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H Oxidase (h + oxidase)
Selected AbstractsRegulation of angiotensin II-stimulated osteopontin expression in cardiac microvascular endothelial cells: Role of p42/44 mitogen-activated protein kinase and reactive oxygen species,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2001Zhonglin Xie Using spontaneously hypertensive and aortic banded rats, we have shown that expression of myocardial osteopontin, an extracellular matrix protein, coincides with the development of heart failure and is inhibited by captopril, suggesting a role for angiotensin II (ANG II). This study tested whether ANG II induces osteopontin expression in adult rat ventricular myocytes and cardiac microvascular endothelial cells (CMEC), and if so, whether induction is mediated via activation of mitogen-activated protein kinases (p42/44 MAPK) and involves reactive oxygen species (ROS). ANG II (1 ,M, 16 h) increased osteopontin expression (fold increase 3.3±0.34, n,=,12, P,<,0.01) in CMEC as measured by northern analysis, but not in ARVM. ANG II stimulated osteopontin expression in CMEC in a time- (within 4 h) and concentration-dependent manner, which was prevented by the AT1 receptor antagonist, losartan. ANG II elicited robust phosphorylation of p42/44 MAPK as measured using phospho-specific antibodies, and increased superoxide production as measured by cytochrome c reduction and lucigenin chemiluminescence assays. These effects were blocked by diphenylene iodonium (DPI), an inhibitor of the flavoprotein component of NAD(P)H oxidase. PD98059, an inhibitor of p42/44 MAPK pathway, and DPI each inhibited ANG II-stimulated osteopontin expression. Northern blot analysis showed basal expression of p22phox, a critical component of NADH/NADPH oxidase system, which was increased 40,60% by exposure to ANG II. These results suggest that p42/44 MAPK is a critical component of the ROS-sensitive signaling pathways activated by ANG II in CMEC and plays a key role in the regulation of osteopontin gene expression. Published 2001 Wiley-Liss, Inc. [source] Aging induces cardiac diastolic dysfunction, oxidative stress, accumulation of advanced glycation endproducts and protein modificationAGING CELL, Issue 2 2005Shi-Yan Li Summary Evidence suggests that aging, per se, is a major risk factor for cardiac dysfunction. Oxidative modification of cardiac proteins by non-enzymatic glycation, i.e. advanced glycation endproducts (AGEs), has been implicated as a causal factor in the aging process. This study was designed to examine the role of aging on cardiomyocyte contractile function, cardiac protein oxidation and oxidative modification. Mechanical properties were evaluated in ventricular myocytes from young (2-month) and aged (24,26-month) mice using a MyoCam® system. The mechanical indices evaluated were peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening/relengthening (± dL/dt). Oxidative stress and protein damage were evaluated by glutathione and glutathione disulfide (GSH/GSSG) ratio and protein carbonyl content, respectively. Activation of NAD(P)H oxidase was determined by immunoblotting. Aged myocytes displayed a larger cell cross-sectional area, prolonged TR90, and normal PS, ± dL/dt and TPS compared with young myocytes. Aged myocytes were less tolerant of high stimulus frequency (from 0.1 to 5 Hz) compared with young myocytes. Oxidative stress and protein oxidative damage were both elevated in the aging group associated with significantly enhanced p47phox but not gp91phox expression. In addition, level of cardiac AGEs was ,2.5-fold higher in aged hearts than young ones determined by AGEs-ELISA. A group of proteins with a molecular range between 50 and 75 kDa with pI of 4,7 was distinctively modified in aged heart using one- or two-dimension SDS gel electrophoresis analysis. These data demonstrate cardiac diastolic dysfunction and reduced stress tolerance in aged cardiac myocytes, which may be associated with enhanced cardiac oxidative damage, level of AGEs and protein modification by AGEs. [source] Inhibition of Nox-4 activity by plumbagin, a plant-derived bioactive naphthoquinoneJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 1 2005Yaxian Ding NAD(P)H oxidase contributes to the pathogenesis of cancer and cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy and heart failure. Plumbagin, a plant-derived naphthoquinone, has been shown to exert anticarcinogenic and anti-atherosclerosis effects in animals. However, the molecular mechanisms underlying these effects remain unknown. It is possible that the beneficial effect of plumbagin is due to the inhibition of NAD(P)H oxidase. Human embryonic kidney 293 (HEK293) and brain tumour LN229 cells express mainly Nox-4, a renal NAD(P)H oxidase. We have examined the effect of plumbagin on Nox-4 activity in HEK293 and LN229 cells using lucigenin-dependent chemiluminescence assay. Plumbagin inhibited the activity of Nox-4 in a time- and dose-dependent manner in HEK293 and LN229 cells. Production of superoxide in HEK293 cells was inhibited by diphenyleneiodonium (DPI), a NAD(P)H oxidase inhibitor. The superoxide production in HEK293 cells was NADPH- and NADH-dependent indicating that the superoxide was generated by a NAD(P)H oxidase in HEK293 cells, but not by the redox-cycling of lucigenin. Furthermore, plumbagin inhibited the superoxide production in Nox-4 transfected COS-7 cells. These results indicated that plumbagin directly interacted with Nox-4 and inhibited its activity. [source] Inhibition of NAD(P)H Oxidase Alleviates Impaired NOS-dependent Responses of Pial Arterioles in Type 1 Diabetes MellitusMICROCIRCULATION, Issue 7 2006WILLIAM G. MAYHAN ABSTRACT Objective: The goal was to identify the role of NAD(P)H oxidase in cerebrovascular dysfunction in type 1 diabetes mellitus (T1D). Methods: In a first series of studies, rats were assigned to nondiabetic, diabetic (streptozotocin; 50 mg/kg IP), nondiabetic-apocynin (40 mg/kg/day in drinking water)-treated and diabetic-apocynin-treated groups. Two to three months later, the authors examined in vivo responses of pial arterioles to nitric oxide synthase (NOS)-dependent (acetylcholine and adenosine diphosphate (ADP)) and -independent (nitroglycerin) agonists. Next, they used Western blot analysis to examine protein levels for subunits of NAD(P)H oxidase in cerebral microvessels and parietal cortex tissue of nondiabetic and diabetic rats. Finally, they measured superoxide production by parietal cortex tissue in nondiabetic and diabetic rats. Results: Acetylcholine- and ADP-induced dilatation of pial arterioles was impaired in diabetic compared to nondiabetic rats. In addition, while apocynin did not alter responses in nondiabetic rats, apocynin alleviated T1D-induced impairment of NOS-dependent vasodilatation. In addition, p47phox and gp91phox proteins were elevated in cerebral microvessels and parietal cortex tissue, respectively, of diabetic compared to nondiabetic rats. Further, basal production of superoxide was increased in diabetic compared to nondiabetic rats and apocynin decreased this basal production. Conclusions: The findings suggest that T1D impairs NOS-dependent reactivity of cerebral arterioles by a mechanism related to the formation of superoxide via activation of NAD(P)H oxidase. [source] Endothelial dysfunction in rat adjuvant-induced arthritis: Vascular superoxide production by NAD(P)H oxidase and uncoupled endothelial nitric oxide synthaseARTHRITIS & RHEUMATISM, Issue 6 2006Yoshisuke Haruna Objective To investigate endothelial function and levels of vascular oxidative stress in rat adjuvant-induced arthritis (AIA), in view of mounting evidence for an association between rheumatoid arthritis (RA) and accelerated vascular disease. Methods Thoracic aortic rings were prepared from AIA and control rats. After preconstriction by norepinephrine, the vasodilatory response to acetylcholine was determined. The amounts of 4-hydroxy-2-nonenal (HNE) and nitrotyrosine in AIA rat aortas were measured by Western blotting. Homogenates of the aortas were incubated with various substrates for superoxide-producing enzymes, and superoxide production was assessed by fluorogenic oxidation of dihydroethidium to ethidium. Expression of endothelial nitric oxide synthase (eNOS) in aortas was examined by real-time reverse transcriptase,polymerase chain reaction and Western blotting. Serum levels of tetrahydrobiopterin (BH4), a critical eNOS cofactor, were determined by high-performance liquid chromatography. Results Endothelium-dependent relaxation of the aortic ring was significantly depressed in AIA rats compared with control rats. The amounts of HNE and nitrotyrosine were increased in AIA rat aortas, indicating overproduction of reactive oxygen species. Incubation of AIA rat aorta homogenates with NADH or L -arginine, a substrate of eNOS, resulted in a significant increase in superoxide production. Endothelial NOS was highly expressed in AIA rat aortas. Serum levels of BH4 were significantly lower in AIA. Treatment of AIA with BH4 reversed the endothelial dysfunction, suggesting that its deficiency may contribute to the uncoupling of eNOS. Conclusion Vascular dysfunction in RA can be partially modeled in animals. NAD(P)H oxidase and uncoupled eNOS are responsible for the increase in vascular oxidative stress, which is likely to be involved in the endothelial dysfunction in AIA. [source] Mechanisms involved in the early increase of serotonin contraction evoked by endotoxin in rat middle cerebral arteriesBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2003Raquel Hernanz The present study investigated the mechanisms involved in the increased 5-hydroxytryptamine (5-HT) vasoconstriction observed in rat middle cerebral arteries exposed in vitro to lipopolysaccharide (LPS, 10 ,g ml,1) for 1,5 h. Functional, immunohistochemical and Western blot analysis and superoxide anion measurements by ethidium fluorescence were performed. LPS exposure increased 5-HT (10 ,M) vasoconstriction only during the first 4 h. In contrast to control tissue, indomethacin (10 ,M), the COX-2 inhibitor NS 398 (10 ,M), the TXA2/PGH2 receptor antagonist SQ 29,548 (1 ,M) and the TXA2 synthase inhibitor furegrelate (1 ,M) reduced 5-HT contraction of LPS-treated arteries from hour one. The iNOS inhibitor aminoguanidine (0.1 mM) increased 5-HT contraction from hour three of LPS incubation. The superoxide anion scavenger superoxide dismutase (SOD, 100 U ml,1) and the H2O2 scavenger catalase (1000 U ml,1), as well as the respective inhibitors of NAD(P)H oxidase and xanthine oxidase, apocynin (0.3 mM) and allopurinol (0.3 mM), reduced 5-HT contraction after LPS incubation. LPS induced an increase in superoxide anion levels that was abolished by PEG-SOD. Subthreshold concentrations of the TXA2 analogue U 46619, xanthine/xanthine oxidase and H2O2 potentiated, whereas those of sodium nitroprusside inhibited, the 5-HT contraction. COX-2 expression was increased at 1 and 5 h of LPS incubation, while that of iNOS, Cu/Zn-SOD and Mn-SOD was only increased after 5 h. All the three vascular layers expressed COX-2 and Cu/Zn-SOD. iNOS expression was detected in the endothelium and adventitia after LPS. In conclusion, increased production of TXA2 from COX-2, superoxide anion and H2O2 enhanced vasoconstriction to 5-HT during the first few hours of LPS exposure; iNOS and SOD expression counteracted that increase at 5 h. These changes can contribute to the disturbance of cerebral blood flow in endotoxic shock. British Journal of Pharmacology (2003) 140, 671,680. doi:10.1038/sj.bjp.0705501 [source] PIOGLITAZONE INHIBITS HOMOCYSTEINE-INDUCED MIGRATION OF VASCULAR SMOOTH MUSCLE CELLS THROUGH A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ,-INDEPENDENT MECHANISMCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 12 2008Li Li SUMMARY 1Peroxisome proliferator-activated receptor (PPAR)-, agonists have been demonstrated to exert protective effects against homocysteine (Hcy)-induced pathogenesis. However, the effects of PPAR-, agonists on Hcy-induced migration are unknown. In the present study, we examined the effect of pioglitazone on the migration of vascular smooth muscle cells (VSMC) induced by Hcy and the possible mechanism involved. 2Vascular smooth muscle cells were isolated from the thoracic aortas of male Sprague-Dawley rats. The migration of VSMC was examined using a transwell technique. The generation of intracellular reactive oxygen species (ROS) was measured using the ROS-sensitive fluoroprobe 2,,7,-dichlorodihydrofluorescein diacetate. The activity of NAD(P)H oxidase was assessed by lucigenin enhanced chemiluminescence. Activation of p38 mitogen-activated protein kinase (MAPK) was determined by western blotting. 3The results showed that pioglitazone dose-dependently inhibited the migration of VSMC induced by Hcy. This was not reversed by the PPAR-, antagonist GW9662. In addition, pretreatment with the NAD(P)H oxidase inhibitor diphenylene iodonium (DPI), the free radical scavenger N -acetylcysteine and the p38 MAPK inhibitor SB202190 blocked Hcy-induced VSMC migration. Furthermore, we observed that pioglitazone suppressed Hcy-induced intracellular ROS production; similar effects were observed with DPI and NAC. Pioglitazone attenuated Hcy-induced activation of NAD(P)H oxidase. Moreover, pioglitazone blocked Hcy-induced p38 MAPK phosphorylation; similar effects were observed for DPI, NAC and SB202190. 4The data demonstrate that pioglitazone inhibits Hcy-induced VSMC migration that is independent of PPAR-,. Furthermore, part of the biological effect of pioglitazone involves a decrease in the levels of NAD(P)H oxidase derived-ROS and p38 MAPK activation. [source] Hepatocyte NAD(P)H oxidases as an endogenous source of reactive oxygen species during hepatitis C virus infection,HEPATOLOGY, Issue 1 2010Nabora Soledad Reyes de Mochel Oxidative stress has been identified as a key mechanism of hepatitis C virus (HCV),induced pathogenesis. Studies have suggested that HCV increases the generation of hydroxyl radical and peroxynitrite close to the cell nucleus, inflicting DNA damage, but the source of reactive oxygen species (ROS) remains incompletely characterized. We hypothesized that HCV increases the generation of superoxide and hydrogen peroxide close to the hepatocyte nucleus and that this source of ROS is reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase 4 (Nox4). Huh7 human hepatoma cells and telomerase-reconstituted primary human hepatocytes, transfected or infected with virus-producing HCV strains of genotypes 2a and 1b, were examined for messenger RNA (mRNA), protein, and subcellular localization of Nox proteins along with the human liver. We found that genotype 2a HCV induced persistent elevations of Nox1 and Nox4 mRNA and proteins in Huh7 cells. HCV genotype 1b likewise elevated the levels of Nox1 and Nox4 in telomerase-reconstituted primary human hepatocytes. Furthermore, Nox1 and Nox4 proteins were increased in HCV-infected human liver versus uninfected liver samples. Unlike Nox1, Nox4 was prominent in the nuclear compartment of these cells as well as the human liver, particularly in the presence of HCV. HCV-induced ROS and nuclear nitrotyrosine could be decreased with small interfering RNAs to Nox1 and Nox4. Finally, HCV increased the level of transforming growth factor beta 1 (TGF,1). TGF,1 could elevate Nox4 expression in the presence of infectious HCV, and HCV increased Nox4 at least in part through TGF,1. Conclusion: HCV induced a persistent elevation of Nox1 and Nox4 and increased nuclear localization of Nox4 in hepatocytes in vitro and in the human liver. Hepatocyte Nox proteins are likely to act as a persistent, endogenous source of ROS during HCV-induced pathogenesis. Hepatology 2010 [source] Involvement of the renin,angiotensin system in the development of vascular damage in a rat model of arthritis: Effect of angiotensin receptor blockersARTHRITIS & RHEUMATISM, Issue 5 2010Takeo Sakuta Objective To explore the involvement of the renin,angiotensin system (RAS) in the development of vascular damage in adjuvant-induced arthritis (AIA) in rats. Methods Angiotensin II (Ang II; 0.25 or 1.0 mg/kg/day) was infused in control rats and rats with AIA for 21 days, and the impact of systemic inflammation on Ang II,induced hypertension, endothelial dysfunction, and vascular hypertrophy was evaluated. Expression of angiotensin II type 1 receptor (AT1R) and angiotensin-converting enzyme (ACE) in the aortas of rats with AIA were examined by real-time polymerase chain reaction (PCR) and Western blot analyses. Losartan (3 mg/kg/day) or irbesartan (5 mg/kg/day), both of which are AT1R blockers, was administered orally to rats with AIA for 21 days. In situ superoxide production in aortas was assessed according to the fluorogenic oxidation of dihydroethidium to ethidium. The expression and activity of NAD(P)H oxidases in aortas were examined by real-time PCR analysis and lucigenin chemiluminescence assay. Endothelial function in rats with AIA treated in vivo or ex vivo with AT1R blockers was also determined. Results The Ang II,induced hypertensive response, endothelial dysfunction, and vascular hypertrophy were exacerbated in rats with AIA. Expression of AT1R and ACE was increased in the aortas of rats with AIA. Both losartan and irbesartan decreased the levels of superoxide and the expression and activity NAD(P)H oxidases in the aortas of rats with AIA. The endothelial dysfunction in AIA was improved by the in vivo or ex vivo treatment with AT1R blockers. Conclusion The locally activated RAS is involved in the increased vascular oxidative stress and endothelial dysfunction in AIA. Our findings have important implications for clinical approaches to the reduction of cardiovascular risk in patients with rheumatoid arthritis. [source] |