NADPH Oxidase Isoforms Research Articles

Selective Recapitulation of Conserved and Nonconserved Regions of Putative NOXA1 Protein Activation Domain Confers Isoform-specific Inhibition of Nox1 Oxidase and Attenuation of Endothelial Cell Migration

Excessive vascular and colon epithelial reactive oxygen species production by NADPH oxidase isoform 1 (Nox1) has been implicated in a number of disease states, including hypertension, atherosclerosis, and neoplasia. A peptide that mimics a putative activation domain of the Nox1 activator subunit NOXA1 (NOXA1 docking sequence, also known as NoxA1ds) potently inhibited Nox1-derived superoxide anion (O2·-) production in a reconstituted Nox1 cell-free system, with no effect on Nox2-, Nox4-, Nox5-, or xanthine oxidase-derived reactive oxygen species production as measured by cytochrome c reduction, Amplex Red fluorescence, and electron paramagnetic resonance. The ability of NoxA1ds to cross the plasma membrane was tested by confocal microscopy in a human colon cancer cell line exclusively expressing Nox1 (HT-29) using FITC-labeled NoxA1ds. NoxA1ds significantly inhibited whole HT-29 carcinoma cell-derived O2·- generation. ELISA and fluorescence recovery after photobleaching experiments indicate that NoxA1ds, but not its scrambled control, binds Nox1. FRET experiments conducted using Nox1-YFP and NOXA1-CFP illustrate that NoxA1ds disrupts the binding interaction between Nox1 and NOXA1, whereas a control peptide did not. Moreover, hypoxia-induced human pulmonary artery endothelial cell O2·- production was completely inhibited by NoxA1ds. Human pulmonary artery endothelial cell migration under hypoxic conditions was also reduced by pretreatment with NoxA1ds. Our data indicate that a peptide recapitulating a putative activation subdomain of NOXA1 (NoxA1ds) is a highly efficacious and selective inhibitor of Nox1 activity and establishes a critical interaction site for Nox1-NOXA1 binding required for enzyme activation.

Hydrogen peroxide generated by DUOX1 regulates the expression levels of specific differentiation markers in normal human keratinocytes

BackgroundRecent studies have demonstrated that the production of reactive oxygen species (ROS) itself plays an indispensable role in the process of differentiation in various tissues. However, it is unclear whether ROS have an effect on the differentiation of keratinocytes essential for the development of the epidermal permeability barrier. ObjectiveThe aim of the study is to determine a major H2O2-generating source by ionomycin in normal human keratinocytes (NHKs), and elucidate the physiological role of H2O2 generated by identified dual oxidase 1 (DUOX1) on differentiation markers of NHKs. MethodsTo detect H2O2 level generated by ionomycin in NHKs, luminal-HRP assays are performed. To examine the effects of DUOX1 on differentiation markers of NHKs, analysis of Q-RT-PCR, siRNA knockdown, and Western blot analysis were performed. ResultsWe found that levels of H2O2 generated by ionomycin, a Ca2+ signal inducer, showed Ca2+ dependence manner. In addition, DPI, an inhibitor of NOXes, significantly reversed the ionomycin-induced H2O2 level, and inhibited the mRNA expression levels of keratin 1, keratin 10, and filaggrin compared with other ROS generating system inhibitors. Interestingly, we demonstrated that extracellular Ca2+ markedly up-regulated mRNA expression levels of DUOX1 among NADPH oxidase (NOX) isoforms. Knockdown of DUOX1 by RNA interference (RNAi) in NHKs significantly antagonized an increase of ionomycin-induced H2O2 level, and specifically decreased the expressions of several keratinocyte differentiation markers such as keratin 1, transglutaminase 3, desmoglein 1, and aquaporin 9. In addition, we also found that formation of cornified envelope was significantly reduced in DUOX1-knockdown NHKs. ConclusionThese results suggest that DUOX1 is the major H2O2-producing source in NHKs stimulated with Ca2+, and plays a significant role in regulating the expression of specific markers necessary for the normal differentiation of keratinocytes.

Response to Comment on: Sukumar et al. Nox2 NADPH Oxidase Has a Critical Role in Insulin Resistance–Related Endothelial Cell Dysfunction. Diabetes 2013;62:2130–2134

We would like to thank Drs. Jandeleit-Dahm and Schmidt (1) for their interesting comments regarding our study examining the role of the Nox2 isoform of NADPH oxidase in insulin resistance–related endothelial cell dysfunction (2). We would also like to congratulate them on their own elegant and comprehensive piece of work examining the role of the Nox isoforms Nox1, Nox2, and Nox4 in the development of advanced type 1 diabetes–related atherosclerosis (3). Based on their findings that Nox1 may be more important in diabetes-related atherosclerosis than other isoforms of NADPH oxidase, the authors raise concerns regarding our conclusions that Nox2 has a critical …

NADPH Oxidase, NOX1, Mediates Vascular Injury in Ischemic Retinopathy

Ischemic retinal diseases such as retinopathy of prematurity are major causes of blindness due to damage to the retinal microvasculature. Despite this clinical situation, retinopathy of prematurity is mechanistically poorly understood. Therefore, effective preventative therapies are not available. However, hypoxic-induced increases in reactive oxygen species (ROS) have been suggested to be involved with NADPH oxidases (NOX), the only known dedicated enzymatic source of ROS. Our major aim was to determine the contribution of NOX isoforms (1, 2, and 4) to a rodent model of retinopathy of prematurity. Using a genetic approach, we determined that only mice with a deletion of NOX1, but not NOX2 or NOX4, were protected from retinal neovascularization and vaso-obliteration, adhesion of leukocytes, microglial accumulation, and the increased generation of proangiogenic and proinflammatory factors and ROS. We complemented these studies by showing that the specific NOX inhibitor, GKT137831, reduced vasculopathy and ROS levels in retina. The source of NOX isoforms was evaluated in retinal vascular cells and neuro-glial elements. Microglia, the immune cells of the retina, expressed NOX1, 2, and 4 and responded to hypoxia with increased ROS formation, which was reduced by GKT137831. Our studies are the first to identify the NOX1 isoform as having an important role in the pathogenesis of retinopathy of prematurity. Our findings suggest that strategies targeting NOX1 have the potential to be effective treatments for a range of ischemic retinopathies.

Metabolic syndrome-induced tubulointerstitial injury: Role of oxidative stress and preventive effects of acetaminophen

The prevalence of metabolic syndrome persistently increases and affects over 30% of U.S. adults. To study how metabolic syndrome may induce tubulointerstitial injury and whether acetaminophen has renal-protective properties, 4-week-old obese Zucker rats were randomly assigned into three groups, control (OC), vehicle dimethyl sulfoxide (OV), and acetaminophen treatment (30mg/kg/day for 26 weeks), and lean Zucker rats served as healthy controls. Significant tubulointerstitial injuries were observed in both OC and OV animals, evidenced by increased tubular cell death, tubular atrophy/dilation, inflammatory cell infiltration, and fibrosis. These tubulointerstitial alterations were significantly reduced by treatment with a chronic but low dose of acetaminophen, which acted to diminish NADPH oxidase isoforms Nox2 and Nox4 and decrease tubulointerstitial oxidative stress (reduced tissue superoxide and macromolecular oxidation). Decreased oxidative stress by acetaminophen was paralleled by the reduction of tubular proapoptotic signaling (diminished Bax/Bcl-2 ratio and caspase 3 activation) and the alleviation of tubular epithelial-to-mesenchymal transition (decreased transforming growth factor β, connective tissue growth factor, α-smooth muscle actin, and laminin). These data suggest that increased oxidative stress plays a critical role in mediating metabolic syndrome-induced tubulointerstitial injury and provide the first evidence suggesting that acetaminophen may be of therapeutic benefit for the prevention of tubulointerstitial injury.

Hemodynamic Regulation of Reactive Oxygen Species: Implications for Vascular Diseases

Arterial blood vessels functionally and structurally adapt to altering hemodynamic forces in order to accommodate changing needs and to provide stress homeostasis. This ability is achieved at the cellular level by converting mechanical stimulation into biochemical signals (i.e., mechanotransduction). Physiological mechanical stress helps maintain vascular structure and function, whereas pathologic or aberrant stress may impair cellular mechano-signaling, and initiate or augment cellular processes that drive disease. Reactive oxygen species (ROS) may represent an intriguing class of mechanically regulated second messengers. Chronically enhanced ROS generation may be induced by adverse mechanical stresses, and is associated with a multitude of vascular diseases. Although a causal relationship has clearly been demonstrated in large numbers of animal studies, an effective ROS-modulating therapy still remains to be established by clinical studies. This review article focuses on the role of various mechanical forces (in the form of laminar shear stress, oscillatory shear stress, or cyclic stretch) as modulators of ROS-driven signaling, and their subsequent effects on vascular biology and homeostasis, as well as on specific diseases such as arteriosclerosis, hypertension, and abdominal aortic aneurysms. Specifically, it highlights the significance of the various NADPH oxidase (NOX) isoforms as critical ROS generators in the vasculature. Directed targeting of defined components in the complex network of ROS (mechano-)signaling may represent a key for successful translation of experimental findings into clinical practice.

Role of NADPH oxidase isoforms NOX1, NOX2 and NOX4 in myocardial ischemia/reperfusion injury

Myocardial reperfusion injury is mediated by several processes including increase of reactive oxygen species (ROS). The aim of the study is to identify potential sources of ROS contributing to myocardial ischemia–reperfusion injury. For this purpose, we investigated myocardial ischemia/reperfusion pathology in mice deficient in various NADPH oxidase isoforms (Nox1, Nox2, Nox4, as well as Nox1/2 double knockout). Following 30min of ischemia and 24h of reperfusion, a significant decrease in the size of myocardial infarct was observed in Nox1-, Nox2- and Nox1/Nox2-, but not in Nox4-deficient mice. However, no protection was observed in a model of chronic ischemia, suggesting that NOX1 and NOX2-mediated oxidative damage occurs during reperfusion. Cardioprotective effect of Nox1 and Nox2 deficiencies was associated with decrease of neutrophil invasion, but, on the other hand an improved reperfusion injury was also observed in isolated perfused hearts (Langendorff model) suggesting that inflammatory cells were not the major source of oxidative damage. A decrease in global post-reperfusion oxidative stress was clearly detected in Nox2-, but not in Nox1-deficient hearts. Analysis of key signaling pathways during reperfusion suggests distinct cardioprotective patterns: increased phosphorylation was seen for Akt and Erk in Nox1-deficient mice and for Stat3 and Erk in Nox2-deficient mice. Consequently, NOX1 and NOX2 represent interesting drug targets for controlling reperfusion damage associated with revascularization in coronary disease.

Contribution of NADPH Oxidase to Membrane CD38 Internalization and Activation in Coronary Arterial Myocytes

The CD38-ADP-ribosylcyclase-mediated Ca2+ signaling pathway importantly contributes to the vasomotor response in different arteries. Although there is evidence indicating that the activation of CD38-ADP-ribosylcyclase is associated with CD38 internalization, the molecular mechanism mediating CD38 internalization and consequent activation in response to a variety of physiological and pathological stimuli remains poorly understood. Recent studies have shown that CD38 may sense redox signals and is thereby activated to produce cellular response and that the NADPH oxidase isoform, NOX1, is a major resource to produce superoxide (O2 ·−) in coronary arterial myocytes (CAMs) in response to muscarinic receptor agonist, which uses CD38-ADP-ribosylcyclase signaling pathway to exert its action in these CAMs. These findings led us hypothesize that NOX1-derived O2 ·− serves in an autocrine fashion to enhance CD38 internalization, leading to redox activation of CD38-ADP-ribosylcyclase activity in mouse CAMs. To test this hypothesis, confocal microscopy, flow cytometry and a membrane protein biotinylation assay were used in the present study. We first demonstrated that CD38 internalization induced by endothelin-1 (ET-1) was inhibited by silencing of NOX1 gene, but not NOX4 gene. Correspondingly, NOX1 gene silencing abolished ET-1-induced O2 ·− production and increased CD38-ADP-ribosylcyclase activity in CAMs, while activation of NOX1 by overexpression of Rac1 or Vav2 or administration of exogenous O2 ·− significantly increased CD38 internalization in CAMs. Lastly, ET-1 was found to markedly increase membrane raft clustering as shown by increased colocalization of cholera toxin-B with CD38 and NOX1. Taken together, these results provide direct evidence that Rac1-NOX1-dependent O2 ·− production mediates CD38 internalization in CAMs, which may represent an important mechanism linking receptor activation with CD38 activity in these cells.

Reactive Oxygen Species from Human Astrocytes Induced Functional Impairment and Oxidative Damage

Reactive oxygen species (ROS) have been shown to be a contributor to aging and disease. ROS also serve as a trigger switch for signaling cascades leading to corresponding cellular and molecular events. In the central nervous system (CNS), microglial cells are likely the main source of ROS production. However, activated astrocytes also appear to be capable of generating ROS. In this study we investigated ROS production in human astrocytes stimulated with interleukin (IL)-1β and interferon (IFN)-γ and its potential harmful effects. Although IFN-γ alone had no effect, it potentiated IL-1β-induced ROS production in a time-dependent manner. One of the sources of ROS in IL-1β-activated astrocytes was from increased superoxide production in mitochondria accompanied by enhanced manganese superoxide dismutase and inhibited catalase expression. NADPH oxidase (NOX) may also contribute to ROS production as astrocytes express NOX isoforms. Glutamate uptake, which represents one of the most important methods of astrocytes to prevent excitotoxicity, was down-regulated in IL-1β-activated astrocytes, and was further suppressed in the presence of IFN-γ; IFN-γ itself exerted minimal effect. Elevated levels of 8-isoprostane in IL-1β ± IFN-γ-activated human astrocytes indicate downstream lipid peroxidation. Pretreatment with diphenyleneiodonium abolished the IL-1β ± IFN-γ-induced ROS production, restored glutamate uptake function and reduced 8-isoprostane to near control levels suggesting that ROS contributes to the dysfunction of activated astrocytes. These results support the notion that dampening activated human astrocytes to maintain the redox homeostasis is vital to preserve their neuroprotective potential in the CNS.

Ethers and esters derived from apocynin avoid the interaction between p47phox and p22phox subunits of NADPH oxidase: evaluation in vitro and in silico

NOX (NADPH oxidase) plays an important role during several pathologies because it produces the superoxide anion (O2•−), which reacts with NO (nitric oxide), diminishing its vasodilator effect. Although different isoforms of NOX are expressed in ECs (endothelial cells) of blood vessels, the NOX2 isoform has been considered the principal therapeutic target for vascular diseases because it can be up-regulated by inhibiting the interaction between its p47phox (cytosolic protein) and p22phox (transmembrane protein) subunits. In this research, two ethers, 4-(4-acetyl-2-methoxy-phenoxy)-acetic acid (1) and 4-(4-acetyl-2-methoxy-phenoxy)-butyric acid (2) and two esters, pentanedioic acid mono-(4-acetyl-2-methoxy-phenyl) ester (3) and heptanedioic acid mono-(4-acetyl-2-methoxy-phenyl) ester (4), which are apocynin derivatives were designed, synthesized and evaluated as NOX inhibitors by quantifying O2•− production using EPR (electron paramagnetic resonance) measurements. In addition, the antioxidant activity of apocynin and its derivatives were determined. A docking study was used to identify the interactions between the NOX2′s p47phox subunit and apocynin or its derivatives. The results showed that all of the compounds exhibit inhibitory activity on NOX, being 4 the best derivative. However, neither apocynin nor its derivatives were free radical scavengers. On the other hand, the in silico studies demonstrated that the apocynin and its derivatives were recognized by the polybasic SH3A and SH3B domains, which are regions of p47phox that interact with p22phox. Therefore this experimental and theoretical study suggests that compound 4 could prevent the formation of the complex between p47phox and p22phox without needing to be activated by MPO (myeloperoxidase), this being an advantage over apocynin.

Fulvene-5 inhibition of Nadph oxidases attenuates activation of epithelial sodium channels in A6 distal nephron cells

Nadph oxidase 4 is an important cellular source of reactive oxygen species (ROS) generation in the kidney. Novel antioxidant drugs, such as Nox4 inhibitor compounds, are being developed. There is, however, very little experimental evidence for the biological role and regulation of Nadph oxidase isoforms in the kidney. Herein, we show that Fulvene-5 is an effective inhibitor of Nox-generated ROS and report the role of Nox isoforms in activating epithelial sodium channels (ENaC) in A6 distal nephron cells via oxidant signaling and cell stretch activation. Using single-channel patch-clamp analysis, we report that Fulvene-5 blocked the increase in ENaC activity that is typically observed with H2O2 treatment of A6 cells: average ENaC NPo values decreased from a baseline level of 1.04 ± 0.18 (means ± SE) to 0.25 ± 0.08 following Fulvene-5 treatment. H2O2 treatment failed to increase ENaC activity in the presence of Fulvene-5. Moreover, Fulvene-5 treatment of A6 cells blocked the osmotic cell stretch response of A6 cells, indicating that stretch activation of Nox-derived ROS plays an important role in ENaC regulation. Together, these findings indicate that Fulvene-5, and perhaps other classes of antioxidant inhibitors, may represent a novel class of compounds useful for the treatment of pathological disorders stemming from inappropriate ion channel activity, such as hypertension.

Compartmentalization of ROS-mediated signal transduction

The localization of signaling molecules close to their targets is the central principle of cell signaling. The colocalization of multicomponent signaling complexes is realized through protein scaffolds that provide better specificity than undirected diffusion ofthe same components. ROS-generating complexes have been suggested to follow this principle by specific intracellular localization of ROS production and the limitation of ROS diffusion distances. However, the lack of adequate methods did not allow direct detection of local ROS production to confirm the model ofredox signaling compartmentalization. Nevertheless, evidences of local ROS production and restriction of diffusion were provided by kinetic modeling and data on the subcellular localization of NADPH-oxidase isoforms, their adapter proteins and local restriction of ROS diffusion. Here we shall discuss the properties of antioxidant system which prevents uncontrolled ROS diffusion from the sites of generation to the adjacent subcellular compartments; the current data of the specific localization NADPH-oxidases activity and its influence on intracellular processes; the recent evidences of the ROS diffusion restriction.

The antioxidant silybin prevents high glucose-induced oxidative stress and podocyte injury in vitro and in vivo

Podocyte injury, a major contributor to the pathogenesis of diabetic nephropathy, is caused at least in part by the excessive generation of reactive oxygen species (ROS). Overproduction of superoxide by the NADPH oxidase isoform Nox4 plays an important role in podocyte injury. The plant extract silymarin is attributed antioxidant and antiproteinuric effects in humans and in animal models of diabetic nephropathy. We investigated the effect of silybin, the active constituent of silymarin, in cultures of mouse podocytes and in the OVE26 mouse, a model of type 1 diabetes mellitus and diabetic nephropathy. Exposure of podocytes to high glucose (HG) increased 60% the intracellular superoxide production, 90% the NADPH oxidase activity, 100% the Nox4 expression, and 150% the number of apoptotic cells, effects that were completely blocked by 10 μM silybin. These in vitro observations were confirmed by similar in vivo findings. The kidney cortex of vehicle-treated control OVE26 mice displayed greater Nox4 expression and twice as much superoxide production than cortex of silybin-treated mice. The glomeruli of control OVE26 mice displayed 35% podocyte drop out that was not present in the silybin-treated mice. Finally, the OVE26 mice experienced 54% more pronounced albuminuria than the silybin-treated animals. In conclusion, this study demonstrates a protective effect of silybin against HG-induced podocyte injury and extends this finding to an animal model of diabetic nephropathy.

Opportunity “Nox”: A Novel Approach to Preventing Endothelial Dysfunction in the Context of Insulin Resistance

Cardiovascular complications are more prevalent in individuals with conditions associated with insulin resistance. Knowledge is evolving concerning the mechanistic link between insulin resistance and vascular disease, but complete clarity does not exist. This is a high-priority area of investigation because individual and societal costs associated with diabetic vascular complications are increasing, and the need to elucidate effective therapeutic targets and intervention strategies with minimal off-target side effects is urgent. Insulin-resistant conditions such as type 2 diabetes and diet-induced obesity are associated with an altered endothelial cell phenotype, i.e., endothelial dysfunction. A crucial component of endothelial cell dysfunction is reduced nitric oxide (NO) bioavailability. Reduced NO bioavailability contributes importantly to pathologies specific to large (atherosclerosis, cardiomyopathy) and small (retinopathy, nephropathy, neuropathy) blood vessels (1). Oxidative stress, hyperglycemia, lipotoxicity, activation of the renin-angiotensin system, and increased proinflammatory cytokines are systemic disturbances in patients with conditions associated with insulin resistance. Each of these factors contributes independently and synergistically to decreasing NO bioavailability by impairing its synthesis and/or by enhancing its degradation (2). In this issue of Diabetes , Sukumar et al. (3) demonstrate that endothelial cell–specific insulin resistance increases NADPH oxidase (Nox) isoform 2 (Nox2) expression to an extent that elevates superoxide anion (O2·−) production and precipitates endothelial dysfunction. Reactive oxygen species (ROS) are highly reactive metabolites of oxygen that participate in oxidation-reduction reactions (4). A traditional definition of oxidative stress is when ROS accumulation overwhelms the cellular antioxidant defense mechanisms and the redox state of the biological compartment is shifted toward one that is more oxidizing (5,6). Cellular sources of ROS include NADPH-dependent oxidases, xanthine oxidase, lipoxygenases, mitochondrial oxidases, and NO synthases (7). Nox is a major source of O2·− in insulin-resistant humans (8,9) and …

Differential roles of NADPH oxidases and associated regulators in polarized growth, conidiation and hyphal fusion in the symbiotic fungus Epichloë festucae

The endophytic fungus Epichloë festucae systemically colonizes the intercellular spaces of temperate grasses to establish mutualistic symbiotic associations. We have previously shown that reactive oxygen species produced by a specific NADPH oxidase isoform, NoxA, and associated regulators, NoxR and RacA, have a critical role in regulating hyphal growth in the host plant to maintain a mutualistic symbiotic interaction. We also identified BemA and Cdc24, homologues of polarity establishment proteins of yeast, as interactors of NoxR. In this study, we investigated culture developmental phenotypes of ‘knockout’ mutants of noxA and noxB and their associated regulators, noxR, racA and bemA. On nutrient-rich medium, all of the mutants except racA, which had undulating hyphae, hyphal swellings and increased branching, had a colony growth phenotype similar to the wild type strain. In contrast, on water agar, noxA, noxR and bemA mutants had disorganized hyphal growth and distorted instead of straight hyphae. These changes in hyphal growth characteristics indicate that NoxA and associated regulators have a crucial role in polarized growth under conditions of nutrient starvation. Conidiation in the noxA mutant was greater than wild type, and further enhanced in the noxA/noxB double mutant suggesting ROS negatively regulates asexual development. In contrast, deletion of noxR had no effect on conidiation. Hyphae of the wild type and noxB mutant of E. festucae had frequent vegetative hyphal fusions, whereas noxA, noxR and racA mutants totally lost this ability and fusions in the bemA mutant were significantly reduced. These results indicate that NoxA, NoxB and their associated regulators have distinct or overlapping functions for the regulation of different hyphal morphogenesis processes.

171 NOX2-DERIVED REACTIVE OXYGEN SPECIES CAUSES VASCULAR DYSFUNCTION IN MURINE MODEL OF ENDOTHELIAL INSULIN SENSITIVITY AND ACTIVATION OF NRF2 TRANSCRIPTION FACTOR

<h3>Introduction</h3> The escalating number of individuals suffering from Type 2 Diabetes is a significant healthcare burden, globally. A critical pathophysiological feature of type 2 diabetes is insulin resistance. It is well-established that insulin stimulates generation of the endothelium-derived anti-inflammatory/anti-atherosclerotic signalling radical, nitric oxide (NO). We investigated the hypothesis that increasing insulin sensitivity specifically, in the endothelium will lead to beneficial effects on NO bioavailability and vascular endothelial function. A novel transgenic mouse over-expressing Type A human Insulin Receptor (HIRECO) in the endothelium, driven by the Tie-2 promoter-enhancer was generated in order to explore the effects of increasing insulin signalling in the vascular bed. <h3>Methods</h3> Various tissues and pulmonary endothelial cells from the HIRECO mice were analysed using RT-PCR to confirm significant levels of human insulin receptor mRNA, while protein expression was confirmed by western blotting. Lucigenin-enhanced chemiluminescence was used to measure superoxide anion levels while; vasomotor function was assessed in thoracic aortic rings mounted in an organ bath. <h3>Results</h3> HIRECO mice demonstrated no significant morphological, metabolic phenotype or blood pressure abnormality compared to wild type (WT) littermates. Plasma insulin levels were similar following an overnight fast, but were decreased in the HIRECO after a glucose challenge. HIRECO mice exhibited significant endothelial dysfunction with a blunted response to acetylcholine (Emax, WT vs. HIRECO: 84±3% vs. 68±3% respectively, p&lt;0.05). The impaired aortic response to acetylcholine was normalized by the NADPH oxidase inhibitor peptide, gp91ds-tat, (Emax: 93±5%; p&lt;0.05), as well as the superoxide dismutase mimetic, MnTmPyP. HIRECO mice had a 1.65-fold increase in the level of superoxide anion production compared to WT. Basal NO bioactivity was decreased in HIRECO compared to WT littermates (Emax upon exposure to eNOS inhibitor, L-NAME in phenylephrine-constricted aorta, WT vs. HIRECO: 144±27.9% vs. 32±33%, n=5, p&lt;0.05). However, basal eNOS and Akt phosphorylation in isolated endothelial cells of HIRECO mice was enhanced 1.56 fold compared to WT littermates. Additionally, increased expressions of NADPH oxidase isoform, Nox2 and redox-sensitive transcription factor, Nrf2 were detected in HIRECO endothelial cells. <h3>Conclusions/Implications</h3> These data clearly suggest that enhanced oxidative stress in a novel murine model of increased endothelial insulin signalling, leads to reduced bioavailability of nitric oxide and vascular dysfunction. These data also demonstrate for the first time, that increased insulin sensitivity in the endothelium, increases the generation of free radical generation and reduces NO bioavailability.

Monocyte- and Macrophage-Targeted NADPH Oxidase Mediates Antifungal Host Defense and Regulation of Acute Inflammation in Mice

Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was >100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and proinflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate β-glucans, whereas inflammation in transgenic and wild-type mice was mild and transient. Taken together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation.

Abstract 4124: The Antioxidant heme oxygenase 1 promotes proliferation and survival of Flt3-ITD-positive AML.

Abstract Acute myelogenous leukemia (AML) afflicts ∼12,330 new patients per year in the United States. Regrettably, only 25% of patients will survive five years past diagnosis. The most common mutation in AML is internal tandem duplication (ITD) of the juxtamembrane domain of the fms-like tyrosine kinase receptor-3 (Flt3), which renders it constitutively active. Flt3-ITD regulates proliferation and survival, and also increases the production of reactive oxygen species (ROS), which act as secondary messengers for oncogenic signaling. ROS can cause the induction of a number of molecules, however, one protein, heme oxygenase 1 (HO-1), a well-known antioxidant, has been connected to both proliferation and drug resistance of various cancers. We hypothesized that Flt3-ITD-dependent signaling and ROS production increase constitutive expression of HO-1, leading to the activation of antioxidant and anti-apoptotic pathways, resulting in proliferation and drug resistance in AML. Western blotting revealed a two-fold increase of HO-1 protein in Flt3-ITD+ cells as compared to Flt3-WT; a four-fold up-regulation of HO-1 mRNA was noted by quantitative real-time PCR suggesting transcriptional control. To determine if this up-regulation was due to the altered redox status of Flt3-ITD+ cells, the flavonoid inhibitor diphenylene iodonium (DPI) was used. Consistent with published results implicating the flavonoid protein complex NADPH oxidase (NOX) as a primary source of ROS in these cells, DPI reduced ROS levels as early as two hours post treatment. This ROS reduction coincided with a decrease in HO-1 protein, suggesting that NOX may be involved in HO-1 up-regulation. Our previous results in chronic myeloid leukemia suggest that a Rac1-dependent isoform of NOX controls HO-1 expression, however, in Flt3-ITD+AML, dominant negative inhibition of Rac1 was insufficient to alter HO-1 expression suggesting that either a Rac1-independent NOX isoform is involved or that another flavonoid protein modulates HO-1 expression in this leukemia subtype. Interestingly, when HO-1 expression was knocked down using RNAi, there was a 50% reduction of proliferation and 40% reduction of viability as measured by trypan blue exclusion in Flt3-ITD+AML cells. These data suggest that the function of HO-1 up-regulation is to promote survival and proliferation of Flt3-ITD+AML. Additionally, we have created a model of acquired resistance to lestaurtinib, a Flt3 kinase inhibitor, by treating with increasing doses of the drug over time. Preliminary results suggest that HO-1 is further elevated in these resistant cells as compared to parental Flt3-ITD cells; thus, we will use this model to test the functional role of HO-1 in acquired resistance. Together, our data suggest that HO-1 is a growth and survival factor in Flt3-ITD+AML; therefore, targeting HO-1 or the mechanisms that control its expression may prove therapeutically valuable. Citation Format: Mary E. Irwin, Joya Chandra. The Antioxidant heme oxygenase 1 promotes proliferation and survival of Flt3-ITD-positive AML. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4124. doi:10.1158/1538-7445.AM2013-4124

Nox1 Oxidase Suppresses Influenza A Virus-Induced Lung Inflammation and Oxidative Stress

Influenza A virus infection is an ongoing clinical problem and thus, there is an urgent need to understand the mechanisms that regulate the lung inflammation in order to unravel novel generic pharmacological strategies. Evidence indicates that the Nox2-containing NADPH oxidase enzyme promotes influenza A virus-induced lung oxidative stress, inflammation and dysfunction via ROS generation. In addition, lung epithelial and endothelial cells express the Nox1 isoform of NADPH oxidase, placing this enzyme at key sites to regulate influenza A virus-induced lung inflammation. The aim of this study was to investigate whether Nox1 oxidase regulates the inflammatory response and the oxidative stress to influenza infection in vivo in mice. Male WT and Nox1-deficient (Nox1−/y) mice were infected with the moderately pathogenic HkX-31 (H3N2, 1×104 PFU) influenza A virus for analysis of bodyweight, airways inflammation, oxidative stress, viral titre, lung histopathology, and cytokine/chemokine expression at 3 and 7 days post infection. HkX-31 virus infection of Nox1−/y mice resulted in significantly greater: loss of bodyweight (Day 3); BALF neutrophilia, peri-bronchial, peri-vascular and alveolar inflammation; Nox2-dependent inflammatory cell ROS production and peri-bronchial, epithelial and endothelial oxidative stress. The expression of pro-inflammatory cytokines including CCL2, CCL3, CXCL2, IL-1β, IL-6, GM-CSF and TNF-α was higher in Nox1−/y lungs compared to WT mice at Day 3, however, the expression of CCL2, CCL3, CXCL2, IFN-γ and the anti-inflammatory cytokine IL-10 were lower in lungs of Nox1−/y mice vs. WT mice at Day 7. Lung viral titre, and airways infiltration of active CD8+ and CD4+ T lymphocytes, and of Tregs were similar between WT and Nox1−/y mice. In conclusion, Nox1 oxidase suppresses influenza A virus induced lung inflammation and oxidative stress in mice particularly at the early phases of the infection. Nox1 and Nox2 oxidases appear to have opposing roles in the regulation of inflammation caused by influenza A viruses.

Superoxide and hydrogen peroxide stimulate renin release from juxtaglomerular cells

Renin is the rate‐limiting enzyme in angiotensin formation, playing a pivotal role in blood pressure regulation. In the kidney, superoxide and hydrogen peroxide (H202) are involved in the development of hypertension by affecting nephron transport and vascular reactivity. However, it is not known whether superoxide and H202 are endogenously produced in juxtaglomerular (JG) cells, the source of these reactive oxygen species and whether they regulate renin release. We hypothesized that superoxide and H2O2 are endogenously produced in JG cells and that they stimulate renin release. Primary cultures of mouse JG cells were treated for 60 min with different inhibitors and renin released to the supernatant measured by radioimmunoassay. Decreasing endogenous superoxide with superoxide dismutase (300U/ml) inhibited renin release by 35±10% (p&lt;0.03). By Western Blot we found that NOX4, one of the NADPH oxidase isoforms is present in JG lysates (n=3). Apocynin (120 μM), a general inhibitor of NADPH oxidases, decreased renin release by 28±7% (p&lt;0.04). Since dismutation of superoxide results in H202 accumulation, we tested the effect of scavenging endogenous H202. Adding catalase (100U/ml) inhibited renin release by 37±10% (p&lt;0.02). We concluded that endogenous superoxide and H202 stimulate renin release from mouse JG cells. These are the first data identifying endogenous sources of superoxide in JG cells. HFHS Award.