Increased NADPH Oxidase Activity Research Articles

Modulation of chronic hypoxia-induced chemoreceptor hypersensitivity by NADPH oxidase subunits in rat carotid body

Previous studies in our laboratory established that reactive oxygen species (ROS) generated by NADPH oxidase (NOX) facilitate the open state of a subset of K+ channels in oxygen-sensitive type I cells of the carotid body. Thus pharmacological inhibition of NOX or deletion of a NOX gene resulted in enhanced chemoreceptor sensitivity to hypoxia. The present study tests the hypothesis that chronic hypoxia (CH)-induced hypersensitivity of chemoreceptors is modulated by increased NOX activity and elevated levels of ROS. Measurements of dihydroethidium fluorescence in carotid body tissue slices showed that increased ROS production following CH (14 days, 380 Torr) was blocked by the specific NOX inhibitor 4-(2-amino-ethyl)benzenesulfonyl fluoride (AEBSF, 3 microM). Consistent with these findings, in normal carotid body AEBSF elicited a small increase in the chemoreceptor nerve discharge evoked by an acute hypoxic challenge, whereas after 9 days of CH the effect of the NOX inhibitor was some threefold larger (P<0.001). Evaluation of gene expression after 7 days of CH showed increases in the isoforms NOX2 (approximately 1.5-fold) and NOX4 (approximately 3.8-fold) and also increased presence of the regulatory subunit p47phox (approximately 4.2-fold). Involvement of p47phox was further implicated in studies of isolated type I cells that demonstrated an approximately 8-fold and an approximately 11-fold increase in mRNA after 1 and 3 days, respectively, of hypoxia in vivo. These findings were confirmed in immunocytochemical studies of carotid body tissue that showed a robust increase of p47phox in type I cells after 14 days of CH. Our findings suggest that increased ROS production by NOX enzymes in type I cells dampens CH-induced hypersensitivity in carotid body chemoreceptors.

Similar effects of resistin and high glucose on P-selectin and fractalkine expression and monocyte adhesion in human endothelial cells

Resistin and high glucose (HG) are concomitantly present at elevated concentration in diabetic’s plasma; both are pro-inflammatory agents acting on vascular cells by mechanisms that are not fully understood. We questioned whether resistin and HG affect the expression of major adhesion molecules, P-selectin and fractalkine in human endothelial cells (HEC). The results showed that in HEC (i) resistin increased P-selectin expression; (ii) HG up-regulated Fk expression; (iii) P-selectin and fractalkine were functional increasing monocyte adhesion to activated cells. Co-stimulation with resistin and HG increased P-selectin and fractalkine mRNA and protein and induced monocyte adhesion, generated an increase in NADPH oxidase activity and of the intracellular reactive oxygen species and activated the NF-kB and AP-1 transcription factors at similar values as those of each activator. In conclusion in HEC, resistin and HG induce the up-regulation of P-selectin and fractalkine and the ensuing increased monocyte adhesion by a mechanism involving oxidative stress and NF-kB and AP-1 activation.

CK2 is a novel negative regulator of NADPH oxidase and a neuroprotectant in mice after cerebral ischemia.

NADPH oxidase is a major complex that produces reactive oxygen species (ROSs) during the ischemic period and aggravates brain damage and cell death after ischemic injury. Although many approaches have been tested for preventing production of ROSs by NADPH oxidase in ischemic brain injury, the regulatory mechanisms of NADPH oxidase activity after cerebral ischemia are still unclear. In this study, we identified casein kinase 2 (CK2) as a critical modulator of NADPH oxidase and elucidated the role of CK2 as a neuroprotectant after oxidative insults to the brain. We found that the protein levels of the catalytic subunits CK2alpha and CK2alpha', as well as the total activity of CK2, are significantly reduced after transient focal cerebral ischemia (tFCI). We also found this deactivation of CK2 caused by ischemia/reperfusion increases expression of Nox2 and translocation of p67(phox) and Rac1 to the membrane after tFCI. Interestingly, we found that the inactive status of Rac1 was captured by the catalytic subunit CK2alpha under normal conditions. However, binding between CK2alpha and Rac1 was immediately diminished after tFCI, and Rac1 activity was markedly increased after CK2 inhibition. Moreover, we found that deactivation of CK2 in the mouse brain enhances production of ROSs and neuronal cell death via increased NADPH oxidase activity. The increased brain infarct volume caused by CK2 inhibition was restored by apocynin, a NADPH oxidase inhibitor. This study suggests that CK2 can be a direct molecular target for modulation of NADPH oxidase activity after ischemic brain injury.

Attenuation of renal excretory responses to ANG II during inhibition of superoxide dismutase in anesthetized rats

To examine the functional interaction between superoxide dismutase (SOD) and NADPH oxidase activity, we assessed renal responses to acute intra-arterial infusion of ANG II (0.5 ng x kg(-1) x min(-1)) before and during administration of a SOD inhibitor, diethyldithiocarbamate (DETC, 0.5 mg x kg(-1) x min(-1)), in enalaprilat-pretreated (33 microg x kg(-1) x min(-1)) rats (n = 11). Total (RBF) and regional (cortical, CBF; medullary; MBF) renal blood flows were determined by Transonic and laser-Doppler flowmetry, respectively. Renal cortical and medullary tissue NADPH oxidase activity in vitro was determined using the lucigenin-chemiluminescence method. DETC treatment alone resulted in decreases in RBF, CBF, MBF, glomerular filtration rate (GFR), urine flow (V), and sodium excretion (U(Na)V) as reported previously. Before DETC, ANG II infusion decreased RBF (-18 +/- 3%), CBF (-16 +/- 3%), MBF [-5 +/- 6%; P = not significant (NS)], GFR (-31 +/- 4%), V (-34 +/- 2%), and U(Na)V (-53 +/- 3%). During DETC infusion, ANG II also caused similar reductions in RBF (-20 +/- 4%), CBF (-19 +/- 3%), MBF (-2 +/- 2; P = NS), and in GFR (-22 +/- 7%), whereas renal excretory responses (V; -12 +/- 2%; U(Na)V; -24 +/- 4%) were significantly attenuated compared with those before DETC. In in vitro experiments, ANG II (100 muM) enhanced NADPH oxidase activity both in cortical [13,194 +/- 1,651 vs. 20,914 +/- 2,769 relative light units (RLU)/mg protein] and in medullary (21,296 +/- 2,244 vs. 30,597 +/- 4,250 RLU/mg protein) tissue. Application of DETC (1 mM) reduced the basal levels and prevented ANG II-induced increases in NADPH oxidase activity in both tissues. These results demonstrate that renal excretory responses to acute ANG II administration are attenuated during SOD inhibition, which seems related to a downregulation of NADPH oxidase in the deficient condition of SOD activity.

Contribution of reactive oxygen species to migration/invasion of human glioblastoma cells U87 via ERK-dependent COX-2/PGE2 activation

In the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation, an increase in the migration/invasion of U87 glioblastoma cells was detected by a wound healing assay, transwell analysis, and spheroid formation assay by inducing matrix metalloproteinase-9 (MMP-9) enzyme activity via a gelatin zymographic analysis. A dose- and time-dependent increase in cyclooxygenase-2 (COX-2) gene expression with elevated prostaglandin E2 (PGE2) production was identified in TPA- but not in 4α-TPA (a respective inactive compound)-treated U87 cells TPA-induced migration/invasion was significantly blocked by adding the COX-2-specific inhibitor, NS398, through a reduction in PGE2 production. Data from the pharmacological studies using specific chemical inhibitors showed that activation of protein kinase C (PKC) and extracellular signal-regulated kinases (ERKs) was involved in TPA-induced migration/invasion, COX-2 protein expression, and MMP-9 activation. Stimulation of intracellular peroxide production by TPA was detected by a DCHF-DA assay, and the addition of superoxide dismutase (SOD) or tempol significantly inhibited TPA-induced migration/invasion and COX-2 protein expression accompanied by a decrease in peroxide production. An increase in NADPH oxidase activity by TPA was examined, and TPA-induced migration/invasion was blocked by adding DPI, an NADPH oxidase inhibitor. Additionally, the natural flavonoids quercetin (QE), baicalein (BE), and myricetin (ME) effectively blocked TPA-induced migration/invasion while simultaneously inhibiting COX-2/PGE2 production, MMP-9 enzyme activity, and peroxide production in U87 cells. The contribution of ROS production to the migration/invasion of U87 glioblastoma cells via ERK-activated COX-2/PGE2 and MMP-9 induction was first investigated here, and agents such as QE, BE, and ME with the ability to block these events possess the potential to be developed for use against migration/invasion by glioblastomas.

Reduced oxidant stress, increased NO‐dependent vasodilatation, and improved endothelial function with voluntary exercise in old mice: another excuse for long walks on the beach

The study by Durrant and coworkers (2009) in this issue of The Journal of Physiology investigates the effect of aerobic exercise on endothelium-dependent (vaso)dilatation (EDD) in B6D2F1 mice, a model of age-associated endothelial dysfunction (Lesniewski et al. 2009). The authors note that increasing age is a major risk factor for cardiovascular disease (Lakatta & Levy, 2003) that is likely to be mediated via increased oxidative stress and reduced NO levels (Cai & Harrison, 2000; Taddei et al. 2001). Current evidence indicates that habitual aerobic exercise improves EDD in older humans by reducing oxidative stress and increasing NO availability (Seals et al. 2008). The multiple beneficial effects of exercise that the authors report in the older mice are consistent with existing studies in humans, but provide more detailed insight into the potential mechanisms by which habitual aerobic exercise may improve EDD with age and potentially reduce age-associated risks of cardiovascular disease. The study evaluates multiple indices of EDD from the whole animal level to the vascular and enzymatic level in young and old mice with and without access to an exercise wheel for voluntary running. The authors report that voluntary exercise ameliorates the decreased whole body sensitivity to systemic infusion of acetylcholine in conscious older mice. The improvement in whole body acetylcholine sensitivity in older mice was accompanied by a restoration of both the maximum capacity for EDD and the NO-dependent component of EDD in the carotid artery, increased eNOS expression and activation (Ser1177 phosphorylation) in the aorta, and reduced nitrotyrosine levels in the aorta. The beneficial effects of voluntary exercise in the older mice are mediated via effects on both pro-oxidant and antioxidant mechanisms. For example, exercise not only increased total SOD activity, MnSOD activity, and Cu/Zn SOD + ecSOD activity in aortas of the older mice, but also ameliorated the increased expression of the p67 subunit of NADPH oxidase and the increased NADPH oxidase activity in aortas of the older mice. An important and striking observation in this study was that the beneficial effects of voluntary exercise in the older mice occurred even though their exercise levels were only about 10% of those in the younger mice. The importance of endothelial dysfunction and vascular oxidant stress in contributing to cardiovascular morbidity and mortality in humans was strongly emphasized in a recent review (Widlansky et al. 2003) citing 10 different studies showing that the presence of endothelial dysfunction is predictive of future adverse cardiovascular events (including death), and suggesting that evaluation of endothelial function in peripheral arteries may be beneficial in guiding the diagnosis and treatment of cardiovascular disease. In this regard, the results of the present study raise the possibility that voluntary exercise might not only reduce the risk of cardiovascular disease in older asymptomatic humans (see Lakatta and Levy, 2003), but also be beneficial in ameliorating more serious conditions such as peripheral artery disease, which increases with age and is associated with increased cardiovascular morbidity and mortality (McDermott et al. 2008; Sakamoto et al. 2009). The latter possibility is supported by studies showing that antioxidant treatment with glutathione infusion increases pain free walking distance and improves several indicators of microcirculatory and whole bed blood flow in humans with peripheral artery disease (Arosio et al. 2002) and that peripheral artery disease patients completing a supervised exercise training programme show reduced cardiovascular morbidity and mortality (Sakamoto et al. 2009).

Role of protein kinase C in NADPH oxidase derived O 2[rad]− -mediated regulation of K V–LVOCC axis under U46619 induced increase in [Ca 2+] i in pulmonary smooth muscle cells

Treatment of bovine pulmonary smooth muscle cells with the TxA 2 mimetic, U46619 stimulated [Ca 2+] i, which was inhibited upon pretreatment with apocynin (NADPH oxidase inhibitor). Pretreatment with cromakalim (K V channel opener) or nifedepine (L-VOCC inhibitor) inhibited U46619 induced increase in [Ca 2+] i, indicating a role of K V–LVOCC axis in this scenario. Neither cromakalim nor nifedepine inhibited U46619 induced increase in NADPH oxidase activity, suggesting that the NADPH oxidase activation is proximal to the K V–LVOCC axis in the cells. Pretreatment with calphostin C (PKC inhibitor) markedly reduced U46619 induced increase in NADPH oxidase activity and [Ca 2+] i in the cells. Calphostin C pretreatment also markedly reduced p 47phox phosphorylation and translocation to the membrane and association with p 22phox, a component of Cyt.b 558 of NADPH oxidase in the membrane. Overall, PKC plays an important role in NADPH oxidase derived O 2 − -mediated regulation of K V–LVOCC axis leading to an increase in [Ca 2+] i by U46619 in the cells.

Role of LOX‐1 in monocyte adhesion‐triggered redox, Akt/eNOS and Ca2+ signaling pathways in endothelial cells

This study was conducted to examine the role of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in monocyte adhesion-induced redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in endothelial cells (ECs). LOX-1 was blocked by an antibody-neutralizing LOX-1 TS92 or small interfering RNA. In cultured human aortic ECs, monocyte adhesion activated Rac1 and p47(phox), and increased NADPH oxidase activity and reactive oxygen species (ROS) generation within 30 min and NF-kappaB phosphorylation within 1 h, resulting in redox-sensitive gene expression. Akt and eNOS phosphorylation was induced 15 min after adding monocytes and returned to control level after 30 min, whereas NO production was not altered by monocyte adhesion. Blockade of LOX-1 blunted the monocyte adhesion-triggered redox-sensitive signaling pathway and Akt/eNOS phosphorylation in ECs. Both endothelial intracellular Ca2+ mobilization and Ca2+ influx caused by monocyte attachment were markedly attenuated by pretreatment of ECs with TS92. This suggests that LOX-1 is involved in redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs independent of oxidized low-density lipoprotein (ox-LDL). Furthermore, blockade of Ca2+ inhibited monocyte adhesion-triggered Rac1 and p47(phox) activation and ROS generation in ECs, whereas Ca2+ signaling was suppressed by blockade of NADPH oxidase and ROS generation. Finally, TS92 blocked the monocyte adhesion to ECs stimulated with or without tumor necrosis factor-alpha or ox-LDL. We provide evidence that LOX-1 plays a role in redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs independent of the ox-LDL-LOX-1 axis.

Enrichment of RAW264.7 macrophages with essential 18-carbon fatty acids affects both respiratory burst and production of immune modulating cytokines

Macrophages play a vital role in the innate immune system. Thereby, production of both reactive oxygen intermediates and immune modulating cytokines is crucial for successful pathogen defense. Fatty acids may interfere with immune response in several ways. In this study, we investigated the influence of essential polyunsaturated fatty acids (PUFA) on key macrophage functions. RAW264.7 macrophages were cultured in a medium supplemented with 2 or 15 μmol/L of the n-6 PUFA linoleic acid (LA) or of the n-3 PUFA α-linolenic acid (LNA), respectively. Cells were tested for incorporation of fatty acids as well as NADPH oxidase activity. Furthermore, supernatants were collected for detection of NO and cytokine release (TNF-α, IL-6, IL-10). Exposure of RAW264.7 macrophages to LA or LNA resulted in incorporation of these fatty acids and their derivatives. Thereby, supplementation with both LA and LNA caused a significant increase in NADPH oxidase activity. In contrast, synthesis of NO was not affected by PUFA supplementation. Moreover, distinct effects could be seen in the release of immune modulating cytokines. Due to enhancement of NADPH oxidase activity, PUFA presumably promote the killing of pathogens crucial in host defense. In addition, the unsaturated fatty acids tested in our study were shown to modulate cytokine release by the macrophages, thus driving immune response into an anti-inflammatory direction. Of note, distinct differences between the n-6 PUFA LA and the n-3 PUFA LNA underline the impact of PUFA family on immune response.

A2A Adenosine Receptor Deficiency Leads to Impaired Tracheal Relaxation via NADPH Oxidase Pathway in Allergic Mice

A(2A) adenosine receptor (A(2A)AR) has been shown to suppress superoxide generation in leukocytes via the cAMP-protein kinase A (PKA) pathway. However, no study has yet explored the role of A(2A)AR in relation to NADPH oxidase in murine tracheas in vitro, which may lead to altered smooth muscle relaxation in asthma. Therefore, the present study evaluated the effects of A(2A)AR deficiency on the NADPH oxidase pathway in tracheas of A(2A) wild-type (WT) and A(2A) knockout (KO) mice. A(2A)WT mice were sensitized with ovalbumin (30 microg i.p.) on days 1 and 6, followed by 5% ovalbumin aerosol challenge on days 11, 12, and 13. A(2A)AR (gene and protein expression), cAMP, and phosphorylated PKA (p-PKA) levels were decreased in A(2A)WT sensitized mice compared with controls. A(2A)KO mice also showed decreased cAMP and p-PKA levels. A(2A)WT sensitized and A(2A)KO control mice had increased gene and protein expression of NADPH oxidase subunits (p47phox and gp91phox) compared with the controls. Tracheal relaxation to specific A(2A)AR agonist, 4-[2-[[6-amino-9-(N-ethyl-beta-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS 21680), decreased in A(2A)WT sensitized mice compared with the controls, although it was absent in A(2A)KO mice. Pretreatment with NADPH oxidase inhibitors apocyanin/diphenyliodonium reversed the attenuated relaxation to CGS 21680 in A(2A)WT sensitized tracheas, whereas specific PKA inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i] [1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) blocked CGS 21680-induced relaxation. Tracheal reactive oxygen species (ROS) generation was also increased in A(2A)WT sensitized and A(2A)KO control mice compared with the controls. In conclusion, this study shows that A(2A)AR deficiency causes increased NADPH oxidase activation leading to decreased tracheal relaxation via altered cAMP-PKA signaling and ROS generation.

NADPH Oxidase Is Required for the Sensory Plasticity of the Carotid Body by Chronic Intermittent Hypoxia

Respiratory motoneuron response to hypoxia is reflex in nature and carotid body sensory receptor constitutes the afferent limb of this reflex. Recent studies showed that repetitive exposures to hypoxia evokes long term facilitation of sensory nerve discharge (sLTF) of the carotid body in rodents exposed to chronic intermittent hypoxia (CIH). Although studies with anti-oxidants suggested the involvement of reactive oxygen species (ROS)-mediated signaling in eliciting sLTF, the source of and the mechanisms associated with ROS generation have not yet been investigated. We tested the hypothesis that ROS generated by NADPH oxidase (NOX) mediate CIH-evoked sLTF. Experiments were performed on ex vivo carotid bodies from rats and mice exposed either to 10 d of CIH or normoxia. Acute repetitive hypoxia evoked a approximately 12-fold increase in NOX activity in CIH but not in control carotid bodies, and this effect was associated with upregulation of NOX2 mRNA and protein, which was primarily localized to glomus cells of the carotid body. sLTF was prevented by NOX inhibitors and was absent in mice deficient in NOX2. NOX activation by CIH required 5-HT release and activation of 5-HT(2) receptors coupled to PKC signaling. Studies with ROS scavengers revealed that H(2)O(2) generated from O(2).(-) contributes to sLTF. Priming with H(2)O(2) elicited sLTF of carotid bodies from normoxic control rats and mice, similar to that seen in CIH-treated animals. These observations reveal a novel role for NOX-induced ROS signaling in mediating sensory plasticity of the carotid body.

Carbon monoxide inhibits NADPH oxidase activation during seizures and prevents loss of postictal cerebral vascular function.

Prolonged seizures cause sustained neuronal injury and loss of cerebral blood flow regulation that may contribute to adverse neurological outcomes in neonates. Oxidative stress is potentially a key mechanism that initiates brain damage during seizures. In a newborn pig model of neonatal glutamatergic seizures, we investigated oxidative stress in the brain. Seizures lasting over 30 min increased the superoxide level in cerebral vessels and in the brain parenchyma. We hypothesize that NADPH oxidase is a source of superoxide during seizures. Apocynin, the NADPH oxidase inhibitor (20 mg/kg iv), inhibited superoxide elevation in response to seizures in cerebral microvessels and in the brain. Glutamate (0.05‐0.2mM) increased NADPH oxidase activity in cerebral microvascular endothelial cells 1.5‐2 fold. Carbon monoxide (CO, as CORM‐A1), reduced the basal enzyme activity and blocked the glutamate stimulation of NADPH oxidase. CORM‐A1 greatly reduced oxidative stress in ictal piglets and prevented sustained postictal loss of endothelial‐dependent cerebral blood flow regulation. Overall, these data suggest that NADPH oxidase is a major contributor to oxidative stress and cerebrovascular injury caused by seizures and glutamate. CO is a gaseous cytoprotective messenger molecule that preserves cerebrovascular integrity by inhibiting NADPH oxidase and preventing oxidative stress in response to seizures.

C.pneumoniae infection increases NADPH oxidase activity in vascular smooth muscle cells

Up‐regulation of NADPH oxidases, leading to increased reactive oxygen species (ROS) formation and oxidative stress is a key feature of atherogenesis. Chlamydia pneumoniae has been detected in human atheromas including vascular smooth muscle cells (VSMCs), suggesting a link between the bacteria and atherosclerosis. We examined whether C.pneumoniae induces NADPH oxidase activity in VSMCs. The mouse VSMC cell line, MOVAS, was infected with C.pneumoniae (104‐106 inclusion forming units) and superoxide production (L‐012 chemiluminescence), expression of NADPH oxidase subunit p47phox (immunoblotting), and C.pneumoniae inclusion counts (immunostaining) was assessed. C.pneumoniae caused a dose‐ and time‐dependent increase in superoxide production over 72 hours (n=6, p&lt;0.001). This response was significantly reduced by superoxide scavengers SOD, PEG SOD and tempol (p&lt;0.001, n=6), as well as the NADPH oxidase inhibitor, apocynin (p&lt;0.05, n=6). In preliminary studies (n=2), infection appeared to have no effect on expression of p47phox, suggesting it is not essential for C.pneumoniae‐induced superoxide production. PEG SOD, SOD and the hydrogen peroxide scavenger, catalase, reduced Chlamydia inclusion counts in VSMCs (by 19%, 25%, and 35% respectively). These data suggest that C.pneumoniae increases NADPH oxidase activity in VSMCs and that ROS may promote bacterial growth in these cells. (NHMRC 436825)

Prolonged Exposure to LPS Increases Iron, Heme, and p22 phox Levels and NADPH Oxidase Activity in Human Aortic Endothelial Cells

Vascular oxidative stress and inflammation are contributing factors in atherosclerosis. We recently found that the iron chelator, desferrioxamine (DFO), suppresses NADPH oxidase-mediated oxidative stress and expression of cellular adhesion molecules in mice treated with lipopolysaccharide (LPS). The objective of the present study was to investigate whether and how LPS and iron enhance, and DFO inhibits, NADPH oxidase activity in human aortic endothelial cells (HAECs). Incubation of HAECs for 24 hours with 5 microg/mL LPS led to a 4-fold increase in NADPH oxidase activity, which was strongly suppressed by pretreatment of the cells for 24 hours with 100 micromol/L DFO. Incubating HAECs with LPS also significantly increased cellular iron and heme levels and mRNA and protein levels of p22phox, a heme-containing, catalytic subunit of NADPH oxidase. All of these effects of LPS on HAECs were strongly inhibited by DFO. Exposing HAECs to 100 micromol/L iron (ferric citrate) for 48 hours exerted similar effects as LPS, and these effects were strongly inhibited by coincubation with DFO. Furthermore, neither LPS nor DFO affected mRNA and protein levels of p47phox a nonheme-containing, regulatory subunit of NADPH oxidase, or the mRNA level of NOX4, an isoform of the principal catalytic subunit of NADPH oxidase in endothelial cells. In contrast, heme oxygenase-1 was strongly suppressed by DFO, both in the absence and presence of LPS or iron. Our data indicate that prolonged exposure to LPS or iron increases endothelial NADPH oxidase activity by increasing p22phox gene transcription and cellular levels of iron, heme, and p22phox protein. Iron chelation by DFO effectively suppresses endothelial NADPH oxidase activity, which may be helpful as an adjunct in reducing vascular oxidative stress and inflammation in atherosclerosis.

Sympathoexcitation by Oxidative Stress in the Brain Mediates Arterial Pressure Elevation in Obesity-Induced Hypertension

Obesity is one of the major risk factors for cardiovascular disease and is often associated with increased oxidative stress and sympathoexcitation. We have already suggested that increased oxidative stress in the brain modulates the sympathetic regulation of arterial pressure in salt-sensitive hypertension, which is often associated with obesity. The present study was performed to determine whether oxidative stress could mediate central sympathoexcitation in the initial stage of obesity-induced hypertension. Four-week-old male Sprague-Dawley rats were fed a high-fat (45% kcal as fat) or low-fat (10% kcal as fat) diet for 6 weeks. Fat loading elicited hypertension and sympathoexcitation, along with visceral obesity. In urethane-anesthetized and artificially ventilated rats, arterial pressure and renal sympathetic nerve activity decreased in a dose-dependent fashion when 53 or 105 mumol/kg tempol, a membrane-permeable superoxide dismutase mimetic, was infused into the lateral cerebral ventricle. Central tempol reduced arterial pressure and renal sympathetic nerve activity to a significantly greater extent in high-fat diet-fed hypertensive rats than in low-fat diet-fed normotensive rats. Intracerebroventricular apocynin or diphenyleneiodonium, a reduced NADPH oxidase inhibitor, also elicited markedly greater reductions in arterial pressure and renal sympathetic nerve activity in the high-fat diet-fed rats. In addition, fat loading increased NADPH oxidase activity and NADPH oxidase subunit p22(phox), p47(phox), and gp91(phox) mRNA expression in the hypothalamus. In obesity-induced hypertension, increased oxidative stress in the brain, possibly via activation of NADPH oxidase, may contribute to the progression of hypertension through central sympathoexcitation.

Oxidative stress in non-small cell lung cancer: Role of nicotinamide adenine dinucleotide phosphate oxidase and glutathione

Background. Cigarette smoke is strongly associated with NSCLC, but the carcinogenesis of NSCLC is poorly understood. Methods. To discover the role of oxidative stress and anti-oxidative defense in NSCLC, we measured NADPH oxidase (NOX) activity, myeloperoxidase activity, 8-OHdG, and glutathione content from lung specimens. These came from 32 patients: 22 NSCLC patients and ten controls without cancer. Results. In NSCLC patients, NOX activity was significantly higher both in the malignant (p = 0.001) and non-malignant (p = 0.044) samples from NSCLC patients, than in the control specimens. Myeloperoxidase activity was lower (p = 0.001) and glutathione content (p = 0.009) higher in malignant tissue. No significant difference was observable in 8-OHdG content between patient groups. Conclusions. Increase in NOX activity in the malignant tissues was independent of smoking history and myeloperoxidase activity, suggesting its independent role in NSCLC pathogenesis.

Cilostazol Attenuates 4-hydroxynonenal-enhanced CD36 Expression on Murine Macrophages via Inhibition of NADPH Oxidase-derived Reactive Oxygen Species Production

Although anti-atherogenic effects of cilostazol have been suggested, its effects on the expression of SR in macrophages are unclear. This study investigated the role of cilostazol on CD36 expression of murine macrophages enhanced by HNE, a byproduct of lipid peroxidation. The stimulation of macrophages with HNE led to an increased expression of CD36, which was significantly attenuated by NAC, an antioxidant. Moreover, the increased production of ROS by HNE was completely abolished by NADPH oxidase inhibitors, DPI and apocynin, as well as by the 5-LO inhibitor, MK886, but not by inhibitors for other oxidases. This suggested that NADPH-oxidase and 5-LO were major sources of ROS induced by HNE. In addition, HNE-enhanced expression of CD36 was reduced by these inhibitors, which indicated a role for NADPH oxidase and 5-LO on CD36 expression. In our present study, cilostazol was a significant inhibitor of ROS production, as well as CD36 expression induced by HNE. An increase in NADPH oxidase activity by HNE was significantly attenuated by cilostazol, however cilostazol had no effect on HNE-enhanced 5-LO activity. Together, these results suggest that cilostazol attenuates HNE-enhanced CD36 expression on murine macrophages thorough inhibition of NADPH oxidase-derived ROS generation.

Roles of NADPH Oxidase in Occurrence of Gastric Damage and Expression of Cyclooxygenase-2 During Ischemia/Reperfusion in Rat Stomachs

NADPH oxidase is an enzyme that converts molecular oxygen into reactive oxygen species, which cause severe damage in several organs. Cyclooxygenase (COX)-2 is an inducible enzyme that is important in gastric mucosal defense and repair processes. It is unclear whether NADPH oxidase is related to COX expression in the gastric mucosa, so we investigated the correlation. Under urethane anesthesia, a male Sprague Dawley rat stomach was mounted in an ex-vivo chamber, and ischemia/reperfusion (I/R) was performed through a cannula in the femoral vein. I/R significantly increased NADPH oxidase activity, H2O2 production, and myeloperoxidase (MPO) activity. In contrast, ischemia alone clearly enhanced both NADPH oxidase activity and H2O2 production but not MPO activity. Pretreatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI) suppressed I/R-induced mucosal damage. On the other hand, the selective COX-2 inhibitor rofecoxib exhibited a tendency to enhance the severity of gastric damage induced by I/R, although the selective COX-1 inhibitor SC-560 and the nonselective COX inhibitor indomethacin had no effect. I/R also increased the expression of COX-2, and this increase was suppressed by pretreatment with DPI. These findings suggest that the increase in NADPH oxidase activity is involved in the occurrence of gastric mucosal damage induced by I/R and that this enzyme activity may be causally related to the upregulation of COX-2 during I/R.

Apocynin attenuates tubular apoptosis and tubulointerstitial fibrosis in transgenic mice independent of hypertension

Angiotensin II stimulates the formation of reactive oxygen species by increased NADPH oxidase activity, which contributes to proapoptotic and profibrotic mechanisms critical in renal injury. Here we determine if apocynin, an inhibitor of NADPH oxidase, interferes with the action of the intrarenal renin-angiotensin system to minimize the progression of renal disease. Transgenic mice that overexpress rat angiotensinogen in their proximal tubule cells were given either apocynin, perindopril, or hydralazine while untreated or apocynin-treated non-transgenic littermates served as controls. Untreated transgenic mice had significant elevations of their systolic blood pressure, albuminuria, reactive oxygen species production, NADPH oxidase activity, tubular apoptosis, active caspase-3, Bax, transforming growth factor-beta1, plasminogen activator inhibitor-1, extracellular matrix proteins, collagen type IV, and phosphorylated p47phox expression compared to untreated non-transgenic mice. Apocynin and perindopril blunted these changes; however, apocynin had no effect on the systolic blood pressure whereas hydralazine prevented hypertension and tubulointerstitial fibrosis but not proximal tubule cell apoptosis. Our study shows that the intrarenal renin-angiotensin system stimulates proximal tubule cell apoptosis and tubulointerstitial fibrosis, in part, by enhanced NADPH oxidase activity and reactive oxygen species generation independent of systemic hypertension.

Nox4 NADPH oxidase mediates oxidative stress and apoptosis caused by TNF-α in cerebral vascular endothelial cells

Inflammatory brain disease may damage cerebral vascular endothelium leading to cerebral blood flow dysregulation. The proinflammatory cytokine TNF-alpha causes oxidative stress and apoptosis in cerebral microvascular endothelial cells (CMVEC) from newborn pigs. We investigated contribution of major cellular sources of reactive oxygen species to endothelial inflammatory response. Nitric oxide synthase and xanthine oxidase inhibitors (N(omega)-nitro-l-arginine and allopurinol) had no effect, while mitochondrial electron transport inhibitors (CCCP, 2-thenoyltrifluoroacetone, and rotenone) attenuated TNF-alpha-induced superoxide (O(2)(*-)) and apoptosis. NADPH oxidase inhibitors (diphenylene iodonium and apocynin) greatly reduced TNF-alpha-evoked O(2)(*-) generation and apoptosis. TNF-alpha rapidly increased NADPH oxidase activity in CMVEC. Nox4, the cell-specific catalytic subunit of NADPH oxidase, is highly expressed in CMVEC, contributes to basal O(2)(*-) production, and accounts for a burst of oxidative stress in response to TNF-alpha. Nox4 small interfering RNA, but not Nox2, knockdown prevented oxidative stress and apoptosis caused by TNF-alpha in CMVEC. Nox4 is colocalized with HO-2, the constitutive isoform of heme oxygenase (HO), which is critical for endothelial protection against TNF-alpha toxicity. The products of HO activity, bilirubin and carbon monoxide (CO, as a CO-releasing molecule, CORM-A1), inhibited Nox4-generated O(2)(*-) and apoptosis caused by TNF-alpha stimulation. We conclude that Nox4 is the primary source of inflammation- and TNF-alpha-induced oxidative stress leading to apoptosis in brain endothelial cells. The ability of CO and bilirubin to combat TNF-alpha-induced oxidative stress by inhibiting Nox4 activity and/or by O(2)(*-) scavenging, taken together with close intracellular compartmentalization of HO-2 and Nox4 in cerebral vascular endothelium, may contribute to HO-2 cytoprotection against inflammatory cerebrovascular disease.