Therapeutic Targeting of Mitochondrial Superoxide in Hypertension

Abstract

(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 ...

Glucose deprivation increases monocarboxylate transporter 1 (MCT1) expression and MCT1-dependent tumor cell migration

“…The general antioxidant and glutathione analogue N-acetyl-L-cysteine (NAC, 5 mM) blocked MCT1 and CD147 overexpression in glucose-starved SiHa cells (Figure 4h). A similar effect was observed with the mitochondriaselective superoxide scavenger MitoTEMPO (50 mM) 23 (Figure 4i), thus confirming that ROS produced by mitochondria when they are deprived from glycolytic fuels trigger the expression of both the lactate transporter and its chaperone. ROS were also key contributors to the response to hypoxia: NAC or the H 2 O 2 scavenger ebselen (20 mM) 24 both blocked hypoxia-induced MCT1 expression (Supplementary Figure 4A).…”

“…While exogenous H 2 O 2 stabilized both MCT1 and CD147 protein expression posttranslationally thus mimicking glucose deprivation, TCA cycle replenishment upon glucose withdrawal reduced mitochondrial ROS production and inhibited protein stabilization, phenocopying antioxidants (Figures 3 and 4). The repressor effect of the specific mitochondrial superoxide scavenger mitoTEMPO 23 and of the glutathione analogue NAC further revealed mitochondria-derived H 2 O 2 as an important mediator of the phenotype. Inhibition was not complete though, suggesting the existence of additional pathways impacting MCT1-CD147 stability (potentially including those active upon glutamine deprivation) 33 that would nevertheless exert a minor contribution under glucose deprivation as in this case NAC almost totally blocked TC migration (Figure 6e).…”

“…Others using capillary electrophoresis coupled to mass spectrometry reported levels of glucose as low as 123±43 nmol/g and 424±131 nmol/g in patient biopsies of colon and stomach cancer, respectively. 17 23 In (h and i), low exposition of the film was used to show the antioxidant effect on MCT1; MCT1 expression with glucose alone was detected with longer exposure times (not shown).…”

Branched‐chain amino acids promote endothelial dysfunction through increased reactive oxygen species generation and inflammation

“…The reasons for the different contribution of mitochondria and NADPH oxidase to the different parameters are unknown and probably rely on a complex regulation of each response or a possible reciprocal relationship between both sources as described earlier 54, 55. Finally, other sources of O 2 ·− such as the uncoupled eNOS, COX‐2 or xanthine oxidase among others, cannot be excluded.…”

Transcription factor NRF2 protects mice against dietary iron-induced liver injury by preventing hepatocytic cell death

“…MPT, in turn, is a cause of both apoptotic and necrotic hepatocytic cell death, via the release of pro-apoptotic factors and a reduction in cellular ATP, respectively [17,21]. To determine if the mitochondria played a significant role in the increased susceptibility of Nrf2 -/-hepatocytes to dietary iron overload, a group of Nrf2 -/-mice on iron-rich diet received daily injections of mito-TEMPOL (10 mg/kg), a derivative of the antioxidant TEMPOL that is targeted to the mitochondria [22,23]. The antioxidant therapy did not affect the uptake of iron from the diet or its deposition in the liver, as the animals displayed similar grading of hepatic siderosis (Fig.…”