Abstract-The hypothesis that the decreased nitric oxide (NO) availability observed in spontaneously hypertensive stroke-prone rats (SHRSP) is due to excess superoxide (O 2 Ϫ ) was examined. O 2 Ϫ generation, measured by lucigenin chemiluminescence, was studied in 12-to 16-week male and female Wistar-Kyoto rats (WKY) Key Words: superoxide Ⅲ endothelium Ⅲ nitric oxide Ⅲ rats, inbred SHR Ⅲ nitric oxide synthase E ndothelial dysfunction and a relative deficiency in nitric oxide (NO) may be associated with hypertension in humans 1,2 and in some models of experimental hypertension. 3,4 In the spontaneously hypertensive stroke-prone rat (SHRSP), a model of genetic hypertension, we have shown an attenuation of functional basal NO despite increased eNOS enzymatic activity. 5 Although endothelial NO synthase (eNOS) enzymatic activity was greater in SHRSP than in Wistar-Kyoto rats (WKY) when examined in vitro the possibility that the actual amount of eNOS was reduced in SHRSP in vivo could not be excluded from these results. Alternatively, eNOS levels could be similar or elevated but NO availability decreased because of more rapid removal after synthesis. Superoxide anion (O 2 Ϫ ) is produced in the vasculature and can scavenge NO forming peroxynitrite. Increased scavenging of NO by O 2 Ϫ could lead to a decrease in NO availability despite increased synthesis. Raised O 2 Ϫ levels have been reported recently in a number of models of endothelial dysfunction including hypertension, induced by either angiotensin infusion 6 or aortic banding. 7 In the majority of cases the source of excess O 2 Ϫ is uncertain, although involvement of NADH/NADPH oxidases 8 and xanthine oxidase 9 have been suggested.The aim of this study was to examine the hypothesis that the decreased NO availability observed in SHRSP is due to excess O 2 Ϫ , to identify the source of this O 2 Ϫ , and to examine other molecular mechanisms involved such as the expression of the gene-encoding enzyme involved in NO generation in the endothelium (eNOS). Methods AnimalsThree-to 4-month-old male and female WKY and SHRSP were obtained from the colonies established in Glasgow by brother and sister mating as previously described. 9 Blood pressure was measured 1 week before study by tail plethysmography according to our published protocol. 10

Abstract-Mitochondria are a major site of reactive oxygen species production, which may contribute to the development of cardiovascular disease. Protecting mitochondria from oxidative damage should be an effective therapeutic strategy; however, conventional antioxidants are ineffective, because they cannot penetrate the mitochondria. This study investigated the role of mitochondrial oxidative stress during development of hypertension in the stroke-prone spontaneously hypertensive rat, using the mitochondria-targeted antioxidant, MitoQ 10 . Eight-week-old male strokeprone spontaneously hypertensive rats were treated with MitoQ 10 (500 mol/L; nϭ16), control compound decyltriphenylphosphonium (decylTPP; 500 mol/L; nϭ8), or vehicle (nϭ9) in drinking water for 8 weeks. Ϫ goes on to produce a range of damaging ROS that lead to nonspecific modification of mitochondrial proteins, lipids, and nucleic acids, thereby altering mitochondrial function. 3-5 Mitochondrial DNA is particularly susceptible to modification by ROS, and this damage can rapidly lead to functional changes in the cell, because it encodes 13 essential polypeptide components of the mitochondrial respiratory chain. 4 Extensive evidence suggests that mitochondrial DNA damage occurs in cardiovascular disease in humans, animal models, and cellular models. 3,[7][8][9] Mitochondria are normally protected from oxidative damage by a multilayer network of mitochondrial antioxidant systems. 10,11 These include the mitochondrial matrix enzyme manganese superoxide dismutase, which converts the O 2 Ϫ anion to hydrogen peroxide, glutathione peroxidase, and peroxiredoxins 3 and 5, which readily convert hydrogen peroxide to water 7,10 and ultimately prevent forms of mitochondrial oxidative damage, eg, lipid peroxidation. Modification of these antioxidant enzymes resulting from the knockout of manganese superoxide dismutase or glutathione peroxidase genes can significantly affect mitochondrial activity and ROS production and has been linked to hypertension and salt sensitivity in mice. [12][13][14][15] The precise contribution of mitochondria to the total ROS production in the vessel wall or other cardiovascular tissues

We undertook these studies to determine whether a deficient nitric oxide production in genetically hypertensive rats could result from its being scavenged by an excess production of superoxide. In one study we used a porphyrinic microsensor to measure nitric oxide concentrations released by cultured endothelial cells from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY). SHRSP cells released only about one third the concentration of nitric oxide as did WKY cells. Treatment of cells with superoxide dismutase increased nitric oxide release, demonstrating that normally nitric oxide is scavenged by endogenous superoxide. The increase in nitric oxide release in response to superoxide dismutase treatment was more than twice as great from SHRSP as from WKY cells, demonstrating the greater amount of superoxide in the hypertensive rats. A direct measure of superoxide with the use of lucigenin demonstrated the presence of 68.1 +/- 7.1 and 27.4 +/- 3.5 nmol/L of this anion in SHRSP and WKY endothelial cells, respectively. The presence of superoxide in the rat aorta was also estimated by quantification of its effect on carbachol relaxation. This relaxation was diminished when endogenous superoxide dismutase was blocked by diethyldithiocarbamic acid. This blockade reduced the relaxation by 51.2 +/- 5.2% in SHRSP aortas and by only 22.0 +/- 8.2% (P = .015) in WKY aortas. Data from these diverse systems are in agreement that superoxide production is excessive in SHRSP tissues. This excess superoxide, by scavenging endothelial nitric oxide, could contribute to the increased vascular smooth muscle contraction and hence to the elevated total peripheral resistance of these rats.

Abstract-There is evidence in humans that hypertension and aging similarly impair endothelial function, although the mechanism remains unclear. Superoxide anion (O 2 Ϫ ) is a major determinant of nitric oxide (NO) bioavailability and thus endothelial function. We sought to determine the relationship between endothelial function, O 2 Ϫ , and age in normotensive Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). Aortic rings were removed from female WKY and SHRSP at 3 to 4 months (young) and 9 to 12 months (old). O 2 Ϫ generation by aortic rings was measured before and after removal of the endothelium or incubation with N G nitro-L-arginine methyl ester, diphenyleneiodonium, or apocynin. Levels of p22phox were studied with immunohistochemistry and used as a marker of NAD (P) Key Words: endothelium Ⅲ nitric oxide Ⅲ hypertension, experimental Ⅲ aging T here is evidence that in animal models and in humans, impaired endothelial function and a decrease in nitric oxide (NO) bioavailability may occur in hypercholesterolemia, 1,2 , diabetes, 3 and hypertension 4 -6 despite normal or increased NO production by the endothelium. 6 A decrease in NO bioavailability may also occur with aging. [7][8][9][10] In a number of animal models of disease, including hypertension 11,12 and hypercholesterolemia, 13 an increase in superoxide (O 2 Ϫ ) occurs concurrent to the decrease in NO bioavailability. O 2 Ϫ rapidly reacts with NO, forming peroxynitrite and decreasing NO bioavailability. 14 Thus, it has been proposed that elevations in O 2 Ϫ levels contribute to the impaired endothelial function associated with atherosclerotic disease. 13,15 Taddei et al 9 proposed that the endothelial dysfunction that occurs in hypertension represents an accelerated form of the dysfunction that occurs with aging. However, the effects of aging on O 2 Ϫ production are less well defined. Huraux and colleagues 16 observed a negative correlation between O 2 Ϫ levels and age in human internal mammary arteries. In contrast, Berry et al 17 found basal O 2 Ϫ production in human internal mammary arteries to be weakly but positively associated with age. Potential vascular sources of O 2 Ϫ are endothelial NO synthase (eNOS), 18 xanthine oxidase, 19 and NAD(P)H oxidase. 20,21 eNOS 18 and NAD(P)H oxidase 22,23 have been proposed to be involved in O 2 Ϫ production in different models of hypertension, whereas xanthine oxidase may be involved in O 2 Ϫ production in hypercholesterolemia. 13 eNOS can be inhibited by arginine analogues such as N G nitro-L-arginine methyl ester (L-NAME). NAD(P)H oxidase is composed of at least 5 subunits, and apocynin can inhibit enzymatic activity by preventing association of the subunits. Diphenyleneiodonium (DPI) is a less specific inhibitor of flavincontaining oxidases, including NAD(P)H oxidase.In this study, the hypothesis that both hypertension and aging result in increased levels of O 2 Ϫ and decreased NO bioavailability in blood vessels from Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertens...

A multitude of studies in experimental animals, together with clinical data, provide evidence that increased production of ROS (reactive oxygen species) are involved in the development and progression of cardiovascular disease. As ROS appear to have a critical role in atherosclerosis, there has been considerable interest in identifying the enzyme systems involved and in developing strategies to reduce oxidative stress. Prospective clinical trials with vitamins and hormone replacement therapy have not fulfilled earlier promises, although there is still interest in other dietary supplements. Superoxide dismutase mimetics, thiols, xanthine oxidase and NAD(P)H oxidase inhibitors are currently receiving much interest, while animal studies using gene therapy show promise, but are still at an early stage. Of the drugs in common clinical use, there is evidence that ACE (angiotensin-converting enzyme) inhibitors and AT1 (angiotensin II type 1) receptor blockers have beneficial effects on oxidative stress above their antihypertensive properties, whereas statins, in addition to improving lipid profiles, may also lower oxidative stress.

Abstract-The NO/superoxide (O 2Ϫ ) balance is a key regulator of endothelial function. O 2 Ϫ levels are elevated in many forms of cardiovascular disease; therefore, decreasing O 2 Ϫ should improve endothelial function. To explore this hypothesis, internal mammary arteries and saphenous veins, obtained from patients undergoing coronary artery revascularization, and aortic and carotid arteries from Wistar-Kyoto and spontaneously hypertensive stroke-prone rats were incubated with O 2 Ϫ dismutase or NAD(P)H oxidase inhibitors. O 2 Ϫ levels were measured using lucigenin chemiluminescence; NO bioavailability was assessed in organ chambers; and mRNA expression of NAD(P)H oxidase components was quantified by use of a Light Cycler. In rat arteries, phenylarsine oxide, 4-(2-aminoethyl)-benzenesulfanyl fluoride, and apocynin all decreased NADH-stimulated O 2 Ϫ production, but only apocynin increased NO bioavailability. In human internal mammary arteries and saphenous veins, apocynin decreased NAD(P)H-stimulated O 2 Ϫ generation and caused vasorelaxation that was endothelium dependent and reversed on addition of the NO synthase inhibitor N G -nitro-L-arginine methyl ester. In addition, it increased NO production from cultured human endothelial saphenous vein cells. Polyethylene-glycolated O 2 Ϫ dismutase also increased NO bioavailability in rat carotid arteries and human blood vessels, but the effects were smaller than those observed with apocynin. NADH-generated O 2 Ϫ and mRNA expression of p22 phox , gp91 phox , and nox-1 were comparable between the 2 strains of rat. This is the first study to demonstrate pharmacological effects of apocynin in human blood vessels. The increases in NO bioavailability shown here suggest that the NAD(P)H oxidase pathway may be a novel target for drug intervention in cardiovascular disease.

Internal mammary arteries (IMAs) and saphenous veins (SVs) were collected at the time of cardiac surgery. Vessels were incubated in Krebs buffer at 37 degrees C.O(2)(-) was measured by lucigenin chemiluminescence. Basal. O(2)(-) concentrations were greater in IMAs than SVs. Inhibitors of NAD(P)H oxidase (10 micromol/L to 200 micromol/L diphenyleneiodonium) and xanthine oxidase (1 mmol/L allopurinol) caused reductions in.O(2)(-) concentrations in both IMAs and SVs. Western blotting of superoxide dismutase proteins demonstrated similar expression in IMAs and SVs. Vessels were also incubated in the presence or absence of Ang II (1 pmol/L to 1 micromol/L). Ang II increased.O(2)(-) production in IMAs at 4 hours of incubation (control, 978+/-117 pmol. min(-1). mg(-1); 1 micromol/L of Ang II, 1690+/-213 pmol. min(-1). mg(-1); n=27, P=0.0001, 95% CI 336, 925) but not in SVs. This effect was completely inhibited by coincubation of IMAs with DPI (100 micromol/L), a nonspecific Ang II antagonist ([sar(1), thre(8)]-Ang II, 1 micromol/L) and a specific Ang II type 1 (AT(1)) receptor antagonist (losartan, 1 micromol/L). Conclusions-. O(2)(-) production is greater in human IMAs than in SVs. NAD(P)H oxidase and xanthine oxidase are sources of.O(2)(-) production in these vessels. The vasoactive peptide Ang II increases.O(2)(-) production in human arteries by an AT(1) receptor-dependent mechanism.

Oxidative stress, a state of excessive reactive oxidative species activity, is associated with vascular disease states such as hypertension. In this review, we discuss the recent advances in the field of reactive oxidative species-mediated vascular damage in hypertension. These include the identification of redox-sensitive tyrosine kinases, the characterization of enzymatic sources of superoxide production in human blood vessels, and their relationship with vascular damage in atherosclerosis and hypertension. Finally, recent developments in the search for strategies to attenuate vascular oxidative stress are reviewed.