The role of reactive oxygen species (ROS) in angiotensin II (AngII) induced endothelial dysfunction, cardiovascular and renal remodeling, inflammation, and fibrosis has been well documented. The ...molecular mechanisms of AngII pathophysiological activity involve the stimulation of NADPH oxidases, which produce superoxide and hydrogen peroxide. AngII also increases the production of mitochondrial ROS, while the inhibition of AngII improves mitochondrial function; however, the specific molecular mechanisms of the stimulation of mitochondrial ROS is not clear.
Interestingly, the overexpression of mitochondrial thioredoxin 2 or mitochondrial superoxide dismutase attenuates AngII-induced hypertension, which demonstrates the importance of mitochondrial ROS in AngII-mediated cardiovascular diseases.
Although mitochondrial ROS plays an important role in normal physiological cell signaling, AngII, high glucose, high fat, or hypoxia may cause the overproduction of mitochondrial ROS, leading to the feed-forward redox stimulation of NADPH oxidases. This vicious cycle may contribute to the development of pathological conditions and facilitate organ damage in hypertension, atherosclerosis, and diabetes.
The development of antioxidant strategies specifically targeting mitochondria could be therapeutically beneficial in these disease conditions.
Abnormal glucose metabolism and enhanced oxidative stress accelerate cardiovascular disease, a chronic inflammatory condition causing high morbidity and mortality. Here, we report that in monocytes ...and macrophages of patients with atherosclerotic coronary artery disease (CAD), overutilization of glucose promotes excessive and prolonged production of the cytokines IL-6 and IL-1β, driving systemic and tissue inflammation. In patient-derived monocytes and macrophages, increased glucose uptake and glycolytic flux fuel the generation of mitochondrial reactive oxygen species, which in turn promote dimerization of the glycolytic enzyme pyruvate kinase M2 (PKM2) and enable its nuclear translocation. Nuclear PKM2 functions as a protein kinase that phosphorylates the transcription factor STAT3, thus boosting IL-6 and IL-1β production. Reducing glycolysis, scavenging superoxide and enforcing PKM2 tetramerization correct the proinflammatory phenotype of CAD macrophages. In essence, PKM2 serves a previously unidentified role as a molecular integrator of metabolic dysfunction, oxidative stress and tissue inflammation and represents a novel therapeutic target in cardiovascular disease.
RATIONALE:Superoxide ( ) has been implicated in the pathogenesis of many human diseases including hypertension; however, commonly used antioxidants have proven ineffective in clinical trials. It is ...possible that these agents are not adequately delivered to the subcellular sites of superoxide production.
OBJECTIVE:Because the mitochondria are important sources of reactive oxygen species, we postulated that mitochondrial targeting of superoxide scavenging would have therapeutic benefit.
METHODS AND RESULTS:In this study, we found that the hormone angiotensin (Ang II) increased endothelial mitochondrial superoxide production. Treatment with the mitochondria-targeted antioxidant mitoTEMPO decreased mitochondrial , inhibited the total cellular , reduced cellular NADPH oxidase activity, and restored the level of bioavailable NO. These effects were mimicked by overexpressing the mitochondrial MnSOD (SOD2), whereas SOD2 depletion with small interfering RNA increased both basal and Ang II–stimulated cellular . Treatment of mice in vivo with mitoTEMPO attenuated hypertension when given at the onset of Ang II infusion and decreased blood pressure by 30 mm Hg following establishment of both Ang II–induced and DOCA salt hypertension, whereas a similar dose of nontargeted TEMPOL was not effective. In vivo, mitoTEMPO decreased vascular , increased vascular NO production and improved endothelial-dependent relaxation. Interestingly, transgenic mice overexpressing mitochondrial SOD2 demonstrated attenuated Ang II–induced hypertension and vascular oxidative stress similar to mice treated with mitoTEMPO.
CONCLUSIONS:These studies show that mitochondrial is important for the development of hypertension and that antioxidant strategies specifically targeting this organelle could have therapeutic benefit in this and possibly other diseases.
Angiotensin II (AngII)-induced superoxide (O2(•-)) 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 O2(•-) and its downstream targets using cultured human aortic endothelial cells and a mouse model of AngII-induced hypertension.
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 O2(•-) production in both mitochondria and cytoplasm. Nox2 depletion in gp91phox knockout mice inhibited AngII-induced cellular and mitochondrial O2(•-) and attenuated hypertension. Inhibition of mitochondrial reverse electron transfer with malonate, malate, or rotenone attenuated AngII-induced cytoplasmic and mitochondrial O2(•-) production. Inhibition of the mitochondrial ATP-sensitive potassium channel (mitoK(+)ATP) with 5-hydroxydecanoic acid or specific PKCɛ peptide antagonist (EAVSLKPT) reduced AngII-induced H2O2 in isolated mitochondria and diminished cytoplasmic O2(•-). The mitoK(+)ATP agonist diazoxide increased mitochondrial O2(•-), cytoplasmic c-Src phosphorylation and cytoplasmic O2(•-) suggesting feed-forward regulation of cellular O2(•-) by mitochondrial reactive oxygen species (ROS). Treatment of AngII-infused mice with malate reduced blood pressure and enhanced the antihypertensive effect of mitoTEMPO. Mitochondria-targeted H2O2 scavenger mitoEbselen attenuated redox-dependent c-Src and inhibited AngII-induced cellular O2(•-), diminished aortic H2O2, and reduced blood pressure in hypertensive mice.
These studies show that Nox2 stimulates mitochondrial ROS by activating reverse electron transfer and both mitochondrial O2(•-) and reverse electron transfer may represent new pharmacological targets for the treatment of hypertension.
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.
Superoxide plays a key role in many pathological processes; however, detection of superoxide by one of the most common methods using dihydroethidium (DHE) may be unspecific because of overlapping ...fluorescence of the superoxide-specific product, 2-OH-ethidium (2OH-E), and the unspecific oxidation product, ethidium. Here, we show a new optimized fluorescence spectroscopy protocol that allows rapid and specific detection of superoxide in cell-free systems and intact cells using DHE. We defined new optimized fluorescent settings to measure the superoxide-specific product and minimize the 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 high-performance liquid chromatography (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.
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 used a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 versus 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 interleukin-17A producing CD4 T cells and both CD4 and CD8 T cells that produce interferon-γ in hypertensive compared with 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.
Hypertension remains a major health problem in Western Societies, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs. Mitochondrial dysfunction contributes ...to hypertension, and mitochondria-targeted agents can potentially improve treatment of hypertension. We have proposed that mitochondrial oxidative stress produces reactive dicarbonyl lipid peroxidation products, isolevuglandins, and that scavenging of mitochondrial isolevuglandins improves vascular function and reduces hypertension. To test this hypothesis, we have studied the accumulation of mitochondrial isolevuglandins-protein adducts in patients with essential hypertension and Ang II (angiotensin II) model of hypertension using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isolevuglandins was tested by the novel mitochondria-targeted isolevuglandin scavenger, mito2HOBA. Mitochondrial isolevuglandins in arterioles from hypertensive patients were 250% greater than in arterioles from normotensive subjects, and ex vivo mito2HOBA treatment of arterioles from hypertensive subjects increased deacetylation of a key mitochondrial antioxidant, SOD2 (superoxide dismutase 2). In human aortic endothelial cells stimulated with Ang II plus TNF (tumor necrosis factor)-α, mito2HOBA reduced mitochondrial superoxide and cardiolipin oxidation, a specific marker of mitochondrial oxidative stress. In Ang II–infused mice, mito2HOBA diminished mitochondrial isolevuglandins-protein adducts, raised Sirt3 (sirtuin 3) mitochondrial deacetylase activity, reduced vascular superoxide, increased endothelial nitric oxide, improved endothelium-dependent relaxation, and attenuated hypertension. Mito2HOBA preserved mitochondrial respiration, protected ATP production, and reduced mitochondrial permeability pore opening in Ang II–infused mice. These data support the role of mitochondrial isolevuglandins in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isolevuglandins may have therapeutic potential in treatment of vascular dysfunction 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.
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