Intermittent hypoxia has organ-specific effects on oxidative stress

Obstructive sleep apnea is characterized by upper airway collapse, leading to intermittent hypoxia (IH). It has been postulated that IH-induced oxidative stress may contribute to several chronic diseases associated with obstructive sleep apnea. We hypothesize that IH induces systemic oxidative stress by upregulating NADPH oxidase, a superoxide-generating enzyme. NADPH oxidase is regulated by a cytosolic p47(phox) subunit, which becomes phosphorylated during enzyme activation. Male C57BL/6J mice were exposed to IH with an inspired O(2) fraction nadir of 5% 60 times/h during the 12-h light phase (9 AM-9 PM) for 1 or 4 wk. In the aorta and heart, IH did not affect lipid peroxidation [malondialdehyde (MDA) level], nitrotyrosine level, or p47(phox) expression and phosphorylation. In contrast, in the liver, exposure to IH for 1 wk resulted in a trend to an increase in MDA levels, whereas IH for 4 wk resulted in a 38% increase in MDA levels accompanied by upregulation of p47(phox) expression and phosphorylation. Administration of an NADPH oxidase inhibitor, apocynin, during IH exposure attenuated IH-induced increases in hepatic MDA. In p47(phox)-deficient mice, MDA levels were higher at baseline and, unexpectedly, decreased during IH. In conclusion, oxidative stress levels and pathways under IH conditions are organ and duration specific.     

Inhibition of NADPH oxidase reduces myocardial oxidative stress and apoptosis and improves cardiac function in heart failure after myocardial infarction

Increases in NADPH oxidase activity, oxidative stress, and myocyte apoptosis coexist in failing hearts. In cardiac myocytes in vitro inhibition of NADPH oxidase reduces apoptosis. In this study, we tested the hypothesis that NADPH oxidase inhibition reduces myocyte apoptosis and improves cardiac function in heart failure after myocardial infarction (MI). Rabbits with heart failure induced by MI and sham-operated animals were randomized to orally receive apocynin, an inhibitor of NADPH oxidase (15 mg per day) or placebo for 4 weeks. Left ventricular (LV) dimension and function were assessed by echocardiography and hemodynamics. Myocardial NADPH oxidase activity was measured by superoxide dismutase-inhibitable cytochrome c reduction assay, NADPH oxidase subunit p47phox expression by Western blot and immunofluorescence analysis, myocardial oxidative stress evaluated by 8-hydroxydeoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (4-HNE) using immunohistochemistry, and myocyte apoptosis by TUNEL assay. MI rabbits exhibited LV dilatation and systolic dysfunction measured by LV fractional shortening and the maximal rate of LV pressure rise (dP/dt). These changes were associated with increases in NADPH oxidase activity, p47phox protein expression, 8-OHdG expression, 4-HNE expression, myocyte apoptosis, and Bax protein and a decrease in Bcl-2 protein. Apocynin reduced NADPH oxidase activity, p47phox protein, oxidative stress, myocyte apoptosis, and Bax protein, increased Bcl-2 protein, and ameliorated LV dilatation and dysfunction after MI. The results suggest that inhibition of NADPH oxidase may represent an attractive therapeutic approach to treat heart failure.     

HSCARG Inhibits NADPH Oxidase Activity through Regulation of the Expression of p47phox

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase catalyzes the transfer of electrons from NADPH to O₂, which is the main source of reactive oxygen species (ROS) in nonphagocytic cells. Excess ROS are toxic; therefore, keeping ROS in homeostasis in cells can protect cells from oxidative damage. It is meaningful to further understand the molecular mechanism by which ROS homeostasis is mediated. Human protein HSCARG is a newly identified oxidative sensor and a negative regulator of NF-κB. Here, we find that HSCARG represses the cellular ROS generation through inhibiting mRNA and protein expression of p47phox, a subunit of NADPH oxidase. In contrast, shRNA-mediated HSCARG knockdown increases endogenous p47phox expression level. And HSCARG has no obvious effect on ROS production in p47phox-depleted cells. Furthermore, HSCARG regulates p47phox through inhibition of NF-κB activity. Our findings identify HSCARG as a novel regulator in regulation of the activity of NADPH oxidase and ROS homeostasis.     

Systemic upregulation of NADPH oxidase in diet-induced obesity in rats

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is upregulated in a variety of tissues in obesity. It is still unclear as to whether NADPH oxidase upregulation in a specific tissue is part of a systemic response. Here we analyzed the expression pattern of NADPH oxidase in vascular, adipose, and kidney tissues in a rat model of diet-induced obesity. After weaning, rats were fed either a normal or high-fat diet for 12 weeks. The high-fat diet resulted in 20% increased body weight. In the aorta, Nox4 expression was increased by three-fold in obese rats. Upregulations of p22phox and p47phox in adipose, and Nox4, p22phox, and p47phox in kidney were observed in obesity. Marked increases in plasma leptin and insulin were observed, with more modest changes in adiponectin in obese rats. The average systolic blood pressure in the obese group was 11 mmHg higher than that of lean rats (P < 0.005). There was a significant correlation between blood pressure and aortic Nox4 expression (P < 0.01). In cultured vascular smooth muscle cells, adiponectin reduced the expression of Nox4 in a protein kinase A-dependent manner. Our results suggest that upregulation of NADPH oxidase in multiple tissues during obesity appears to be a systemic response. At least in vitro, adiponectin may have a protective antioxidant role by suppressing vascular NADPH oxidase expression. The association between NADPH oxidase Nox4 expression in the vasculature and the elevated blood pressure in obesity requires further investigation.     

The vascular NADPH oxidase subunit p47phox is involved in redox-mediated gene expression

An NADPH oxidase is thought to be a main source of vascular superoxide (O(2)(-)) production. The functional role of this oxidase, however, and the contribution of the different subunits of the enzyme to cellular signaling are still incompletely understood. We determined the role of the p47phox subunit of the oxidase in O(2)(-) generation and signaling in aortic rings and cultured smooth muscle cells (SMC) from wild-type (WT) and p47phox-deficient (p47phox -/-) mice. Basal O(2)(-) levels in aortae of p47phox -/- mice were lower than those in WT aortae. Infusion of [val(5)]-angiotensin II increased O(2)(-) levels in aortae from WT more than in aortae from p47phox -/- mice. O(2)(-) generation was similar in quiescent SMC from WT and p47phox -/- mice. However, exposure to thrombin selectively increased O(2)(-) generation in VSMC from WT, but not from p47phox -/- mice. Thrombin-activated redox-mediated signal transduction and gene expression was attenuated in VSMC from p47phox -/- compared to cells from WT mice as determined by p38 MAP kinase activation and VEGF gene expression. We conclude that p47phox is important for vascular ROS production and redox-modulated signaling and gene expression in VSMC.     

Cytochrome P4502E1 primes macrophages to increase TNF-alpha production in response to lipopolysaccharide

Kupffer cells become activated in response to elevated levels of LPS during ethanol feeding, but the role of ethanol in the molecular processes of activation remains unclear. Because cytochrome P4502E1 (CYP2E1) is upregulated in Kupffer cells after ethanol, we hypothesized that this effect primes Kupffer cells, sensitizing them to increase TNF-alpha production in response to LPS. However, cultured Kupffer cells rapidly lose their CYP2E1. This difficulty was overcome by transfecting CYP2E1 to RAW 264.7 macrophages. Macrophages with stable increased CYP2E1 expression (E2) displayed increased levels of CD14/Toll-like receptor 4, NADPH oxidase and H2O2, accompanied by activation of ERK1/2, p38, and NF-kappaB. These increases primed E2 cells, sensitizing them to LPS stimuli, with amplification of LPS signaling, resulting in increased TNF-alpha production. Diphenyleneiodonium, a NADPH oxidase inhibitor, and diallyl sulfide, a CYP2E1 inhibitor, decreased approximately equally H2O2 levels in E2 cells, suggesting that NADPH oxidase and CYP2E1 contribute equally to H2O2 generation. Because CYP2E1 expression also enhanced the levels of the membrane localized NADPH oxidase subunits p47phox and p67phox, thereby contributing to the oxidase activation, it may augment H2O2 generation via this mechanism. H2O2, derived in part from NADPH and CYP2E1, activated ERK1/2 and p38. ERK1/2 stimulated TNF-alpha production via activation of NF-kappaB, whereas p38 promoted TNF-alpha production by stabilizing TNF-alpha mRNA. Oxidant generation after CYP2E1 overexpression appears to be central to macrophage priming and their sensitization to LPS. Accordingly, CYP2E1 priming could explain the sensitization of Kupffer cells to LPS activation by ethanol, a critical early step in alcoholic liver disease.     

Trichinella spiralis infection affects p47(phox) protein expression in guinea-pig alveolar macrophages

To establish whether NADPH oxidase activation, responsible for previously demonstrated Trichinella spiralis-induced respiratory burst, results from assembling of membrane and cytosolic NADPH oxidase components and/or increased expression of the oxidase complex proteins, the superoxide anion production and expression of the regulatory p47(phox) subunit were measured in cultured alveolar macrophages obtained during T. spiralis infection of guinea pigs. The results demonstrate for the first time helminth parasite-infection-induced stimulation of NADPH oxidase p47(phox) subunit protein expression, with the effect being decreased by in vivo treatment with cyclosporin A, previously shown to inhibit T. spiralis infection-induced respiratory burst in guinea-pig alveolar macrophages. However, although the expression of the p47(phox) subunit protein remained induced during secondary infection, it was accompanied by superoxide anion production that was significantly suppressed in comparison with that observed during primary infection, suggesting suppressive action of T. spiralis on host's alveolar macrophage immune response, presumably connected with NADPH oxidase complex activity attenuation.     

p47(phox) PX domain of NADPH oxidase targets cell membrane via moesin-mediated association with the actin cytoskeleton

Activation of phagocytic NADPH oxidase requires association of its cytosolic subunits with the membrane-bound flavocytochrome. Extensive phosphorylation of the p47(phox) subunit of NADPH oxidase marks the initiation of this activation process. The p47(phox) subunit then translocates to the plasma membrane, bringing the p67(phox) subunit to cytochrome b558 to form the active NADPH oxidase complex. However, the detailed mechanism for targeting the p47(phox) subunit to the cell membrane during activation still remains unclear. Here, we show that the p47(phox) PX domain is responsible for translocating the p47(phox) subunit to the plasma membrane for subsequent activation of NADPH oxidase. We also demonstrate that translocation of the p47(phox) PX domain to the plasma membrane is not due to interactions with phospholipids but rather to association with the actin cytoskeleton. This association is mediated by direct interaction between the p47(phox) PX domain and moesin.     

NADPH oxidase and apoptosis in cerulein-stimulated pancreatic acinar AR42J cells

Apoptosis linked to oxidative stress has been implicated in pancreatitis. We investigated whether NADPH oxidase mediates apoptosis in cerulein-stimulated pancreatic acinar AR42J cells. We report here that cerulein treatment resulted in the activation of NADPH oxidase, as determined by ROS production, translocation of cytosolic subunits p 47(phox) and p 67(phox) to the membrane, and interaction between NADPH oxidase subunits. Cerulein induced Ca(2+) oscillation, the expression of apoptotic genes p53 and bax, and apoptotic indices (DNA fragmentation, TUNEL staining, caspase 3 activity, decrease in cell viability) in AR42J cells. Treatment with a Ca(2+) chelator, BAPTA-AM, or transfection with antisense oligonucleotides for NADPH oxidase subunits p22(phox) and p 47(phox) inhibited cerulein-induced ROS production, translocation of NADPH oxidase cytosolic subunits p 47(phox) and p 67(phox) to the membrane, and the expression of apoptotic genes and apoptotic indices, as compared to the cells without treatment and those transfected with the corresponding sense oligonucleotides. These results indicate that NADPH oxidase may mediate ROS-induced apoptosis in pancreatic acinar cells in a Ca(2+)-dependent manner.     

Role of components of the phagocytic NADPH oxidase in oxygen sensing.

It has been hypothesized that O(2) sensing in type I cells of the carotid body and erythropoietin (EPO)-producing cells of the kidney involves protein components identical to the NADPH oxidase system responsible for the respiratory burst of phagocytes. In the present study, we evaluated O(2) sensing in mice with null mutant genotypes for two components of the phagocytic oxidase. Whole body plethysmography was used to study unanesthetized, unrestrained mice. When exposed to an acute hypoxic stimulus, gp91(phox)-null mutant and wild-type mice increased their minute ventilation by similar amounts. In contrast, p47(phox)-null mutant mice demonstrated increases in minute ventilation in response to hypoxia that exceeded that of their wild-type counterparts: 98.0 +/- 18.0 vs. 20.0 +/- 13.0% (n = 11, P = 0.003). In vitro recordings of carotid sinus nerve (CSN) activity demonstrated that resting (basal) neural activity was marginally elevated in p47(phox)-null mutant mice. With hypoxic challenge, mean CSN discharge was 1.5-fold greater in p47(phox)-null mutant than in wild-type mice: 109.61 +/- 13.29 vs. 72.54 +/- 7.65 impulses/s (n = 8 and 7, respectively, P = 0.026). Consequently, the hypoxia-evoked CSN discharge (stimulus-basal) was approximately 58% larger in p47(phox)-null mutant mice. Quantities of EPO mRNA in kidney were similar in gp91(phox)- and p47(phox)-null mutant mice and their respective wild-type controls exposed to hypobaric hypoxia for 72 h. These findings confirm the previous observation that absence of the gp91(phox) component of the phagocytic NADPH oxidase does not alter the O(2)-sensing mechanism of the carotid body. However, absence of the p47(phox) component significantly potentiates ventilatory and chemoreceptor responses to hypoxia. O(2) sensing in EPO-producing cells of the kidney appears to be independent of the gp91(phox) and p47(phox) components of the phagocytic NADPH oxidase.     

Role of NADPH oxidase in the brain injury of intracerebral hemorrhage

The major risk factors for intracerebral hemorrhage (ICH) are hypertension and aging. A fundamental mechanism for hypertension- and aging-induced vascular injury is oxidative stress. We hypothesize that oxidative stress has a crucial role in ICH. To test our hypothesis, we used bacterial collagenase to produce ICH in wild-type C57BL/6 and gp91phox knockout (gp91phox KO) mice (deficient in gp91phox subunit of the superoxide-producing enzyme NADPH oxidase). All animals were studied at 20-35 weeks of age, resembling an older patient population. We found that collagenase produced less bleeding in gp91phox KO mice than wild-type mice. Total oxidative product was lower in gp91phox KO mice than in wild-type mice, both under basal conditions and after ICH. Consistent with the ICH volume, brain edema formation, neurological deficit and a high mortality rate was noted in wild-type but not in gp91phox KO mice. This ICH-induced brain injury in wild-type mice is associated with enhanced expression of the gp91phox subunit of NADPH oxidase. In conclusion, the oxidative stress resulting from activation of NADPH oxidase contributes to ICH induced by collagenase and promotes brain injury.     

Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta activation

Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233-240] demonstrated that Hcy (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hcy treatment (20-100 microM) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCalpha, PKCbeta and PKCgamma) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCbeta oligonucleotide, but not antisense PKCalpha oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCbeta inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.     

Expression of isoforms of NADPH oxidase components in rat pancreatic islets

Increased oxidative stress plays a role in the pathogenesis of beta-cell dysfunction and death. We studied isoforms of NADPH oxidase components in islets of Langerhans isolated from rat pancreas and tumoral rat beta-cell line RINm5F cells by RT-PCR and sequencing of its products. RT-PCR revealed that isolated islets constitutively expressed mRNA of NADPH oxidase components, Nox1, Nox2, Nox4 and p22(phox) as membrane-associated components and p47(phox), Noxo1 (homologue of p47(phox)), Noxa1 (homologue of p67(phox)), and p40(phox) as cytosolic components. RINm5F cells showed a similar pattern of expression but Nox2 mRNA was not detected. Expression of Nox1, Nox4, Noxo1 and Noxa1 was confirmed by sequencing the PCR products. Immunohistochemistry revealed the expression of NADPH oxidase component in beta-cells of rat pancreatic islets. Glucose-stimulated insulin secretion from isolated islets was suppressed by diphenyleneiodonium, a flavocytochrome inhibitor, but not by apocynin, an inhibitor of p47(phox) translocation to membranes. Our results suggest that the functional significance of NADPH oxidase in insulin secretion may merit further investigation.     

Acute tumor necrosis factor alpha signaling via NADPH oxidase in microvascular endothelial cells: role of p47phox phosphorylation and binding to TRAF4

Tumor necrosis factor alpha (TNF-alpha) receptor-associated factors (TRAFs) play important roles in TNF-alpha signaling by interacting with downstream signaling molecules, e.g., mitogen-activated protein kinases (MAPKs). However, TNF-alpha also signals through reactive oxygen species (ROS)-dependent pathways. The interrelationship between these pathways is unclear; however, a recent study suggested that TRAF4 could bind to the NADPH oxidase subunit p47phox. Here, we investigated the potential interaction between p47phox phosphorylation and TRAF4 binding and their relative roles in acute TNF-alpha signaling. Exposure of human microvascular endothelial cells (HMEC-1) to TNF-alpha (100 U/ml; 1 to 60 min) induced rapid (within 5 min) p47phox phosphorylation. This was paralleled by a 2.7- +/- 0.5-fold increase in p47phox-TRAF4 association, membrane translocation of p47phox-TRAF4, a 2.3- +/- 0.4-fold increase in p47phox-p22phox complex formation, and a 3.2- +/- 0.2-fold increase in NADPH-dependent O2- production (all P < 0.05). TRAF4-p47phox binding was accompanied by a progressive increase in extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38(MAPK) activation, which was inhibited by an O2- scavenger, tiron. TRAF4 predominantly bound the phosphorylated form of p47phox, in a protein kinase C-dependent process. Knockdown of TRAF4 expression using siRNA had no effect on p47phox phosphorylation or binding to p22phox but inhibited TNF-alpha-induced ERK1/2 activation. In coronary microvascular EC from p47phox-/- mice, TNF-alpha-induced NADPH oxidase activation, ERK1/2 activation, and cell surface intercellular adhesion molecule 1 (ICAM-1) expression were all inhibited. Thus, both p47phox phosphorylation and TRAF4 are required for acute TNF-alpha signaling. The increased binding between p47phox and TRAF4 that occurs after p47phox phosphorylation could serve to spatially confine ROS generation from NADPH oxidase and subsequent MAPK activation and cell surface ICAM-1 expression in EC.     

NOX in liver fibrosis

NADPH oxidase is a multi-protein complex producing reactive oxygen species (ROS) both in phagocytic cells, being essential in host defense, and in non-phagocytic cells, regulating intracellular signalling. In the liver, NADPH oxidase plays a central role in fibrogenesis. A functionally active form of the NADPH oxidase is expressed not only in Kupffer cells (phagocytic cell type) but also in hepatic stellate cells (HSCs) (non-phagocytic cell type), suggesting a role of the non-phagocytic NADPH oxidase in HSC activation. Consistent with this concept, profibrogenic agonists such as Angiotensin II (Ang II) and platelet derived growth factor (PDGF), or apoptotic bodies exert their activity through NADPH oxidase-activation in HSCs. Both pharmacological inhibition with DPI and genetic studies using p47(phox) knockout mice provided evidence for a central role of NADPH oxidase in the regulation of HSC-activity and liver fibrosis. In addition to the p47(phox) component, only Rac1 has been identified as a functional active component of the NADPH oxidase complex in HSCs.     

Oxidative stress and increased eNOS and NADPH oxidase expression in mouse microvessel endothelial cells

Elevated oxidative stress plays a key role in diabetes-associated vascular disease. In this study, we tested the hypothesis that high glucose-induced oxidative stress was associated with changes in the expression of NADPH oxidase, superoxide dismutase (SOD) and endothelial nitric oxide synthase (eNOS). Oxidative stress was assessed in cell cultures of mouse microvessel endothelial cells (MMECs) by fluorescence labelling with dihydroethidium, lucigenin-enhanced chemiluminescence and determining NADPH oxidase subunit and eNOS expression with real-time polymerase chain reaction protocol and Western blotting. Oxidative stress and expression of the NADPH oxidase subunit, p22phox, were both increased, SOD1 and 3 expression lowered and eNOS significantly elevated in MMECs treated with 40 mM glucose for 72 h compared to low glucose medium. Oxidative stress, p22phox mRNA, eNOS mRNA, and protein were lowered by concurrent incubation with sepiapterin. When eNOS protein expression in endothelial cells was significantly decreased by eNOS siRNA treatment, superoxide generation was significantly higher in the MMECs grown in low glucose, but reduced in those grown in high glucose for 72 h. Thus, exposure of MMECs to high glucose results in increased oxidative stress that is associated with increased eNOS and NADPH oxidase subunit expression, notably p22phox, and decreased expression of SOD1 and 3.     

Systemic upregulation of NADPH oxidase in diet-induced obesity in rats

Abstract

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is upregulated in a variety of tissues in obesity. It is still unclear as to whether NADPH oxidase upregulation in a specific tissue is part of a systemic response. Here we analyzed the expression pattern of NADPH oxidase in vascular, adipose, and kidney tissues in a rat model of diet-induced obesity. After weaning, rats were fed either a normal or high-fat diet for 12 weeks. The high-fat diet resulted in 20% increased body weight. In the aorta, Nox4 expression was increased by three-fold in obese rats. Upregulations of p22phox and p47phox in adipose, and Nox4, p22phox, and p47phox in kidney were observed in obesity. Marked increases in plasma leptin and insulin were observed, with more modest changes in adiponectin in obese rats. The average systolic blood pressure in the obese group was 11 mmHg higher than that of lean rats (P < 0.005). There was a significant correlation between blood pressure and aortic Nox4 expression (P < 0.01). In cultured vascular smooth muscle cells, adiponectin reduced the expression of Nox4 in a protein kinase A-dependent manner. Our results suggest that upregulation of NADPH oxidase in multiple tissues during obesity appears to be a systemic response. At least in vitro, adiponectin may have a protective antioxidant role by suppressing vascular NADPH oxidase expression. The association between NADPH oxidase Nox4 expression in the vasculature and the elevated blood pressure in obesity requires further investigation.     

17beta-estradiol inhibits NADPH oxidase activity through the regulation of p47phox mRNA and protein expression in THP-1 cells

In this report, we demonstrate that NADPH oxidase is activated by tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma) in human monocytic cells (THP-1 cells) differentiated with phorbol ester (PMA) and that physiological concentration of 17beta-estradiol inhibits NADPH oxidase activity in THP-1 cells stimulated with TNF-alpha plus IFN-gamma. This effect is mediated by estrogen receptor based on estrogen receptor antagonist (ICI 182, 780) that diminishes inhibition by 17beta-estradiol. This inhibition is specific in 17beta-estradiol because 17alpha-estradiol, testosterone and progesterone do not inhibit NADPH oxidase activity. Activation of NADPH oxidase induced by TNF-alpha plus IFN-gamma is caused by up-regulation of p47(phox) (cytosolic component of NADPH oxidase) expression. 17beta-Estradiol prevents the up-regulation of p47(phox) mRNA and protein expression. This prevention of p47(phox) expression depends on the inhibition of NF-kappaB activation. Our results implicate that 17beta-estradiol has an anti-atherosclerotic effects through the improvement of nitric oxide (NO) bioavailability caused by the regulation of superoxide (O(2)(-)) production.     

Expression and activity of polymorphisms in the 67-kDa protein of the NADPH oxidase system

The NADPH oxidase system plays a central role in the antimicrobial activity of phagocytes. This system is initiated by the translocation of cytosolic proteins p67phox, p47phox and p40phox to be in close contact with membrane flavocytochrome b558. This event begins the electron transfer from cytosolic NADPH to molecular oxygen to produce superoxide anions. Herein, a functional analysis is presented of p67phox polymorphisms identified from healthy humans. Mutations were generated in the p67phox cDNA by site-directed mutagenesis and then transiently expressed in COS7 cells that also expressed gp91phox, p22phox, and p47phox from stable transgenes. The changes Va1166lle, Pro329Ser and His389Gln correspond to possible polymorphisms identified in healthy individuals revealed a functional activity similar to COSphox cells transiently transfected with WT p67phox; therefore, these modifications are not associated with genetic deficiencies in NADPH oxidase. In conclusion, the COSphox system represents an easily transfectable model for analysis of NADPH oxidase function in intact cells. The analysis of mutant derivatives of p67phox provides insight into molecular mechanisms by which this subunit regulates the NADPH oxidase.     

Inhibition of NADPH oxidase promotes alternative and anti-inflammatory microglial activation during neuroinflammation

Like macrophages, microglia are functionally polarized into different phenotypic activation states, referred as classical and alternative. The balance of the two phenotypes may be critical to ensure proper brain homeostasis, and may be altered in brain pathological states, such as Alzheimer's disease. We investigated the role of NADPH oxidase in microglial activation state using p47(phox) and gp91(phox) -deficient mice as well as apocynin, a NADPH oxidase inhibitor during neuroinflammation induced by an intracerebroventricular injection of LPS or Aβ????. We showed that NADPH oxidase plays a critical role in the modulation of microglial phenotype and subsequent inflammatory response. We demonstrated that inhibition of NADPH oxidase or gene deletion of its functional p47(phox) subunit switched microglial activation from a classical to an alternative state in response to an inflammatory challenge. Moreover, we showed a shift in redox state towards an oxidized milieu and that subpopulations of microglia retain their detrimental phenotype in Alzheimer's disease brains. Microglia can change their activation phenotype depending on NADPH oxidase-dependent redox state of microenvironment. Inhibition of NADPH oxidase represents a promising neuroprotective approach to reduce oxidative stress and modulate microglial phenotype towards an alternative state.