Gp91phox mRNA Levels Research Articles

NADPH Oxidase Isoform 2 (NOX2) Is Involved in Drug Addiction Vulnerability in Progeny Developmentally Exposed to Ethanol.

Ethanol exposure increases oxidative stress in developing organs, including the brain. Antioxidant treatment during maternal ethanol ingestion improves behavioral deficits in rodent models of fetal alcohol spectrum disorder (FASD). However, the impact of general antioxidant treatment in their adult offspring and the Specific Reactive Species (ROS)-dependent mechanism, are not fully understood. We hypothesized that pre and early postnatal ethanol exposure (PEE) modifies redox homeostasis, in particular NOX2 function during reward signaling in the mesocorticolimbic pathway, which reinforces the effects of alcohol. We developed a FASD rat model which was evaluated during adolescence (P21) and adulthood (P70). We first studied whether redox homeostasis is affected in PEE animals, by analyzing mRNA expression of SOD1, CAT, and Gpx1. We found that PEE reduced the mRNA levels of these three anti-oxidant enzymes in PFC and HIPP at P21 and in the VTA at P70. We also analyzed basal mRNA and protein expression of NOX2 subunits such as gp91phox, p22 phox, and p47 phox, in mesocorticolimbic brain areas of PEE rat brains. At P21, gp91 phox, and p47 phox levels in the VTA were decreased. At P70, gp91 phox mRNA levels was decreased in HIPP and both mRNA and protein levels were decreased in PFC. Since NOX2 is regulated by the N-methyl-D-aspartate Receptor (NMDAR), we analyzed NMDAR mRNA expression and found differential expression of NMDAR subunits (NR1 and NR2B) in the PFC that was age dependent, with levels decreased at P21 and increased at P70. The analysis also revealed decreased NR2B mRNA expression in HIPP and VTA at P70. Offspring from maternal ethanol users consumed 25% more ethanol in a free choice alcohol consumption test than control rats, and showed place preference for an alcohol-paired compartment. In vivo inhibition of NOX2 using apocynin in drinking water, or infusion of blocked peptide gp91 phox ds in the VTA normalized alcohol place preference, suggesting that NOX2 plays an important role in addictive like behavior. Taken together, PEE significantly affects the expression of antioxidant enzymes, NOX2, NMDAR in an age, and brain region dependent manner. Moreover, we demonstrate that NOX2 regulates alcohol seeking behavior.

41 - Chronic GVHD Could Ameliorate the Impact of Adverse Somatic Mutations in Patients with Myelodysplastic Syndromes and Hematopoietic Stem Cell Transplantation

Our aim was to assess the use of peripheral blood mononuclear cells (PBMC) as an in vivo cellular model to evaluate diet-induced changes in the oxidative stress status by analyzing the gene expression pattern of NADPH-oxidase subunits and antioxidant genes. A randomized, controlled trial assigned metabolic syndrome patients to 4 diets for 12 weeks each: (i) high-saturated fatty acid (HSFA), (ii) high-monounsaturated fatty acid, and (iii), (iv) two low-fat, high-complex carbohydrate diets supplemented with n-3 polyunsaturated fatty acids or placebo. A fat challenge reflecting the fatty acid composition as the original diets was conducted post-intervention. The mRNA levels of gp91phox (P < 0.001), p22phox (P = 0.005), p47phox (P = 0.001) and p40phox (P < 0.001) increased at 2 h after the intake of the HSFA meal. The expression of SOD1, SOD2, GSR, GPx1, GPX4, TXN, TXNRD1 and Nrf2 increased after the HSFA meal (p < 0.05). In contrast, the expression of these genes remained unaltered in response to the other dietary interventions. Our results suggest that the increased expression of antioxidant genes in PBMC seems to be due to the response to the postprandial oxidative stress generated mainly in adipose tissue after the consumption of an HSFA diet.

Ingested Capsaicinoids Can Prevent Low‐Fat/High‐Carbohydrate diet and High‐Fat Diet‐Induced Obesity by Regulating the Expression of Genes for Metabolism

BACKGROUNDWe previously found that ingested capsaicinoids (CAPs) inhibit pancreatic lipase and the mRNA levels of adipogenic genes such as peroxisome proliferator‐activated receptor‐gamma (PPAR‐gamma) and CCAAT enhancer‐binding protein‐alpha (C/EBP‐alpha) decreased in in vitro models. The aim of this study was to investigate the capsaicinoids (CAPs) effects on regulating the gene protein levels in low fat/high carbohydrate diet (LF/HCD) and high fat diet (HFD) induced obesity in rats.METHODSEight week male Wistar rats were fed on a LF/HCD and HFD with and without capsaicinoids compared with control group (CTL, +/− CAPs) for 8 weeks to induce obesity. Subsequently they were divided into different groups and were maintained on a CTL (+/− 0.2mg CAPs, 10 mg/kg bw CapsimaxTM, LF/HCD (+/− 0.2mg CAPs, 10 mg/kg bw CapsimaxTM) and HFD (+/−0.2 mg CAPs, 10 mg/kg bw CapsimaxTM) for 8 weeks. Changes in the gene protein levels of gp91phox (NOX2), Sirtuin 1 (SIRT1), nuclear factor NF‐κB, endothelial NO synthase (eNOS), NFkB, p22phox Protein (CYBA) and nuclear factor erythroid 2–related factor 2 (Nrf2) were analyzed. Serum and liver TBARS and total antioxidant capacity (TAC) were analyzed in all treatments.RESULTSExperimental diets supplemented with CAPs lowered the levels of mRNA levels of gp91phox (NOX2), NFkB, p22phox Protein (CYBA) and However, the mRNA levels of SIRT an important regulator of energy metabolism, Nrf2 an emerging regulator of cellular resistance to oxidant, eNOS a vasoprotective molecule nitric oxide (NO·) were significantly increased. CAPs treated groups had significant decrease in TBARS in serum and liver tissue and an increase in serum TAC were observed.CONCLUSIONThese results suggest that CAPs effectively reduces TBARS and increased TAC. These effects might be at least partially mediated via regulation of the gene proteins involved in energy metabolism, oxidative stress and vasoprotection.Support or Funding InformationOmniActive Health Technologies Inc.

TALEN-mediated functional correction of X-linked chronic granulomatous disease in patient-derived induced pluripotent stem cells

X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of the immune system. It is characterized by a defect in the production of reactive oxygen species (ROS) in phagocytic cells due to mutations in the NOX2 locus, which encodes gp91phox. Because the success of retroviral gene therapy for X-CGD has been hampered by insertional activation of proto-oncogenes, targeting the insertion of a gp91phox transgene into potential safe harbor sites, such as AAVS1, may represent a valid alternative. To conceptually evaluate this strategy, we generated X-CGD patient-derived induced pluripotent stem cells (iPSCs), which recapitulate the cellular disease phenotype upon granulocytic differentiation. We examined AAVS1-specific zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for their efficacy to target the insertion of a myelo-specific gp91phox cassette to AAVS1. Probably due to their lower cytotoxicity, TALENs were more efficient than ZFNs in generating correctly targeted iPSC colonies, but all corrected iPSC clones showed no signs of mutations at the top-ten predicted off-target sites of both nucleases. Upon differentiation of the corrected X-CGD iPSCs, gp91phox mRNA levels were highly up-regulated and the derived granulocytes exhibited restored ROS production that induced neutrophil extracellular trap (NET) formation. In conclusion, we demonstrate that TALEN-mediated integration of a myelo-specific gp91phox transgene into AAVS1 of patient-derived iPSCs represents a safe and efficient way to generate autologous, functionally corrected granulocytes.

L-Arginine Attenuates Cardiac Dysfunction, But Further Down-Regulates α-Myosin Heavy Chain Expression in Isoproterenol-Induced Cardiomyopathy.

In view of previously reported increased capacity for nitric oxide production, we suggested that l-arginine (ARG), the nitric oxide synthase (NOS) substrate, supplementation would improve cardiac function in isoproterenol (ISO)-induced heart failure. Male Wistar rats were treated with ISO for 8days (5mg/kg/day, i.p.) or vehicle. ARG was given to control (ARG) and ISO-treated (ISO+ARG) rats in water (0.4g/kg/day). ISO administration was associated with 40% mortality, ventricular hypertrophy, decreased heart rate, left ventricular dysfunction, fibrosis and ECG signs of ischaemia. RT-PCR showed increased mRNA levels of cardiac hypertrophy marker atrial natriuretic peptide, but not BNP, decreased expression of myosin heavy chain isoform MYH6 and unaltered expression of pathological MYH7. ISO increased the protein levels of endothelial nitric oxide synthase, but at the same time it markedly up-regulated mRNA and protein levels of gp91phox, a catalytical subunit of superoxide-producing NADPH oxidase. Fibrosis was markedly increased by ISO. ARG treatment moderately ameliorated left ventricular dysfunction, but was without effect on cardiac hypertrophy and fibrosis. Combination of ISO and ARG led to a decrease in cav-1 expression, a further increase in MYH7 expression and a down-regulation of MYH6 that inversely correlated with gp91phox mRNA levels. Although ARG, at least partially, improved ISO-impaired basal left ventricular systolic function, it failed to reduce cardiac hypertrophy, fibrosis, oxidative stress and mortality. The protection of contractile performance might be related to increased capacity for nitric oxide production and the up-regulation of MYH7 which may compensate for the marked down-regulation of the major MYH6 isoform.

Age-related hypertension and salt sensitivity are associated with unique cortico-medullary distribution of D1R, AT1R, and NADPH-oxidase in FBN rats

We examined effects of normal (NS) and high salt (HS) on blood pressure (BP) and cortico-medullary distribution of dopamine D1 receptor (D1R), angiotensin AT1 receptor (AT1R), NADPH oxidase-gp91phox, and sodium transporters (NHE-3, Na, K ATPase) in adult and aged rats. Aged rats fed with NS diet had higher BP, which further increased with HS. HS increased D1R mRNA and protein levels in cortex and medulla of adult rats. NS or HS fed-aged rats had higher AT1R and gp91phox mRNA levels in cortex and medulla. Aged rats fed with NS diet had higher gp91phox protein levels in cortex. HS diet increased AT1R and gp91phox protein levels in medulla of aged rats. Aged rats fed with NS or HS diet had higher NHE-3 protein levels in medulla. HS increased Na, K ATPase protein levels in medulla of aged rats. HS increased urinary kidney injury molecule-1 (KIM-1) but not protein or albumin levels in aged rats. These results suggest that cortical gp91phox and medullary NHE-3 contribute to age-related hypertension. Whereas D1R (cortical and medullary) together with medullary AT1R, gp91phox and Na, K-ATPase contribute to salt sensitivity in aged rats. And, KIM-1 may be a better marker for kidney damage.

Peripheral blood mononuclear cells as in vivo model for dietary intervention induced systemic oxidative stress

Our aim was to assess the use of peripheral blood mononuclear cells (PBMC) as an in vivo cellular model to evaluate diet-induced changes in the oxidative stress status by analyzing the gene expression pattern of NADPH-oxidase subunits and antioxidant genes. A randomized, controlled trial assigned metabolic syndrome patients to 4 diets for 12weeks each: (i) high-saturated fatty acid (HSFA), (ii) high-monounsaturated fatty acid, and (iii), (iv) two low-fat, high-complex carbohydrate diets supplemented with n-3 polyunsaturated fatty acids or placebo. A fat challenge reflecting the fatty acid composition as the original diets was conducted post-intervention. The mRNA levels of gp91phox (P<0.001), p22phox (P=0.005), p47phox (P=0.001) and p40phox (P<0.001) increased at 2h after the intake of the HSFA meal. The expression of SOD1, SOD2, GSR, GPx1, GPX4, TXN, TXNRD1 and Nrf2 increased after the HSFA meal (p<0.05). In contrast, the expression of these genes remained unaltered in response to the other dietary interventions. Our results suggest that the increased expression of antioxidant genes in PBMC seems to be due to the response to the postprandial oxidative stress generated mainly in adipose tissue after the consumption of an HSFA diet.

Abstract 481: Age-related Hypertension and Salt-sensitivity Are Associated With Specific Kidney Cortico-medullary Distribtion of D1R, AT1R And NADPH-oxidase

We recently reported that oxidative stress has a causal role in increasing kidney angiotensin II AT1 receptor (AT1R) and decreasing dopamine D1 receptor (D1R) functions in the aging Fischer Brown Norway (FBN) rats. These renal receptors by counter-regulating each other’s functions maintain sodium homeostasis and normal blood pressure (BP). Here, we examined systolic BP and kidney distribution (cortex vs medulla) of these receptors as well as of an oxidant producing enzyme (gp 91phox -NADPH-oxidase) in response to 4-week normal-salt (NS, 0.4% NaCl) and high-salt (HS, 8% NaCl) feeding in adult (3-month) and old (21-month) FBNs. BP in conscious animals was measured by radio-telemetry. Distribution of D1R, AT1R and gp 91phox were determined by RT-PCR and western blotting. We found that NS-fed old rats had higher BP (adult vs old: 105±1 vs 118 ±1 mmHg), which further increased with HS feeding in these rats (adult vs old: 111±1 vs 124 ±1 mmHg). The levels of D1R mRNA in cortex or in medulla were not different between NS-fed adult and old rats, but decreased in both cortex [adult vs old: 67 ± 15 vs 54±16 density unit (DU)] and medulla (adult vs old: 22±15 vs 11±3 DU) only in HS-fed old rats. Contrary to this, AT1R mRNA levels were higher only in cortex of old rats fed either NS (adult vs old: 81 ± 23 vs 102± 34 DU) or HS (adult vs old: 80 ± 17vs 98 ± 36 DU). Moreover, there were similar gp 91phox mRNA levels in the cortex and medulla of adult and old rats fed either NS or HS. But, its protein levels were higher in the cortex of old rats fed NS (adult vs old: 60±7 vs 112±8 DU) and HS (adult vs old: 61±3 vs 94±15 DU), and increased only in the medulla with HS feeding in these rats (adult vs old: 12±1 vs 28±2 DU). Our results suggest that there exists a specific cortico-medullary expression pattern of D1R, AT1R and gp 91phox with NS and HS feeding in old rats, which might be contributing to age-related hypertension and salt-sensitivity in FBNs.

Statins Inhibit NADPH Oxidase Activity by Interference with Membrane Raft Clustering Independent of Rac1 Inactivation in Endothelial Cells

Statins, the inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase, inhibit mevalonate pathway to block cholesterol biosynthesis, thereby lowering plasma cholesterol and being used for prevention and treatment of atherosclerotic diseases. Our previous studies have shown that statins including pravastatin and simvastatin interfere with the formation of OxLDL‐induced membrane raft (MR)‐redox signaling platforms in human coronary arterial endothelial cells (HCAECs) and in mouse coronary arterial endothelium. However, it remains unknown how statins produce this action that is independent of plasma cholesterol lowering. Here, we tested a hypothesis that inhibition of enhanced MR clustering is essential for the inhibitory effect of statins on Rac1 and NADPH oxidase (NOX) activity. We first demonstrated that statins had no effect on protein expression of NOX subunits, gp91phox or p47phox and Rac1 in HCAECs and mRNA levels of gp91phox or p47phox in coronary arteries of mice, confirming that statins primarily inhibit NADPH oxidase activation rather than alter their gene expression. Overexpression of oncogenic Rac1 in HCAECs was unable to reverse the inhibitory effects of statins on OxLDL‐induced MR clustering suggesting that the effect of statins on MR clustering does not require Rac1 activation. Electron spin resonance spectrometry demonstrated that incubation of HCAECs with sphingomyelinase, a strong stimulator of MR clustering significantly increased O2•− production, which was abolished by pravastatin and simvastatin or by a MR disruptor methyl‐β‐cyclodextrin. Taken together, these results indicate that statins inhibit NOX activity by interference with MR clustering independent of Rac1 activation. This action of statins on MR clustering may be associated with reduction of cholesterol in cell membrane of ECs (supported by NIH grants HL057244, HL091464 and HL075316).

Acute and chronic endurance exercise effects on nitric oxide, superoxide, and redox‐related gene expression in circulating CD34+ cells

The angiogenic potential of circulating CD34+ progenitor cells depends largely on the balance between intracellular nitric oxide (NO) and superoxide (O2−) levels. Our objective was to determine intracellular NO and O2− levels and expression of redox-related genes [endothelial nitric oxide synthase (eNOS), NADPH oxidase subunits p47phox and gp91phox, and superoxide dismutase-2 (SOD2)] in CD34+ cells isolated before and after acute exercise in age- and BMI-matched endurance-trained (ET) and sedentary (S) men. ET had lower baseline CD34+ cell NO and O2− levels than S (P < 0.05), and baseline eNOS and p47phox mRNA levels were higher in ET than S (P < 0.05). Acute exercise normalized group differences in NO and O2−, and p47phox mRNA decreased significantly in both ET and S (P < 0.05). gp91phox mRNA levels were similar between groups at baseline and decreased in S only (P < 0.05). We observed no differences in SOD2 mRNA levels between groups or with acute exercise. CONCLUSIONS The higher NO and lower p47phox mRNA levels observed in the S group's CD34+ cells were unexpected, as our previous work indicated that exercise enhanced NO dynamics in circulating angiogenic cells. However, given the lower O2− levels and higher eNOS mRNA in the ET group's cells, it appears that exercise training enhances intracellular conditions for vasoprotective angiogenic actions of CD34+ cells.

Effects of Exercise and l-Arginine on Ventricular Remodeling and Oxidative Stress

Our aim was to characterize the changes in messenger RNA (mRNA) abundance, protein, and activity levels of the enzymatic antioxidants, superoxide dismutase (SOD), glutathione peroxidase, and catalase by exercise training combined with L-arginine after myocardial infarction (MI). L-Arginine (1 g x kg(-1) x d(-1)) and N(G)-nitro-L-arginine methyl ester (L-NAME; 10 mg x kg(-1) x d(-1)) were administered in drinking water for 8 wk. Sprague-Dawley rats were randomized to the following groups: sham-operated control (Sham); MI sedentary (Sed); MI exercise (Ex); MI sedentary + L-arginine (Sed + LA); MI exercise + L-arginine (Ex + LA); MI sedentary + L-NAME (Sed + L-NAME); and MI exercise + L-NAME (Ex + L-NAME). The glutathione peroxidase, catalase, and gp91(phox) mRNA levels were comparable among all the groups. The SOD mRNA level was significantly increased in the Ex group (5.43 +/- 0.87) compared with the Sed group (1.74 +/- 0.29), whereas this effect was pronouncedly down-regulated by the L-NAME intervention (2.51 +/- 1.17, P < 0.05). The protein levels of SOD in the Sed and Ex groups were both significantly decreased with the administration of L-NAME. The protein levels of catalase were significantly higher in the Ex and Ex + LA groups than that in the Sed, Sed + LA, and L-NAME-treated groups. The collagen volume fraction was significantly lowered by the exercise and/or L-arginine treatment when compared with the Sed group. Fractional shortening was significantly preserved in the trained groups compared with their corresponding sedentary groups with or without drug treatments. However, the beneficial effect was not further improved by L-arginine treatment. Our results suggest that exercise training exerts antioxidative effects and attenuates myocardial fibrosis in the MI rats. These improvements, in turn, alleviate cardiac stiffness and preserve post-MI cardiac function. In addition, L-arginine appears to have no additive effect on cardiac function or expression of enzymatic antioxidants.

Administration of pigment epithelium-derived factor (PEDF) inhibits cold injury-induced brain edema in mice

Brain edema is the most life-threatening complication that occurs as a result of a number of insults to the brain. However, its therapeutic options are insufficiently effective. We have recently found that administration of pigment epithelium-derived factor (PEDF) inhibits retinal hyperpermeability in rats by counteracting biological effects of vascular endothelial growth factor (VEGF). In this study, we investigated whether PEDF could inhibit cold injury-induced brain edema in mice. Cold injury was induced by applying a pre-cooled metal probe on the parietal skull. VEGF and its receptor Flk-1 gene and/or protein expressions were up-regulated in the cold-injured brain. Cold injury induced brain edema, which was reduced by intraperitoneal injection of VEGF antibodies (Abs) or apocynin, an inhibitor of NADPH oxidase. PEDF mRNA and protein levels were up-regulated in response to cold injury. PEDF dose-dependently inhibited the brain edema, whose effect was neutralized by simultaneous treatments with anti-PEDF Abs. Although VEGF and Flk-1 gene and/or protein expressions were not suppressed by PEDF, PEDF or anti-VEGF Abs inhibited the cold injury-induced NADPH oxidase activity in the brain. Further, PEDF treatment inhibited activation of Rac-1, an essential component of NADPH oxidase in the cold-injured brain, while it did not affect mRNA levels of gp91phox, p22phox, or Rac-1. These results demonstrate that PEDF could inhibit the cold injury-induced brain edema by blocking the VEGF signaling to hyperpermeability through the suppression of NADPH oxidase via inhibition of Rac-1 activation. Our present study suggests that PEDF may be a novel therapeutic agent for the treatment of brain edema.

P1792 Atorvastatin decreases vascular superoxide production by reducing NAD(P)H oxidase activity without modification in gp91phox MRNa levels

P1792 Atorvastatin decreases vascular superoxide production by reducing NAD(P)H oxidase activity without modification in gp91phox MRNa levels

Cutting edge: infection by the agent of human granulocytic ehrlichiosis prevents the respiratory burst by down-regulating gp91phox.

The agent of human granulocytic ehrlichiosis (HGE) is an emerging tick-borne pathogen that resides in neutrophils and can be cultured in a promyelocytic (HL-60) cell line. In response to microbes, polymorphonuclear leukocytes normally activate the NADPH oxidase enzyme complex and generate superoxide anion (O2-). However, HL-60 cells infected with HGE bacteria did not produce O2- upon activation with PMA. RT-PCR demonstrated that HGE organisms inhibited mRNA expression of a single component of NADPH oxidase, gp91phox, and FACS analysis showed that plasma membrane-associated gp91phox protein was reduced on the infected cells. Infection with HGE organisms also decreased gp91phox mRNA levels in splenic neutrophils in a murine model of HGE, demonstrating this phenomenon in vivo. Therefore, HGE bacteria repress the respiratory burst by down-regulating gp91phox, the first direct inhibition of NADPH oxidase by a pathogen.

Cooperative effects of interferon-γ on the induction of NADPH oxidase by retinoic acid or 1,25(OH) 2-vitamin D 3 in monocytic U937 cells

The effect of retinoic acid (RA), 1,25-dihydroxyvitamin D 3 (1,25-D 3) or human recombinant interferon-γ (IFN-γ) on the induction of NADPH oxidase was studied in premonocytic U937 cells. Differentiation with the combination of either RA (1 μM) or 1,25-D 3 (10 nM) with IFN-γ (100 IU/ml) induced NADPH oxidase activity as demonstrated by increased superoxide anion (O 2 −) generation in response to stimulation with phorbol myristate acetate (PMA, 100 nM). Induction of NADPH oxidase activity was preceded by increases in mRNA levels of p47-phox, p67-phox and gp91-phox, which encode three subunits of the enzyme, and immunoblot analysis of the p47-phox and p67-phox proteins revealed that the increases in mRNA levels were equally reflected by increases in protein levels. In contrast, RA, 1,25-D 3 or IFN-γ alone did not induce NADPH oxidase activity which correlated with their failure to increase p67-phox and gp91-phox mRNA levels. The mRNA of p21 racl, a GTP-binding protein that regulates NADPH oxidase activity in macrophages, was constitutively expressed in undifferentiated cells and was not affected by differentiation. These data indicate that induction of a functional NADPH oxidase in premonocytic U937 cells requires the cooperative actions of IFN-γ plus RA or 1,25-D 3 and is reflected in the increased expression of p67-phox and gp91-phox.