Role of pollen NAD(P)H oxidase in allergic inflammation

PURPOSE OF REVIEW: Plant pollens are one of the most common outdoor allergens. Pollen grains and subpollen particles can reach lower airways and induce symptoms of seasonal asthma and allergic rhinitis. Plants possess NAD(P)H oxidase activity that generates reactive oxygen species for physiological functions such as root-hair and pollen-tube growth, defense against microbial infections and cell signaling. The presence of NAD(P)H oxidases in pollens and their role in induction of airway inflammation have not been described until recently. RECENT FINDINGS: We discovered the presence of NAD(P)H oxidase in ragweed and other plant pollens. These oxidases induce reactive oxygen species in mucosal cells (signal 1) independent of adaptive immunity. This reactive oxygen species facilitates antigen (signal 2)-induced allergic inflammation. Inhibiting signal 1 by administration of antioxidants attenuated ragweed extract-induced allergic inflammation. Likewise, abrogating signal 2 by antigen challenge in mice lacking T cells failed to induce allergic inflammation. SUMMARY: Reactive oxygen species generated by pollen NAD(P)H oxidase play a major role in pathogenesis of allergic airway inflammation and airway hypersensitivity. Based on our findings, we propose a 'two signal hypothesis of allergic inflammation' in which both signal 1 (reactive oxygen species) and signal 2 (antigen presentation) are required in order to induce full-blown allergic inflammation.     

Inhibition of NAD(P)H oxidase activity blocks vascular endothelial growth factor overexpression and neovascularization during ischemic retinopathy

Because oxidative stress has been strongly implicated in up-regulation of vascular endothelial growth factor (VEGF) expression in ischemic retinopathy, we evaluated the role of NAD(P)H oxidase in causing VEGF overexpression and retinal neovascularization. Dihydroethidium imaging analyses showed increased superoxide formation in areas of retinal neovascularization associated with relative retinal hypoxia in a mouse model for oxygen-induced retinopathy. The effect of hypoxia in stimulating superoxide formation in retinal vascular endothelial cells was confirmed by in vitro chemiluminescence assays. The superoxide formation was blocked by specific inhibitors of NAD(P)H oxidase activity (apocynin, gp91ds-tat) indicating that NAD(P)H oxidase is a major source of superoxide formation. Western blot and immunolocalization analyses showed that retinal ischemia increased expression of the NAD(P)H oxidase catalytic subunit gp91phox, which localized primarily within vascular endothelial cells. Treatment of mice with apocynin blocked ischemia-induced increases in oxidative stress, normalized VEGF expression, and prevented retinal neovascularization. Apocynin and gp91ds-tat also blocked the action of hypoxia in causing increased VEGF expression in vitro, confirming the specific role of NAD(P)H oxidase in hypoxia-induced increases in VEGF expression. In conclusion, NAD(P)H oxidase activity is required for hypoxia-stimulated increases in VEGF expression and retinal neovascularization. Inhibition of NAD(P)H oxidase offers a new therapeutic target for the treatment of retinopathy.     

Regional NAD(P)H:quinone oxidoreductase activity in Alzheimer's disease

Converging evidence supports the role of oxidative stress in the pathology of Alzheimer's disease (AD). This notion is further supported by recent findings of increased NAD(P)H:quinone oxidodreductase (NQO1) activity, a potent antioxidant system, in association with hippocampal AD pathology. If increased NQO1 activity is truly related to the AD process, however, we would expect to see regional co-localization of NQO1 activity with AD pathology throughout affected brain regions and the absence of NQO1 activity in regions unaffected by AD. We examined this hypothesis by measuring NQO1 enzymatic activity and NQO1 immunohistochemical staining in regions commonly affected by the AD process such as frontal cortex and compared this to regions generally unaffected by the AD process such as occipital cortex, cerebellum, and substantia nigra for a group of AD patients and controls. The ratio of frontal to cerebellar NQO1 enzymatic activity was significantly increased in patients with AD (2.07 +/- 1.90) versus controls (0.60 +/- 0.31; P < 0.03). Moreover, regional immunohistochemical staining revealed specific localization of NQO1 staining to astrocytes and neurites surrounding senile plaques. The extent of immunohistochemical staining also closely correlated with the extent of local AD pathology across the various brain regions examined. Neuronal NQO1 staining seen in frontal cortex of AD patients was absent in frontal cortex of controls, but was found to the same extent in neurons of the substantia nigra of both AD patients and controls. We conclude that NQO1 activity co-localizes closely with AD pathology supporting a presumed role as an antioxidant system upregulated in response to the oxidative stress of the AD process. The antioxidant role for NQO1 is further supported by finding increased neuronal NQO1 activity in substantia nigra neurons of both AD patients and controls as this neuronal population is known to be under constant oxidative stress. While requiring further study, these findings, in conjunction with previous work, suggest that increased NQO1 activity may be neuroprotective, may offer novel insights into the pathophysiology of AD and may also provide possible avenues for future treatment.     

Statin attenuates high glucose-induced and angiotensin II-induced MAP kinase activity through inhibition of NAD(P)H oxidase activity in cultured mesangial cells

An increased oxidative stress may contribute to the development of diabetic nephropathy. We have recently reported that high glucose level stimulated superoxide production through protein kinase C (PKC)-dependent activation of NAD(P)H oxidase in cultured vascular cells. Here we show that 3-hydroxy-3-methylglutaryl CoA reductase inhibitor (statin) attenuates both high glucose level-induced and angiotensin II (Ang II)-induced activation of p42/44 mitogen-activated kinase (MAP kinase) in cultured human mesangial cells through inhibition of NAD(P)H oxidase activity. The intracellular oxidative stress in cultured mesangial cells was evaluated by electron spin resonance (ESR) measurement. MAP kinase activity was evaluated by western blot analysis using anti phospho-specific MAP kinase antibody and anti-ERK-1 antibody. Exposure of the cells to high glucose level (450 mg/dl) for 72 hrs significantly increased MAP kinase activity as compared to normal glucose level (100 mg/dl). This increase was completely blocked by the treatment of pitavastatin (5x10(-7) M) as well as a NAD(P)H oxidase inhibitor (diphenylene iodonium, 10(-5) M) in parallel with the attenuation of oxidative stress. Ang II-induced activation of MAP kinase was also completely blocked by pitavastatin as well as a diphenylene iodonium in parallel with the attenuation of oxidative stress. In conclusion, pitavastatin attenuated high glucose-induced and Ang II- induced MAP kinase activity in mesangial cells through inhibition of NAD(P)H oxidase. Thus, statins may have a potential as a therapeutic tool for early diabetic nephropathy.     

Haplotype analysis of NAD(P)H oxidase p22 phox polymorphisms in end-stage renal disease

NAD(P)H oxidase is one of the most important sources of reactive oxygen species and has been demonstrated to be upregulated by angiotensin II in the kidney. Given the effect of angiotensin-converting enzyme inhibitors on the progression of both diabetic and non-diabetic renal disease, we hypothesized that the polymorphisms of NAD(P)H oxidase are associated with development of end-stage renal disease (ESRD). We examined five polymorphisms in the CYBA gene encoding the p22 phox component of NAD(P)H oxidase, including 242C/T and 640A/G polymorphisms in 467 ESRD patients and 490 healthy individuals. The T allele of the 242C/T polymorphism showed a protective effect against ESRD only in the nondiabetic (non-DM) group (P=0.0095), and haplotype estimation revealed that the frequency of 242C–640A was higher in the non-DM group (46.7%) than in the control group (39.7%). The CC–AA genotype was still significantly associated with ESRD without diabetes after adjusting for confounding factors (P=0.035). In contrast, there was no difference between the DM group and the control group. In conclusion, we identified a risk haplotype for nondiabetic ESRD in the CYBA gene using haplotype analysis. Haplotype analysis proved useful for elucidating the genetic contribution of NAD(P)H oxidase p22 phox to ESRD.     

NAD(P)H:quinone oxidoreductase activity is increased in hippocampal pyramidal neurons of patients with Aalzheimer's disease

NAD(P)H:quinone oxidoreductase (QR) catalyzes the two-electron reduction of quinones, preventing their participation in redox cycling and subsequent generation of reactive oxygen species. Pretreatment of neuroblastoma cells with compounds, such as tert-butylhydroquinone and dimethyl fumarate, that increase QR expression protect cells from oxidative stress-induced cell death by glutamate, H(2)O(2,) and dopamine. The potential neuroprotective role of QR as well as the evidence for oxidative stress-induced neuronal cell death in Alzheimer's disease (AD) led us to examine the expression pattern of QR from AD and control patients. Histochemical staining of hippocampal sections from AD patients revealed QR activity in pyramidal neurons. The presence of QR protein in these neurons also was confirmed by immunoreactivity. In control patients, hippocampal pyramidal neurons were negative for both QR enzymatic activity and QR immunoreactivity. In addition, the QR positive neurons of AD patients were selectively located in areas where neuronal populations exhibited tau immunostaining. Our data demonstrate that QR is up-regulated in hippocampal pyramidal neurons of AD patients. We hypothesize that this is part of a neuroprotective system up-regulated in response to the AD process. Understanding this system may lead to further insights into the pathogenesis and potential new avenues of treatment for AD.     

Arachidonic acid-dependent activation of a p22(phox)-based NAD(P)H oxidase mediates angiotensin II-induced mesangial cell protein synthesis and fibronectin expression via Akt/PKB

Angiotensin II (Ang II) induces protein synthesis and hypertrophy through arachidonic acid (AA)- and redoxdependent activation of the serine-threonine kinase Akt/PKB in mesangial cells (MCs). The role of NAD(P)H oxidase component p22( phox ) was explored in this signaling pathway and in Ang II-induced expression of the extracellular matrix protein fibronectin. Ang II causes activation of Akt/PKB and induces fibronectin protein expression, effects abrogated by phospholipase A(2) inhibition and mimicked by AA. Ang II and AAalso elicited an increase in fibronectin expression that was reduced with a dominant negative mutant of Akt/PKB. Exposure of the cells to hydrogen peroxide stimulates Akt/PKB activity and fibronectin synthesis. The antioxidant N-acetylcysteine abolished Ang II- and AA-induced Akt/PKB activation and fibronectin expression. Western blot analysis revealed high levels of p22( phox ) in MCs. Antisense (AS) but not sense oligonucleotides for p22( phox ) prevented ROS generation in response to Ang II and AA. AS p22( phox ) inhibited Ang II- or AA-induced Akt/PKB as well as protein synthesis and fibronectin expression. These data provide the first evidence, in MCs, of activation by AAof a p22( phox )-based NAD(P)H oxidase and subsequent generation of ROS. Moreover, this pathway mediates the effect of Ang II on Akt/PKB-induced protein synthesis and fibronectin expression.     

Pollen NAD(P)H oxidases and their contribution to allergic inflammation

This article provides an overview of NADPH oxidase and its role in allergic inflammation. A background and historical perspectives of NADPH oxidase are first provided, followed by a detailed overview of mammalian NADPH oxidase subunits and their functional organization. Plant NADPH oxidase, the authors' discovery of NADPH oxidase in pollens, and their contribution to allergic inflammation are then discussed, concluding with a discussion of future directions and outstanding questions that require attention.     

Chronic high pressure-induced arterial oxidative stress: involvement of protein kinase C-dependent NAD(P)H oxidase and local renin-angiotensin system

Regardless of the underlying pathological mechanisms oxidative stress seems to be present in all forms of hypertension. Thus, we tested the hypothesis that chronic presence of high pressure itself elicits increased arterial O(2)(.-) production. Hypertension was induced in rats by abdominal aortic banding (Ab). Rats with Ab had elevated pressure in vessels proximal and normal pressure in vessels distal to the coarctation, yet both vascular beds were exposed to the same circulating factors. Compared to normotensive hind limb arteries (HLAs) hypertensive forelimb arteries (FLAs) exhibited 1) impaired dilations to acetylcholine and the nitric oxide donor S-nitroso-N-acetyl-D,L-penicillamine that were restored by administration of superoxide dismutase; 2) an increased production of O(2)(.-) (measured by lucigenin chemiluminescence and ethidium bromide fluorescence) that was inhibited or reduced by superoxide dismutase, the NAD(P)H oxidase inhibitors diphenyleneiodonium and apocynin, or the protein kinase C (PKC) inhibitors chelerythrine and staurosporine or by the angiotensin-converting enzyme (ACE) inhibitor captopril; and 3) increased ACE activity. In organ culture, exposure of isolated arteries of normotensive rats to high pressure (160 mmHg, for 24 hours) significantly increased O(2)(.-) production compared to that in arteries exposed to 80 mmHg. High pressure-induced O(2)(.-) generation was reduced by inhibitors of ACE and PKC. Incubation of cultured arteries with angiotensin II elicited significantly increased O(2)(.-) generation that was inhibited by chelerythrine. Thus, we propose that chronic presence of high pressure itself can elicit arterial oxidative stress, primarily by activating directly a PKC-dependent NAD(P)H oxidase pathway, but also, in part, via activation of the local renin-angiotensin system.     

NAD(P)H oxidase in human fetal membrane chorion laeve trophoblasts with or without chorioamnionitis: ultrastructural enzyme histochemical study

We examined the subcellular localizations of NAD(P)H oxidase, a reactive oxygen species (ROS)-producing enzyme, in fetal membrane chorion laeve trophoblasts from preterm or term pregnant women with or without chorioamnionitis (CAM). Ultrastructural enzyme histochemistry for NAD(P)H oxidase was used. In fetal membranes without CAM, approximately one quarter of the chorion laeve trophoblasts (25.6%) showed NAD(P)H oxidase activity on their surface plasma membranes and microvillous membranes. In mild CAM, the proportion of these NAD(P)H oxidase-positive cells significantly increased, reaching about half (51.0%). Enzyme activity appeared on the plasma and microvillous membranes and also on both phagosomal membranes and intracellular vesico-tubular structures. Appearance of NAD(P)H oxidase on surface plasma membranes, phagosomal membranes, and vesico-tubular structures is strong cytochemical evidence of phagocytic cell activation. These observations indicate that chorion laeve trophoblasts possess NAD(P)H oxidase activity, and therefore that fetal membranes themselves have ROS-generating capacity. Further, in fetal membrane inflammation, chorion laeve trophoblasts exhibited enzyme distribution characteristic of activated professional phagocytes. Similar to phagocytes infiltrating to the intrauterine environment, chorion laeve trophoblast NAD(P)H oxidase may play a role both in the defence of chorioamnion against infection and in the pathogenesis or pathophysiology of CAM-related preterm delivery.     

Cytosolic NAD(P)H regulation of redox signaling and vascular oxygen sensing

This article considers how regulation of signaling controlled by cytosolic NADPH and NADH redox systems contained within the vascular smooth muscle cell may contribute to coordinating alterations in force generation elicited by acute changes in oxygen tension. Additional important issues considered include defining when oxidases generating reactive oxygen species (ROS), such as Nox oxidases, or ROS metabolizing activities which utilize cytosolic NADH and/or NADPH are key participants in eliciting responses that are observed, and assessing how mitochondria can potentially contribute to the regulation that is seen. Many important signaling mechanisms potentially involved in vascular oxygen sensing such as potassium channels, systems regulating intracellular calcium, and the sensitivity of the contractile apparatus to calcium, and the control of cGMP-mediated relaxation by soluble guanylate cyclase appear to be regulated by cytosolic NAD(P)H redox and or ROS. Differences in the processes controlling the maintenance of cytosolic NADPH redox by the pentose phosphate pathway of glucose metabolism are hypothesized to be a key factor in controlling the expression of a relaxation to hypoxia seen in systemic arteries compared to the hypoxic contractile response observed in pulmonary arterial smooth muscle.     

Non-invasive live-cell measurement of changes in macrophage NAD(P)H by two-photon microscopy

Two-photon microscopy allows determination of UV-excitable fluorophores using long-wavelength light. We aimed to determine NAD(P)H autofluorescence as a measure for macrophage NADPH-oxidase activation. RAW264.7 macrophages were grown on glass coverslips and kept in HBSS for microscopic investigation. Cells were excited with 710 nm light and NAD(P)H autofluorescence was detected. Glucose as well as NaCN evoked an increase of NAD(P)H autofluorescence. Activators of NADPH oxidase lead to significantly decreased NAD(P)H autofluorescence. Therefore, this work shows the suitability of two-photon microscopy as a non-invasive method determining changes in phagocyte NAD(P)H upon activation.     

No linkage of P187S polymorphism in NAD(P)H: Quinone oxidoreductase (NQO1/DIA4) and type 1 diabetes in the Danish population

Recent genome screening studies have identified novel regions of possible interest for susceptibility to type 1 diabetes. One of these is a 30‐35 cM region mapping to 16q22‐q24 (D16S515‐D16S520), where also the gene encoding NAD(P)H: quinone oxidoreductase (NQO1) maps. Data has suggested association of a polymorphism (P187S) in the NQO1 gene and type 1 diabetes. NQO1 is involved in protection against oxidative stress, which is likely to be involved in β‐cell destruction. By use of the transmission disequilibrium test (TDT), we analyzed the P187S polymorphism for association to type 1 diabetes in a population‐based sample of 247 Danish nuclear type 1 diabetic families. Random transmission patterns were observed to all affected offspring (p_tdt = 0.82), to index cases (p_tdt = 0.77), as well as to unaffected offspring (p_tdt = 0.93). Hence, the NQO1 polymorphism is not likely to be an etiological mutation underlying the reported linkage of the 16q22‐q24 region. Hum Mutat 14:67–70, 1999. © 1999 Wiley‐Liss, Inc.     

Expression of NAD(P)H:quinone oxidoreductase in the normal and Parkinsonian substantia nigra

Dopamine (DA) autooxidation, and consequent formation of neurotoxic DA-derived quinones and reactive oxygen species, has been implicated in dopaminergic cell death and, hence, in the pathogenesis of Parkinson's disease (PD). Stimulation of pathways involved in the detoxication of DA-quinones in the brain is hypothesized to be an effective means to limit oxidative stress and to confer neuroprotection in PD. In this respect, the inducible flavoprotein NAD(P)H:quinone oxidoreductase (NQO1) is of particular interest as it is directly implicated in the detoxication of DA-quinones and, in addition, has broad spectrum anti-oxidant properties. To study the potential pathophysiological role of NQO1 in PD, the cellular expression of NQO1 was examined in the mesencephalon of PD patients and age-matched controls. In the substantia nigra pars compacta (SNpc), NQO1 was found to be expressed in astroglial and endothelial cells and, albeit less frequently, also in dopaminergic neurons. Moreover, while overt NQO1 immunoreactivity was absent in the surrounding nervous tissue, in the Parkinsonian SNpc a marked increase in the astroglial and neuronal expression of NQO1 was consistently observed.     

单胺氧化酶B和NAD(P)H醌氧化还原酶基因多态性与帕金森病的关系

目的 探讨参与多巴胺代谢的单胺氧化酶B（monoamine oxidase B,MAO-B)内含子13A／G多态性,NAD（P）H醌氧化还原酶基因[NAD(P)H:quinone oxidoreductase,NQO1]cDNA609C/T多态性与帕金森病（Parkinson's disease,PD)遗传易感性的关系。方法 应用等位基因特异PCR扩增分析MAO－B基因的多态性,用PCR－RFLP分析NQO1基因多态性。结果 MAO－BA／G等位基因频率、各基因型频率PD组与对照组差异无显著性。NQO1基因T等位基因频率PD组（52％）高于正常对照组（43％）带T碱基的NQO1基因型频率PD组显著高于正常对照组（P＜0．05）,其患PD的相对危险度（odds ratio,OR)为3．8。在带有A等位基因MAO－B基因型的个体,带有T碱基因的T等位基因是PD的危险因素。MAO－B基因的A等位基因型与NQO1基因的T等位基因的基因型可相互协同,增加PD发生的风险。