Nox2-containing NADPH Oxidase Research Articles

P47phox-Dependent Oxidant Signalling through ASK1, MKK3/6 and MAPKs in Angiotensin II-Induced Cardiac Hypertrophy and Apoptosis.

The p47phox is a key regulatory subunit of Nox2-containing NADPH oxidase (Nox2) that by generating reactive oxygen species (ROS) plays an important role in Angiotensin II (AngII)-induced cardiac hypertrophy and heart failure. However, the signalling pathways of p47phox in the heart remains unclear. In this study, we used wild-type (WT) and p47phox knockout (KO) mice (C57BL/6, male, 7-month-old, n = 9) to investigate p47phox-dependent oxidant-signalling in AngII infusion (0.8 mg/kg/day, 14 days)-induced cardiac hypertrophy and cardiomyocyte apoptosis. AngII infusion resulted in remarkable high blood pressure and cardiac hypertrophy in WT mice. However, these AngII-induced pathological changes were significantly reduced in p47phox KO mice. In WT hearts, AngII infusion increased significantly the levels of superoxide production, the expressions of Nox subunits, the expression of PKCα and C-Src and the activation of ASK1 (apoptosis signal-regulating kinase 1), MKK3/6, ERK1/2, p38 MAPK and JNK signalling pathways together with an elevated expression of apoptotic markers, i.e., γH2AX and p53 in the cardiomyocytes. However, in the absence of p47phox, although PKCα expression was increased in the hearts after AngII infusion, there was no significant activation of ASK1, MKK3/6 and MAPKs signalling pathways and no increase in apoptosis biomarker expression in cardiomyocytes. In conclusion, p47phox-dependent redox-signalling through ASK1, MKK3/6 and MAPKs plays a crucial role in AngII-induced cardiac hypertrophy and cardiomyocyte apoptosis.

Atrial nitroso-redox balance and refractoriness following on-pump cardiac surgery: a randomized trial of atorvastatin.

AimsSystemic inflammation and increased activity of atrial NOX2-containing NADPH oxidases have been associated with the new onset of atrial fibrillation (AF) after cardiac surgery. In addition to lowering LDL-cholesterol, statins exert rapid anti-inflammatory and antioxidant effects, the clinical significance of which remains controversial.Methods and resultsWe first assessed the impact of cardiac surgery and cardiopulmonary bypass (CPB) on atrial nitroso-redox balance by measuring NO synthase (NOS) and GTP cyclohydrolase-1 (GCH-1) activity, biopterin content, and superoxide production in paired samples of the right atrial appendage obtained before (PRE) and after CPB and reperfusion (POST) in 116 patients. The effect of perioperative treatment with atorvastatin (80 mg once daily) on these parameters, blood biomarkers, and the post-operative atrial effective refractory period (AERP) was then evaluated in a randomized, double-blind, placebo-controlled study in 80 patients undergoing cardiac surgery on CPB. CPB and reperfusion led to a significant increase in atrial superoxide production (74% CI 71–76%, n = 46 paired samples, P < 0.0001) and a reduction in atrial tetrahydrobiopterin (BH4) (34% CI 33–35%, n = 36 paired samples, P < 0.01), and in GCH-1 (56% CI 55–58%, n = 26 paired samples, P < 0.001) and NOS activity (58% CI 52–67%, n = 20 paired samples, P < 0.001). Perioperative atorvastatin treatment prevented the effect of CPB and reperfusion on all parameters but had no significant effect on the postoperative right AERP, troponin release, or NT-proBNP after cardiac surgery.ConclusionPerioperative statin therapy prevents post-reperfusion atrial nitroso-redox imbalance in patients undergoing on-pump cardiac surgery but has no significant impact on postoperative atrial refractoriness, perioperative myocardial injury, or markers of postoperative LV function.Clinical Trial Registration https://clinicaltrials.gov/ct2/show/NCT01780740

Building a Career in Cardiovascular Research: A Conversation With Barbara Casadei, MD, DPhil.

Building a Career in Cardiovascular Research: A Conversation With Barbara Casadei, MD, DPhil.

Icariin ameliorates angiotensin II-induced cerebrovascular remodeling by inhibiting Nox2-containing NADPH oxidase activation.

Cerebrovascular smooth muscle cells (SMCs) hyperplasia is an important contributor to cerebrovascular remodeling during hypertension. The aim of present study was to investigate the effects of Icariin on cerebrovascular SMCs proliferation and remodeling and the underlying mechanisms. The results revealed that Icariin administration attenuated the enhanced basilar artery constriction in angiotensin II (AngII)-induced hypertension rat model, as well as the inhibition of basilar artery diameter reduction in response to AngII and phenylephrine. In addition, histological analyses showed that Icariin also significantly ameliorated basilar artery remodeling in AngII hypertensive rats. In human brain vascular SMCs (HBVSMCs), AngII-induced cell proliferation, migration and invasion were markedly inhibited by Icariin treatment. Moreover, Icariin treatment largely limited AngII-induced the increase of reactive oxygen species (ROS) production in HBVSMCs, which was closely associated with cell proliferation. Analysis of the mechanisms showed that Icariin decreased ROS production via inhibiting NADPH oxidase activity but not mitochondria-derived ROS production. Further, Icariin promoted Nox2 degradation and consequently reduced its protein expression. In conclusion, these findings demonstrate that Icariin attenuates cerebrovascular SMCs hyperplasia and subsequent remodeling through inhibiting Nox2-containing NADPH oxidase activation, suggesting Icariin may be a potential therapeutic agent to prevent the onset and progression of stroke.

Targeting reactive oxygen species for respiratory infection: Fact or fancy?

Targeting reactive oxygen species for respiratory infection: Fact or fancy?

Abstract 054: Genetic Deficiency in Nox2-containing NADPH Oxidase Decreases Susceptibility to Venous Thrombosis in Mice

Activated neutrophils release neutrophil extracellular traps (NETs) via a pathway dependent on reactive oxygen species (ROS). NETs are enriched in extracellular DNA and histones, and contribute to venous thrombosis. Nox2-containing NADPH oxidase is a major upstream mediator of ROS-dependent release of NETs. Neutrophils from mice deficient in Nox2 or patients with X-linked chronic granulomatous disease due to Nox2 deficiency do not produce NETs, suggesting a role for Nox2 in venous thrombosis. We hypothesized that mice genetically deficient in Nox2 are protected from venous thrombosis. We studied male and female mice deficient in Nox2 (Nox2-KO) and their wild type (WT) littermates at 10-14 weeks of age (n=7-13/group). Using electron spin resonance and H2C-DCFH-DA staining, we confirmed that neutrophils from Nox2-KO mice do not produce ROS at baseline or upon PMA-activation. Neutrophils from WT mice displayed a dose-dependent release of NETs upon PMA-stimulation (SYTOX green staining using fluorescence microscopy), whereas Nox2-KO mice did not produce NETs. We next examined susceptibility to venous thrombosis in a stasis model of inferior vena cava (IVC) ligation. After 48 hours of ligation, both male and female Nox2-KO mice developed significantly smaller thrombi compared with WT mice (P&lt;0.05). Markers for NETs were measured in plasma (nucleosome levels using ELISA) and venous thrombi (staining for citrullinated histones, Cit-H3). Levels of nucleosomes in the mice that did not develop thrombi were similar to those in baseline samples collected before IVC ligation. All of the mice that developed thrombi had significant elevation of plasma nucleosome levels compared with mice that did not develop thrombi (P&lt;0.05), but nucleosome levels did not differ between WT and Nox2-KO mice. Likewise, the heterogeneous pattern of NET formation within thrombi detected by Cit-H3 staining was similar in WT and Nox2-KO mice. These findings suggest that Nox2 is an important determinant of thrombus size during IVC ligation. Interestingly, our study also provides evidence for Nox2-independent pathways for NET release during venous thrombosis.

Abstract 305: Genetic Deficiency in Nox2-containing NADPH Oxidase Decreases Susceptibility to Venous Thrombosis in Mice

Activated neutrophils release neutrophil extracellular traps (NETs) via a pathway dependent on reactive oxygen species (ROS). NETs are enriched in extracellular DNA and histones, and contribute to venous thrombosis. Nox2-containing NADPH oxidase is a major upstream mediator of ROS-dependent release of NETs. Neutrophils from mice deficient in Nox2 or patients with X-linked chronic granulomatous disease due to Nox2 deficiency do not produce NETs, suggesting a role for Nox2 in venous thrombosis. We hypothesized that mice genetically deficient in Nox2 are protected from venous thrombosis. We studied male and female mice deficient in Nox2 (Nox2-KO) and their wild type (WT) littermates at 10-14 weeks of age (n=7-13/group). Using electron spin resonance and H2C-DCFH-DA staining, we confirmed that neutrophils from Nox2-KO mice do not produce ROS at baseline or upon PMA-activation. Neutrophils from WT mice displayed a dose-dependent release of NETs upon PMA-stimulation (SYTOX green staining using fluorescence microscopy), whereas Nox2-KO mice did not produce NETs. We next examined susceptibility to venous thrombosis in a stasis model of inferior vena cava (IVC) ligation. After 48 hours of ligation, both male and female Nox2-KO mice developed significantly smaller thrombi compared with WT mice (P&lt;0.05). Markers for NETs were measured in plasma (nucleosome levels using ELISA) and venous thrombi (staining for citrullinated histones, Cit-H3). Levels of nucleosomes in the mice that did not develop thrombi were similar to those in baseline samples collected before IVC ligation. All of the mice that developed thrombi had significant elevation of plasma nucleosome levels compared with mice that did not develop thrombi (P&lt;0.05), but nucleosome levels did not differ between WT and Nox2-KO mice. Likewise, the heterogeneous pattern of NET formation within thrombi detected by Cit-H3 staining was similar in WT and Nox2-KO mice. These findings suggest that Nox2 is an important determinant of thrombus size during IVC ligation. Interestingly, our study also provides evidence for Nox2-independent pathways for NET release during venous thrombosis.

153 Metabolic disorder-induced global nox2 activation and endothelial dysfunction in ageing

Ageing is an independent risk factor of cardiovascular diseases which involves oxidative stress derived from a Nox2-containing NADPH oxidase. However, the mechanism of endothelial Nox2 activation in normal ageing process...

218 Knockout p47phox reduces angiotensin ii-induced cardiac oxidative stress and hypertrophy

Oxidative stress due to the activation of a Nox2-containing NADPH oxidase is involved in Angiotensin II (AngII)-induced cardiovascular dysfunction. p47phox is a key regulatory subunit of Nox2. However, the role...

Brain Perivascular Macrophages Initiate the Neurovascular Dysfunction of Alzheimer Aβ Peptides.

Increasing evidence indicates that alterations of the cerebral microcirculation may play a role in Alzheimer disease, the leading cause of late-life dementia. The amyloid-β peptide (Aβ), a key pathogenic factor in Alzheimer disease, induces profound alterations in neurovascular regulation through the innate immunity receptor CD36 (cluster of differentiation 36), which, in turn, activates a Nox2-containing NADPH oxidase, leading to cerebrovascular oxidative stress. Brain perivascular macrophages (PVM) located in the perivascular space, a major site of brain Aβ collection and clearance, are juxtaposed to the wall of intracerebral resistance vessels and are a powerful source of reactive oxygen species. We tested the hypothesis that PVM are the main source of reactive oxygen species responsible for the cerebrovascular actions of Aβ and that CD36 and Nox2 in PVM are the molecular substrates of the effect. Selective depletion of PVM using intracerebroventricular injection of clodronate abrogates the reactive oxygen species production and cerebrovascular dysfunction induced by Aβ applied directly to the cerebral cortex, administered intravascularly, or overproduced in the brain of transgenic mice expressing mutated forms of the amyloid precursor protein (Tg2576 mice). In addition, using bone marrow chimeras, we demonstrate that PVM are the cells expressing CD36 and Nox2 responsible for the dysfunction. Thus, deletion of CD36 or Nox2 from PVM abrogates the deleterious vascular effects of Aβ, whereas wild-type PVM reconstitute the vascular dysfunction in CD36-null mice. The data identify PVM as a previously unrecognized effector of the damaging neurovascular actions of Aβ and unveil a new mechanism by which brain-resident innate immune cells and their receptors may contribute to the pathobiology of Alzheimer disease.

Aging-associated metabolic disorder induces Nox2 activation and oxidative damage of endothelial function

Oxidative stress attributable to the activation of a Nox2-containing NADPH oxidase is involved in the development of vascular diseases and in aging. However, the mechanism of Nox2 activation in normal aging remains unclear. In this study, we used age-matched wild-type (WT) and Nox2 knockout (KO) mice at 3–4 months (young); 11–12 months (middle-aged) and 21–22 months (aging) to investigate age-related metabolic disorders, Nox2 activation and endothelial dysfunction. Compared to young mice, middle-aged and aging WT mice had significant hyperglycaemia, hyperinsulinaemia, increased systemic oxidative stress and higher blood pressure. Endothelium-dependent vessel relaxation to acetylcholine was significantly impaired in WT aging aortas, and this was accompanied by increased Nox2 and ICAM-1 expressions, MAPK activation and decreased insulin receptor expression and signaling. However, these aging-associated disorders were significantly reduced or absent in Nox2KO aging mice. The effect of metabolic disorder on Nox2 activation and endothelial dysfunction was further confirmed using high-fat diet-induced obesity and insulin resistance in middle-aged WT mice treated with apocynin (a Nox2 inhibitor). In vitro experiments showed that in response to high glucose plus high insulin challenge, WT coronary microvascular endothelial cells increased significantly the levels of Nox2 expression, activation of stress signaling pathways and the cells were senescent, e.g. increased p53 and β–galactosidase activity. However, these changes were absent in Nox2KO cells. In conclusion, Nox2 activation in response to aging-associated hyperglycaemia and hyperinsulinaemia plays a key role in the oxidative damage of vascular function. Inhibition or knockout of Nox2 preserves endothelial function and improves global metabolism in old age.

Abstract 113: Glucose Metabolic Disorders in Aging-Associated Microglial NADPH Oxidase 2 Activation and Oxidative Damage of Cerebral Microvasculature and Neurons

Aging has been recognised to be a major risk factor for the development of cardiovascular and neurodegenerative diseases and growing evidence suggests a role for oxidative stress. A Nox2-containing NADPH oxidase has been reported to be a major source of reactive oxygen species (ROS) generation in the vascular system and in the brain. However, the role of Nox2 enzyme in aging-related metabolic disorders and vascular neurodegeneration remains unclear. In this study, we used age-matched wild-type (WT) and Nox2-deficient (Nox2 -/- ) mice on a C57BL/6 background at young (3-4 month) and aging (20-24 month) to investigate the role of Nox2 in aging-related oxidative stress, metabolic disorders and cerebral vascular dysfunction. There was an aging-related increase in blood pressure in WT mice (126 mmHg for young and 148 mmHg for aging) (P&lt;0.05); however the blood pressure was well maintained without significant change in Nox2 -/- aging mice. Compared to young WT mice, WT aging mice had significantly high levels of fasting serum insulin and this was accompanied with delayed clearance of glucose (P&lt;0.05) indicating insulin resistance. In contrast, there was no indication of insulin resistance for Nox2 -/- aging mice. We then examined aging-related brain oxidative stress. Compared to WT young mice, there were significant increases (2.7±0.7 folds) in the levels of ROS production by WT aging brain tissue homogenates as detected by lucigenin-chemiluminescence and DHE fluorescence. Increased ROS production in WT aging brain was accompanied by a significant increase (1.8±0.3 folds) in the Nox2 expression detected mainly in the microglial cells (labelled by Iba-1) and decreases in brain capillaries (labelled by CD31) (2.4±0.8 folds) and neurons (labelled by Neu-N) (2.9±0.5 folds) (all P&lt;0.05). Knockout of Nox2 abolished aging-associated increases in brain ROS production and significantly reduced the aging-related pathophysiological changes in the brain. In conclusion, aging-associated metabolic disorders play a crucial role in aging-associated Nox2 activation and vascular neurodegeneration. Nox2-containing NADPH oxidase represents a valuable therapeutic target for oxidative stress-related brain microvascular damage and neurodegeneration.

Abstract 362: Knockout of NADPH Oxidase 2 Improves Global Metabolism and Endothelial Function in Aging Mice

Oxidative stress attributable to the activation of a Nox2-containing NADPH oxidase has been suggested to play a crucial role in the development of aging-associated vascular diseases. However, the mechanism of endothelial Nox2 activation in normal aging process remains unclear. In this study, we investigated the therapeutic potential of targeting Nox2 in improving global metabolism and endothelial function at old age by using age-matched wild-type and Nox2 knockout mice at 3-4 months (young); 11-12 months (middle aged) and 21-22 months (aging). Compared to young mice, middle-aged and ageing wild-type mice had significantly higher blood pressure, hyperglycaemia, hyperinsulinaemia. These were accompanied by oxidative stress in multiple organs including the lung, the liver, the heart and the vessels. The vessel motor function was examined in an organ bath using aortas isolated from these mice. Endothelium-dependent vessel relaxation to acetylcholine was significantly impaired in aortas of wild-type aging mice, and this was accompanied by increased expressions of Nox2 and markers of inflammation, activation of MAPK and Akt and decreased insulin receptor expression and function. However, these aging-associated disorders in aortas were significantly reduced by knocking out Nox2 in mice. In response to high glucose plus high insulin challenge, coronary microvascular endothelial cells isolated from wild-type mice displayed significantly increased Nox2 expression, oxidative stress and cell senescence, e.g. increased p53 expression and β-galactosidase activity. However, these responses were absent or significantly reduced in the endothelial cells isolated from Nox2 knockout mice. In conclusion, metabolic disorders in particular hyperglycaemia and insulin resistance play an important role in mediating Nox2 activation and oxidative stress in multiple organs in aging. Nox2 is involved in normal aging process-associated vascular inflammation and oxidative damage of endothelial dysfunction.

TMEM16A Contributes to Endothelial Dysfunction by Facilitating Nox2 NADPH Oxidase-Derived Reactive Oxygen Species Generation in Hypertension.

Ca2+-activated Cl- channels play a crucial role in various physiological processes. However, the role of TMEM16A in vascular endothelial dysfunction during hypertension is unclear. In this study, we investigated the specific involvement of TMEM16A in regulating endothelial function and blood pressure and the underlying mechanism. Reverse transcription-polymerase chain reaction, Western blotting, coimmunoprecipitation, confocal imaging, patch-clamp recordings, and TMEM16A endothelial-specific transgenic and knockout mice were used. We found that TMEM16A was expressed abundantly and functioned as a Ca2+-activated Cl- channel in endothelial cells. Angiotensin II induced endothelial dysfunction with an increase in TMEM16A expression. The knockout of endothelial-specific TMEM16A significantly lowered the blood pressure and ameliorated endothelial dysfunction in angiotensin II-induced hypertension, whereas the overexpression of endothelial-specific TMEM16A resulted in the opposite effects. These results were related to the increased reactive oxygen species production, Nox2-containing NADPH oxidase activation, and Nox2 and p22phox protein expression that were facilitated by TMEM16A on angiotensin II-induced hypertensive challenge. Moreover, TMEM16A directly bound with Nox2 and reduced the degradation of Nox2 through the proteasome-dependent degradation pathway. Therefore, TMEM16A is a positive regulator of endothelial reactive oxygen species generation via Nox2-containing NADPH oxidase, which induces endothelial dysfunction and hypertension. Modification of TMEM16A may be a novel therapeutic strategy for endothelial dysfunction-associated diseases.

NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells.

NADPH oxidase-generated reactive oxygen species (ROS) from immune cells are well known to be important for pathogen killing in response to TLR ligands. Here, we investigated a new aspect of NADPH oxidase in the TLR2/6-induced release of the immunologically relevant GM-CSF by endothelial cells. Stimulation of human endothelial cells with TLR2/6 agonist, MALP-2 (macrophage-activating lipopeptide of 2 kDa), induced NADPH oxidase activation and ROS formation. Inhibition by ROS scavengers and NADPH oxidase inhibitors blocked MALP-2-induced GM-CSF release. NADPH oxidase activators or ROS donors alone did not result in GM-CSF secretion; however, additional superoxide supply augmented MALP-2-induced GM-CSF secretion and restored GM-CSF levels after NADPH oxidase inhibition. MALP-2-dependent NF-ĸB activation was suppressed by NADPH oxidase inhibition, and inhibition of NF-κB completely blunted MALP-2-induced GM-CSF release. Vascular explants from mice that were deficient for the NADPH oxidase subunit p47 phox showed diminished intimal superoxide production and GM-CSF release after ex vivo stimulation with MALP-2. Moreover, an increase in circulating progenitor cells after MALP-2 injection was completely abolished in p47phox-knockout mice. Finally, MALP-2 stimulation increased mRNA expression of the major subunit NADPH oxidase, (Nox)2, in endothelial cells, and Nox2 inhibition prevented MALP-2-induced GM-CSF release. Our findings identify a Nox2-containing NADPH oxidase as a crucial regulator of the immunologic important growth factor GM-CSF after TLR2/6 stimulation in endothelial cells.-Schuett, J., Schuett, H., Oberoi, R., Koch, A.-K., Pretzer, S., Luchtefeld, M., Schieffer, B., Grote, K. NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells.

NADPH oxidase 2 does not contribute to early reperfusion-associated reactive oxygen species generation following transient focal cerebral ischemia.

Excess production of reactive oxygen species (ROS) critically contributes to occurrence of reperfusion injury, the paradoxical response of ischemic brain tissue to restoration of cerebral blood flow. However, the enzymatic sources of ROS generation remain to be unclear. This study examined Nox2-containing NADPH oxidase (Nox2) expression and its activity in ischemic brain tissue following post-ischemic reperfusion to clarify the mechanism of enzymatic reaction of ROS. Male Sprague-Dawley rats were subjected to 90-minute middle cerebral artery occlusion, followed by 3 or 22.5 hours of reperfusion. Quantitative reverse transcriptase PCR and western blot assay were performed to measure mRNA and protein expression of Nox2. Lucigenin fluorescence assays were performed to assess Nox activity. Our data showed that Nox2 mRNA and protein expression levels were significantly increased (3.7-fold for mRNA and 3.6-fold for protein) in ischemic brain tissue at 22.5 hours but not at 3 hours following post-ischemic reperfusion. Similar results were obtained for the changes of NADPH oxidase activity in ischemic cerebral tissue at the two reperfusion time points. Our results suggest that Nox2 may not contribute to the early burst of reperfusion-related ROS generation, but is rather an important source of ROS generation during prolonged reperfusion.

Therapeutic inhibition of mitochondrial reactive oxygen species with mito-TEMPO reduces diabetic cardiomyopathy

AimsThe mitochondria are important sources of reactive oxygen species (ROS) in the heart. Mitochondrial ROS production has been implicated in the pathogenesis of diabetic cardiomyopathy, suggesting that therapeutic strategies specifically targeting mitochondrial ROS may have benefit in this disease. We investigated the therapeutic effects of mitochondria-targeted antioxidant mito-TEMPO on diabetic cardiomyopathy. MethodsThe mitochondria-targeted antioxidant mito-TEMPO was administrated after diabetes onset in a mouse model of streptozotocin-induced type-1 diabetes and type-2 diabetic db/db mice. Cardiac adverse changes were analyzed and myocardial function assessed. Cultured adult cardiomyocytes were stimulated with high glucose, and mitochondrial superoxide generation and cell death were measured. ResultsIncubation with high glucose increased mitochondria superoxide generation in cultured cardiomyocytes, which was prevented by mito-TEMPO. Co-incubation with mito-TEMPO abrogated high glucose-induced cell death. Mitochondrial ROS generation, and intracellular oxidative stress levels were induced in both type-1 and type-2 diabetic mouse hearts. Daily injection of mito-TEMPO for 30 days inhibited mitochondrial ROS generation, prevented intracellular oxidative stress levels, decreased apoptosis and reduced myocardial hypertrophy in diabetic hearts, leading to improvement of myocardial function in both type-1 and type-2 diabetic mice. Incubation with mito-TEMPO or inhibition of Nox2-containing NADPH oxidase prevented oxidative stress levels and cell death in high glucose-stimulated cardiomyocytes. Mechanistic study revealed that the protective effects of mito-TEMPO were associated with down-regulation of ERK1/2 phosphorylation. ConclusionsTherapeutic inhibition of mitochondrial ROS by mito-TEMPO reduced adverse cardiac changes and mitigated myocardial dysfunction in diabetic mice. Thus, mitochondria-targeted antioxidants may be an effective therapy for diabetic cardiac complications.

P47phox contributes to albuminuria and kidney fibrosis in mice

Reactive oxygen species (ROS) play an important pathogenic role in the development of many diseases, including kidney disease. Major ROS generators in the glomerulus of the kidney are the p47phox-containing NAPDH oxidases NOX1 and NOX2. The cytosolic p47phox subunit is a key regulator of the assembly and function of NOX1 and NOX2 and its expression and phosphorylation are up-regulated in the course of renal injury, and have been shown to exacerbate diabetic nephropathy. However, its role in non-diabetic-mediated glomerular injury is unclear. To address this, we subjected p47phox-null mice to either adriamycin-mediated or partial renal ablation-mediated glomerular injury. Deletion of p47phox protected the mice from albuminuria and glomerulosclerosis in both injury models. Integrin α1-null mice develop more severe glomerulosclerosis than wild type mice in response to glomerular injury mainly due to increased production of ROS. Interestingly, the protective effects of p47phox knockout were more profound in p47phox/integrin α1 double knockout mice. In vitro analysis of primary mesangial cells showed that deletion of p47phox led to reduced basal levels of superoxide and collagen IV production. Thus, p47phox-dependent NADPH oxidases are a major glomerular source of ROS, contribute to kidney injury, and are potential targets for antioxidant therapy in fibrotic disease.

NADPH OXIDASE ACTIVATION AND OXIDATIVE STRESS IN HIGH-FAT DIET-INDUCED HYPERTENSION AND METABOLIC DISORDERS

Dietary obesity is a major risk factor for the development of cardiovascular diseases. However, the mechanism of high fat diet (HFD)-induced vascular dysfunction remains unclear. In this study we investigated...

Chronic aldosterone administration causes Nox2-mediated increases in reactive oxygen species production and endothelial dysfunction in the cerebral circulation.

An elevated plasma aldosterone level is an independent cardiovascular risk factor. Although excess aldosterone promotes cardiovascular disease, no studies have examined the effect of increased plasma aldosterone on the cerebral circulation. A major source of vascular reactive oxygen species (ROS) during cardiovascular disease is the NADPH oxidases. Because Nox2-containing NADPH oxidase (Nox2 oxidase) is highly expressed in the cerebral endothelium, we postulated that it might contribute to ROS generation and vascular dysfunction in response to aldosterone. Here, we examined the effect of aldosterone and Nox2 oxidase on ROS production and endothelial dysfunction in the cerebral circulation, and whether the effects of aldosterone are exacerbated in aged mice. In adult (average age ∼24-25 weeks) wild-type and Nox2-deficient (Nox2(/y)) mice, neither vehicle nor aldosterone (0.28 mg/kg per day for 14 days) affected blood pressure (measured using tail-cuff). By contrast, aldosterone treatment reduced dilation of the basilar artery (measured using myography) to the endothelium-dependent agonist acetylcholine in wild-type mice (P < 0.05), but had no such effect in Nox2(/y) mice (P > 0.05). Aldosterone increased basal and phorbol dibutyrate-stimulated superoxide production (measured using L-012-enhanced chemiluminesence) in cerebral arteries from wild-type but not from Nox2(/y) mice. In aged wild-type mice (average age ∼70 weeks), aldosterone treatment increased blood pressure, but had a similar effect on cerebral artery superoxide levels as in adult wild-type mice. These data indicate that Nox2 oxidase mediates aldosterone-induced increases in ROS production and endothelial dysfunction in cerebral arteries from adult mice independently of blood pressure changes. Aldosterone-induced hypertension is augmented during aging.