Repetitive sulfur dioxide exposure in mice models post-deployment respiratory syndrome.

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than nondeployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the subjects in this cohort reported exposure to sulfur dioxide (SO2), we developed a model of repetitive exposure to SO2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular (PV) disease. Although abnormalities in small airways were not sufficient to alter lung mechanics, PV remodeling resulted in the development of pulmonary hypertension and reduced exercise tolerance in SO2-exposed mice. SO2 exposure led to increased formation of isolevuglandins (isoLGs) adducts and superoxide dismutase 2 (SOD2) acetylation in endothelial cells, which were attenuated by treatment with the isoLG scavenger 2-hydroxybenzylamine acetate (2-HOBA). In addition, 2-HOBA treatment or Siruin-3 overexpression in a transgenic mouse model prevented vascular remodeling following SO2 exposure. In summary, our results indicate that repetitive SO2 exposure recapitulates many aspects of PDRS and that oxidative stress appears to mediate PV remodeling in this model. Together, these findings provide new insights regarding the critical mechanisms underlying PDRS.NEW & NOTEWORTHY We developed a mice model of "post-deployment respiratory syndrome" (PDRS), a condition in Veterans with unexplained exertional dyspnea. Our model successfully recapitulates many of the pathological and physiological features of the syndrome, revealing involvement of the ROS-isoLGs-Sirt3-SOD2 pathway in pulmonary vasculature pathology. Our study provides additional knowledge about effects and long-term consequences of sulfur dioxide exposure on the respiratory system, serving as a valuable tool for future PDRS research.

Repetitive Sulfur Dioxide Exposure in Mice Models Post-Deployment Respiratory Syndrome.

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than non-deployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the deployers in this cohort reported exposure to sulfur dioxide (SO 2 ), we developed a model of repetitive exposure to SO 2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular disease (PVD). Although abnormalities in small airways were not sufficient to alter lung mechanics, PVD was associated with the development of pulmonary hypertension and reduced exercise tolerance in SO 2 exposed mice. Further, we used pharmacologic and genetic approaches to demonstrate a critical role for oxidative stress and isolevuglandins in mediating PVD in this model. In summary, our results indicate that repetitive SO 2 exposure recapitulates many aspects of PDRS and that oxidative stress may mediate PVD in this model, which may be helpful for future mechanistic studies examining the relationship between inhaled irritants, PVD, and PDRS.

Pathological mechanisms of cigarette smoking, dietary, and sedentary lifestyle risks in vascular dysfunction: mitochondria as a common target of risk factors.

In the past century, the lifespan of the human population has dramatically increased to the 80 s, but it is hindered by a limited health span to the 60 s due to an epidemic increase in the cardiovascular disease which is a main cause of morbidity and mortality. We cannot underestimate the progress in understanding the major cardiovascular risk factors which include cigarette smoking, dietary, and sedentary lifestyle risks. Despite their clinical significance, these modifiable risk factors are still the major contributors to cardiovascular disease. It is, therefore, important to understand the specific molecular mechanisms behind their pathological effects to develop new therapies to improve the treatment of cardiovascular disease. In recent years, our group and others have made a progress in understanding how these risk factors can promote endothelial dysfunction, smooth muscle dysregulation, vascular inflammation, hypertension, lung, and heart diseases. These factors, despite differences in their nature, lead to stereotypical alterations in vascular metabolism and function. Interestingly, cigarette smoking has a tremendous impact on a very distant site from the initial epithelial exposure, namely circulation and vascular cells mediated by a variety of stable cigarette smoke components which promote vascular oxidative stress and alter vascular metabolism and function. Similarly, dietary and sedentary lifestyle risks facilitate vascular cell metabolic reprogramming promoting vascular oxidative stress and dysfunction. Mitochondria are critical in cellular metabolism, and in this work, we discuss a new concept that mitochondria are a common pathobiological target for these risk factors, and mitochondria-targeted treatments may have a therapeutic effect in the patients with cardiovascular disease.

Protein adduction causes non-mutational inhibition of p53 tumor suppressor.

p53 is a key tumor suppressor that is frequently mutated in human tumors. In this study, we investigated how p53 is regulated in precancerous lesions prior to mutations in the p53 gene. Analyzing esophageal cells in conditions of genotoxic stress that promotes development of esophageal adenocarcinoma, we find that p53 protein is adducted with reactive isolevuglandins (isoLGs), products of lipid peroxidation. Modification of p53 protein with isoLGs diminishes its acetylation and binding to the promoters of p53 target genes causing modulation of p53-dependent transcription. It also leads to accumulation of adducted p53 protein in intracellular amyloid-like aggregates that can be inhibited by isoLG scavenger 2-HOBA in vitro and in vivo. Taken together, our studies reveal a posttranslational modification of p53 protein that causes molecular aggregation of p53 protein and its non-mutational inactivation in conditions of DNA damage that may play an important role in human tumorigenesis.

SOLUBLE GUANYLYL CYCLASE ACTIVATION RESCUES HYPEROXIA-INDUCED DYSFUNCTION OF VASCULAR RELAXATION.

ABSTRACT: Introduction: Perioperative alterations in perfusion lead to ischemia and reperfusion injury, and supplemental oxygen is administered during surgery to limit hypoxic injury but can lead to hyperoxia. We hypothesized that hyperoxia impairs endothelium-dependent and endothelium-independent vasodilation but not the vasodilatory response to heme-independent soluble guanylyl cyclase activation. Methods: We measured the effect of oxygen on vascular reactivity in mouse aortas. Mice were ventilated with 21% (normoxia), 60% (moderate hyperoxia), or 100% (severe hyperoxia) oxygen during 30 minutes of renal ischemia and 30 minutes of reperfusion. After sacrifice, the thoracic aorta was isolated, and segments mounted on a wire myograph. We measured endothelium-dependent and endothelium-independent vasodilation with escalating concentrations of acetylcholine (ACh) and sodium nitroprusside (SNP), respectively, and we measured the response to heme-independent soluble guanylyl cyclase activation with cinaciguat. Vasodilator responses to each agonist were quantified as the maximal theoretical response ( Emax ) and the effective concentration to elicit 50% relaxation (EC 50 ) using a sigmoid model and nonlinear mixed-effects regression. Aortic superoxide was measured with dihydroethidium probe and high-performance liquid chromatography quantification of the specific superoxide product 2-hydroxyethidium. Results: Hyperoxia impaired endothelium-dependent (ACh) and endothelium-independent (SNP) vasodilation compared with normoxia and had no effect on cinaciguat-induced vasodilation. The median ACh Emax was 76.4% (95% confidence interval = 69.6 to 83.3) in the normoxia group, 53.5% (46.7 to 60.3) in the moderate hyperoxia group, and 53.1% (46.3 to 60.0) in the severe hyperoxia group ( P < 0.001, effect across groups), while the ACh EC 50 was not different among groups. The SNP Emax was 133.1% (122.9 to 143.3) in normoxia, 128.3% (118.1 to 138.6) in moderate hyperoxia, and 114.8% (104.6 to 125.0) in severe hyperoxia ( P < 0.001, effect across groups), and the SNP EC 50 was 0.38 log M greater in moderate hyperoxia than in normoxia (95% confidence interval = 0.18 to 0.58, P < 0.001). Cinaciguat Emax and EC 50 were not different among oxygen treatment groups (median range Emax = 78.0% to 79.4% and EC 50 = -18.0 to -18.2 log M across oxygen groups). Aorta 2-hydroxyethidium was 1419 pmol/mg of protein (25th-75th percentile = 1178-1513) in normoxia, 1993 (1831-2473) in moderate hyperoxia, and 2078 (1936-2922) in severe hyperoxia ( P = 0.008, effect across groups). Conclusions: Hyperoxia, compared with normoxia, impaired endothelium-dependent and endothelium-independent vasodilation but not the response to heme-independent soluble guanylyl cyclase activation, and hyperoxia increased vascular superoxide production. Results from this study could have important implications for patients receiving high concentrations of oxygen and at risk for ischemia reperfusion-mediated organ injury.

Critical role of Interleukin 21 and T follicular helper cells in hypertension and vascular dysfunction.

T and B cells have been implicated in hypertension, but the mechanisms by which they produce a coordinated response is unknown. T follicular helper (Tfh) cells that produce interleukin 21 (IL21) promote germinal center (GC) B cell responses leading to immunoglobulin (Ig) production. Here we investigate the role of IL21 and Tfh cells in hypertension. In response to angiotensin (Ang) II-induced hypertension, T cell IL21 production is increased, and Il21-/- mice develop blunted hypertension, attenuated vascular end-organ damage, and decreased interleukin 17A (IL17A) and interferon gamma production. Tfh-like cells and GC B cells accumulate in the aorta and plasma IgG1 is increased in hypertensive WT but not Il21-/-mice. Furthermore, Tfh cell deficient mice develop blunted hypertension and vascular hypertrophy in response to Ang II infusion. Importantly, IL21 neutralization reduces blood pressure (BP) and reverses endothelial dysfunction and vascular inflammation. Moreover, recombinant IL21 impairs endothelium-dependent relaxation ex vivo and decreases nitric oxide production from cultured endothelial cells. Finally, we show in humans that peripheral blood T cell production of IL21 correlates with systolic BP and IL17A production. These data suggest that IL21 may be a novel therapeutic target for the treatment of hypertension and its micro- and macrovascular complications.

Endothelial nitric oxide synthase modulates Toll-like receptor 4-mediated IL-6 production and permeability via nitric oxide-independent signaling.

Endothelial dysfunction, characterized by changes in eNOS, is a common finding in chronic inflammatory vascular diseases. These states are associated with increased infectious complications. We hypothesized that alterations in eNOS would enhance the response to LPS-mediated TLR4 inflammation. Human microvascular endothelial cells were treated with sepiapterin or N-nitro-L-arginine methylester (L-NAME) to alter endogenous NO production, and small interfering RNA to knockdown eNOS. Alterations of endogenous NO by sepiapterin, and L-NAME provided no significant changes to LPS inflammation. In contrast, eNOS knockdown greatly enhanced endothelial IL-6 production and permeability in response to LPS. Knockdown of eNOS enhanced LPS-induced p38. Inhibition of p38 with SB203580 prevented IL-6 production, without altering permeability. Knockdown of p38 impaired NF-κB activation. Physical interaction between p38 and eNOS was demonstrated by immunoprecipitation, suggesting a novel, NO-independent mechanism for eNOS regulation of TLR4. In correlation, biopsy samples in patients with systemic lupus erythematous showed reduced eNOS expression with associated elevations in TLR4 and p38, suggesting an in vivo link. Thus, reduced expression of eNOS, as seen in chronic inflammatory disease, was associated with enhanced TLR4 signaling through p38. This may enhance the response to infection in patients with chronic inflammatory conditions.-Stark, R. J., Koch, S. R., Choi, H., Mace, E. H., Dikalov, S. I., Sherwood, E. R., Lamb, F. S. Endothelial nitric oxide synthase modulates Toll-like receptor 4-mediated IL-6 production and permeability via nitric oxide-independent signaling.

Electron Paramagnetic Resonance Measurements of Reactive Oxygen Species by Cyclic Hydroxylamine Spin Probes.

SIGNIFICANCE: Oxidative stress contributes to numerous pathophysiological conditions such as development of cancer, neurodegenerative, and cardiovascular diseases. A variety of measurements of oxidative stress markers in biological systems have been developed; however, many of these methods are not specific, can produce artifacts, and do not directly detect the free radicals and reactive oxygen species (ROS) that cause oxidative stress. Electron paramagnetic resonance (EPR) is a unique tool that allows direct measurements of free radical species. Cyclic hydroxylamines are useful and convenient molecular probes that readily react with ROS to produce stable nitroxide radicals, which can be quantitatively measured by EPR. In this work, we critically review recent applications of various cyclic hydroxylamine spin probes in biology to study oxidative stress, their advantages, and the shortcomings. Recent Advances: In the past decade, a number of new cyclic hydroxylamine spin probes have been developed and their successful application for ROS measurement using EPR has been published. These new state-of-the-art methods provide improved selectivity and sensitivity for in vitro and in vivo studies. CRITICAL ISSUES: Although cyclic hydroxylamine spin probes EPR application has been previously described, there has been lack of translation of these new methods into biomedical research, limiting their widespread use. This work summarizes "best practice" in applications of cyclic hydroxylamine spin probes to assist with EPR studies of oxidative stress. FUTURE DIRECTIONS: Additional studies to advance hydroxylamine spin probes from the "basic science" to biomedical applications are needed and could lead to better understanding of pathological conditions associated with oxidative stress. Antioxid. Redox Signal. 28, 1433-1443.

Physiological Levels of Nitric Oxide Diminish Mitochondrial Superoxide. Potential Role of Mitochondrial Dinitrosyl Iron Complexes and Nitrosothiols.

Mitochondria are the major source of superoxide radicals and superoxide overproduction contributes to cardiovascular diseases and metabolic disorders. Endothelial dysfunction and diminished nitric oxide levels are early steps in the development of these pathological conditions. It is known that physiological production of nitric oxide reduces oxidative stress and inflammation, however, the precise mechanism of "antioxidant" effect of nitric oxide is not clear. In this work we tested the hypothesis that physiological levels of nitric oxide diminish mitochondrial superoxide production without inhibition of mitochondrial respiration. In order to test this hypothesis we analyzed effect of low physiological fluxes of nitric oxide (20 nM/min) on superoxide and hydrogen peroxide production by ESR spin probes and Amplex Red in isolated rat brain mitochondria. Indeed, low levels of nitric oxide substantially attenuated both basal and antimycin A-stimulated production of reactive oxygen species in the presence of succinate or glutamate/malate as mitochondrial substrates. Furthermore, slow releasing NO donor DPTA-NONOate (100 µM) did not change oxygen consumption in State 4 and State 3. However, the NO-donor strongly inhibited oxygen consumption in the presence of uncoupling agent CCCP, which is likely associated with inhibition of the over-reduced complex IV in uncoupled mitochondria. We have examined accumulation of dinitrosyl iron complexes and nitrosothiols in mitochondria treated with fast-releasing NO donor MAHMA NONOate (10 µM) for 30 min until complete release of NO. Following treatment with NO donor, mitochondria were frozen for direct detection of dinitrosyl iron complexes using Electron Spin Resonance (ESR) while accumulation of nitrosothiols was measured by ferrous-N-Methyl-D-glucamine dithiocarbamate complex, Fe(MGD)2, in lysed mitochondria. Treatment of mitochondria with NO-donor gave rise to ESR signal of dinitrosyl iron complexes while ESR spectra of Fe(MGD)2 supplemented mitochondrial lysates showed presence of both dinitrosyl iron complexes and nitrosothiols. We suggest that nitric oxide attenuates production of mitochondrial superoxide by post-translational modifications by nitrosylation of protein cysteine residues and formation of protein dinitrosyl iron complexes with thiol-containing ligands and, therefore, nitric oxide reduction in pathological conditions associated with endothelial dysfunction may increase mitochondrial oxidative stress.

Activation of NADPH oxidases leads to DNA damage in esophageal cells.

Gastroesophageal reflux disease (GERD) is the strongest known risk factor for esophageal adenocarcinoma. In the center of tumorigenic events caused by GERD is repeated damage of esophageal tissues by the refluxate. In this study, we focused on a genotoxic aspect of exposure of esophageal cells to acidic bile reflux (BA/A). Analyzing cells generated from patients with Barrett's esophagus and human esophageal specimens, we found that BA/A cause significant DNA damage that is mediated by reactive-oxygen species. ROS originate from mitochondria and NADPH oxidases. We specifically identified NOX1 and NOX2 enzymes to be responsible for ROS generation. Inhibition of NOX2 and NOX1 with siRNA or chemical inhibitors significantly suppresses ROS production and DNA damage induced by BA/A. Mechanistically, our data showed that exposure of esophageal cells to acidic bile salts induces phosphorylation of the p47phox subunit of NOX2 and its translocation to the cellular membrane. This process is mediated by protein kinase C, which is activated by BA/A. Taken together, our studies suggest that inhibition of ROS induced by reflux can be a useful strategy for preventing DNA damage and decreasing the risk of tumorigenic transformation caused by GERD.

Prevention of DNA damage in Barrett's esophageal cells exposed to acidic bile salts.

Esophageal adenocarcinoma (EA) is one of the fastest rising tumors in the USA. The major risk factor for EA is gastroesophageal reflux disease (GERD). During GERD, esophageal cells are exposed to refluxate which contains gastric acid frequently mixed with duodenal bile. This may lead to mucosal injury and Barrett's metaplasia (BE) that are important factors contributing to development of EA. In this study, we investigated DNA damage in BE cells exposed to acidic bile salts and explored for potential protective strategies. Exposure of BE cells to acidic bile salts led to significant DNA damage, which in turn, was due to generation of reactive oxygen species (ROS). We found that acidic bile salts induce a rapid increase in superoxide radicals and hydrogen peroxide, which were determined using electron paramagnetic resonance spectroscopy and Amplex Red assay. Analyzing a panel of natural antioxidants, we identified apocynin to be the most effective in protecting esophageal cells from DNA damage induced by acidic bile salts. Mechanistic analyses showed that apocynin inhibited ROS generation and increases the DNA repair capacity of BE cells. We identified BRCA1 and p73 proteins as apocynin targets. Downregulation of p73 inhibited the protective effect of apocynin. Taken together, our results suggest potential application of natural compounds such as apocynin for prevention of reflux-induced DNA damage and GERD-associated tumorigenesis.

Mitochondrial Cyclophilin D in Vascular Oxidative Stress and Hypertension.

Vascular superoxide (O˙2 (-)) and inflammation contribute to hypertension. The mitochondria are an important source of O˙2 (-); however, the regulation of mitochondrial O˙2 (-) and the antihypertensive potential of targeting the mitochondria remain poorly defined. Angiotensin II and inflammatory cytokines, such as interleukin 17A and tumor necrosis factor-α (TNFα) significantly contribute to hypertension. We hypothesized that angiotensin II and cytokines co-operatively induce cyclophilin D (CypD)-dependent mitochondrial O˙2 (-) production in hypertension. We tested whether CypD inhibition attenuates endothelial oxidative stress and reduces hypertension. CypD depletion in CypD(-/-) mice prevents overproduction of mitochondrial O˙2 (-) in angiotensin II-infused mice, attenuates hypertension by 20 mm Hg, and improves vascular relaxation compared with wild-type C57Bl/6J mice. Treatment of hypertensive mice with the specific CypD inhibitor Sanglifehrin A reduces blood pressure by 28 mm Hg, inhibits production of mitochondrial O˙2 (-) by 40%, and improves vascular relaxation. Angiotensin II-induced hypertension was associated with CypD redox activation by S-glutathionylation, and expression of the mitochondria-targeted H2O2 scavenger, catalase, abolished CypD S-glutathionylation, prevented stimulation mitochondrial O˙2 (-), and attenuated hypertension. The functional role of cytokine-angiotensin II interplay was confirmed by co-operative stimulation of mitochondrial O˙2 (-) by 3-fold in cultured endothelial cells and impairment of aortic relaxation incubated with combination of angiotensin II, interleukin 17A, and tumor necrosis factor-α which was prevented by CypD depletion or expression of mitochondria-targeted SOD2 and catalase. These data support a novel role of CypD in hypertension and demonstrate that targeting CypD decreases mitochondrial O˙2 (-), improves vascular relaxation, and reduces hypertension.

Contribution of mitochondrial oxidative stress to hypertension.

PURPOSE OF REVIEW: In 1954 Harman proposed the free radical theory of aging, and in 1972 he suggested that mitochondria are both the source and the victim of toxic free radicals. Interestingly, hypertension is an age-associated disease and clinical data show that by age 70, 70% of the population has hypertension and this is accompanied by oxidative stress. Antioxidant therapy, however, is not currently available and common antioxidants such as ascorbate and vitamin E are ineffective in preventing hypertension. The present review focuses on the molecular mechanisms of mitochondrial oxidative stress and the therapeutic potential of targeting mitochondria in hypertension. RECENT FINDINGS: Over the past several years, we have shown that the mitochondria become dysfunctional in hypertension and have defined a novel role of mitochondrial superoxide radicals in this disease. We have shown that genetic manipulation of mitochondrial antioxidant enzyme superoxide dismutase affects blood pressure, and have developed mitochondria-targeted therapies such as mitochondrial superoxide dismutase mimetics that effectively lower blood pressure. However, the specific mechanism of mitochondrial oxidative stress in hypertension remains unclear. Recent animal and clinical studies have demonstrated several hormonal, metabolic, inflammatory, and environmental pathways contributing to mitochondrial dysfunction and oxidative stress. SUMMARY: Nutritional supplements, calorie restriction, and life style change are the most effective preventive strategies to improve mitochondrial function and reduce mitochondrial oxidative stress. Aging associated mitochondrial dysfunction, however, reduces the efficacy of these strategies. Therefore, we propose that new classes of mitochondria-targeted antioxidants can provide a high therapeutic potential to improve endothelial function and reduce hypertension.

Reactive γ-ketoaldehydes promote protein misfolding and preamyloid oligomer formation in rapidly-activated atrial cells.

Rapid activation causes remodeling of atrial myocytes resembling that which occurs in experimental and human atrial fibrillation (AF). Using this cellular model, we previously observed transcriptional upregulation of proteins implicated in protein misfolding and amyloidosis. For organ-specific amyloidoses such as Alzheimer's disease, preamyloid oligomers (PAOs) are now recognized to be the primary cytotoxic species. In the setting of oxidative stress, highly-reactive lipid-derived mediators known as γ-ketoaldehydes (γ-KAs) have been identified that rapidly adduct proteins and cause PAO formation for amyloid ß1-42 implicated in Alzheimer's. We hypothesized that rapid activation of atrial cells triggers oxidative stress with lipid peroxidation and formation of γ-KAs, which then rapidly crosslink proteins to generate PAOs. To investigate this hypothesis, rapidly-paced and control, spontaneously-beating atrial HL-1 cells were probed with a conformation-specific antibody recognizing PAOs. Rapid stimulation of atrial cells caused the generation of cytosolic PAOs along with a myocyte stress response (e.g., transcriptional upregulation of Nppa and Hspa1a), both of which were absent in control, unpaced cells. Rapid activation also caused the formation of superoxide and γ-KA adducts in atriomyocytes, while direct exposure of cells to γ-KAs resulted in PAO production. Increased cytosolic atrial natriuretic peptide (ANP), and the generation of ANP oligomers with exposure to γ-KAs and rapid atrial HL-1 cell stimulation, strongly suggest a role for ANP in PAO formation. Salicylamine (SA) is a small molecule scavenger of γ-KAs that can protect proteins from modification by these reactive compounds. PAO formation and transcriptional remodeling were inhibited when cells were stimulated in the presence of SA, but not with the antioxidant curcumin, which is incapable of scavenging γ-KAs. These results demonstrate that γ-KAs promote protein misfolding and PAO formation as a component of the atrial cell stress response to rapid activation, and they provide a potential mechanistic link between oxidative stress and atrial cell injury.

Nox2-induced production of mitochondrial superoxide in angiotensin II-mediated endothelial oxidative stress and hypertension.

AIMS: 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. 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 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. INNOVATION AND CONCLUSIONS: 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.

Nox2 as a potential target of mitochondrial superoxide and its role in endothelial oxidative stress.

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.

Mitochondrial ROS in the prohypertensive immune response.

In the past decade, it has become clear that reactive oxygen species (ROS) and inflammation play an important role in the development of hypertension. Scavenging of mitochondrial superoxide and blocking either IL-17 or tumor necrosis factor-α (TNF-α) attenuates hypertension. T-cells, critical for development of hypertension, once activated intensively produce cytokines, proliferate, and differentiate. Thus T-cell activation leads to expanded energy demand. To fulfill these needs, T-cells through tightly regulated mechanisms, supported by mitochondrial ROS (mtROS), alter their metabolic phenotype. In this review we summarize data and show evidence supporting new concept that mtROS directly contributes to prohypertensive response of immune cells.

Does scavenging of mitochondrial superoxide attenuate cancer prosurvival signaling pathways?

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.

Menaquinone biosynthesis potentiates haem toxicity in Staphylococcus aureus.

Staphylococcus aureus is a pathogen that infects multiple anatomical sites leading to a diverse array of diseases. Although vertebrates can restrict the growth of invading pathogens by sequestering iron within haem, S. aureus surmounts this challenge by employing high-affinity haem uptake systems. However, the presence of excess haem is highly toxic, necessitating tight regulation of haem levels. To overcome haem stress, S. aureus expresses the detoxification system HrtAB. In this work, a transposon screen was performed in the background of a haem-susceptible, HrtAB-deficient S. aureus strain to identify the substrate transported by this putative pump and the source of haem toxicity. While a recent report indicates that HrtAB exports haem itself, the haem-resistant mutants uncovered by the transposon selection enabled us to elucidate the cellular factors contributing to haem toxicity. All mutants identified in this screen inactivated the menaquinone (MK) biosynthesis pathway. Deletion of the final steps of this pathway revealed that quinone molecules localizing to the cell membrane potentiate haem-associated superoxide production and subsequent oxidative damage. These data suggest a model in which membrane-associated haem and quinone molecules form a redox cycle that continuously generates semiquinones and reduced haem, both of which react with atmospheric oxygen to produce superoxide.