Nox4 Protein Levels Research Articles

Protective effect of soybeans as protein source in the diet against cadmium-aorta redox and morphological alteration

We investigated the effects of cadmium exposition on thoracic aorta redox status and morphology, and the putative protective effect of soybeans in the diet.Male Wistar rats were separated into 6 groups: 3 fed with a diet containing casein and 3 containing soybeans, as protein source. Within each protein group, one was given tap water (control) and the other two tap water containing 15 and 100ppm of Cd2+, respectively, for two months.In rats fed with casein diet, 15ppm of Cd induced an increase of thiobarbituric acid-reactive substances (TBARS), and of the catalase (CAT) and glutathione peroxidase (GPx) activities, which were even higher with 100ppm of Cd2+, in aorta.Also, 100ppm Cd2+ exposure increased superoxide dismutase (CuZnSOD) activity; CAT, GPX, SOD, Nrf2 and metallothioneine II mRNA expressions and CAT, GPx and NOX-2 protein levels, compared with control. Aorta endothelial and cytoplasmic alterations were observed.However, with the soybeans diet, 15 and 100ppm of Cd2+ did not modify TBARS levels; CAT, GPX and Nrf2 mRNA expressions; CAT, GPx and NOX-2 protein; and the aorta morphology, compared with control.The soybean diet attenuates the redox changes and protects against morphological alterations induced, in a dose-dependent way, by Cd in aorta.

Mitochondrial-localized NADPH oxidase 4 is a source of superoxide in angiotensin II-stimulated neurons

Angiotensin II (ANG II) plays an important role in the central regulation of systemic cardiovascular function. ANG II-mediated intraneuronal signaling has been shown to be predicated by an increase in mitochondrial superoxide (O₂∙-), yet the source of this reactive oxygen species (ROS) production remains unclear. NADPH oxidase 4 (Nox4), a member of the NADPH oxidase family, has been reported to be localized in mitochondria of various cell types and has been implicated in brain angiotensinergic signaling. However, the subcellular localization and function of Nox4 in neurons has not been fully elucidated. In this study, we hypothesized that Nox4 is expressed in neuron mitochondria and is involved in ANG II-dependent O₂∙--mediated intraneuronal signaling. To query this, Nox4 immunofluorescent staining and mitochondrial enrichment were performed in a mouse catecholaminergic neuronal cell model (CATH.a). Nox4 was shown to be present in neuron mitochondria as evidenced by colocalization with both the mitochondrial-localized protein manganese superoxide dismutase (MnSOD) and dye MitoTracker Red. Moreover, Nox4 expression was significantly increased in enriched mitochondrial fractions compared with whole cell lysates. Additionally, adenoviral-encoded small interfering RNA for Nox4 (AdsiNox4) caused a robust knockdown in Nox4 mRNA and protein levels, which led to the attenuation of ANG II-induced mitochondrial O₂∙- production. Finally, in the subfornical organ (SFO) of the brain, Nox4 not only demonstrated mitochondrial localization but was induced by chronic, peripheral infusion of ANG II. Collectively, these data suggest that Nox4 is a source of O₂∙- in neuron mitochondria that contributes to ANG II intraneuronal signaling.

Toll-Like Receptor 4 Mutation Protects Obese Mice Against Endothelial Dysfunction by Decreasing NADPH Oxidase Isoforms 1 and 4

To analyze the role of toll-like receptor 4 in modulating metabolism and endothelial function. Type 2 diabetic mice with mutated toll-like receptor 4 (DWM) were protected from hyperglycemia and hypertension, despite an increased body weight. Isometric tension was measured in arterial rings with endothelium. Relaxations to acetylcholine were blunted in aortae and mesenteric arteries of Lepr(db/db) mice, but not in DWM mice; the endothelial NO synthase dimer/monomer ratio and endothelial NO synthase phosphorylation levels were higher in DWM preparations. These differences were abolished by apocynin. Contractions to acetylcholine (in the presence of L-NAME) were larger in carotid arteries from Lepr(db/db) mice than from DWM mice and were inhibited by indomethacin and SC560, demonstrating involvement of cyclooxygenase-1. The release of 6-ketoprostaglandin F1α was lower in DWM mice arteries, implying lower cyclooxygenase-1 activity. Apocynin, manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin, catalase, and diethyldithiocarbamate inhibited endothelium-dependent contractions. The mRNA and protein levels of NADPH oxidase isoforms NOX1 and NOX4 were downregulated in DWM mice arteries. The in vivo and in vitro administration of lipopolysaccharide caused endothelial dysfunction in the arteries of wild-type, but not toll-like receptor 4-mutated mice. Toll-like receptor 4 plays a key role in obesity and diabetes-associated endothelial dysfunction by increasing oxidative stress.

Abstract 366: Nox1-Derived Superoxide Anion Mediates Stretch-induced Phenotypic Switching in Smooth Muscle Cells

Hemodynamic forces in the vessel wall have profound influences on function, structure and pathology of vascular cells. The predominant mechanical force influencing vascular smooth muscle cells (VSMC) structure and signaling is cyclic stretch (CS). Aberrant CS can direct VSMC to a synthetic phenotype (SP) characterized by increased VSMC migration, loss of contractility and matrix production. Some evidence suggests that CS is capable of inducing reactive oxygen species (ROS); however, the mechanisms by which stretch-derived ROS can induce a VSMC SP are poorly understood. Here we postulate that the NADPH oxidase isoform Nox1, which is an important source of ROS in vascular disease, is involved in a CS-mediated SP. In VSMC subjected to CS (10%, 1Hz) there was an increase in Nox1 mRNA levels (3.9 ± 0.14 fold of increase after 6 hr of CS), Nox1 protein levels (1.44 ± 0.04 fold increase after 24 hr CS) and Nox1-derived superoxide production (CS: 13.82±1.34 vs static: 6.61±0.67 pmol of superoxide/min/mg protein after 24 hr). Using our recently-developed specific peptide inhibitor of Nox1 (NoxA1ds), CS-induced superoxide production abated to baseline levels (6.61±0.67, 13.82±1.34 and 5.06±0.76 pmol of superoxide/min/mg protein; for static, CS and CS+NoxA1ds respectively). Furthermore, osteopontin protein levels, a VSMC SP marker, were increased by CS for 24 hrs (1.25±0.04 fold higher than static) and abolished by NoxA1ds pretreatment (0.96±0.19 fold of static). On the other hand, calponin protein levels, a VSMC contractile phenotype marker, were decreased by CS and restored by pretreatment with NoxA1ds (0.67±0.05- and 0.93±0.12-fold change from static respectively). Finally, we demonstrate that stretch-induced migration of smooth muscle cells is promoted by Nox1-derived superoxide (46.36±1.47, 56.16±1.74 and 40.98±2.46 % of scratch wound closure for static, CS and CS+NoxA1ds treatments respectively). These results suggest that Nox1-derived superoxide mediates a synthetic phenotype in vascular smooth muscle under CS, and identify Nox1 as a potential therapeutic target for the treatment of diseases such as atherosclerosis and hypertension which are characterized by VMSC SP.

Abstract 2059: NADPH Oxidase 4 induced ROS regulates anoikis resistance through activation of Src and EGFR.

Abstract Since cancer cells must overcome the anoikis for the metastasis, anoikis resistance is an important process for tumor progression and metastasis. To identify anoikis resistance mechanism, we performed mRNA microarray analysis with attached and suspended human lung epithelial adenocarcinoma A549 cells. Our microarray data reveals that Nox4 mRNA is up-regulated in the suspended cells. We evaluated both mRNA and protein levels of Nox4 and found that Nox4 is up-regulated in both mRNA and protein levels. In addition, higher levels of ROS are detected and Src activation was enhanced in suspended cells than attached cells. Interestingly, increased level of ROS in suspended cells was decreased by ROS scavengers or Nox4 inhibitors, leading to inactivation of Src and EGFR. Furthermore, knock-down of Nox4 by si-RNA specific for Nox4 resulted in inactivation of Src and EGFR. Because Src activation is not inhibited by EGFR inhibitor, Src activation appears to be independent on EGFR. In addition, si-RNA for Nox4 transfection decreased viability of cells in suspension. These results suggest that upon cell detachment, Nox4 expression is up-regulated and thus ROS levels are increased, which are important for activation of Src and EGFR, leading to sustained cell viability in the suspended cells. Therefore, we postulate that ROS generation through the Nox4 is an important process for anoikis resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2059. doi:1538-7445.AM2012-2059

Ethanol Induces Oxidative Stress in Alveolar Macrophages via Upregulation of NADPH Oxidases

Chronic alcohol abuse is a comorbid variable of acute respiratory distress syndrome. Previous studies showed that, in the lung, chronic alcohol consumption increased oxidative stress and impaired alveolar macrophage (AM) function. NADPH oxidases (Noxes) are the main source of reactive oxygen species in AMs. Therefore, we hypothesized that chronic alcohol consumption increases AM oxidant stress through modulation of Nox1, Nox2, and Nox4 expression. AMs were isolated from male C57BL/6J mice, aged 8-10 wk, which were treated with or without ethanol in drinking water (20% w/v, 12 wk). MH-S cells, a mouse AM cell line, were treated with or without ethanol (0.08%, 3 d) for in vitro studies. Selected cells were treated with apocynin (300 μM), a Nox1 and Nox2 complex formation inhibitor, or were transfected with Nox small interfering RNAs (20-35 nM), before ethanol exposure. Human AMs were isolated from alcoholic and control patients' bronchoalveolar lavage fluid. Nox mRNA levels (quantitative RT-PCR), protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red analysis), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol increased Nox expression and oxidative stress in mouse AMs in vivo and in vitro. Experiments using apocynin and Nox small interfering RNAs demonstrated that ethanol-induced Nox4 expression, oxidative stress, and AM dysfunction were modulated through Nox1 and Nox2 upregulation. Further, Nox1, Nox2, and Nox4 protein levels were augmented in human AMs from alcoholic patients compared with control subjects. Ethanol induces AM oxidative stress initially through upregulation of Nox1 and Nox2 with downstream Nox4 upregulation and subsequent impairment of AM function.

Nox2 and Nox4 mediate tumour necrosis factor-α-induced ventricular remodelling in mice

Reactive oxygen species (ROS) and pro-inflammatory cytokines are crucial in ventricular remodelling, such as inflammation-associated myocarditis. We previously reported that tumour necrosis factor-α (TNF-α)-induced ROS in human aortic smooth muscle cells is mediated by NADPH oxidase subunit Nox4. In this study, we investigated whether TNF-α-induced ventricular remodelling was mediated by Nox2 and/or Nox4. An intravenous injection of murine TNF-α was administered to a group of mice and saline injection was administered to controls. Echocardiography was performed on days 1, 7 and 28 post-injection. Ventricular tissue was used to determine gene and protein expression of Nox2, Nox4, ANP, interleukin (IL)-1β, IL-2, IL-6, TNF-α and to measure ROS. Nox2 and Nox4 siRNA were used to determine whether or not Nox2 and Nox4 mediated TNF-α-induced ROS and upregulation of IL-1β and IL-6 in adult human cardiomyocytes. Echocardiography showed a significant increase in left ventricular end-diastolic and left ventricular end-systolic diameters, and a significant decrease in the ejection fraction and fractional shortening in mice 7 and 28 days after TNF-α injection. These two groups of mice showed a significant increase in ventricular ROS, ANP, IL-1β, IL-2, IL-6 and TNF-α proteins. Nox2 and Nox4 mRNA and protein levels were also sequentially increased. ROS was significantly decreased by inhibitors of NADPH oxidase, but not by inhibitors of other ROS production systems. Nox2 and Nox4 siRNA significantly attenuated TNF-α-induced ROS and upregulation of IL-1β and IL-6 in cardiomyocytes. Our study highlights a novel TNF-α-induced chronic ventricular remodelling mechanism mediated by sequential regulation of Nox2 and Nox4 subunits.

Simvastatin protects osteoblast against H2O2-induced oxidative damage via inhibiting the upregulation of Nox4

Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, have been used clinically as a cholesterol-lowering drug to treat hyperlipidemia. In recent years, accumulating evidence indicates the possible beneficial effect of statins on osteoporosis. However, the underlying molecular mechanism remains to be elucidated. In the present study, we investigated the therapeutic effects of simvastatin on cell viability, apoptosis, and alkaline phosphatase activity in murine osteoblastic MC3T3-E1 cells treated by hydrogen peroxide (H(2)O(2), 100 μM). It was shown that simvastatin suppressed H(2)O(2)-induced oxidative stress and attenuated H(2)O(2)-induced cell injury including increasing osteoblastic viability, inhibiting apoptosis, and promoting differentiation. Then, we examined the effects of simvastatin (10(-7) M) on Nox1, Nox2, and Nox4 expressions in osteoblastic cells treated by H(2)O(2) (100 μM). We found that in MC3T3-E1 cells, H(2)O(2)-induced upregulation of Nox4 expression was inhibited by simvastatin, which was restored by farnesyl pyrophosphate (5 μM) as well as geranylgeranyl pyrophosphate (5 μM). RNAi approach was used to reduce Nox4 protein levels in osteoblastic cells to explore its biological effects against H(2)O(2)-induced oxidative damage. When Nox4 expression was reduced in osteoblastic cells, H(2)O(2)-induced cell injury was attenuated markedly. We concluded that simvastatin protected osteoblast against H(2)O(2)-induced oxidative damage, at least in part, via inhibiting the upregulation of Nox4.

Angiotensin II stimulates neural stem cell proliferation through Nox4‐generated superoxide

How reactive oxygen species (ROS) affect neural stem cell (NSC) self‐renewal is poorly understood, but might be important for normal development and during neurodegenerative conditions. We investigated whether production of superoxide via activation of NADPH oxidase (Nox) affects NSC proliferation. In the vascular smooth muscle cells, angiotensin II (Ang II) activates Nox‐driven superoxide generation to induce proliferation. We hypothesized that Ang II activates one of the Nox isoforms to regulate NSC self‐renewal. In a murine neural stem cell line, C17.2, Ang II (100nM) stimulated superoxide production detected by dihydroethidium (DHE) fluorescence. Ang II increased proliferation and an antioxidant N‐acetyl‐cysteine blocked Ang II‐induced increase in cell numbers. Confocal microscopy using DHE and a mitochondrial marker (MitoTracker) revealed that C17.2 cells contain two sources of ROS: mitochondrial and extramitochondrial, indicating that Nox might be involved. Nox4 was found to be constitutively expressed and Ang II increased protein levels of Nox4. SiRNA targeting Nox4 reduced Nox4 expression and attenuated Ang II‐induced ROS production and proliferation. Our findings suggest that Ang II‐induced proliferation of NSCs requires Nox4‐generated superoxide production. This mechanism might be important for normal and stress‐ or disease‐modified neurogenesis. Support: AA016654 and HL64917.

248 APOCYNIN AND DIPHENYLENE IODIUM(DPI) FUNCTION AS INHIBITORS OF CELL MIGRATION, AND PROLIFERATION BUT DO NOT INHIBIT NADPH OXIDASE IN RENAL CELLS CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research1 Apr 2011248 APOCYNIN AND DIPHENYLENE IODIUM(DPI) FUNCTION AS INHIBITORS OF CELL MIGRATION, AND PROLIFERATION BUT DO NOT INHIBIT NADPH OXIDASE IN RENAL CELLS CARCINOMA Jung-Sik Huh, Fernando J. Kim, Hyeon Ju Kim, and Hari Koul Jung-Sik HuhJung-Sik Huh Aurora, CO More articles by this author , Fernando J. KimFernando J. Kim Aurora, CO More articles by this author , Hyeon Ju KimHyeon Ju Kim Aurora, CO More articles by this author , and Hari KoulHari Koul Aurora, CO More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.317AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Renal cell carcinoma, which forms in tubules of the kidney, accounts for approximately 3% of adult malignancies and 90-95% of neoplasms arising from the kidney. Reactive oxygen species(ROS), which originate mainly from NADPH oxidase and are highly reactive O2 metabolites, have physiological and pathological processes. ROX and the oxidative stress have been associated with tumor formation. To respond oxidative stress, inhibition of NADPH oxidase represents a target for the treatment of many diseases. DPI and apocynin has been used as an efficient inhibitor of the complex NADPH oxidase in phagocytic and non-phagocytic cells. We investigated the effects of NOX4 inhibitors in renal cell carcinoma. METHODS We tested the effects of Apocynin and DPI against RCC cell lines (A498, Caki-2, KIKO-12), and the underlying mechanisms. Western Blot analysis was used to measure protein levels of Nox4, Akt and Erk signal molecules. Fluoremetric assays were used to measure ROS generation as well as NADPH oxidase activities. Cell growth and viability as well as migration were measured by standard methods. RESULTS Results show that treatment of RCC cells with apocynin and DPI resulted in a dose and time dependent inhibition of cell growth and proliferation. These treatments however did not induce apoptosis. Apocynin or DPI treatment had no effect on ROS (hydrogen peroxide and superoxide anion) generation in RCC cells. No changes were observed in NOX4, Akt, and Erk signal protein expression or activity change in the presence of apocynin and DPI. But apocynin and DPI interrupted and decreased cell migration of RCC cells. There was no change of actin cytoskeleton in treated and untreated A498 renal cell cancer cell line. CONCLUSIONS Apocynin and DPI, so called specific inhibitors of NADPH oxidase system acts as inhibitors of migration and proliferation but these effects are independent of NADPH oxidase inhibition in RCC cells as these agents did not inhibit Nox activity or ROS generation. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e100 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jung-Sik Huh Aurora, CO More articles by this author Fernando J. Kim Aurora, CO More articles by this author Hyeon Ju Kim Aurora, CO More articles by this author Hari Koul Aurora, CO More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Tyrphostin AG490 reduces NAPDH oxidase activity and expression in the aorta of hypercholesterolemic apolipoprotein E-deficient mice

Oxidative stress-induced vascular injury represents a major contributor to the pathoetiology of atherosclerosis. Elevated NADPH oxidase (Nox) activity promotes oxidative injury of the cardiovascular cells. Janus-tyrosine-kinase (Jak) family regulate various aspects of the atherosclerotic process e.g., inflammation, cellular growth, proliferation, and migration. Here, we investigated the potential of Jak2 inhibition to counteract Nox-dependent O 2 •− formation in atherogenesis in hypercholesterolemic apolipoprotein E-deficient (ApoE −/−) mice. Male ApoE −/− mice fed a high-fat, cholesterol-rich diet were treated for 5 weeks with either vehicle or tyrphostin AG490 (1 mg/kg), a specific Jak2 inhibitor. Lucigenin-enhanced-chemiluminescence assay, real-time PCR and Western blot analysis revealed that Nox-derived O 2 •− generation, Nox1, Nox2, and Nox4 mRNA and protein levels were significantly elevated in the aortas of ApoE −/− mice fed a high-fat diet compared to ApoE −/− mice fed a normal diet. Treatment with tyrphostin AG490 significantly reduced the up-regulated Nox activity, the expression of each Nox subtype, as well as the protein level of CD68, a macrophage-specific marker. Morphometric analysis showed a marked reduction of atherosclerotic lesions in the aorta of AG490-treated animals. These data provide new insights into the regulation of vascular Nox by tyrphostins in the cardiovascular system. Since Jak2 transduces the signals of various cardiovascular risk factors, pharmacological manipulation of this signaling pathway may represent a novel strategy to reduce oxidative stress in atherosclerosis.

Inhibition of NADPH Oxidases Prevents Chronic Ethanol-Induced Bone Loss in Female Rats

Previous in vitro data suggest that ethanol (EtOH) activates NADPH oxidase (Nox) in osteoblasts leading to accumulation of reactive oxygen species (ROS). This might be a mechanism underlying inhibition of bone formation and increased bone resorption observed in vivo after EtOH exposure. In a rat model in which cycling females were infused intragastrically with EtOH-containing liquid diets, EtOH significantly decreased bone formation and stimulated osteoblast-dependent osteoclast differentiation. These effects were reversed by exogenous 17-β-estradiol coadministration. Moreover, coadministration of N-acetyl cysteine (NAC), an antioxidant, or diphenylene iodonium (DPI), a specific Nox inhibitor, also abolished chronic EtOH-associated bone loss. EtOH treatment up-regulated mRNA levels of Nox1, 2, 4, and the receptor activator of nuclear factor-κB ligand (RANKL), an essential factor for differentiation of osteoclasts in bone. Protein levels of Nox4, a major Nox isoform expressed in nonphagocytic cells, was also up-regulated by EtOH in bone. 17-β-Estradiol, NAC, and DPI were able to normalize EtOH-induced up-regulation of Nox and RANKL. In vitro experiments demonstrated that EtOH directly up-regulated Nox expression in osteoblasts. Pretreatment of osteoblasts with DPI eliminated EtOH-induced RANKL promoter activity. Furthermore, EtOH induced RANKL gene expression, and RANKL promoter activation in osteoblasts was ROS-dependent. These data suggest that inhibition of Nox expression and activity may be critical for prevention of chronic EtOH-induced osteoblast-dependent bone loss.

Regulation of NADPH Oxidase Activity Is Associated with miRNA-25-Mediated NOX4 Expression in Experimental Diabetic Nephropathy

Background/Aims: Although numerous studies have explored the mechanisms regulating the enzyme activity of NADPH oxidase in diabetic nephropathy (DN), little information is available for the contribution of microRNAs (miRNAs) to the regulation of NADPH oxidase expression. Therefore, the present study was to test whether miRNAs importantly contribute to the regulation of NOX4 expression, a major catalytic subunit of NADPH oxidase under hyperglycemia. Methods: Diabetic rats were induced by streptozotocin. miRNA microarray, Western blot, real-time RT-PCR and luciferase reporter assays were employed in this study. Results: Among 5 miRNAs, which are predicted to have a binding capacity to rat NOX4, the miRNA-25 level was significantly reduced both in the kidney from diabetic rats and in high glucose-treated mesangial cells, accompanied by the increases in NOX4 expression levels. In an in vitrostudy, we found that NADPH activity was increased by 226.2% in miRNA-25 inhibitor transfected cells and decreased by 51.0% in miRNA-25 precursor transfected cells. miR-25 inhibitor dramatically increased both NOX4 mRNA and protein levels. We then showed that miR-25 negatively regulated NOX4 expression by directly targeting the 3′-UTR by luciferase reporter assays. It was found that transfection of miR-25 precursor significantly decreased the luciferase activity of NOX4 3′-UTR by 39.5%, whereas the mutant sequence restored levels to 79.4%. Finally, our results indicated that the miR-25-mediated NOX4 mRNA level may result from the regulation of mRNA stability. Conclusions: These findings for the first time indicate that miRNA-25 may serve as an endogenous gene silencing factor and contributes to the regulation of NOX4 expression and function in DN.

NADPH oxidase overexpression in human colon cancers and rat colon tumors induced by 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)

NADPH oxidase/dual-oxidase (Nox/Duox) family members have been implicated in nuclear factor kappa-B (NFκB)-mediated inflammation and inflammation-associated pathologies. We sought to examine, for the first time, the role of Nox/Duox and NFκB in rats treated with the cooked meat heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). In the PhIP-induced colon tumors obtained after 1 year, Nox1, Nox4, NFκB-p50 and NFκB-p65 were all highly overexpressed compared with their levels in adjacent normal-looking colonic mucosa. Nox1 and Nox4 mRNA and protein levels also were markedly elevated in a panel of primary human colon cancers, compared with their matched controls. In HT29 human colon cancer cells, Nox1 knockdown induced G1 cell cycle arrest, whereas in Caco-2 cells there was a strong apoptotic response, with increased levels of cleaved caspase-3, -6, -7 and poly(ADP-ribose)polymerase. Nox1 knockdown blocked lipopolysaccharide-induced phosphorylation of IκB kinase, inhibited the nuclear translocation of NFκB (p50 and p65) proteins, and attenuated NFκB DNA binding activity. There was a corresponding reduction in the expression of downstream NFκB targets, such as MYC, CCND1 and IL1β. The results provide the first evidence for a role of Nox1, Nox4 and NFκB in PhIP-induced colon carcinogenesis, including during the early stages before tumor onset. Collectively, the findings from this investigation and others suggest that further work is warranted on the role of Nox/Duox family members and NFκB in colon cancer development.

P59 Can we predict different phenotypes of preeclampsia in low risk nulliparous women using first trimester angiogenic/antiangiogenic biomarkers

The roles of cerebrovascular oxidative stress in vascular functional remodeling have been described in hindlimb-unweighting (HU) rats. However, the underlying mechanism remains to be established.We investigated the generation of vascular reactive oxygen species (ROS), Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and manganese superoxide dismutase (MnSOD) and glutathione peroxidase-1 (GPx-1) mRNA levels in cerebral and mesenteric smooth muscle cells (VSMCs) of HU rats.ROS production increased in cerebral but not in mesenteric VSMCs of HU rats compared with those in control rats. Nox2 and Nox4 protein and mRNA levels were increased significantly but MnSOD/GPx-1 mRNA levels decreased in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly more in cerebral but not in mesenteric arteries of HU rats. NADPH oxidase inhibition with apocynin attenuated cerebrovascular ROS production and partially restored Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and MnSOD/GPx-1 mRNA levels in cerebral VSMCs of HU rats.These results suggest that vascular NADPH oxidases regulate cerebrovascular redox status and participate in vascular oxidative stress injury during simulated microgravity.

Upregulation of Nox4 by TGFβ1 Oxidizes SERCA and Inhibits NO in Arterial Smooth Muscle of the Prediabetic Zucker Rat

Vascular smooth muscle cell (SMC) migration is an important pathological process in several vascular occlusive diseases, including atherosclerosis and restenosis, both of which are accelerated by diabetes mellitus. To determine the mechanisms of abnormal vascular SMC migration in type 2 diabetes, the obese Zucker rat (ZO), a model of obesity and insulin resistance, was studied. In culture, ZO aortic SMCs showed a significant increase in Nox4 mRNA and protein levels compared with the control lean Zucker rat (ZL). The sarco-/endoplasmic reticulum Ca(2+) ATPase (SERCA) nitrotyrosine-294,295 and cysteine-674 (C674)-SO(3)H were increased in ZO SMCs, indicating oxidant stress. Unlike ZL SMC, nitric oxide (NO) failed to inhibit serum-induced SMC migration in ZO. Transfection of Nox4 small interference RNA or overexpression of SERCA2b wild type, but not C674S mutant SERCA, restored the response to NO. Knockdown of Nox4 also decreased SERCA oxidation in ZO SMCs. In addition, transforming growth factor-β1 via Smad2 was necessary and sufficient to upregulate Nox4, oxidize SERCA, and block the antimigratory action of NO in ZO SMCs. Corresponding to the results in cultured SMCs, immunohistochemistry confirmed that Nox4 and SERCA C674-SO(3)H were significantly increased in ZO aorta. After common carotid artery injury, knockdown of Nox4 by adenoviral Nox4 short hairpin RNA decreased Nox4 and SERCA C674-SO(3)H staining and significantly decreased injury-induced neointima. These studies indicate that the upregulation of Nox4 by transforming growth factor-β1 in ZO SMCs is responsible for the impaired response to NO by a mechanism involving the oxidation of SERCA C674. Knockdown of Nox4 inhibits oxidation of SERCA, as well as neointima formation, after ZO common carotid artery injury.

Vascular oxidative stress and inflammation increase with age: ameliorating effects of α‐lipoic acid supplementation

Increased oxidative stress and inflammation causally contribute to cardiovascular diseases, for which advanced age is a major risk factor. We found that indicators of oxidative stress, including NADPH oxidase activity and superoxide levels, were significantly increased in aortas of old (22-24 months) versus young (3-4 months) male F344 rats, whereas superoxide dismutase (SOD) activity was decreased. Aortic mRNA and protein levels of NOX4, the principal catalytic subunit of NADPH oxidase in vascular cells, also were increased with age, but not NOX2 and p22(phox). Indicators of inflammation, including activation of NFkappaB and upregulation of vascular cell adhesion molecule-1 (VCAM-1) in aorta, and monocyte chemotactic protein-1 (MCP-1) in plasma, also were significantly increased in old rats. Supplementation with 0.2% (wt/wt) (R)-alpha-lipoic acid (LA) for 2 weeks caused a nonsignificant decrease in NADPH oxidase activity in aged aorta and a significant decrease in mRNA--but not protein--levels of NOX4 and VCAM-1. Furthermore, LA reversed the age-dependent changes in aortic SOD activity and plasma MCP-1 levels. Hence, vascular oxidative stress and inflammation increase with age and are ameliorated by LA supplementation.

The NADPH Oxidase Subunit NOX4 Is a New Target Gene of the Hypoxia-inducible Factor-1

NADPH oxidases are important sources of reactive oxygen species (ROS), possibly contributing to various disorders associated with enhanced proliferation. NOX4 appears to be involved in vascular signaling and may contribute to the response to hypoxia. However, the exact mechanisms controlling NOX4 levels under hypoxia are not resolved. We found that hypoxia rapidly enhanced NOX4 mRNA and protein levels in pulmonary artery smooth-muscle cells (PASMCs) as well as in pulmonary vessels from mice exposed to hypoxia. This response was dependent on the hypoxia-inducible transcription factor HIF-1alpha because overexpression of HIF-1alpha increased NOX4 expression, whereas HIF-1alpha depletion prevented this response. Mutation of a putative hypoxia-responsive element in the NOX4 promoter abolished hypoxic and HIF-1alpha-induced activation of the NOX4 promoter. Chromatin immunoprecipitation confirmed HIF-1alpha binding to the NOX4 gene. Induction of NOX4 by HIF-1alpha contributed to maintain ROS levels after hypoxia and hypoxia-induced proliferation of PASMCs. These findings show that NOX4 is a new target gene of HIF-1alpha involved in the response to hypoxia. Together with our previous findings that NOX4 mediates HIF-1alpha induction under normoxia, these data suggest an important role of the signaling axis between NOX4 and HIF-1alpha in various cardiovascular disorders under hypoxic and also nonhypoxic conditions.

Effect of Angiotensin II on Oxidative Stress in Female Borderline Hypertensive Rats

Oxidative stress is a contributing factor to cardiovascular tissue injury. Angiotensin II (Ang II) produces oxidative stress in male animals, but it's effect in females in unknown. The focus of this study was to determine the effect of chronic Ang II on oxidative stress levels in the kidneys of female Borderline Hypertensive Rats (BHR). Following two weeks of Ang II infusion (200ng/kg/min), BHR mean arterial pressure rose from 111±3 to 135±7 mmHg. Protein levels in the urine, which corresponds to kidney tissue damage, increased by 17±3 mg/day in Ang II‐treated BHRs. Whole body oxidative stress was assessed through observations of H2O2 levels in urine. Ang II treated animals showed a 72±29 mg/ml increase in H2O2 in urine as compared to control animals. Protein expression of NOX4, gp91, p22, p67, CuSOD, MnSOD, ECSOD and catalase was determined by western blot. Ang‐II treated BHRs renal tissue showed a decrease in MnSOD and catalase, and no significant differences in NOX4, gp91, p22, p67, CuSOD, and ECSOD protein levels. We found that chronic Ang II treatment resulted in hypertension, kidney tissue damage and elevation in whole body oxidative stress. Research was supported by funds from grant number 1 RO1 HL093271‐O1A1.

Role of caveolin and heat shock protein 70 interaction in the antioxidative effects of an angiotensin II type 1 receptor blocker in spontaneously hypertensive rats proximal tubules

Role of caveolin and heat shock protein 70 interaction in the antioxidative effects of an angiotensin II type 1 receptor blocker in spontaneously hypertensive rats proximal tubules