compared with control subjects (7.7 [+ or -] 0.7%). EIV was decreased in T2 (15.0 [+ or -] 1.2%, P < 0.01) but not T1 subjects (18.5 [+ or -] 2.3%, P = 0.3) compared with controls (21.8 [+ or -] 1.8%). Administration of antioxidant vitamins increased EDV in T1 (by 3.4 [+ or -] 1.4%, P = 0.023) but not T2 subjects (by 0.5. [+ or -] 0.4%, P = 0.3). Antioxidant therapy had no significant affect on EIV. Oral antioxidant therapy improves EDV in T1 but not T2 diabetes. These results are consistent with the lack of clinical benefit in studies that have included primarily T2 diabetic patients. endothelium; nitric oxide

Nitric oxide can oxidize proteins, which may be a risk factor for cardiovascular disease. This was the conclusion of a study of 100 patients with cardiovascular disease and 108 healthy people. This study also showed that a group of drugs called statins can decrease the amount of these oxidized proteins, in addition to lowering blood cholesterol levels. Nitric oxide is a natural substance in the body that relaxes blood vessels.

Nitric oxide (NO) plays an important role in the control of numerous vascular functions including basal [Na.sup.+]-[K.sup.+]-ATPase activity in arterial tissue. Hyperglycemia inhibits [Na.sup.+]-[K.sup.+]-ATPase activity in rabbit aorta, in part, through diminished bioactivity of NO. The precise mechanism(s) for such observations, however, are not yet clear. The purpose of this study was to examine the role of superoxide in modulating NO-mediated control of [Na.sup.+]-[K.sup.+]-ATPase in response to hyperglycemia. Rabbit aorta incubated with hyperglycemic glucose concentrations (44 mM) demonstrated a 50% reduction in [Na.sup.+]-[K.sup.+]-ATPase activity that was abrogated by superoxide dismutase. Hyperglycemia also produced a 50% increase in steady-state vascular superoxide measured by lucigenin-enhanced chemiluminescence that was closely associated with reduced [Na.sup.+]-[K.sup.+]-ATPase activity. Specifically, the hyperglycemia-induced increase in vascular superoxide was endothelium dependent, inhibited by L-arginine, and stimulated by [N.sup.[omega]]-nitro-L-arginine. Aldose reductase inhibition with zopolrestat also inhibited the hyperglycemia-induced increase in vascular superoxide. In each manipulation of vascular superoxide, a reciprocal change in [Na.sup.+]-[K.sup.+]-ATPase activity was observed. Finally, a commercially available preparation of [Na.sup.+]-[K.sup.+]-ATPase was inhibited by pyrogallol, a superoxide generator. These data suggest that hyperglycemia induces an increase in endothelial superoxide that inhibits the stimulatory effect of NO on vascular [Na.sup.+]-[K.sup.+]-ATPase activity. sodium-potassium adenosine 5'-triphosphatase; nitric oxide; endothelium; glucose

A substantial number of epidemiologic studies have demonstrated that antioxidants can reduce the incidence of coronary artery disease. However, the mechanism for this effect is still not clear. The oxidative-modification hypothesis states that antioxidants prevent the oxidation of low density lipoproteins that can cause cholesterol deposits to build up inside arteries. However, some studies have failed to show a regression of atherosclerosis in patients taking antioxidants. Coronary artery disease also involves plaque rupture, clot formation and blood vessel constriction. These could also be affected by antioxidants.

The effects of NO on the normal beta-adrenergic cardiac function and myocardial contractility were analyzed in anesthetized dogs. Inhibition of NOS activity by NG-nitro-L-arginine methyl ester in anesthetized, autonomically blocked dogs augmented cardiac peripheral inotropic response to beta-adrenergic receptor activation. However, basal myocardial contractility was not altered by dobutamine- and isoproterenol-mediated cardiac NOS inhibition.

Maxadilan is a potent vasodilator suggested to share pharmacological and immunological properties with the calcitonin gene-related peptide. Exposure of vessels from isolated rabbit thoracic and abdominal aorta, carotid artery and iliac artery caused dose-dependent and endothelium-independent arterial relaxation. Pharmacological probes revealed that maxadilan-mediated arterial relaxation involved the activation of adenosine 3',5'-cyclic monophosphate but was inhibited by protein kinase A inhibitor H-89.

Elevated blood cholesterol levels may cause atherosclerosis and resulting ischemic heart disease. Ischemia refers to local anemia caused by obstructed or limited blood supply, and atherosclerosis is the irregular distribution of fat deposits in arteries. High levels of cholesterol may cause fat to be deposited in the arteries and restrict blood flow. Primary and secondary trials to prevent high levels of cholesterol show that reducing the level of cholesterol may significantly decrease the number of adverse cardiovascular events. Angiographic (x-ray studies of blood vessels) trials show that although cholesterol reduction may not have much effect on the progression of arterial lesions, it may improve the function of the heart lining and discourage the rupture of plaque on arterial walls. Cholesterol-reducing drugs, such as simvastatin, cholestyramine, gemfibrozil, and lovastatin may be helpful in lowering high levels of cholesterol, which in turn may improve the tone and function of blood vessels.

Analyses of the function of dietary antioxidants in retaining normal endothelial cell vasodilator function in cholesterol-fed rabbits revealed that patients with atherogenesis may benefit from dietary antioxidants. The analyses also focused on the likely influence on serum lipoproteins and low density lipoprotein oxidation. Dietary antioxidants use a process which is linked to vascular tissue antioxidant content, to retain endothelial vasodilator function.

Unstable angina seems to involve an inflammation in the coronary arteries, according to a study published in 2002. However, the inflammation does not necessarily correspond to a plaque in the artery that might rupture. Most heart attacks are believed to be caused by a ruptured plaque that stimulates blood clot formation.

Exercise may reduce the risk of heart disease by improving endothelial function. The endothelium is the specialized layer of tissue inside blood vessels. There is much evidence that abnormal endothelial function is the underlying cause of coronary artery disease. A study showed that men who exercised had greater blood flow and less constriction in their coronary arteries compared to men who did not exercise. It was clear that exercise affected the endothelium.

The bioactivity of endothelium-derived nitric oxide (NO) is an important component of vascular homeostasis that is sensitive to intracellular redox status. Because glutathione (GSH) is a major determinant of intracellular redox state, we sought to define its role in modulating endothelial NO bioactivity. In porcine aortic endothelial cells (PAECs), we depleted intracellular GSH ([is greater than] 70%) using 1) buthionine-(S,R)-sulfoximine (BSO), which inhibits GSH synthesis; 2) diamide, which oxidizes thiols; or 3) 1-chloro-2,4-dinitrobenzene (CDNB), which putatively depletes GSH through glutathione S-transferase activity. Cellular GSH depletion with BSO had no effect on endothelial NO bioactivity measured as A-23187-induced cGMP accumulation. In contrast, oxidation of intracellular thiols with diamide inhibited both A-23187-induced cGMP accumulation and the cGMP response to exogenous NO. Diamide treatment of either PAECs, PAEC membrane fractions, or purified endothelial nitric oxide synthase (eNOS) resulted in significant inhibition (~75%) of eNOS catalytic activity measured as L-[[sup.3]H]arginine-to-L-[[sup.3]H]citrulline conversion. This effect appeared related to oxidation of eNOS thiols as it was completely reversed by dithiothreitol. Glutathione depletion with CDNB inhibited A-23187-stimulated cGMP accumulation but not the cGMP response to exogenous NO. Rather, CDNB treatment impaired eNOS catalytic activity in intact PAECs, and this effect was reversed by excess NADPH in isolated purified eNOS assays. Consistent with these results, we found spectral evidence that CDNB reacts with NADPH and renders it inactive as a cofactor for either eNOS or glutathione reductase. Thus thiol-modulating agents exert pleiotropic effects on endothelial NO bioactivity, and these data may help to resolve a number of conflicting previous studies linking GSH status with endothelial cell NO bioactivity. thiols; nitric oxide; antioxidants; endothelium; nitric oxide synthase; glutathione

Effect of ascorbic acid treatment on conduit vessel endothelial dysfunction in patients with hypertension. Am J Physiol Heart Circ Physiol 280: H528-H534, 2001.--Hypertension is associated with low plasma ascorbic acid levels and impaired endothelial function. Recent evidence suggests that increased vascular oxidative stress contributes to the pathophysiology of endothelial dysfunction and hypertension. We recently showed that chronic oral ascorbic acid therapy lowers blood pressure in hypertensive patients. We hypothesized that it would also improve endothelial vasomotor function. In a randomized, double-blind, placebo-controlled study, we examined the effect of acute (2 g po) and chronic (500 mg/day for 1 mo) ascorbic acid treatment on brachial artery flow-mediated dilation in 39 patients with hypertension. Compared with 82 age- and gender-matched normotensive controls, these patients had impaired endothelium-dependent, flow-mediated dilation of the brachial artery [8.9 [+ or -] 6.1 vs. 11.2 [+ or -] 5.7% (SD), P [is less than] 0.04]. After therapy, plasma ascorbic acid concentrations increased acutely from 50 [+ or -] 12 to 149 [+ or -] 51 [micro]mol/l and were maintained at 99 [+ or -] 33 [micro]mol/l with chronic treatment (both P [is less than] 0.001). As previously reported, chronic ascorbic acid therapy reduced systolic and mean blood pressure in these patients. However, acute or chronic ascorbic acid treatment had no effect on brachial artery endothelium-dependent, flow-mediated dilation or on endothelium-independent, nitroglycerin-mediated dilation. These results demonstrate that conduit vessel endothelial dysfunction secondary to hypertension is not reversed by acute or chronic treatment with oral ascorbic acid. The effects of this treatment on resistance vessel vasomotor function warrant further investigation. antioxidants; endothelium-derived factors; nitric oxide