(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.