(O 2 . ). In several common conditions, such as atherosclerosis, 1,2 ischemia/reperfusion injury and aging, 3-7 the mitochondria become dysfunctional and this leak of electrons is increased. 1 The mitochondria contain a unique form of superoxide dismutase (SOD), the manganesecontaining mitochondrial SOD (SOD2), which is critical in protecting against excessive production of O 2 . . Mice lacking this enzyme die of a cardiomyopathy within 10 days of birth and mice lacking one allele of SOD2 (SOD2 ϩ/Ϫ mice) develop hypertension with aging and in response to a high salt diet. 8 The development of hypertension in SOD2 ϩ/Ϫ mice is in keeping with a role of reactive oxygen species (ROS) in the pathogenesis of this and many other vascular diseases. 9 Hypertension has been associated with increased ROS production in the vasculature, the kidney and in portions of the central nervous system that control blood pressure. The hormone angiotensin II (Ang II), commonly implicated in hypertension, increases ROS production in the sites. Moreover, ROS overproduction leads to decreased bioavailability of NO, impairs endothelium-dependent vasodilatation, and promotes vasoconstriction. These alterations occur early in the development of vascular disease. 10 There is substantial interest in the enzymatic source of ROS in hypertension. Ang II stimulates the NADPH oxidase in many mammalian cells via pathways involving protein kinase C and the tyrosine kinase c-Src. 11 Ang II also activates the NADPH oxidase in vivo and mice lacking components of this enzyme are resistant to both Ang II and salt-dependent hypertension. Specific inhibitors of the NADPH oxidase have antihypertensive effects. 12,13 Another potential source of ROS in hypertension is the Original received December 8, 2009; revision received April 23, 2010; accepted April 27, 2010. In March 2010 MethodsAn expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. ReagentsMitoTEMPO, mitoTEMPO-H, 1-hydroxy-3-carboxy-pyrrolidine (CPH), and nitroxide 3-carboxy-proxyl (CP) were purchased from Alexis Corporation (San Diego, Calif). Xanthine oxidase was purchased from Roche Molecular Biochemicals (Indianapolis, Ind). All other reagents were obtained from Sigma (St Louis, Mo). Cell CultureBovine aortic endothelial cells (BAECs) (passage 4 to 8) were cultured on 100-mm plates in media 199 containing 10% FCS supplemented with 2 mmol/L L-glutamine and 1% vitamins. Confluent cells were used for the experiments. 16 Human aortic endothelial cells (HAECs) purchased from Lonza (Chicago, Ill) and cultured in EGM-2 medium supplemented with 2% FBS but without antibiotics. On the day before the study, the FBS concentration was reduced to 1%. In preliminary experiments, we examined the effect of varying doses of Ang II on cellular O 2 . production. We found that 4 hours of Ang II increased cellular O 2 . in a dose-dependent manner with maximum stimulation at 200 nmol/L (Online Figure I). This concentration was therefore used in the remainder of the ...

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NADPH oxidases are major sources of superoxide (O2∸) and hydrogen peroxide (H2O2) in vascular cells. Production of these reactive oxygen species (ROS) is essential for cell proliferation and differentiation, while ROS overproduction has been implicated in hypertension and atherosclerosis. It is known that the heme-containing catalytic subunits Nox1 and Nox4 are responsible for oxygen reduction in vascular smooth muscle cells from large arteries. However, the exact mechanism of ROS production by NADPH oxidases is not completely understood. We hypothesized that Nox1 and Nox4 play distinct roles in basal and angiotensin II (AngII)-stimulated production of O2∸ and H2O2. Nox1 and Nox4 expression in rat aortic smooth muscle cells (RASMCs) was selectively reduced by treatment with siNox4 or antisense Nox1 adenovirus. Production of O2∸ and H2O2 in intact RASMCs was analyzed by dihydroethidium and Amplex Red assay. Activity of NADPH oxidases was measured by NADPH-dependent O2∸ and H2O2 production using electron spin resonance (ESR) and 1-hydroxy-3-carboxy-pyrrolidine (CPH) in the membrane fraction in the absence of cytosolic superoxide dismutase. It was found that production of O2∸ by quiescent RASMC NADPH oxidases was five times less than H2O2 production. Stimulation of cells with AngII led to a 2-fold increase of O2∸ production by NADPH oxidases, with a small 15 to 30% increase in H2O2 formation. Depletion of Nox4 in RASMC led to diminished basal H2O2 production, but did not affect O2∸ or H2O2 production stimulated by AngII. In contrast, depletion of Nox1 in RASMC inhibited production of O2∸ and AngII-stimulated H2O2 in the membrane fraction and intact cells. Our data suggest that Nox4 produces mainly H2O2, while Nox1 generates mostly O2∸ that is later converted to H2O2. Therefore, Nox4 is responsible for basal H2O2 production, while O2∸ production in non-stimulated and AngII-stimulated cells depends on Nox1. The difference in the products generated by Nox1 and Nox4 may help to explain the distinct roles of these NADPH oxidases in cell signaling. These findings also provide important insight into the origin of H2O2 in vascular cells, and may partially account for the limited pharmacological effect of antioxidant treatments with O2∸ scavengers that do not affect H2O2.

Aims: Angiotensin II (AngII)-induced superoxide (O 2 -) production by the NADPH oxidases and mitochondria has been implicated in the pathogenesis of endothelial dysfunction and hypertension. In this work, we investigated the specific molecular mechanisms responsible for the stimulation of mitochondrial O 2 -and its downstream targets using cultured human aortic endothelial cells and a mouse model of AngII-induced hypertension. Results: Western blot analysis showed that Nox2 and Nox4 were present in the cytoplasm but not in the mitochondria. Depletion of Nox2, but not Nox1, Nox4, or Nox5, using siRNA inhibits AngII-induced O 2 -production in both mitochondria and cytoplasm. Nox2 depletion in gp91phox knockout mice inhibited AngIIinduced cellular and mitochondrial O 2 -and attenuated hypertension. Inhibition of mitochondrial reverse electron transfer with malonate, malate, or rotenone attenuated AngII-induced cytoplasmic and mitochondrial O 2 -production. Inhibition of the mitochondrial ATP-sensitive potassium channel (mitoK

Abstract-Excess food intake leads to obesity and diabetes, both of which are well-known independent risk factors for atherosclerosis, and both of which are growing epidemics in an aging population. We hypothesized that aging enhances the metabolic and vascular effects of high fat diet (HFD) and therefore examined the effect of age on atherosclerosis and insulin resistance in lipoprotein receptor knockout (LDLR Ϫ/Ϫ ) mice. We found that 12-month-old (middle-aged) LDLR Ϫ/Ϫ mice developed substantially worse metabolic syndrome, diabetes, and atherosclerosis than 3-month-old (young) LDLR Ϫ/Ϫ mice when both were fed HFD for 3 months, despite similar elevations in total cholesterol levels. Microarray analyses were performed to analyze the mechanism responsible for the marked acceleration of atherosclerosis in middle-aged mice. Chow-fed middle-aged mice had greater aortic expression of multiple antioxidant genes than chow-fed young mice, including glutathione peroxidase-1 and -4, catalase, superoxide dismutase-2, and uncoupling protein-2. Aortic expression of these enzymes markedly increased in young mice fed HFD but decreased or only modestly increased in middle-aged mice fed HFD, despite the fact that systemic oxidative stress and vascular reactive oxygen species generation, measured by plasma F2␣ isoprostane concentration (systemic) and dihydroethidium conversion and p47phox expression (vascular), were greater in middle-aged mice fed HFD. Thus, the mechanism for the accelerated vascular injury in older LDLR Ϫ/Ϫ mice was likely the profound inability to mount an antioxidant response. This effect was related to a decrease in vascular expression of 2 key transcriptional pathways regulating the antioxidant response, DJ-1 and forkhead box, subgroup O family (FOXOs). Treatment of middle-aged mice fed HFD with the antioxidant apocynin attenuated atherosclerosis, whereas treatment with the insulin sensitizer rosiglitazone attenuated both metabolic syndrome and atherosclerosis. Both treatments decreased oxidative stress. A novel effect of rosiglitazone was to increase expression of Nrf2 (nuclear factor [erythroid-derived 2]-like 2), a downstream target of DJ-1 contributing to enhanced expression of vascular antioxidant enzymes. This investigation underscores the role of oxidative stress when multiple atherosclerotic risk factors, particularly aging, converge on the vessel wall and emphasizes the need to develop effective strategies to inhibit oxidative stress to protect aging vasculature. (Circ Res. 2009;104:e42-e54.)

Superoxide plays a key role in many pathological processes; however, detection of superoxide by one of the most common methods using dihydroethidium may be unspecific due to overlapping fluorescence of the superoxide specific product, 2-OH-ethidium (2OH-E), and the unspecific oxidation product, ethidium. Here, we show new optimized fluorescence spectroscopy protocol that allows rapid and specific detection of superoxide in cell free systems and intact cells using dihydroethydium (DHE). We defined new optimized fluorescent settings to measure superoxide specific product and minimize interference of unspecific DHE oxidation products. Using this protocol we studied real time superoxide production by xanthine oxidase and menadione-treated cultured cells. Specificity of the plate reader-based superoxide measurements was confirmed by the inhibition of fluorescence with superoxide dismutase and HPLC analysis. We show that limitations of the HPLC-based analysis can be overcome by the optimized fluorescence spectroscopy.

Superoxide (O2(·-)) production by the NADPH oxidases is implicated in the pathogenesis of many cardiovascular diseases, including hypertension. We have previously shown that activation of NADPH oxidases increases mitochondrial O2(·-) which is inhibited by the ATP-sensitive K(+) channel (mitoKATP) inhibitor 5-hydroxydecanoic acid and that scavenging of mitochondrial or cytoplasmic O2(·-) inhibits hypertension. We hypothesized that mitoKATP-mediated mitochondrial O2(·-) potentiates cytoplasmic O2(·-) by stimulation of NADPH oxidases. In this work we studied Nox isoforms as a potential target of mitochondrial O2(·-). We tested contribution of reverse electron transfer (RET) from complex II to complex I in mitochondrial O2(·-) production and NADPH oxidase activation in human aortic endothelial cells. Activation of mitoKATP with low dose of diazoxide (100 nM) decreased mitochondrial membrane potential (tetramethylrhodamine methyl ester probe) and increased production of mitochondrial and cytoplasmic O2(·-) measured by site-specific probes and mitoSOX. Inhibition of RET with complex II inhibitor (malonate) or complex I inhibitor (rotenone) attenuated the production of mitochondrial and cytoplasmic O2(·-). Supplementation with a mitochondria-targeted SOD mimetic (mitoTEMPO) or a mitochondria-targeted glutathione peroxidase mimetic (mitoEbselen) inhibited production of mitochondrial and cytoplasmic O2(·-). Inhibition of Nox2 (gp91ds) or Nox2 depletion with small interfering RNA but not Nox1, Nox4, or Nox5 abolished diazoxide-induced O2(·-) production in the cytoplasm. Treatment of angiotensin II-infused mice with RET inhibitor dihydroethidium (malate) significantly reduced blood pressure. Our study suggests that mitoKATP-mediated mitochondrial O2(·-) stimulates cytoplasmic Nox2, contributing to the development of endothelial oxidative stress and hypertension.

It has been previously suggested that overexpression of mitochondrial superoxide dismutase (SOD) attenuates cancer development; however, the exact mechanism remains unclear. In this work, we have studied the direct effect of the mitochondria-targeted superoxide scavenger, (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO), on B16-F0 mouse melanoma cells and tumor growth in a nude mouse model of human melanoma. We show that scavenging of mitochondrial superoxide inhibited cell growth, reduced viability, and induced apoptosis in melanoma cells, but did not affect nonmalignant skin fibroblasts. Diminished mitochondrial superoxide inhibited redox-dependent Akt, restored activity of mitochondrial pyruvate dehydrogenase, and reduced HIF1-α and lactate dehydrogenase expression in cancer cells. Suppression of glycolysis in mitoTEMPO-treated melanoma cells resulted in a significant drop of cellular adenosine-5'-triphosphate and induced cell death. In vivo mitoTEMPO treatment effectively suppressed growth of established tumor in the mouse model of human melanoma. Therefore, our data lead to the hypothesis that scavenging of mitochondrial superoxide selectively inhibits redox-sensitive survival and metabolic pathways, resulting in cancer cell death. In contrast to existing anticancer therapies, inhibition of mitochondrial superoxide may represent a novel specific anticancer treatment with reduced cytotoxic side effects.