Role of neutrophil NADPH oxidase in the mechanism of tumor necrosis factor-alpha -induced NF-kappa B activation and intercellular adhesion molecule-1 expression in endothelial cells.

In this study, we explored a novel function of polymorphonuclear neutrophils (PMN) NAD(P)H oxidase in the mechanism of tumor necrosis factor-alpha (TNFalpha)-induced NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells. Studies were made in mice lacking the p47(phox) subunit of NAD(P)H oxidase as well as in cultured mouse lung vascular endothelial cells (MLVEC) from these mice. In response to TNFalpha challenge, NF-kappaB activation and ICAM-1 expression were significantly attenuated in lungs of p47(phox)(-/-) mice as compared with wild-type (WT) mice. The attenuated NF-kappaB activation in p47(phox)(-/-) mice was secondary to inhibition of NIK activity and subsequent IkappaBalpha degradation. Induction of neutropenia using anti-PMN serum prevented the initial TNFalpha-induced NF-kappaB activation and ICAM-1 expression in WT mice, indicating the involvement of PMN NAD(P)H oxidase in signaling these responses. Moreover, the responses were restored upon repletion with PMN obtained from WT mice but not with PMN from p47(phox)(-/-) mice. These findings were recapitulated in MLVEC co-cultured with PMN, suggesting that NF-kappaB activation and resultant ICAM-1 expression in endothelial cells occurred secondary to oxidants generated by the PMN NAD(P)H oxidase complex. The functional relevance of the PMN NAD(P)H oxidase in mediating TNFalpha-induced ICAM-1-dependent endothelial adhesivity was evident by markedly reduced adhesion of p47(phox)(-/-) PMN in co-culture experiments. Thus, oxidant signaling by the PMN NAD(P)H oxidase complex is an important determinant of TNFalpha-induced NF-kappaB activation and ICAM-1 expression in endothelial cells.     

gp91phox-containing NAD(P)H oxidase mediates attenuation of nitric oxide-dependent control of myocardial oxygen consumption by ANG II

We have previously reported that ANG II stimulation increased superoxide anion (O2-) through the activation of NAD(P)H oxidase and inhibited nitric oxide (NO)-dependent control of myocardial oxygen consumption (MVo2) by scavenging NO. Our objective was to investigate the role of NAD(P)H oxidase, especially the gp91phox subunit, in the NO-dependent control of MVo2. MVo2 in mice with defects in the expression of gp91phox [gp91(phox)(-/-)] was measured with a Clark-type oxygen electrode. Baseline MVo2 was not significantly different between wild-type (WT) and gp91(phox)(-/-) mice. Stimulation of NO production by bradykinin (BK) induced significant decreases in MVo2 in WT mice. BK-induced reduction in MVo2 was enhanced in gp91(phox)(-/-) mice. BK-induced reduction in MVo2 in WT mice was attenuated by 10(-8) mol/l ANG II, which was restored by coincubation with Tiron or apocynin. In contrast to WT mice, BK-induced reduction in MVo2 in gp91(phox)(-/-) mice was not altered by ANG II. There was a decrease in lucigenin (5 x 10(-6) mol/l)-detectable O2- in gp91(phox)(-/-) mice compared with WT mice. ANG II resulted in significant increases in O2- production in WT mice, which was inhibited by coincubation with Tiron or apocynin. However, ANG II had no effect on O2- production in gp91(phox)(-/-) mice. Histological examination showed that the development of abscesses and/or the invasion of inflammatory cells occurred in lungs and livers but not in hearts and kidneys from gp91(phox)(-/-) mice. These results indicate that the gp91(phox) subunit of NAD(P)H oxidase mediates O2- production through the activation of NAD(P)H oxidase and attenuation of NO-dependent control of MVo2 by ANG II.     

NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-alpha

We tested the hypothesis that the NAD(P)H oxidase-dependent generation of superoxide anion (O2-*) mediates tumor necrosis factor-alpha (TNF)-induced alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The NAD(P)H oxidase subcomponents p47phox and p22phox were assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species O2-* was measured by the fluorescence of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetatedi(acetoxymethyl ester), 5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester, and dihydroethidium. TNF treatment (50 ng/ml for 4.0 h) induced 1) p47phox translocation, 2) an increase in p22phox protein, 3) increased localization of p47phox with p22phox, 4) O2-* generation, and 5) increased permeability to albumin. p22phox antisense oligonucleotide prevented the TNF-induced effect on p22phox, p47phox, O2-*, and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in O2-* and permeability to albumin was also prevented by the O2-* scavenger Cu-Zn superoxide dismutase (100 U/ml). The results indicate that the activation of NAD(P)H oxidase, via the generation of O2-*, mediates TNF-induced barrier dysfunction in PMEM.     

NAD(P)H oxidase-derived reactive oxygen species contribute to age-related impairments of endothelium-dependent dilation in rat soleus feed arteries

We tested the hypothesis that age-related endothelial dysfunction in rat soleus muscle feed arteries (SFA) is mediated in part by NAD(P)H oxidase-derived reactive oxygen species (ROS). SFA from young (4 mo) and old (24 mo) Fischer 344 rats were isolated and cannulated for examination of vasodilator responses to flow and acetylcholine (ACh) in the absence or presence of a superoxide anion (O(2)(-)) scavenger (Tempol; 100 μM) or an NAD(P)H oxidase inhibitor (apocynin; 100 μM). In the absence of inhibitors, flow- and ACh-induced dilations were attenuated in SFA from old rats compared with young rats. Tempol and apocynin improved flow- and ACh-induced dilation in SFA from old rats. In SFA from young rats, Tempol and apocynin had no effect on flow-induced dilation, and apocynin attenuated ACh-induced dilation. To determine the role of hydrogen peroxide (H(2)O(2)), dilator responses were assessed in the absence and presence of catalase (100 U/ml) or PEG-catalase (200 U/ml). Neither H(2)O(2) scavenger altered flow-induced dilation, whereas both H(2)O(2) scavengers blunted ACh-induced dilation in SFA from young rats. In old SFA, catalase improved flow-induced dilation whereas PEG-catalase improved ACh-induced dilation. Compared with young SFA, in response to exogenous H(2)O(2) and NADPH, old rats exhibited blunted dilation and constriction, respectively. Immunoblot analysis revealed that the NAD(P)H oxidase subunit gp91phox protein content was greater in old SFA compared with young. These results suggest that NAD(P)H oxidase-derived reactive oxygen species contribute to impaired endothelium-dependent dilation in old SFA.     

Airway smooth muscle relaxation is impaired in mice lacking the p47phox subunit of NAD(P)H oxidase

NAD(P)H oxidase is one of the critical enzymes mediating cellular production of reactive oxygen species and has a central role in airway smooth muscle (ASM) cell proliferation. Since reactive oxygen species also affect ASM contractile response, we hypothesized a regulatory role of NAD(P)H oxidase in ASM contractility. We therefore studied ASM function in wild-type mice (C57BL/6J) and mice deficient in a component (p47phox) of NAD(P)H oxidase. In histological sections of the trachea, we found that the area occupied by ASM was 17% more in p47(phox-/-) than in wild-type mice. After correcting for the difference in ASM content, we found that force generation did not vary between the two genotypes. Similarly, their ASM shortening velocity, maximal power, and sensitivity to acetylcholine, as well as airway responsiveness to methacholine in vivo, were not significantly different. The main finding of this study was a significantly reduced ASM relaxation in p47phox-/- compared with wild-type mice both during the stimulus and after the end of stimulation. The tension relaxation attained at the 20th second of electric field stimulation was, respectively, 17.6 +/- 2.4 and 9.2 +/- 2.3% in null and wild-type mice (P <0.01 by t-test). Similar significant differences were found in the rate of tension relaxation and the time required to reduce tension by one-half. Our data suggest that NAD(P)H oxidase may have a role in the structural arrangement and mechanical properties of the airway tissue. Most importantly, we report the first evidence that the p47phox subunit of NAD(P)H oxidase plays a role in ASM relaxation.     

Upregulation of p67(phox) and gp91(phox) in aortas from angiotensin II-infused mice

Although NAD(P)H oxidase-derived superoxide (O(2)(-)) is increased during the development of angiotensin II (ANG II)-dependent hypertension, vascular regulation at the protein level has not been reported. We have shown that four major components of NAD(P)H oxidase are located primarily in the vascular adventitia as a primary source of vascular O(2)(-). Here we compare vascular levels of O(2)(-) and NAD(P)H oxidase in normotensive and ANG II-infused hypertensive mice and show that, after 7 days of ANG II infusion (750 microg. kg(-1). day(-1) ip) in C57B1/6 mice, systolic blood pressure was increased compared with that after sham infusion, concomitant with increased O(2)(-) in the thoracic aorta as measured using lucigenin (25 microM)-enhanced chemiluminescence. Both p67(phox) and gp91(phox) were detectable by Western blotting in aortic homogenates, and we observed increased protein levels of NAD(P)H oxidase subunits. These ANG II-induced increases were normalized by simultaneous treatment with the AT(1) receptor antagonist losartan. Moreover, the primary location of these subunits was the adventitia as detected immunohistochemically. Our results suggest that ANG II-induced increases in O(2)(-) are due to increased adventitial NAD(P)H oxidase activity, brought about by the heightened expression and interaction of its components.     

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.     

Upregulation of vascular NAD(P)H oxidase subunit gp91phox and impairment of the nitric oxide signal transduction pathway in hypertension

In this study we analyzed the role of vascular NAD(P)H oxidase in the generation of O(2)(-) and the endothelial impairment of NO signal transduction pathway in hypertension. In aortic rings of 15-month-old stroke-prone spontaneously hypertensive rats (SHR15) we found a 10-fold increased expression of NAD(P)H oxidase subunit gp91phox mRNA associated with a 3-fold increased production of O(2)(-) compared to age-matched Wistar rats (WIS15). Vasorelaxation studies in aortas of SHR15 showed a strongly diminished response to acetylcholine, NO-donor S-nitroso-N-acetyl-d,l-penicillamine, and organic nitrate glyceryl trinitrate compared to WIS15. Soluble guanylate cyclase (sGC) activity and sGC beta(1)-subunit protein expression was downregulated in aortas and lungs of SHR15. These data suggest an upregulation of vascular NAD(P)H oxidase and an impairment of the NO signal transduction pathway in hypertension.     

Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress

Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.     

Enhanced oxidative stress in kidneys of salt-sensitive hypertension: role of sensory nerves

To determine the mechanism(s) underlying enhanced oxidative stress in kidneys of salt-sensitive hypertension, neonatal Wistar rats were given vehicle or capsaicin (CAP, 50 mg/kg sc) on the first and second days of life. After being weaned, male rats were assigned into four groups and treated for 2 wk with the following: vehicle + a normal sodium diet (NS, 0.4%, CON-NS), vehicle + a high-sodium diet (HS, 4%, CON-HS), CAP + NS (CAP-NS), and CAP + HS (CAP-HS). Systolic blood pressure was significantly increased in CAP-HS but not CAP-NS or CON-HS rats. Plasma and urinary 8-iso-prostaglandin F(2alpha) levels increased by approximately 40% in CON-HS and CAP-HS rats compared with their respective controls fed a NS diet (P < 0.05), and these parameters were higher in CAP-HS compared with CON-HS rats. Superoxide (O(2)(-)*) levels in the renal cortex and medulla increased by approximately 45% in CAP-HS compared with CON-HS, CON-NS, and CAP-NS rats (P < 0.05). Enhanced O(2)(-)* levels in the cortex and medulla in CAP-HS rats were prevented by preincubation of renal tissues with apocynin, a selective NAD(P)H oxidase inhibitor. Protein expression of NAD(P)H oxidase subunits, including p47(phox) and gp91(phox) in the renal cortex and medulla, was significantly increased in CAP-HS compared with CON-HS, CON-NS, and CAP-NS rats. In contrast, protein expression and activities of Cu/Zn SOD and Mn SOD were significantly increased in the renal medulla in both CAP-HS and CON-HS but in the cortex in CAP-HS rats only. Creatinine clearance decreased by approximately 45% in CAP-HS rats compared with CON-HS, CON-NS, and CAP-NS rats (P < 0.05). O(2)(-)* levels in the renal cortex of CAP-HS rats negatively correlated with creatinine clearance (r = -0.76; P < 0.001). Therefore, regardless of enhanced SOD activity to suppress oxidative stress, increased oxidative stress in the kidney of CAP-treated rats fed a HS diet is likely the result of increased expression and activities of NAD(P)H oxidase, which may contribute to decreased renal function and increased blood pressure in these rats. Our results suggest that sensory nerves may play a compensatory role in attenuating renal oxidative stress during HS intake.     

Src-mediated tyrosine phosphorylation of p47phox in hyperoxia-induced activation of NADPH oxidase and generation of reactive oxygen species in lung endothelial cells

Superoxide (O(2)(-)) production by nonphagocytes, similar to phagocytes, is by activation of the NADPH oxidase multicomponent system. Although activation of neutrophil NADPH oxidase involves extensive serine phosphorylation of p47(phox), the role of tyrosine phosphorylation of p47(phox) in NADPH oxidase-dependent O(2)(-) production is unclear. We have shown recently that hyperoxia-induced NADPH oxidase activation in human pulmonary artery endothelial cells (HPAECs) is regulated by mitogen-activated protein kinase signal transduction. Here we provided evidence on the role of nonreceptor tyrosine kinase, Src, in hyperoxia-induced tyrosine phosphorylation of p47(phox) and NADPH oxidase activation in HPAECs. Exposure of HPAECs to hyperoxia for 1 h resulted in increased O(2)(-) and reactive oxygen species (ROS) production and enhanced tyrosine phosphorylation of Src as determined by Western blotting with phospho-Src antibodies. Pretreatment of HPAECs with the Src kinase inhibitor PP2 (1 mum) or transient expression of a dominant-negative mutant of Src attenuated hyperoxia-induced tyrosine phosphorylation of Src and ROS production. Furthermore, exposure of cells to hyperoxia enhanced tyrosine phosphorylation of p47(phox) and its translocation to cell peripheries that were attenuated by PP2. In vitro, Src phosphorylated recombinant p47(phox) in a time-dependent manner. Src immunoprecipitates of cell lysates from control cells revealed the presence of immunodetectable p47(phox) and p67(phox), suggesting the association of oxidase components with Src under basal conditions. Moreover, exposure of HPAECs to hyperoxia for 1 h enhanced the association of p47(phox), but not p67(phox), with Src. These results indicated that Src-dependent tyrosine phosphorylation of p47(phox) regulates hyperoxia-induced NADPH oxidase activation and ROS production in HPAECs.     

Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin. Evidence that p47(phox) may participate in forming this oxidase in vitro and in vivo.

Thrombin is a potent vascular smooth muscle cell (VSMC) mitogen. Because recent evidence implicates reactive oxygen intermediates (ROI) in VSMC proliferation in general and atherogenesis in particular, we investigated whether ROI generation is necessary for thrombin-induced mitogenesis. Treatment of human aortic smooth muscle cells with thrombin increased DNA synthesis, an effect that was antagonized by diphenyleneiodonium but not by other inhibitors of cellular oxidase systems. This effect of thrombin was accompanied by increased O-2 and H2O2 generation and NADH/NADPH consumption. ROI generation in response to thrombin pretreatment could also be blocked by diphenyleneiodonium, suggesting that the NAD(P)H oxidase was necessary for ROI generation and thrombin-induced mitogenesis. Because of observed differences between the VSMC and neutrophil oxidase, we examined whether the cytosolic components of the phagocytic NAD(P)H oxidase were present in VSMC. p47(phox) and Rac2 were present in VSMC. Furthermore, thrombin increased expression of p47(phox) and Rac2 and stimulated their translocation to the cell membrane. We examined whether p47(phox) might be similarly regulated in vivo in a rat aorta balloon injury model and found that p47(phox) protein was increased after injury. Immunocytochemistry localized expression of p47(phox) to the neointima and media of injured arteries. Our data demonstrate that generation of O-2 and H2O2 is required for thrombin-mediated mitogenesis in VSMC and that p47(phox) is regulated by thrombin in vitro and is associated with vascular lesion formation in vivo.     

Oscillatory shear stress stimulates endothelial production of O2- from p47phox-dependent NAD(P)H oxidases,leading to monocyte adhesion

Arterial regions exposed to oscillatory shear (OS) in branched arteries are lesion-prone sites of atherosclerosis, whereas those of laminar shear (LS) are relatively well protected. Here, we examined the hypothesis that OS and LS differentially regulate production of O2- from the endothelial NAD(P)H oxidase, which, in turn, is responsible for their opposite effects on a critical atherogenic event, monocyte adhesion. We used aortic endothelial cells obtained from C57BL/6 (MAE-C57) and p47phox-/- (MAE-p47-/-) mice, which lack a component of NAD(P)H oxidase. O2- production was determined by dihydroethidium staining and an electron spin resonance using an electron spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Chronic exposure (18 h) to an arterial level of OS (+/- 5 dynes/cm2) increased O2- (2-fold) and monocyte adhesion (3-fold) in MAE-C57 cells, whereas chronic LS (15 dynes/cm2, 18 h) significantly decreased both monocyte adhesion and O2- compared with static conditions. In contrast, neither LS nor OS were able to induce O2- production and monocyte adhesion to MAE-p47-/-. Treating MAE-C57 with a cell-permeable superoxide dismutase compound, polyethylene glycol-superoxide dismutase, also inhibited OS-induced monocyte adhesion. In addition, over-expressing p47phox in MAE-p47-/- restored OS-induced O2- production and monocyte adhesion. These results suggest that chronic exposure of endothelial cells to OS stimulates O2- and/or its derivatives produced from p47phox-dependent NAD(P)H oxidase, which, in turn, leads to monocyte adhesion, an early and critical atherogenic event.     

Induction of heme oxygenase-1 inhibits NAD(P)H oxidase activity by down-regulating cytochrome b558 expression via the reduction of heme availability

Heme-oxygenase-1 (HO-1), the rate-limiting enzyme of heme degradation, has powerful anti-oxidant properties related to the production of the reactive oxygen species scavenger bilirubin. However, some data suggest that HO-1 could also inhibit the cellular production of reactive oxygen species. Therefore, we investigated whether the anti-oxidant properties of HO-1 could be mediated by modulation of the activity and/or expression of the heme-containing NAD(P)H oxidase, the main source of the superoxide anion (O(2)(-)) in phagocytic cells. Increasing HO-1 expression in RAW 264.7 macrophages effectively decreased NAD(P)H oxidase activity and expression of gp91(phox), its heme-containing catalytic component, because of deficient protein maturation and increased degradation. Loading cells with heme reversed the decrease in O(2)(-) production and gp91(phox) expression induced by HO-1 overexpression. Similar results were obtained in vivo in rat alveolar macrophages after pharmacological modulation of HO-1 expression or activity. These results show that a decrease in heme content due to HO-1 activation limits heme availability for maturation of the gp91(phox) subunit and assembly of the functional NAD(P)H oxidase. This study provides a new mechanism to explain HO-1 anti-oxidant properties.     

阿托伐他汀通过RGS6改善糖尿病心肌病大鼠心功能的作用及其机制研究

目的:探讨阿托伐他汀通过调节RGS6/NAD(P)H氧化酶/活性氧生成通路保护糖尿病心肌病大鼠心功能的药理作用机制。方法:40只6周龄雄性Wistar大鼠按随机数字表法随机分为对照组,糖尿病心肌病模型组,低剂量阿托伐他汀组,高剂量阿托伐他汀组,每组10只。实验过程中动态监测大鼠体质量及血脂水平;实验结束后脉冲多普勒检测各组大鼠心功能指标;组织活性氧检测试剂盒检测心肌组织中活性氧的水平;免疫组化法检测大鼠心肌组织中RGS6的表达;Western blot法检测大鼠心肌组织中RGS6及NAD (P)H氧化酶活性亚单位p47phox和p67phox的水平。结果:与对照组相比,糖尿病心肌病模型大鼠体质量明显减少(P0.01),血脂水平明显升高(P0.01),心脏E/A、LVEF、FS值降低(P0.01),心肌组织活性氧生成明显增多(P0.01),心肌组织RGS6及p47phox、p67phox表达明显上调(P0.01),而不同剂量阿托伐他汀干预均可有效逆转上述指标的改变。结论:阿托伐他汀对糖尿病心肌病大鼠的心脏具有明显保护作用,其机制可能与对RGS6/NAD(P)H氧化酶/活性氧生成通路的抑制有关。     

Hyperoxia-induced NAD(P)H oxidase activation and regulation by MAP kinases in human lung endothelial cells

Hyperoxia increases reactive oxygen species (ROS) production in vascular endothelium; however, the mechanisms involved in ROS generation are not well characterized. We determined the role and regulation of NAD(P)H oxidase in hyperoxia-induced ROS formation in human pulmonary artery endothelial cells (HPAECs). Exposure of HPAECs to hyperoxia for 1, 3, and 12 h increased the generation of superoxide anion, which was blocked by diphenyleneiodonium but not by rotenone or oxypurinol. Furthermore, hyperoxia enhanced NADPH- and NADH-dependent and superoxide dismutase- or diphenyleneiodonium-inhibitable ROS production in HPAECs. Immunohistocytochemistry and Western blotting revealed the presence of gp91, p67 phox, p22 phox, and p47 phox subcomponents of NADPH oxidase in HPAECs. Transfection of HPAECs with p22 phox antisense plasmid inhibited hyperoxia-induced ROS production. Exposure of HPAECs to hyperoxia activated p38 MAPK and ERK, and inhibition of p38 MAPK and MEK1/2 attenuated the hyperoxia-induced ROS generation. These results suggest a role for MAPK in regulating hyperoxia-induced NAD(P)H oxidase activation in HPAECs.     

Platelet-associated NAD(P)H oxidase contributes to the thrombogenic phenotype induced by hypercholesterolemia

Elevated cholesterol levels promote proinflammatory and prothrombogenic responses in venules and impaired endothelium-dependent arteriolar dilation. Although NAD(P)H oxidase-derived superoxide has been implicated in the altered vascular responses to hypercholesterolemia, it remains unclear whether this oxidative pathway mediates the associated arteriolar dysfunction and platelet adhesion in venules. Platelet and leukocyte adhesion in cremasteric postcapillary venules and arteriolar dilation responses to acetylcholine were monitored in wild-type (WT), Cu,Zn-superoxide dismutase transgenic (SOD-TgN), and NAD(P)H oxidase-knockout (gp91(phox-/-)) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 weeks. HC elicited increased platelet and leukocyte adhesion in WT mice versus ND. Cytosolic subunits of NAD(P)H oxidase (p47phox and p67phox) were expressed in platelets. This was not altered by hypercholesterolemia; however, platelets and leukocytes from HC mice exhibited elevated generation of reactive oxygen species compared to ND mice. Hypercholesterolemia-induced leukocyte recruitment was attenuated in SOD-TgN-HC and gp91(phox-/-)-HC mice. Recruitment of platelets derived from WT-HC mice in venules of SOD-TgN-HC or gp91(phox-/-)-HC recipients was comparable to ND levels. Adhesion of SOD-TgN-HC platelets paralleled the leukocyte response and was attenuated in SOD-TgN-HC recipients, but not in WT-HC recipients. However, gp91(phox-/-)-HC platelets exhibited low levels of adhesion comparable to those of WT-ND in both hypercholesterolemic gp91(phox-/-) and WT recipients. Arteriolar dysfunction was evident in WT-HC mice, compared to WT-ND. Overexpression of SOD or, to a lesser extent, gp91(phox) deficiency restored arteriolar vasorelaxation responses toward WT-ND levels. These findings reveal a novel role for platelet-associated NAD(P)H oxidase in producing the thrombogenic phenotype in hypercholesterolemia and demonstrate that NAD(P)H oxidase-derived superoxide mediates the HC-induced arteriolar dysfunction.     

Activation of NADPH oxidase and extracellular superoxide production in seizure-induced hippocampal damage

We sought to determine whether the extracellular compartment contributed to seizure-induced superoxide (O2*-) production and to determine the role of the NADPH oxidase complex as a source of this O2*- production. The translocation of NADPH oxidase subunits (p47phox, p67phox and rac1) was assessed by immunoblot analysis and NADPH-driven O2*- production was measured using 2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazo [1,2-alpha] pyrazin-3-one-enhanced chemiluminescence. Kainate-induced status epilepticus resulted in a time-dependent translocation of NADPH oxidase subunits (p47phox, p67phox and rac-1) from hippocampal cytosol to membrane fractions. Hippocampal membrane fractions from kainate-injected rats showed increased NADPH-driven and diphenylene iodonium-sensitive O2*- production in comparison to vehicle-treated rats. The time-course of kainate-induced NADPH oxidase activation coincided with microglial activation in the rat hippocampus. Finally, kainate-induced neuronal damage and membrane oxygen consumption were inhibited in mice overexpressing extracellular superoxide dismutase. These results suggest that seizure activity activates the membrane NADPH oxidase complex resulting in increased formation of O2*-.     

Increased expression of NAD(P)H oxidase subunit p67(phox) in the renal medulla contributes to excess oxidative stress and salt-sensitive hypertension

NAD(P)H oxidase has been shown to be important in?the development of salt-sensitive hypertension. Here, we show that the expression of a subunit of NAD(P)H oxidase, p67(phox), was increased in response to a high-salt diet in the outer renal medulla of the Dahl salt-sensitive (SS) rat, an animal model for human salt-sensitive hypertension. The higher expression of p67(phox), not the other subunits observed, was associated with higher NAD(P)H oxidase activity and salt sensitivity in SS rats compared with a salt-resistant strain. Genetic mutations of the SS allele of p67(phox) were found in the promoter region and contributed to higher promoter activity than that of the salt-resistant strain. To verify the importance of p67(phox), we disrupted p67(phox) in SS rats using zinc-finger nucleases. These rats exhibited a significant reduction of salt-sensitive hypertension and renal medullary oxidative stress and injury. p67(phox) could represent a target for salt-sensitive hypertension therapy.