Role Of Nox4 Research Articles

Docosahexaenoic Acid Protects Traumatic Brain Injury by Regulating NOX2 Generation via Nrf2 Signaling Pathway

Docosahexaenoic acid (DHA) is verified to have neuroprotective effects on traumatic brain injury (TBI) rats by activating Nrf2 signaling pathway, but the role of NOX2 in this effect has not been illuminated. So this study explored the role of NOX2 in TBI models treated with DHA, aiming to complete the mechanism of DHA. TBI rat models were constructed with or without DHA treatment, and H2O2-induced hippocampal neurons were pretreated with DHA alone or in combination with Nrf2 inhibitor brusatol. The neurological function, cognitive ability, and cerebral edema degree of rats were assessed. The apoptosis rate and viability of cells was measured. The generation of NOX2, Nrf2, HO-1 and NQO-1 expression levels, and ROS content in hippocampal CA1 region and hippocampal neurons were detected. DHA could not only improve the neurological function, brain edema and cognitive ability in TBI rats, but also decrease effectively the contents of NOX2 and ROS in hippocampal CA1 region and hippocampal neurons. DHA promoted the nuclear transposition of Nrf2 and the expression levels of HO-1 and NQO-1 in hippocampal CA1 region and hippocampal neurons. On the contrary, Nrf2 inhibitor brusatol inhibited the nuclear transposition of Nrf2 and the expression levels of HO-1 and NQO-1 in hippocampal neurons, promoted the generation of ROS and NOX2, and accelerated cell apoptosis. Both in vivo and in vitro experiments demonstrated that DHA treated TBI by reducing NOX2 generation that might function on Nrf2 signaling pathway, providing a potential evidence for its clinical application.

NADPH oxidase 5 activation; a novel approach to human sperm cryoinjury.

Sperm cryopreservation leads to various structural and functional damages, some of which induce by oxidative stress. The reactive oxygen species (ROS) generates by mitochondria and membrane NADPH oxidases (NOXs). Among the NOXs, only NOX5 has been identified in the cell membrane of human sperm. This study was designed to clarify the possible role of NOX5 on sperm cryoinjury. Forty human semen samples were washed and randomly divided into fresh and cryopreserved groups. Each group was divided into 4 subgroups containing Ham's F10 (control), 0.1% DMSO (vehicle), 100nM of PMA (phorbol 12-myristate 13-acetate) and 1µM of DPI (diphenyleneiodonium), as NOX5 activator and inhibitor. The samples of cryopreserved groups were preserved in liquid nitrogen for 1month. The sperm kinematics, membrane integrity, ROS production, apoptosis rate, mitochondrial membrane potential (MMP), intracellular ATP and calcium concentration [Ca2+]i were evaluated. The percent of sperm with intact membrane and motile sperm reduced significantly after thawing (p ≤ 0.01). The ROS production (p ≤ 0.01) and the apoptotic rate increased, MMP dissipated, and the percentage of live cells with high [Ca2+]i decreased significantly in the cryopreserved control group relative to the fresh control group. DPI, in contrast to PMA, improved sperm progressive motility (p ≤ 0.01), membrane integrity in fresh and cryopreserved groups and reduced the ROS amount in cryopreserved group (p ≤ 0.01). Apoptotic rate, [Ca2+]i, ATP, and MMP did not change with DPI and PMA in cryopreserved groups. We conclude that NOX5 activity in fresh sperm is low, and it increases during cryopreservation. NOX5 inhibition improves the cryopreserved sperm quality.

NOX2 Is Critical to Endocardial to Mesenchymal Transition and Heart Development.

NADPH oxidases (NOX) are a major source of reactive oxygen species (ROS) production in the heart. ROS signaling regulates gene expression, cell proliferation, apoptosis, and migration. However, the role of NOX2 in embryonic heart development remains elusive. We hypothesized that deficiency of Nox2 disrupts endocardial to mesenchymal transition (EndMT) and results in congenital septal and valvular defects. Our data show that 34% of Nox2−/− neonatal mice had various congenital heart defects (CHDs) including atrial septal defects (ASD), ventricular septal defects (VSD), atrioventricular canal defects (AVCD), and malformation of atrioventricular and aortic valves. Notably, Nox2−/− embryonic hearts show abnormal development of the endocardial cushion as evidenced by decreased cell proliferation and an increased rate of apoptosis. Additionally, Nox2 deficiency disrupted EndMT of atrioventricular cushion explants ex vivo. Furthermore, treatment with N-acetylcysteine (NAC) to reduce ROS levels in the wild-type endocardial cushion explants decreased the number of cells undergoing EndMT. Importantly, deficiency of Nox2 was associated with reduced expression of Gata4, Tgfβ2, Bmp2, Bmp4, and Snail1, which are critical to endocardial cushion and valvoseptal development. We conclude that NOX2 is critical to EndMT, endocardial cushion cell proliferation, and normal embryonic heart development.

P0525ROLE OF NADPH OXIDASE 4 IN ACUTE KIDNEY INJURY ASSOCIATED TO MASSIVE INTRAVASCULAR HEMOLYSIS

Abstract Background and Aims Massive intravascular hemolysis is a common condition of several pathologies. It is associated with acute kidney injury (AKI) and progressive impairment of renal function. In this context, free hemoglobin (Hb) can exert harmful effects by accumulating in the kidney, where induces oxidative stress and it becomes cytotoxic. NADPH oxidase 4 (Nox4) is the principal source of reactive oxygen species (ROS) in the kidney. Nox4 is mostly expressed in proximal tubular cells with lower levels in glomerulus. The role of Nox4 in renal damage is not clear, with studies reporting beneficial or deleterious actions depending of the environmental conditions. For that reason we aimed to investigate the role of Nox4 in massive intravascular hemolysis-associated AKI. Method To study the role of Nox4 in AKI caused by massive intravascular hemolysis, we performed an experimental model of intravascular hemolysis by intraperitoneal injection of phenylhydrazine (200 mg/kg) in wild type (Nox4+/+) and Nox4 knockout mice (Nox4-/-). Mice were sacrificed 24 and 72 hours after intravascular hemolysis induction. We collected serum, urine and tissues sample. We analyzed renal function, oxidative stress, cell death and inflammation in these samples. In other experiments, wild type mice were treated with GKT137831 (10mg/kg/day), a potent Nox4 and Nox1 inhibitor, and mice were sacrificed 72h after induction of hemolysis. We also performed in vitro experiments in murine tubular epithelial cells (MCT) and murine podocytes cells to investigate the regulation of Nox4 in Hb-stimulated cells treated or not with GKT137831. Results Induction of intravascular hemolysis in Nox4+/+ mice increased creatinine and BUN levels and enhanced the expression of tubular injury markers, such as NGAL. These pathological effects were reduced in Nox4 knockout mice. Then, we analyzed oxidative stress in our experimental model thought determination of HO-1, ferritin, GSH and lipid peroxidation levels. All of these oxidative markers were reduced in Nox4-/- mice with intravascular hemolysis as compared with Nox4+/+ mice. We also observed that inflammatory markers such as IL-6, cell death and podocytes injury markers were reduced in Nox4-/- mice than in wild type mice, specially 72 hours after phenylhydrazine injection. In line with these results, GKT137831 administration ameliorated intravascular hemolysis-associated renal function impairment. Moreover, oxidative stress, tubular injury markers and podocyte injury were reduced in hemolytic mice treated with GKT137831. GKT137831 also reduced Hb- and heme-mediated oxidative stress in MCT and podocytes. Conclusion Our results show the important role of Nox4 in renal injury associated to massive intravascular hemolysis. Moreover, the inhibition of Nox4 may be a potential therapeutic target to prevent renal damage associated to Hb accumulation. These findings provide new insights into novel aspects of Hb-toxicity and may have important pathogenic and therapeutic implications for intravascular hemolysis related diseases

SARS-CoV-2 and myocardial injury: a role for Nox2?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be complicated by myocardial injury but at-risk patients as well as mechanism of disease are unclear. We gathered data regarding troponin levels in the so far reported SARS-CoV-2 patients and found a large variability in terms of troponin levels, patients with more severe disease, as those treated by ICU, presenting with higher percentage of troponin elevation. However, lack of prospective studies hampers adequate analysis of risk factors of myocardial damage. Previous study demonstrated that Nox2 is up-regulated in pneumonia and closely associated with troponin elevation suggesting Nox2 activation as mechanism eliciting myocardial damage; data in SARS-CoV-2 are still lacking. We hypothesize that SARS-Cov-2 may induce myocardial injury via Nox2-related ROS production and that analysis and eventually targeting Nox2 may be a novel approach to manage SARS-CoV-2.

Role of NOX2 in the BDNF dysregulation and cognitive deficits after sepsis (4106)

Role of NOX2 in the BDNF dysregulation and cognitive deficits after sepsis (4106)

Gender differences in the effect of blackberry supplementation in vascular senescence and atherosclerosis in ApoE−/− mice

As the cardiovascular system ages, it becomes more vulnerable to the effects of oxidative stress and inflammation. The aging process, along with external factors such as radiation exposure and lifestyle, induces vascular senescence and accelerates atherosclerotic plaque accumulation. Expression of nicotinamide adenine dinucleotide phosphate oxidase 1 (Nox1), which produces superoxide, is associated with senescence in vascular smooth muscle cells in vitro and atherosclerosis in ApoE−/− mice in vivo. However, it is unknown whether Nox1 could be down-regulated by nutritional interventions aimed to reduce atherosclerosis. Here we study the effect of blackberry supplementation in Nox1 expression and atherosclerosis. Four-month-old ApoE−/− male and female mice were fed low-fat, high-fat or high-fat supplemented with 2% freeze-dried blackberry powder diets for 5 weeks. Analysis of the aorta showed that diet supplemented with blackberry significantly decreased plaque accumulation, senescence associated-β-galactosidase and Nox1 expression in the aorta of male but not female mice. The lipid profile was unchanged by blackberry in both female and male animals. Thus, the known role of Nox1 in atherosclerosis suggests that the atheroprotective effect of blackberry is mediated by Nox1 down-regulation in male mice and that Nox1 is regulated in a gender-dependent manner in females.

Role of NOX1 in intestinal barrier dysfunction

Role of NOX1 in intestinal barrier dysfunction

Celastrol Alleviates Aortic Valve Calcification Via Inhibition of NADPH Oxidase 2 in Valvular Interstitial Cells

This study sought to investigate whether reactive oxygen species (ROS)-generating reduced nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) contributes to calcific aortic valve disease (CAVD) or whether celastrol, a natural Nox2 inhibitor, may provide potential therapeutic target for CAVD. CAVD is an active and cellular-driven fibrocalcific process characterized by differentiation of aortic valvular interstitial cells (AVICs) toward an osteogenic-like phenotype. ROS levels increase in calcified aortic valves, while the sources of ROS and their roles in the pathogenesis of CAVD are elusive. The roles of Nox2 and the effects of celastrol were studied using cultured porcine AVICs invitro and a rabbit CAVD model invivo. Nox2 proteins were significantly upregulated in human aortic valves with CAVD. Invitro, Nox2 was markedly induced upon stimulation of AVICs with osteogenic medium, along with the increases in ROS production and calcium nodule formation. Celastrol significantly decreased calcium deposition of AVICs by 35%, with a reduction of ROS generation. Knockdown of endogenous Nox2 substantially suppressed AVIC calcification by 39%, the inhibitory effect being similar to celastrol treatment. Mechanistically, either celastrol treatment or knockdown of Nox2 significantly inhibited glycogen synthase kinase 3 beta/β-catenin signaling, leading to attenuation of fibrogenic and osteogenic responses of AVICs. In a rabbit CAVD model, administration of celastrol significantly reduced aortic valve ROS production, fibrosis, calcification, and severity of aortic stenosis, with less left ventricular dilatation and better preserved contractile function. Upregulation of Nox2 is critically involved in CAVD. Celastrol is effective to alleviate CAVD, likely through the inhibition of Nox2-mediated glycogen synthase kinase 3 beta/β-catenin pathway in AVICs.

Abstract 074: Vascular Dysfunction in Boys With Androgen Insufficiency - Role of Nox5 and Calcium

Hypospadias is usually associated with androgen insufficiency. As androgens regulate Ca 2+ influx, Nox activity and redox signalling, we sought to ascertain where these may be implicated in vascular dysfunction seen in boys with hypospadias. Peripheral arteries dissected from excess foreskin tissue from hypospadias (cases) or circumcision (controls) were used. Vascular function was assessed by wire myography.VSMCs were cultured and ROS production was measured by amplex red and chemiluminescence. mRNA expression was measured by qPCR and Ca 2+ influx by fluorescence microscopy. 20 boys with hypospadias and 29 age-matched controls were enrolled in this study (median age 1.9 (range 1.3, 12.2) years). Arteries from cases demonstrated increased constriction to U46619 vs controls (Emax %KCl: 175.6 vs 66.3 p<0.05). Expression of Nox5 was increased in cases (2.6 fold,p<0.05), while a decrease in Nox4 expression was observed (9.5 fold, p<0.05). VSMC superoxide anion (5.3 fold) production and H 2 0 2 (3.3 fold) levels were increased in cases compared to controls (p<0.05), an effect inhibited by melittin. Melittin, a Nox5 inhibitor, inhibited contraction in arteries from boys with hypospadias (Emax %KCl: 137.9 vs 62.9, p<0.05). There was no difference in mRNA expression of androgen receptors ( AR and GPRC6A) between cases and controls but cases had higher expression of the oestrogen receptors, ESR1 (2.7 fold), ESR2 (2.6 fold) and GPR30 (2.9 fold) (p<0.05).Boys with hypospadias demonstrated increased CACNA1C (3.9 fold), SERCA (3.1 fold), TRPM2 (2.0 fold) and Ryr1 (3.5 fold) mRNA expression (p<0.05).There was no basal difference between Ca 2+ influx between controls and boys with hypospadias.Testosterone and oestradiol stimulation reduced expression of Ca 2+ channels (p<0.05). Moreover, testosterone (p=0.04) and oestradiol (p=0.02) reduced Ca 2+ influx in boys with hypospadias. Vascular dysfunction observed in small arteries from boys with hypospadias may be due to Nox5 activation and altered Ca 2+ regulation. These data suggest a role for androgens in the regulation of vascular Nox5-Ca 2+ signaling, which when perturbed may predispose to cardiovascular risk.

Abstract 141: Enhanced Hydrogen Peroxide-Dependent Renin and Pro-Renin Release in Juxtaglomerular Cells from Diabetic Mice: Role of NOX1

A large fraction of patients with diabetes mellitus develop diabetic nephropathy and are hypertensive. The Akita mice is one of the few mouse models of diabetes mellitus with features of the human disease including hypertension and kidney damage. Previous data showed increased levels of reactive oxygen species in the kidneys of Akita mice. We found that hydrogen peroxide (H 2 O 2 ) is a potent stimulus for renin release from the juxtaglomerular (JG) cells in vitro and in vivo . Plasma pro-renin levels are elevated in diabetic patients and thought to contribute to hypertension and renal damage. Thus, we hypothesized that H 2 O 2 levels are higher in JG cells from Akita mice leading to increased renin and pro-renin release in diabetes. We measured H 2 O 2 by transducing primary cultures of JG cells with the fluorescent protein H 2 O 2 sensor Hyper, under control of the renin1 promoter. JG cells isolated from Akita mice had higher baseline levels of H 2 O 2 (C57= 100, Akita=222.5±79%, p <0.05). We also found that pro-renin expression was higher in JG cells from Akita by 79±21% ( p <0.05). In addition, JG cells from Akita mice have higher pro-renin (n=4; p <0.05) and renin (C57= 100, Akita=219.2±26%; p <0.04, n=6) release to the media when compared to JG cells from C57. Western Blots showed that the NADPH oxidase isoform NOX1 is upregulated in JG cells from Akita mice (C57=100%; Akita=147±10%; n=4; p <0.05), whereas NOX2 or NOX4 were not different. NOX1 co-localized with renin-containing granules in kidney sections analyzed by confocal microscopy (n=4). Silencing NOX1 in JG cells from Akita mice decreased renin release to C57 control levels; (n=6; p <0.01) while silencing NOX4 had no effect (n=6). Expression of Catalase, which scavenges H 2 O 2 , was reduced in JG cells from Akita mice (C57=100%; Akita= 36±9.2%; n=4; p <0.05) and overexpression of Catalase in JG cells from Akita decreased baseline renin release by 35±8% ( p <0.05). We conclude that renin cells from diabetic Akita mice have higher endogenous H 2 O 2 leading to increased renin and pro-renin release via enhanced NOX1 expression and decreased catalase. Enhanced H 2 O 2 -dependent renin release in Akita JG cells could be involved in renal damage and hypertension in diabetes mellitus.

Abstract 089: Role of Nox5 in Systemic Vascular Dysfunction in Ischemic Heart Disease

Patients with coronary microvascular dysfunction (CMD), a potential cause of heart ischemia, have systemic vascular dysfunction, characterized by increased vascular contraction to ET-1 and a thromboxane A2 analogue (U46619). Nox5 regulates vascular contraction and is involved in cardiovascular diseases. In our study, we questioned whether Nox5 plays a role in systemic vascular dysfunction in heart ischemia. As Nox5 expression has been described in the cardiovascular system of rabbits, a model of ischaemic cardiomyopathy (IC) was used. Coronary artery ligation was performed in Male New Zealand White rabbits. After 8 weeks, skin and mesenteric arteries were isolated and vascular function assessed by wire myography. Vascular contraction to NA (EMax %KCl: 122±4 vs sham 97±3.7) and U46619 (EMax %KCl: 82±3 vs sham 67±4) were exacerbated in skin arteries from IC (p<0.05); an effect blocked by tiron (antioxidant, 10 μM) and melittin (nox5 inhibitor, 0.1 μM). In mesenteric arteries from IC animals, NA (EMax %KCl: 108±3 vs sham 98±7) and ET-1 (EMax %KCl: 103±3 vs sham 81±4) induced contraction were increased in a Nox5-ROS-dependent manner (p<0.05). No differences were observed in mRNA levels of Cav1.2 and IP3R Ca 2+ channels, but an increase in RyR was observed (2^-ddCT: 1.67±0.15 vs sham 0.98±0.08) in VSMCs isolated from IC animals. Peroxiredoxin (Prdx), antioxidant, mRNA was increased in IC (2^-ddCT: 1.95±0.4 vs sham 0.88±0.1, p<0.05). Conoidin A, an inhibitor of Prdx oxidation, reduced vascular contraction to NA in arteries from IC animals (EMax %KCl: 95±4, p<0.05). In subjects with CMD, we assessed total number of microparticles (MP) as biomarkers of vascular dysfunction. MPs were increased in CMD subjects (x10 11 /mL: 4.8±0.6 vs control 1.75±0.2), where Nox5 expression was also increased (AU: 0.11±0.02 vs control MP 0.03±0.006) (p<0.05). In separate studies, we exposed WT control arteries to MPs from WT and Nox5-expressing mice before assessing contraction. MPs from Nox5 mice increased contraction to a higher level of that observed with MPs from WT mice (EMax %KCl: 106±2 vs WT 96±2, p<0.05). In our study, we identify Nox5 as a regulator of systemic vascular dysfunction in ischemic heart diseases, through mechanisms that may involve ROS, Prdx oxidation and MPs.

Abstract 068: Interplay Between Nox5 and Endoplasmic Reticulum Stress Regulates Vascular Signalling in Human Hypertension

Nox5, a major ROS-generating oxidase in human vessels, regulates vascular contraction. We demonstrated an ER-perinuclear Nox5 localization and questioned the role of ER stress in Nox5 regulation. Vascular smooth muscle cells (VSMC) were isolated from small arteries from subcutaneous fat from normotensive (NT) and hypertensive (HT) subjects. Nox5 compartmentalization (cell fractionation); ROS generation (chemiluminescence); peroxiredoxin/DJ-1 oxidation, activation of ER stress and contractile signalling (IRE1α, Src, PKC, MLC phosphorylation; immunoblotting) and actin cytoskeleton organization (phalloidin staining) were assessed. In hypertension, ROS levels (139±27% vs NT, p<0.05), oxidation of peroxiredoxin (870.4±188.7% vs NT, p<0.05) and DJ-1 (125±34% vs NT, p<0.05) were increased. IRE1α phosphorylation was increased in the HT group (58±21% vs NT, p<0.05). ER stress inhibition (4-PBA, 1mM) reduced ROS levels in HT subjects (20±6% vs NT, p<0.05), suggesting association between ER and oxidative stress. Nox5 expression was increased in the HT group (103±23% vs NT, p<0.05) in a compartment specific manner: Nox5 levels were reduced in plasma membrane (45±7% vs NT, p<0.05), but increased in the ER/nuclear fraction (46±13% vs NT, p<0.05). IRE1 inhibition (STF083010, 60μM) decreased Nox5 expression in the HT group (65±5% vs Ctl, p<0.05), while induction of ER stress (tunicamycin, 5μg/ml, 24h) increased Nox5 expression in cells from both groups (p<0.05). To investigate the role of Nox5 on contractile signalling, cells were treated with mellitin (100nM), a Nox5 inhibitor. ROS generation and phosphorylation of c-Src, PKC and MLC 20 induced by Ang II were reduced by mellitin in both groups (p<0.05 vs Ctl). In contrast, silencing of p22phox increased ROS and activation of c-Src, PKC and MLC 20 in both groups, an effect blocked by mellitin (p<0.05 vs Ctl). VSMC from hypertensive subjects had increased number of stress fibres, an effect attenuated by mellitin. Our findings demonstrate that Nox5 is upregulated in a compartment specific manner and is regulated by ER stress in hypertension. Nox5 upregulation influences pro-contractile signalling and cytoskeleton reorganization in VSMC, processes that contribute to vascular dysfunction in hypertension.

Clinical Significance of NADPH Oxidase 5 in Human Colon Cancer.

Recent studies have reported the involvement of NADPH oxidases (NOXs) in tumor progression. However, the role of NOX5 in colon cancer is unclear. We examined the clinical significance of NOX5 expression in colon cancer. NOX5 expression was evaluated by immunohistochemistry in 119 patients with stage II or III colon cancer, and the relationship between NOX5 expression and clinicopathological data was analyzed. Of all tissues, 39.5% were negative and 60.5% were positive for NOX5 expression. Positive expression was significantly associated with undifferentiated histology (p=0.037) and lymph node metastasis (p=0.023). The 5-year progression-free survival rate of NOX5-positive patients was significantly worse than that of NOX5-negative patients (p=0.046). The rates of local recurrence observed in NOX5-positive patients were higher than that in NOX5-negative patients. NOX5 expression may be related to poor prognostic factors and could be useful as a prognostic biomarker.

The role of Nox5 in diabetic complications

The role of Nox5 in diabetic complications

The role of Nox-4 in gastric mucosal inflammation induced by chronic restraint stress in mice

Objective To investigate the expression of NADPH oxidase Nox-4 induced by stress in gastric mucosa and its role in inflammation. Methods Twenty male SPF Kunming mice were randomly divided into chronic restraint stress group(stress group) and control group.Stress mice were restrained in self-made restraint device for 2 hours each day.The rest of the time, the mice in the two groups had free access to food and water normally, experiment lasted 14 days.The histopathological changes of gastric mucosa were assessed by HE staining under light microscope.The expression of Nox-4 in gastric mucosa of mice was carried out by immunohistochemical method.The relative expression levels of Nox-4, antioxidant protein(Mn-SOD, GSH, Catalase) and inflammatory factors(IL-8, IL-1β, TNF-α) in gastric mucosa were detected by real-time quantitative RT-PCR and ELISA. Results Basal cell proliferation, neutrophil, eosinophil and plasma cell infiltration and inflammatory changes were observed in the lamina propria and glandular epithelium of stress mice, while no obvious abnormalities were found in control mice.The expression of Nox-4 in stress group was deeper and more abundant than that in control group, mainly expressed in lamina propria and glandular epithelium.The mRNA expression levels of Nox-4 in gastric mucosa of stress group was(2.42±0.51) times higher than that of control group, and blood concentration of stress group was(2.23±0.67) times higher than that of control group(t=-46.32, P<0.001). The RT-PCR of antioxidant proteins in gastric mucosa showed that the transcription levels of Mn SOD, GSH and Catalase in stress group were significantly lower than that of control group(Mn-SOD: 0.59±0.10, GSH: 0.58±0.11, Catalase: 0.57±0.09), and there were significant differences between the two groups(t=13.57, 11.67, 15.01, P<0.01). RT-PCR results showed that the transcription levels of IL-8, IL-1β, TNF-α in stress group were significantly higher than those in control group(IL-8: 1.47±0.34, IL-1β: 1.48±0.42, TNF-α: 1.51±0.37), and there were significant differences in two groups(t=-18.45, -19.14, -20.85, P<0.01). ELISA results showed that the serum levels of inflammatory factors in stress group were significantly higher than those in control group(2.25±0.37, 3.59±0.45, 3.41±0.34), and the differences were statistically significant(t=-47.11, -79.36, -96.32, P<0.01). Pearson correlation analysis showed that there was a positive correlation between serum concentration of Nox-4 and inflammatory factors(IL-8, IL-1β, TNF-α) in stress group(r=0.97, 0.99, 0.98, P<0.01). Spearman rank correlation analysis showed that the grade of gastric mucosal inflammation was positively correted with serum levels of Nox-4 and inflammatory factors(IL-8, IL-1β, TNF-α)(r=0.96, 0.92, 0.91, 0.94, all P<0.01) Conclusion Stress may lead to gastric mucosal lesion by overexpression of proinflammatory factors through destroying the balance of oxidation/antioxidant system in gastric mucosa. Key words: Chronic restraint stress; Gastric mucosal lesion; Nox-4; Anti-oxidant proteins; Inflammatory cytokines; Mice

Role of Nox4 in Angiotensin II‐Mediated Changes in Volume Dynamics and Nitric Oxide Production in Podocytes

Nitric oxide (NO) is a powerful vasodilator that serves important functions in the kidney such as regulation of renal hemodynamics, pressure natriuresis, and diuresis. Significant reduction in NO production has been found to play a role in the pathogenesis of hypertension. Our recent studies revealed that Angiotensin II (Ang II), in addition to causing a release of calcium, stimulates NO production in the glomerular podocytes. This signaling pathway can be also triggered by extracellular application of hydrogen peroxide (H2O2) and is significantly reduced with the development of salt‐sensitive hypertension and kidney injury in Dahl salt‐sensitive (SS) rats (Palygin et al., Nitric Oxide, 2018). Recently, using a knock out of NADPH oxidase 4 (Nox4) in SS rats (SSNox4−/−), one of the primary source of renal reactive oxygen species (ROS) and specifically H2O2 we described the reduction in intracellular calcium activation in podocytes as well as H2O2 production in isolated glomeruli in response to Ang II (Ilatovskaya et al., J Am Soc Nephrol. 2018). Overall, SSNox4−/− rats displayed a protective phenotype with a significant reduction of albuminuria, glomeruli injury and reduction in the elevation of blood pressure under high salt challenge (Cowley et al., Hypertension, 2016). Here we used this model to characterize the effects of alterations in the balance in the relationship between ROS and NO production in podocytes and establish the relation between Nox4 and glomerular/podocyte volume dynamics. The in vivo concentration of H2O2, measured by biosensors amperometry as catalase sensitive current, was significantly reduced in both cortex and medullar parts of the SSNox4−/− rat kidney compared to wild type SS rats, indicating a direct association between Nox4 and the production of renal ROS. We found that acute application of Ang II promoted opposite changes in glomeruli and podocyte volumes, where glomeruli volume decreased, and podocyte volume increased approximately by 18% in SS rats. Overall, SSNox4−/− rats exhibited an increase in total NO production (by 16% to compare with SS), calculated using confocal microscopy as a total integral of NO fluorescent transient (DAF‐FM dye) in the population of podocytes within a freshly isolated glomerulus. In SSNox4−/− rats the elevation in NO together with declining in H2O2 levels directly correlated with an increase of podocyte volume in response to Ang II (13% vs. SS control). We anticipate a rapid podocyte volume change dependent on intracellular NO production and reflecting the ability of these cells to dynamically regulate the permeability of glomeruli filtration barrier. Thus far, we can conclude that in the absence of Nox4 the podocytes shift the ROS production in response to Ang II towards the increase of intracellular NO bioavailability and a decrease of renal H2O2 production. This mechanism may play a key role in the protection of glomeruli form oxidative stress and elevation of blood pressure during the development of salt‐sensitive hypertension.Support or Funding InformationNHLBI R35 HL135749; P01 HL116264; AHA 18PRE34030127; ADA 1‐15‐BS‐172; DK‐020595 Pilot &amp; Feasibility projectThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The association of NOX4 mediated nuclear colocalization and overall survival in renal cell carcinoma.

639 Background: NOX4 protein are involved in cell differentiation, migration and apoptosis. We recently demonstrated that NOX4 in renal cell carcinoma (RCC) is an energetic sensor and couples energy metabolism to drug-resistance. In this study we demonstrate that enhanced nuclear localization of NOX4 is correlated with poor survival. Methods: We identified 350 RCC patients with (n = 85) and without (n = 265) venous tumor thrombus (VTT) who underwent radical nephrectomy with or without thrombectomy at our institution between 2013-2016. Fifty patients with RCC and VTT were matched 1:1 to patients with RCC and no VTT. Tissue Microarray (TMA) consisting of primary renal tumor, adjacent renal tumor and VTT were created. These matched TMA (n = 174) were stained with NOX4 antibodies; scanned using the Aperio scanning system and analyzed with the ImageScope software. The RCC nucleus was evaluated for roundness, elongation and size. NOX4 nuclear staining was categorized based on intensity as either strong positive, moderate, weak or no localization. Differential protein expression between the primary tumor, adjacent normal parenchyma and VTT was assessed using the Chi-squared test or t-test. Progression-free survival (PFS) and overall-survival (OS), stratified by NOX4 staining intensity, was estimated using the Kaplan-Meier method and compared with the log-rank test. Cox regression model was used to investigate effect of NOX4 staining on survival in multivariate analyses. Results: NOX4 nuclear colocalization was higher in patients who had progression and death from RCC. On univariate analysis, the intensity of NOX4 protein expression in the nucleus was significantly associated with PFS (HR 1.67, 95%CI (1.08-2.57; p = 0.02) and OS (HR 2.31, 95%CI (1.26-4.24; p = 0.007). On multivariate analyses the association between NOX4 protein expression and OS remains statistical significance (HR 2.09, 95%CI (1.05-4.18, p = 0.037). Conclusions: We provide evidence that in high grade/high stage cancer, NOX4 is colocalized within the nucleus of RCC. The prognostic role of NOX4 was determined at the protein level. We provide rationale for further investigation of NOX4, which may serve as a checkpoint in RCC.

The Role of NOX4 in Parkinson's Disease with Dementia.

The neuropathology of Parkinson’s disease with dementia (PDD) has been reported to involve heterogeneous and various disease mechanisms. Alpha-synuclein (α-syn) and amyloid beta (Aβ) pathology are associated with the cognitive status of PDD, and NADPH oxidase (NOX) is known to affect a variety of cognitive functions. We investigated the effects of NOX on cognitive impairment and on α-syn and Aβ expression and aggregation in PDD. In the 6-hydroxydopamine (6-OHDA)-injected mouse model, cognitive and motor function, and the levels of α-syn, Aβ, and oligomer A11 after inhibition of NOX4 expression in the hippocampal dentate gyrus (DG) were measured by the Morris water maze, novel object recognition, rotation, and rotarod tests, as well as immunoblotting and immunohistochemistry. After 6-OHDA administration, the death of nigrostriatal dopamine neurons and the expression of α-syn and NOX1 in the substantia nigra were increased, and phosphorylated α-syn, Aβ, oligomer A11, and NOX4 were upregulated in the hippocampus. 6-OHDA dose-dependent cognitive impairment was observed, and the increased cognitive impairment, Aβ expression, and oligomer A11 production in 6-OHDA-treated mice were suppressed by NOX4 knockdown in the hippocampal DG. Our results suggest that increased expression of NOX4 in the hippocampal DG in the 6-OHDA-treated mouse induces Aβ expression and oligomer A11 production, thereby reducing cognitive function.

The NADPH Oxidases Nox1 and Nox2 Differentially Regulate Volatile Organic Compounds, Fungistatic Activity, Plant Growth Promotion and Nutrient Assimilation in Trichoderma atroviride.

In eukaryotic systems, membrane-bound NADPH oxidases (Nox) generate reactive oxygen species (ROS) as a part of normal physiological functions. In the soil-borne mycoparasitic and plant facultative symbiont Trichoderma atroviride, Nox1 and the regulator NoxR are involved in differentiation induced by mechanical damage, while the role of Nox2 has not been determined. The knock-out strains Δnox1, ΔnoxR and Δnox2 were compared to the parental strain (WT) in their ability to grow and conidiate under a series of stress conditions (osmotic, oxidative, membrane, and cell-wall stresses). All three genes were differentially involved in the stress-response phenotypes. In addition, several interactive experiments with biotic factors (plant seedlings and other fungi) were performed comparing the mutant phenotypes with the WT, which was used as the reference strain. Δnox1 and ΔnoxR significantly reduced the antagonistic activity of T. atroviride against Rhizoctonia solani and Sclerotinia sclerotiorum in direct confrontation assays, but Δnox2 showed similar activity to the WT. The Δnox1, ΔnoxR, and Δnox2 mutants showed quantitative differences in the emission of several volatile organic compounds (VOCs). The effects of a blend of these volatiles on plant-growth promotion of Arabidopsis thaliana seedlings were determined in closed-chamber experiments. The increase in root and shoot biomass induced by T. atroviride VOCs was significantly lowered by ΔnoxR and Δnox1, but not by Δnox2. In terms of fungistatic activity at a distance, Δnox2 had a significant reduction in this trait against R. solani and S. sclerotiorum, while fungistasis was highly increased by ΔnoxR and Δnox1. Identification and quantification of individual VOCs in the blends emitted by the strains was performed by GC-MS and the patterns of variation observed for individual volatiles, such as 6-Pentyl-2H-pyran-2-one (6PP-1) and (E)-6-Pent-1-enylpyran-2-one (6PP-2) were consistent with their negative effects in plant-growth promotion and positive effects in fungistasis at a distance. Nox1 and NoxR appear to have a ubiquitous regulatory role of in a variety of developmental and interactive processes in T. atroviride either as positive or negative modulators. Nox2 may also have a role in regulating production of VOCs with fungistatic activity.