Reduced NADPH Oxidase Activity Research Articles

Sesamin Ameliorates Advanced Glycation End Products-Induced Pancreatic β-Cell Dysfunction and Apoptosis.

Advanced glycation end products (AGEs), the direct modulators of β-cells, have been shown to cause insulin-producing β-cell dysfunction and apoptosis through increase of intracellular reactive oxygen species (ROS) production. Sesamin has been demonstrated to possess antioxidative activity. This study was designed to investigate whether sesamin protects against AGEs-evoked β-cell damage via its antioxidant property. The effects of sesamin were examined in C57BL/6J mice and MIN6 cell line. In in vivo studies, mice were intraperitoneally injected with AGEs (120 mg/kg) and orally treated with sesamin (160 mg/kg) for four weeks. Intraperitoneal glucose tolerance and insulin releasing tests were performed. Insulin content, ROS generation and β-cell apoptosis in pancreatic islets were also measured. In in vitro studies, MIN6 cells were pretreated with sesamin (50 or 100 μM) and then exposed to AGEs (200 mg/L) for 24 h. Insulin secretion, β-cell death, ROS production as well as expression and activity of NADPH oxidase were determined. Sesamin treatment obviously ameliorated AGE-induced β-cell dysfunction and apoptosis both in vivo and in vitro. These effects were associated with decreased ROS production, down-regulated expression of p67phox and p22phox, and reduced NADPH oxidase activity. These results suggest that sesamin protects β-cells from damage caused by AGEs through suppressing NADPH oxidase-mediated oxidative stress.

Abstract 526: The Influence of Endothelium on Smooth Muscle-Derived α1-Integrin, ROS and Collagen IV Expression: Effects of High Glucose and Diabetes

The abnormal metabolism in diabetes facilitates the development of atherosclerotic cardiovascular disease. Consequently, can HUVECs (human-umbilical-vascular-endothelial-cells) transiently primed by high glucose (HG) or diabetic serum (DM) change HuVSMCs (Human-Vascular-Smooth- Muscle-Cells)-derived α1-integrin, ROS (Reactive Oxygen Species) and collagen IV expression? Is there a relationship between HuVSMCs-created α1-integrin expression, ROS production and collagen IV accumulation? Conditioned medium (CM) from HUVECs, transiently primed by HG (CM HG ) or DM (CM DM ), was added to HuVSMCs. The expression of HuVSMCs-derived α1-integrin was measured with an in situ ELISA and the quantity of HuVSMCs-produced ROS was evaluated applying dihydro-rhodamine. The accumulation of HuVSMCs-secreted collagen IV was estimated in the medium by an ELISA. The isolated effect of reduced HuVSMCs-derived α1-integrin expression was elucidated by knock-down using siRNA whereas the impact of NADPH oxidase on HuVSMCs-derived ROS production was estimated using NADPH inhibition (Diphenyleneiodonium(DPI)). CM HG and CM DM reduced HuVSMCs-derived α1-intergin expression while collagen IV and ROS production was increased. Immunopurified HSP90α from CM HG gave similar results whereas treatment with recombinant HSP90α has no impact. However, knock-down of α1-intergin increased HuVSMCs-derived collagen IV and ROS accumulation whereas reduced NADPH oxidase activity decreased ROS and collagen IV production but increased α1-intergin expression. Previously, we showed that CM HG has increased pHSP90α (phosphorylated HSP90α) and now our observations suggest that HUVECs-derived pHSP90α down-regulate the expression of HuVSMCs-derived α1-integrin. The reduced presences of α1-integrin increased ROS and collagen IV expression from HuVSMCs. Consequently, HG and DM might via HUVECs-secreted pHSP90α act as potent modifiers of injury-induced changes within the arterial wall in diabetes.

The probiotic Lactobacillus coryniformis CECT5711 reduces the vascular pro-oxidant and pro-inflammatory status in obese mice

Obesity is associated with intestine dysbiosis and is characterized by a low-grade inflammatory status, which affects vascular function. In the present study, we evaluated the effects of a probiotic with immunomodulatory properties, Lactobacillus coryniformis CECT5711, in obese mice fed on an HFD (high-fat diet). The probiotic treatment was given for 12 weeks, and it did not affect the weight evolution, although it reduced basal glycaemia and insulin resistance. L. coryniformis administration to HFD-induced obese mice induced marked changes in microbiota composition and reduced the metabolic endotoxaemia as it decreased the LPS (lipopolysaccharide) plasma level, which was associated with a significant improvement in gut barrier disruption. Furthermore, it lowered TNFα (tumour necrosis factor α) expression in liver, improving the inflammatory status, and thus the glucose metabolism. Additionally, the probiotic reversed the endothelial dysfunction observed in obese mice when endothelium- and NO (nitric oxide)-dependent vasodilatation induced by acetylcholine in aortic rings was studied. It also restored the increased vessel superoxide levels observed in obese mice, by reducing NADPH oxidase activity and increasing antioxidant enzymes. Moreover, chronic probiotic administration for 2 weeks also improved endothelial dysfunction and vascular oxidative stress induced by in vivo administration of LPS in control mice fed on a standard chow diet. The results of the present study demonstrate an endothelial-protective effect of L. coryniformis CECT5711 in obese mice by increasing NO bioavailability, suggesting the therapeutic potential of this gut microbiota manipulation to prevent vasculopathy in obesity.

Protection of NAD(P)H:quinone oxidoreductase 1 against renal ischemia/reperfusion injury in mice

Ischemia/reperfusion (I/R) is the most common cause of acute renal injury. I/R-induced reactive oxygen species (ROS) are thought to be a major factor in the development of acute renal injury by promoting the initial tubular damage. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a well-known antioxidant protein that regulates ROS generation. The purpose of this study was to investigate whether NQO1 modulates the renal I/R injury (IRI) associated with NADPH oxidase (NOX)-derived ROS production in an animal model. We analyzed renal function, oxidative stress, and tubular apoptosis after IRI. NQO1−/− mice showed increased blood urea nitrogen and creatinine levels, tubular damage, oxidative stress, and apoptosis. In the kidneys of NQO1−/− mice, the cellular NADPH/NADP+ ratio was significantly higher and NOX activity was markedly higher than in those of NQO1+/+ mice. The activation of NQO1 by β-lapachone (βL) significantly improved renal dysfunction and reduced tubular cell damage, oxidative stress, and apoptosis by renal I/R. Moreover, the βL treatment significantly lowered the cellular NADPH/NADP+ ratio and dramatically reduced NOX activity in the kidneys after IRI. From these results, it was concluded that NQO1 has a protective role against renal injury induced by I/R and that this effect appears to be mediated by decreased NOX activity via cellular NADPH/NADP+ modulation. These results provide convincing evidence that NQO1 activation might be beneficial for ameliorating renal injury induced by I/R.

Perivascular adipose tissue supresses superoxide production in internal mammary artery grafts by regulating Rac1-mediated activation of vascular NADPH-oxidase

Background: Internal mammary artery (IMA) is used as a graft in bypass surgery (CABG), but it is unclear whether it should be harvested with or without its perivascular adipose tissue (PVAT). Given that PVAT produces the antiatherogenic adipokine adiponectin (AdN), we examined the role of AdN as a signalling molecule between PVAT and IMA grafts. Methods: In Study 1, IMA segments were obtained from 364 patients undergoing CABG and used to determine NADPH-oxidase activity by lucigenin chemiluminescence (+/-NADPH 100μM). Mesothoracic (Mt-AT), subcutaneous (Sc-AT) and PVAT were obtained for gene expression studies. In Study 2, IMA segments from 17 patients were exposed ex vivo to AdN (10 μg/ml) for 6 hours ± wortmannin (a PI3K/Akt inhibitor); changes to NADPH-stimulated and Vas2870 (specific NADPH-oxidase inhibitor)-inhibitable O2- as well as the activation and membrane translocation of Rac1 (NADPH-oxidase subunit responsible for its activation) were determined. Results: Elevated circulating AdN was related with reduced NADPH-stimulated O2- (A). Increased expression of ADIPOQ in Mt- and Sc-AT was related with reduced NADPH-oxidase activity, however in PVAT it was associated with increased NADPH-stimulated O2-(B). AdN directly suppressed NADPH-stimulated (C) and Vas2870-inihibitable (D) O2- in the IMA. This was due to reduced activation (E)/membrane translocation (F) of Rac1, which was reversed by wortmannin. ![Figure][1] Conclusions: We demonstrate for the first time in humans, that AdN directly suppresses NADPH-oxidase through an Akt-mediated reduction of Rac1 activation and translocation to the membrane. The observed increase of AdN expression in PVAT in the presence of high oxidative stress may represent a novel, local defence mechanism of IMA against oxidative stress, that warrants further investigation. [1]: pending:yes

Impaired NADPH oxidase activity in peripheral blood lymphocytes of galactosemia patients

Galactosemia is an autosomal recessive disorder with a wide range of clinical abnormalities. Cellular oxidative stress is considered as one of the pathogenic mechanisms of galactosemia. In this study, we examined the activity of NADPH oxidase (NOX), a major superoxide-generating enzyme system, in peripheral blood lymphocytes (PBL) from galactosemia patients. PBL were isolated from galactosemia patients and healthy control subjects and used for cell culture studies and biochemical assays. PBL were cultured in the presence or absence of galactose or galactose-1-phosphate (Gal-1-P), and enzyme activities and/or gene expression of NOX, catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured in the cell homogenates. PBL isolated from galactosemia patients showed significantly reduced (P < 0.01) activities of catalase and GPx; however SOD activity remained unaltered. Galactosemia patients were found to have significantly (P < 0.01) increased levels of malondialdehyde (MDA) in blood lymphocytes. Enzymatic activity of NOX was significantly (P < 0.001) reduced in galactosemia patients; however, Western blotting revealed that NOX-1 protein was not significantly altered. Interestingly, levels of NOX activity in lymphocytes isolated from galactosemia patients significantly increased but remained subnormal when cultured in galactose-deficient medium for two weeks, indicating a galactose-mediated inhibition of NOX. Lymphocytes isolated from control subjects were found to have significantly (P < 0.01) reduced NOX activity when cultured in the presence of galactose or Gal-1-P for two weeks. These results show that galactose-induced cellular oxidative stress is not NOX mediated. However, impairment of the NOX system might be responsible for some of the clinical complications in galactosemia patients.

Rapid Determination of Chimerism Status Using Dihydrorhodamine Assay in a Patient with X-linked Chronic Granulomatous Disease Following Hematopoietic Stem Cell Transplantation

Chronic granulomatous disease (CGD) is a rare genetic disease, which is caused by defects in the NADPH oxidase complex (gp91phox, p22phox, p40phox, p47phox, and p67phox) of phagocytes. This defect results in impaired production of superoxide anions and other reactive oxygen species (ROS), which are necessary for killing bacterial and fungal microorganisms and leads to recurrent, life-threatening bacterial and fungal infections and granulomatous inflammation. The dihydrorhodamine (DHR) flow cytometry assay is a useful diagnostic tool for CGD that can detect absent or reduced NADPH oxidase activity in stimulated phagocytes. We report a patient with X-linked CGD carrying a novel mutation of the CYBB gene whose chimerism status following hematopoietic stem cell transplantation (HSCT) has been rapidly determined using the DHR assay. The level of DHR activity correlates well with short tandem repeat PCR analysis. Considering the advantages of this simple, rapid, and cost-effective procedure, serial measurement of DHR assay would facilitate the rapid determination of a patient's engraftment status, as a supplementary monitoring tool of chimerism status following HSCT.

Effects of peroxisome proliferator-activated receptor-β activation in endothelin-dependent hypertension

We analysed the chronic effects of the peroxisome proliferator-activated receptor β/δ (PPAR-β) agonist GW0742 on the renin-independent hypertension induced by deoxycorticosterone acetate (DOCA)-salt. Rats were treated for 5 weeks with: control-vehicle, control-GW0742 (5 or 20 mg kg(-1) day(-1)), DOCA-vehicle, DOCA-GW0742 (5 or 20 mg kg(-1) day(-1)), DOCA-GSK0660 (1 mg kg(-1) day(-1)), and DOCA-GSK0660-GW0742. Rats receiving DOCA-vehicle showed increased systolic blood pressure, left ventricular and kidney weight indices, endothelin-1 (ET-1), and malondialdehyde plasma levels, urinary iso-PGF2α excretion, impaired endothelium-dependent relaxation to acetylcholine, and contraction to ET-1 when compared with controls. Aortic reactive oxygen species content, NADPH oxidase activity, and p47(phox), p22(phox), NOX-4, glutathione peroxidase 1, hemeoxygenase-1, and preproET-1 expression were increased, whereas catalase and regulators of G protein-coupled signalling proteins (RGS)5 expression were decreased in the DOCA-vehicle group. GW0742 prevented the development of hypertension in a dose-dependent manner but the reduction of renal and cardiac hypertrophy, systemic and vascular oxidative stress markers, and improvement of endothelial dysfunction were only observed after the higher dose. GW0742, at 20 mg kg(-1) day(-1), attenuated ET-1 contraction by increasing RGS5 expression and restored the intracellular redox balance by reducing NADPH-oxidase activity, and by increasing the antioxidant genes expression. The PPAR-β antagonist GSK0660 prevented all vascular changes induced by GW0742 but not its antihypertensive effects. Vascular protective effects of GW0742 operate via PPAR-β by interference with the ET-1 signalling as a result of increased expression of RGS5 and up-regulation of antioxidant genes and via PPAR-β-independent mechanisms to decrease blood pressure.

Diabetes Mellitus and Vascular Disease

Obesity, impaired glucose tolerance, and diabetes mellitus are associated with a substantially increased prevalence of hypertension, cardiovascular (CVD), and chronic renal disease. The prevalence of hypertension in patients who have type 2 diabetes mellitus is up to 3 times higher than in patients without diabetes mellitus. Further, the coexistence of hypertension in diabetic patients greatly enhances their likelihood of developing CVD and chronic kidney disease.1–4 Indeed, studies conducted in the Framingham population with diabetes mellitus1 indicated that the presence of hypertension in these participants was a resulting risk factor for the presence of CVD. This data and other studies suggest a 2-fold increased risk of CVD events and deaths in diabetic persons with hypertension compared with those with normal blood pressures. The increased association between hypertension and diabetes mellitus can be explained, in part, by the presence of a maladaptive interaction of factors, such as excessive caloric intake/decreased activity and associated insulin resistance (IR), chronic activation of the renin–angiotensin (Ang II)–aldosterone system (RAAS), the sympathetic nervous system, and abnormalities of innate immunity, inflammation, and oxidative stress1–21 (Figure). The epidemic of obesity and sedentary lifestyle, and the aging of populations worldwide have contributed to the current high prevalence of diabetes mellitus and hypertension. Figure. Systemic and metabolic factors that promote coexistent diabetes mellitus, hypertension, cardiovascular, and chronic kidney disease. Hypertension is more common in diabetic patients than in the general population.1–4 In the Strong Heart Study, baseline measurements which independently predicted incident hypertension, included waist circumference, elevations in baseline systolic blood pressure, and left ventricular mass and diabetes mellitus.2 Further, a recent longitudinal Japanese study of 5198 subjects showed that parental hypertension has an age-independent impact on both male and female offspring in elevations in blood pressure, plasma glucose, and triglyceride …

P9: Nitrite reduces angiotensin II-mediated contractions of the renal microcirculation by reducing NADPH oxidase activity and increasing nitric oxide bioavailability

Nitrite reduces angiotensin II-mediated contractions of the renal microcirculation by reducing NADPH oxidase activity and increasing nitric oxide bioavailability

N‐acetylcysteine administration modulates NADPH oxidase in skeletal muscle of rats with heart failure

This study aimed to evaluate the influence of antioxidant N‐acetylcysteine on NADPH oxidase (NOX) activity and gene expression in soleus muscle of rats with myocardial infarctioninduced heart failure.MethodsMyocardial infarction (MI) was induced by left coronary occlusion. Four months later, rats were assigned to three groups: Sham (n=8), MI‐C (MI without treatment, n=8), and MI‐NAC (MI treated with N‐acetylcysteine, 120 mg/kg/day, n=8). Rats with small MI were excluded. Six months after surgery, NOX activity was assessed by HPLC analysis of dihydroethidium oxidation fluorescent products in soleus muscle. Gene expression of NOX complex subunits was evaluated by RT‐PCR.ResultsMI size did not differ between MI‐C and MI‐NAC groups (p&gt;;0.05, Student's t test). NADPH oxidase activity was not different between MI‐C and Sham, but it was reduced in MI‐NAC compared to MI‐C group. Table shows the RT‐PCR results (ANOVA and Bonferroni; *p&lt;0.05 vs. Sham, #p&lt;0.05 vs. MI‐C):ConclusionN‐acetylcysteine administration reduces NADPH oxidase activity and gene expression in soleus muscle during heart failure. Support: FAPESP and CNPq.

Treatment with exogenous hydrogen sulfide attenuates hyperoxia-induced acute lung injury in mice

The aim of this work was to test the effect of treatment with hydrogen sulfide (H2S) on hyperoxia-induced acute lung injury in mice. Mice were exposed to room air or 95 % O2, and treated with NaHS (intraperitoneal injection of 0.1 ml/kg/day of 0.56 mol/l NaHS). Treatment with H2S partly restored the reduced H2S levels in plasma and lungs of mice exposed to hyperoxia. Treatment with H2S attenuated hyperoxia-induced acute lung injury marked by reduced ratio of lung weight to body weight, ratio of lung wet weight to dry weight, and cell numbers and protein content in bronchoalveolar lavage (BAL) and decreased apoptosis. Treatment with H2S markedly prolonged the survival of mice under oxygen exposure. Treatment with H2S abated hyperoxia-induced oxidative stress marked by reduced malondialdehyde and peroxynitrite formation, reduced NADPH oxidase activity, enhanced translocation of nuclear factor E2-related factor (Nrf2) into nucleus and increased activity of HO-1. Treatment with H2S decreased IL-1β, MCP-1, and MIP-2, and increased IL-10 expression in lungs of mice exposed to hyperoxia. Treatment with H2S decreased NFκB activity and iNOS expression in lungs, and reduced NOx content in BAL of mice exposed to hyperoxia. Treatment with H2S reduced lung permeability and suppressed VEGF release and VEGFR2 expression in lungs of mice under oxygen exposure. Treatment with exogenous H2S attenuated hyperoxia-induced acute lung injury through abating oxidative stress, suppressing inflammation, and reducing lung permeability in mice.

Water restriction increases renal inner medullary manganese superoxide dismutase (MnSOD)

Oxidative stress damages cells. NaCl and urea are high in renal medullary interstitial fluid, which is necessary to concentrate urine, but which causes oxidative stress by elevating reactive oxygen species (ROS). Here, we measured the antioxidant enzyme superoxide dismutases (SODs, MnSOD, and Cu/ZnSOD) and catalase in mouse kidney that might mitigate the oxidative stress. MnSOD protein increases progressively from the cortex to the inner medulla, following the gradient of increasing NaCl and urea. MnSOD activity increases proportionately, but MnSOD mRNA does not. Water restriction, which elevates renal medullary NaCl and urea, increases MnSOD protein, accompanied by a proportionate increase in MnSOD enzymatic activity in the inner medulla, but not in the cortex or the outer medulla. In contrast, Cu/ZnSOD and TNF-α (an important regulator of MnSOD) do not vary between the regions of the kidney, and expression of catalase protein actually decreases from the cortex to the inner medulla. Water restriction increases activity of mitochondrial enzymes that catalyze production of ROS in the inner medulla, but reduces NADPH oxidase activity there. We also examined the effect of high NaCl and urea on MnSOD in Madin-Darby canine kidney (MDCK) cells. High NaCl and high urea both increase MnSOD in MDCK cells. This increase in MnSOD protein apparently depends on the elevation of ROS since it is eliminated by the antioxidant N-acetylcysteine, and it occurs without raising osmolality when ROS are elevated by antimycin A or xanthine oxidase plus xanthine. We conclude that ROS, induced by high NaCl and urea, increase MnSOD activity in the renal inner medulla, which moderates oxidative stress.

(Pro)renin receptor over‐expression induces angiotensin II‐independent NADPH oxidase activation through PI3K ‐ ERK signaling in Neuronal cells

We previously showed that (pro)renin receptor (PRR) mediates angiotensin II‐independent NADPH oxidase (NOX) activation and oxidative stress in neuronal cells. To investigate the signaling cascade of these events, Neuro‐2A cells were infected with adeno‐associated virus coding for human PRR and control virus. Cells were then treated with phosphoinositide 3‐kinase (PI3K,) inhibitor (wortmannin, 2uM) or extracellular signal‐regulated kinases 1/2(ERK1/2) inhibitor (U0126, 20uM). NOX activity, NOX2 and NOX4 expression levels were determined. The PI3K and ERK1/2 inhibitor decreased NOX2 (wortmannin: 2.685±0.11 vs. 6.346±0.532, P&lt;0.05; U0126:0.373±0.03 vs. 6.346±0.53, P&lt;0.05) and NOX4 (wortmannin:1.803±0.04 vs. 2.658±0.13, P&lt;0.05; U0126:1.391±0.01 vs. 2.658±0.13, P&lt;0.05) mRNA levels (Relative fold change) compare to vehicle following PRR over‐expression. Furthermore, PI3K (0.965±0.02 vs. 1.280±0.03, P&lt;0.05) or ERK1/2 (0.923±0.03 vs. 1.280±0.03, P&lt;0.05) inhibition significantly reduced NOX activity (Relative luminescence units) compared to vehicle following PRR over‐expression. These data suggested that PRR over‐expression induces oxidative stress in neuronal cells through activation of PI3K ‐ERK1/2 signaling cascade, and thus up‐regulation of NOX2 and NOX4 levels, indicating a new pathway for oxidative stress in the central nervous system.(NIH1P30HL101285, AHA11SDG73600050)

Reduction of NADPH-Oxidase Activity Ameliorates the Cardiovascular Phenotype in a Mouse Model of Williams-Beuren Syndrome

A hallmark feature of Williams-Beuren Syndrome (WBS) is a generalized arteriopathy due to elastin deficiency, presenting as stenoses of medium and large arteries and leading to hypertension and other cardiovascular complications. Deletion of a functional NCF1 gene copy has been shown to protect a proportion of WBS patients against hypertension, likely through reduced NADPH-oxidase (NOX)–mediated oxidative stress. DD mice, carrying a 0.67 Mb heterozygous deletion including the Eln gene, presented with a generalized arteriopathy, hypertension, and cardiac hypertrophy, associated with elevated angiotensin II (angII), oxidative stress parameters, and Ncf1 expression. Genetic (by crossing with Ncf1 mutant) and/or pharmacological (with ang II type 1 receptor blocker, losartan, or NOX inhibitor apocynin) reduction of NOX activity controlled hormonal and biochemical parameters in DD mice, resulting in normalized blood pressure and improved cardiovascular histology. We provide strong evidence for implication of the redox system in the pathophysiology of the cardiovascular disease in a mouse model of WBS. The phenotype of these mice can be ameliorated by either genetic or pharmacological intervention reducing NOX activity, likely through reduced angII–mediated oxidative stress. Therefore, anti-NOX therapy merits evaluation to prevent the potentially serious cardiovascular complications of WBS, as well as in other cardiovascular disorders mediated by similar pathogenic mechanism.

Translating the effects of statins: From redox regulation to suppression of vascular wall inflammation

Vascular oxidative stress is a key feature of atherogenesis, and targeting vascular redox signalling is a rational therapeutic goal in vascular disease pathogenesis. 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors or statins are potent lipid-lowering drugs that improve cardiovascular outcomes. It is now widely accepted that cardiovascular disease prevention by statins is dependent not only on their lipid lowering effects, but also on their beneficial effects on vascular redox signalling. Cell culture and animal models have provided important findings on the effects of statins on vascular redox and nitric oxide bioavailability. Recent evidence from studies on human vessels has further enhanced our understanding of the "pleiotropic" effects of statins on vascular wall. Reversal of endothelial dysfunction in human vessels by statins is dependent on the mevalonate pathway and Rac1 inhibition. These critical steps are responsible for reducing NADPH-oxidase activity and improving tetrahydrobiopterin bioavailability and nitric oxide synthase (NOS) coupling in human vessels. However, mevalonate pathway inhibition has been also held responsible for some of the side effects observed after statin treatment. In this review we summarise the existing knowledge on the effects of statins on vascular biology by discussing key findings from basic science as well as recent evidence from translational studies in humans. Finally, we discuss emerging aspects of statin pleiotropy, such as their effects on adipose tissue biology and adipokine synthesis that may light additional mechanistic links between statin treatment and improvement of clinical outcome in primary and secondary prevention.

Prevention of salt-induced renal injury by activation of NAD(P)H:quinone oxidoreductase 1, associated with NADPH oxidase

NADPH oxidase (NOX) is a predominant source of reactive oxygen species (ROS), and the activity of NOX, which uses NADPH as a common rate-limiting substrate, is upregulated by prolonged dietary salt intake. β-Lapachone (βL), a well-known substrate of NAD(P)H:quinone oxidoreductase 1 (NQO1), decreases the cellular NAD(P)H/NAD(P)+ ratio via activation of NQO1. In this study, we evaluated whether NQO1 activation by βL modulates salt-induced renal injury associated with NOX-derived ROS regulation in an animal model. Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet were used to investigate the renoprotective effect of NQO1 activation. βL treatment significantly lowered the cellular NAD(P)H:NAD(P)+ ratio and dramatically reduced NOX activity in the kidneys of HS diet-fed DS rats. In accordance with this, total ROS production and expression of oxidative adducts also decreased in the βL-treated group. Furthermore, HS diet-induced proteinuria and glomerular damage were markedly suppressed, and inflammation, fibrosis, and apoptotic cell death were significantly diminished by βL treatment. This study is the first to demonstrate that activation of NQO1 has a renoprotective effect that is mediated by NOX activity via modulation of the cellular NAD(P)H:NAD(P)+ ratio. These results provide strong evidence that NQO1 might be a new therapeutic target for the prevention of salt-induced renal injury.

Interleukin-10 Released by CD4 + CD25 + Natural Regulatory T Cells Improves Microvascular Endothelial Function Through Inhibition of NADPH Oxidase Activity in Hypertensive Mice

We previously demonstrated that a reduced number of CD(4+)CD(25+)-regulatory T cells (Tregs) was associated with microvascular dysfunction in hypertension. However, the underlying mechanism by which Tregs regulate vascular endothelial function remains unknown. Control and interleukin (IL)-10(-/-) knockout mice were infused with angiotensin II (400 ng/kg/min) for 2 weeks (hypertensive [HT] and HT-IL-10(-/-)). Endothelium-dependent relaxation (EDR) in response to acetylcholine was significantly reduced in mesenteric resistance artery (MRA) from HT and HT-IL-10(-/-) compared with control and IL-10(-/-) mice. Importantly, the incubation of MRA from HT mice with the conditioned media of cultured Tregs, isolated from control mice, reduced NADPH oxidase activity and improved EDR, whereas no effect was observed in MRA from control mice incubated with the same media. These effects were reversed when MRAs were preincubated with IL-10 antibody or IL-10 receptor antagonist, whereas incubation with transforming growth factor-β receptor antagonist had no effect. The transfer of cultured Tregs, isolated from control mice, into HT-IL-10(-/-) mice reduced systolic blood pressure (SBP) and NADPH oxidase activity and improved EDR in MRA compared with untreated HT-IL-10(-/-) mice. In vivo treatment of HT mice with IL-10 (1000 ng/mouse) significantly reduced SBP and NADPH oxidase activity and improved EDR in MRA compared with untreated HT mice. The transfer of cultured Tregs, isolated from IL-10(-/-) mice, into HT mice did not reduce SBP or NADPH oxidase activity or improve EDR. The incubation of MRA from HT mice with apocynin improved EDR, whereas NADPH oxidase substrate attenuated EDR in MRA from control mice, which was reversed with exogenous IL-10. These data demonstrate that IL-10 released from Tregs attenuates NADPH oxidase activity, which is a critical process in the improvement of microvascular endothelial function in hypertension, suggesting that Tregs/IL-10 could be a therapeutic target for treatment of vasculopathy in hypertension.

Endothelium-Dependent Coronary Vasodilatation Requires NADPH Oxidase-Derived Reactive Oxygen Species

To determine the functional significance of physiological reactive oxygen species (ROS) levels in endothelium-dependent nitric oxide (NO)-mediated coronary vasodilatation. Endothelium-derived NO is important in regulating coronary vascular tone. Excess ROS have been shown to reduce NO bioavailability, resulting in endothelial dysfunction and coronary diseases. NADPH oxidase is a major source of ROS in endothelial cells (ECs). By using lucigenin-based superoxide production and dichlorfluorescein diacetate (DCFH-DA) fluorescence-activated cell sorter assays, we found that mouse heart ECs from NADPH oxidase-knockdown (p47(phox-/-)) animals have reduced NADPH oxidase activity (>40%) and ROS levels (>30%) compared with wild-type mouse heart ECs. Surprisingly, a reduction in ROS did not improve coronary vasomotion; rather, endothelium-dependent vascular endothelial growth factor-mediated coronary vasodilatation was reduced by greater than 50% in p47(phox-/-) animals. Western blots and L-citrulline assays showed a significant reduction in Akt/protein kinase B (PKB) and endothelial NO synthase phosphorylation and NO synthesis, respectively, in p47(phox-/-) coronary vessels and mouse heart ECs. Adenoviral expression of constitutively active endothelial NO synthase restored vascular endothelial growth factor-mediated coronary vasodilatation in p47(phox-/-) animals. Endothelium-dependent vascular endothelial growth factor regulation of coronary vascular tone may require NADPH oxidase-derived ROS to activate phosphatidylinositol 3-kinase-Akt-endothelial NO synthase axis.

NADPH oxidase inhibition ameliorates cardiac dysfunction in rabbits with heart failure

Increased NADPH oxidase activity is found in both experimental and clinical HF. Here, we investigated the effects and mechanisms of NADPH oxidase inhibition on cardiac function in rabbits with HF. HF was induced by combined volume and pressure overload. Rabbits with HF or sham operation were randomized to orally receive apocynin, an inhibitor of NADPH oxidase (15 mg per day) or placebo for 8 weeks. Echocardiography was performed to examine the cardiac function and structure of the rabbits. Cardiac fibrosis was evaluated by masson's trichrome staining. The transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-9 (MMP-9) expression were measured by real-time PCR. The expression of SERCA2a and phospholamban (PLB) was detected by reverse transcription-polymerase chain reaction and Western Blot. SERCA2a activity was evaluated by measuring the Pi liberated from ATP hydrolysis. Rabbits with HF exhibited cardiac dysfunction and fibrosis. These changes were associated with significant increases in myocardial NADPH oxidase activity and oxidative stress. Compared with sham-operated rabbits, the TGF-β, CTGF, MMP-2, and MMP-9 mRNA expression significantly increased, the expression of SERCA2a and PLB dramatically decreased, and the SERCA2a activity was lower in HF rabbits. Apocynin reduced NADPH oxidase activity and oxidative stress, decreased TGF-β, CTGF, MMP-2, and MMP-9 expression, attenuated cardiac fibrosis, increased SERCA2a and PLB expression, restored SERCA2a activity, and thereby ameliorated cardiac dysfunction. Thus, chronic NADPH oxidase inhibition ameliorated cardiac dysfunction by decreasing cardiac fibrosis and preserving SERCA2a expression and activity.