(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 ...

Shirai et al. show that the glycolytic enzyme PKM2 serves as a molecular integrator of metabolic dysfunction, oxidative stress and tissue inflammation in macrophages from patients with atherosclerotic coronary artery disease.

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

The development of antioxidant strategies specifically targeting mitochondria could be therapeutically beneficial in these disease conditions.

Tamoxifen is an anticancer drug that induces oxidative stress and apoptosis via mitochondria-dependent and nitric oxide (NO)-dependent pathways. The present report shows that tamoxifen increases intramitochondrial ionized Ca 2+ concentration and stimulates mitochondrial NO synthase (mtNOS) activity in the mitochondria from rat liver and human breast cancer MCF-7 cells. By stimulating mtNOS, tamoxifen hampers mitochondrial respiration, releases cytochrome c, elevates mitochondrial lipid peroxidation, increases protein tyrosine nitration of certain mitochondrial proteins, decreases the catalytic activity of succinyl-CoA:3-oxoacid CoA-transferase, and induces aggregation of mitochondria. The present report suggests a critical role for mtNOS in apoptosis induced by tamoxifen. [Cancer Res 2007;67(3):1282-90]

Rationale Clinical studies have shown that Sirt3 expression declines by 40% by age 65 paralleling the increased incidence of hypertension and metabolic conditions further inactivate Sirt3 due to increased NADH and acetyl-CoA levels. Sirt3 impairment reduces the activity of a key mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2), due to hyperacetylation. Objective In this study we examined if loss of Sirt3 activity increases vascular oxidative stress due to SOD2 hyperacetylation and promotes endothelial dysfunction and hypertension. Methods and Results Hypertension was markedly increased in Sirt3 knockout (Sirt3−/−) and SOD2 depleted (SOD2+/−) mice in response to low dose of angiotensin II (0.3 mg/kg/day) compared with wild-type C57Bl/6J mice. Sirt3 depletion increased SOD2 acetylation, elevated mitochondrial O2•, and diminished endothelial nitric oxide. Angiotensin II induced hypertension was associated with Sirt3 S-glutathionylation, acetylation of vascular SOD2 and reduced SOD2 activity. Scavenging of mitochondrial H2O2 in mCAT mice prevented Sirt3 and SOD2 impairment and attenuated hypertension. Treatment of mice after onset of hypertension with a mitochondria-targeted H2O2 scavenger, mitoEbselen, reduced Sirt3 S-glutathionylation, diminished SOD2 acetylation and reduced blood pressure in wild-type but not in Sirt3−/− mice while an SOD2 mimetic, mitoTEMPO, reduced blood pressure and improved vasorelaxation both in Sirt3−/− and wild type mice. SOD2 acetylation had an inverse correlation with SOD2 activity and a direct correlation with the severity of hypertension. Analysis of human subjects with essential hypertension showed 2.6-fold increase in SOD2 acetylation and 1.4-fold decrease in Sirt3 levels while SOD2 expression was not affected. Conclusions Our data suggest that diminished Sirt3 expression and redox inactivation of Sirt3 lead to SOD2 inactivation and contributes to the pathogenesis of hypertension.

Emerging evidence supports an important role for T cells in the genesis of hypertension. Because this work has predominantly been performed in experimental animals, we sought to determine whether human T cells are activated in hypertension. We employed a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 mm Hg vs. 116 mm Hg for sham treated animals. Flow cytometry of thoracic lymph nodes, thoracic aorta and kidney revealed increased infiltration of human leukocytes (CD45+) and T lymphocytes (CD3+ and CD4+) in response to angiotensin II infusion. Interestingly, there was also an increase in the memory T cells (CD3+/CD45RO+) in the aortas and lymph nodes. Prevention of hypertension using hydralazine and hydrochlorothiazide prevented the accumulation of T cells in these tissues. Studies of isolated human T cells and monocytes indicated that angiotensin II had no direct effect on cytokine production by T cells or the ability of dendritic cells to drive T cell proliferation. We also observed an increase in circulating IL-17A producing CD4+ T cells and both CD4+ and CD8+ T cells that produce IFN-γ in hypertensive compared to normotensive humans. Thus, human T cells become activated and invade critical end-organ tissues in response to hypertension in a humanized mouse model. This response likely reflects the hypertensive milieu encountered in vivo and is not a direct effect of the hormone angiotensin II.

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.