Fibrotic response induced by angiotensin-II requires NAD(P)H oxidase-induced reactive oxygen species (ROS) in skeletal muscle cells

Fibrotic disorders are typified by excessive connective tissue and extracellular matrix (ECM) deposition that precludes normal healing processes in different tissues. Angiotensin-II (Ang-II) is involved in the fibrotic response. Several muscular dystrophies are characterized by extensive fibrosis. However, the exact role of Ang-II in skeletal muscle fibrosis is unknown. Here we show that myoblasts responded to Ang-II by increasing protein levels of connective tissue growth factor (CTGF/CCN2), collagen-III and fibronectin. These Ang-II-induced pro-fibrotic effects were mediated by AT-1 receptors. Remarkably, Ang-II induced reactive oxygen species (ROS) via a NAD(P)H oxidase-dependent mechanism, as shown by inhibition of ROS production via the NAD(P)H oxidase inhibitors diphenylene iodonium (DPI) and apocynin. This increase in ROS is critical for Ang-II-induced fibrotic effects, as indicated by the decrease in Ang-II-induced CTGF and fibronectin levels by DPI and apocynin. We also show that Ang-II-induced ROS production and fibrosis require PKC activity as indicated by the generic PKC inhibitor chelerythrine.These results strongly suggest that the fibrotic response induced by Ang-II is mediated by AT-1 receptor and requires NAD(P)H-induced ROS in skeletal muscle cells.     

Tumor-induced endothelial cell apoptosis: roles of NAD(P)H oxidase-derived reactive oxygen species

Many tumor cells are capable of migrating through endothelial cell (EC) junctions and disintegrating sub-endothelial extracellular matrix to achieve extravasation. We demonstrate in this study that certain solid tumor cells can induce EC apoptosis to facilitate their escape from the circulation. The EC apoptosis is triggered by elevated intracellular reactive oxygen species (ROS) levels and direct contacts with tumor cells are required. Treating ECs with antioxidants, such as ascorbate and N-acetyl-L-cysteine (NAC), and a glutathione precursor can rescue the ECs from tumor-induced apoptosis and reduce the number of tumor cells migrating across endothelial barriers. NAD(P)H oxidase was identified as the major ROS producer in the event since inhibitors and small interference RNA specific to the enzyme could abrogate the tumor-induced ROS production and hence EC death. This study also provides evidence showing that the interaction between tumor and EC increases intracellular Ca(2+) concentration and activates protein kinase C (PKC) activity, which leads to NAD(P)H oxidase activation through the serine-phosphorylation of p47(phox) subunit. These findings suggest that blocking the tumor-induced EC apoptosis is a potential way to prevent tumor metastasis.     

Caveolin-1 is a negative regulator of NADPH oxidase-derived reactive oxygen species

Changes in the expression and function of caveolin-1 (Cav-1) have been proposed as a pathogenic mechanism underlying many cardiovascular diseases. Cav-1 binds to and regulates the activity of numerous signaling proteins via interactions with its scaffolding domain. In endothelial cells, Cav-1 has been shown to reduce reactive oxygen species (ROS) production, but whether Cav-1 regulates the activity of NADPH oxidases (Noxes), a major source of cellular ROS, has not yet been shown. Herein, we show that Cav-1 is primarily expressed in the endothelium and adventitia of pulmonary arteries (PAs) and that Cav-1 expression is reduced in isolated PAs from multiple models of pulmonary artery hypertension (PH). Reduced Cav-1 expression correlates with increased ROS production in the adventitia of hypertensive PA. In vitro experiments revealed a significant ability of Cav-1 and its scaffolding domain to inhibit Nox1–5 activity and it was also found that Cav-1 binds to Nox5 and Nox2 but not Nox4. In addition to posttranslational actions, in primary cells, Cav-1 represses the mRNA and protein expression of Nox2 and Nox4 through inhibition of the NF-κB pathway. Last, in a mouse hypoxia model, the genetic ablation of Cav-1 increased the expression of Nox2 and Nox4 and exacerbated PH. Together, these results suggest that Cav-1 is a negative regulator of Nox function via two distinct mechanisms, acutely through direct binding and chronically through alteration of expression levels. Accordingly, the loss of Cav-1 expression in cardiovascular diseases such as PH may account for the increased Nox activity and greater production of ROS.     

Fibroblast-to-myofibroblast switch is mediated by NAD(P)H oxidase generated reactive oxygen species

Tumour–stroma interaction is a prerequisite for tumour progression in skin cancer. Hereby, a critical step in stromal function is the transition of tumour-associated fibroblasts to MFs (myofibroblasts) by growth factors, for example TGFβ (transforming growth factor beta(). In this study, the question was addressed of whether fibroblast-associated NAD(P)H oxidase (NADH/NADPH oxidase), known to be activated by TGFβ1, is involved in the fibroblast-to-MF switch. The up-regulation of αSMA (alpha smooth muscle actin), a biomarker for MFs, is mediated by a TGFβ1-dependent increase in the intracellular level of ROS (reactive oxygen species). This report demonstrates two novel aspects of the TGFβ1 signalling cascade, namely the generation of ROS due to a biphasic NAD(P)H oxidase activity and a ROS-dependent downstream activation of p38 leading to a transition of dermal fibroblasts to MFs that can be inhibited by the selective NAD(P)H oxidase inhibitor apocynin. These data suggest that inhibition of NAD(P)H oxidase activity prevents the fibroblast-to-MF switch and may be important for chemoprevention in context of a ‘stromal therapy’ which was described earlier.     

Interaction of myeloperoxidase with vascular NAD(P)H oxidase-derived reactive oxygen species in vasculature: implications for vascular diseases

Vascular NAD(P)H oxidase-derived reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) have emerged as important molecules in the pathogenesis of atherosclerosis, hypertension, and diabetic vascular complications. Additionally, myeloperoxidase (MPO), a transcytosable heme protein that is derived from leukocytes, is also believed to play important roles in the above-mentioned inflammatory vascular diseases. Previous studies have shown that MPO-induced vascular injury responses are H2O2 dependent. It is well known that MPO can use leukocyte-derived H2O2; however, it is unknown whether the vascular-bound MPO can use vascular nonleukocyte oxidase-derived H2O2 to induce vascular injury. In the present study, ANG II was used to stimulate vascular NAD(P)H oxidases and increase their H2O2 production in the vascular wall, and vascular dysfunction was used as the vascular injury parameter. We demonstrated that vascular-bound MPO has sustained activity in the vasculature. MPO could use the vascular NAD(P)H oxidase-derived H2O2 to produce hypochlorus acid (HOCl) and its chlorinating species. More importantly, MPO derived HOCl and chlorinating species amplified the H2O2-induced vascular injury by additional impairment of endothelium-dependent relaxation. HOCl-modified low-density lipoprotein protein (LDL), a specific biomarker for the MPO-HOCl-chlorinating species pathway, was expressed in LDL and MPO-bound vessels with vascular NAD(P)H oxidase-derived H2O2. MPO-vascular NAD(P)H oxidase-HOCl-chlorinating species may represent a common pathogenic pathway in vascular diseases and a new mechanism involved in exacerbation of vascular diseases under inflammatory conditions.     

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.     

Ceramide-induced preconditioning involves reactive oxygen species

INTRODUCTION: Ceramide induces programmed cell death and it is thought to contribute to cardiac ischemia/reperfusion (I/R) injury. In contrast, we have demonstrated that administration of low doses of ceramide engenders cardiac preconditioning (PC). Ceramide is known to generate reactive oxygen species (ROS) in cells. Since mechanisms triggering the ceramide-induced cardioprotection remain unknown, we investigated the role of ROS in the genesis of this protective mechanism. METHODS: Using an isolated Langendorff-perfused rat heart model, four groups (n > or = 6 in each group) were considered: Control hearts underwent 30 min index regional ischemia and 120 min of reperfusion. In the ceramide group, hearts were preconditioned with c2-ceramide 1 microM for 7 min followed by 10 min washout prior to the I/R insult. In additional groups, MPG (1 mM), a synthetic antioxidant was given for 15 min alone or bracketing the ceramide perfusion. In each group, infarct size was determined at the end of the reperfusion period and superoxide dismutases (CuZnSOD and MnSOD) and catalase activities were evaluated. RESULTS: Ceramide preconditioning reduced the infarct/area at risk (I/AAR) ratio (8.3 +/- 1.1% for ceramide vs. 36.4 +/- 1.2% for control, p < 0.001). Perfusion with MPG abolished the preconditioning effect of ceramide (I/AAR ratio = 36.7 +/- 4.9%). Ceramide was also associated with a 29% and 38% increase in catalase and CuZnSOD activities, respectively, compared with control group. CONCLUSION: Production of reactive oxygen species following ceramide preconditioning of the ischemic-reperfused heart appears to play a role in the cardioprotective effect of ceramide.     

Epidermal growth factor rescues trophoblast apoptosis induced by reactive oxygen species

Pre-eclampsia and intrauterine growth restriction are associated with increased apoptosis of placental villous trophoblast. This may result from placental hypoperfusion, leading to the generation of reactive oxygen species (ROS). Apoptosis can be induced in villous trophoblast following exposure to oxidative stress. Epidermal growth factor (EGF) reduces trophoblast apoptosis resulting from exposure to hypoxia. We hypothesised that exposure to hydrogen peroxide, a potent generator of ROS, would induce apoptosis in term placental villous explants and that this could be reduced by treatment with EGF. Placental explants were taken from normal term pregnancies and exposed to increasing doses of hydrogen peroxide (0–1,000 μM) or to a combination of increasing doses of hydrogen peroxide and EGF (0–100 ng/ml) for either 6 or 48 h. Apoptosis was assessed by TUNEL, proliferation by Ki-67 immunostaining, necrosis by lactate dehydrogenase activity and trophoblast differentiation by human chorionic gonadotrophin (hCG) secretion in conditioned culture media. Immunoperoxidase staining was performed to identify phosphorylated-AKT (p-AKT) and phosphorylated-PI3 kinase (p-PI3k). Exposure to 1,000 μM hydrogen peroxide for 48 h induced apoptosis in placental explants. The increase in TUNEL positive nuclei predominantly localised to syncytiotrophoblast. The amount of apoptosis was reduced to control levels by treatment with 10 and 100 ng/ml EGF. Proliferation of cytotrophoblasts within villous explants was significantly reduced following exposure to 1,000 μM hydrogen peroxide, this was restored to control levels by simultaneous treatment with 10 or 100 ng/ml EGF. Neither exposure to hydrogen peroxide or EGF altered the amount of necrosis. There was increased immunostaining for pPI3K following treatment with EGF. This study shows that apoptosis may be induced in villous trophoblast following exposure to ROS, and demonstrates the anti-apoptotic effect of EGF in trophoblast, the maintenance of which is essential for normal pregnancy.     

Glucose down-regulation of cGMP-dependent protein kinase I expression in vascular smooth muscle cells involves NAD(P)H oxidase-derived reactive oxygen species

Reduced levels of cGMP-dependent protein kinase I (PKG-I) in vasculature have been shown to contribute to diabetic vascular dysfunctions. However, the underlying mechanisms remain unknown. In this report, using primary rat aortic smooth muscle cells (VSMC), we investigated the mechanisms of glucose-mediated regulation of PKG-I expression. Our data showed that high glucose (30 mM glucose) exposure significantly reduced PKG-I production (protein and mRNA levels) as well as PKG-I activity in cultured VSMC. Glucose-mediated decreases in PKG-I levels were inhibited by a superoxide scavenger (tempol) or NAD(P)H oxidase inhibitors (diphenylene iodonium or apocynin). High glucose exposure time-dependently increased superoxide production in VSMC, which was abolished by tempol or apocynin treatment, but not by other inhibitors of superoxide-producing enzymes (L-NAME, rotenone, or oxypurinol). Total protein levels and phosphorylated levels of p47phox (an NADPH oxidase subunit) were increased in VSMC after high glucose exposure. Transfection of cells with siRNA-p47phox abolished glucose-induced superoxide production and restored PKG-I protein levels in VSMC. Treatment of cells with PKC inhibitor prevented glucose-induced p47phox expression/phosphorylation and superoxide production and restored the PKG-I levels. Decreased PKG-I protein levels were also found in femoral arteries from diabetic mice, which were associated with the decreased DEA-NONOate-induced vasorelaxation. Taken together, the present results suggest that glucose-mediated down-regulation of PKG-I expression in VSMC occurs through PKC-dependent activation of NAD(P)H oxidase-derived superoxide production, contributing to diabetes-associated vessel dysfunctions.     

The effect of reactive oxygen species on cardiomyocyte differentiation of pluripotent stem cells

The coordination of metabolic shift with genetic circuits is critical to cell specification, but the metabolic mechanisms that drive cardiac development are largely unknown. Reactive oxygen species (ROS) are not only the by-product of mitochondrial metabolism, but play a critical role in signalling cascade of cardiac development as a second messenger. Various levels of ROS appear differential and even oppose effect on selfrenewal and cardiac differentiation of pluripotent stem cells (PSCs) at each stage of differentiation. The intracellular ROS and redox balance are meticulous regulated by several systems of ROS generation and scavenging, among which mitochondria and the NADPH oxidase (NOX) are major sources of intracellular ROS involved in cardiomyocyte differentiation. Some critical signalling modulators are activated or inactivated by oxidation, suggesting ROS can be involved in regulation of cell fate through these downstream targets. In this review, the literatures about major sources of ROS, the effect of ROS level on cardiac differentiation of PSCs, as well as the underlying mechanism of ROS in the control of cardiac fate of PSC are summarised and discussed.     

Peroxisomes and reactive oxygen species,a lasting challenge

Oxidases generating and enzymes scavenging H₂O₂ predestine peroxisomes (PO) to a pivotal organelle in oxygen metabolism. Catalase, the classical marker enzyme of PO, exhibits both catalatic and peroxidatic activity. The latter is responsible for the staining with 3,3′-diamino-benzidine, which greatly facilitated the visualization of the organelle and promoted further studies on PO. d-Amino acid oxidase catalyzes with strict stereospecificity the oxidative deamination of d-amino acids. The oxidase is significantly more active in the kidney than in liver and more in periportal than pericentral rat hepatocytes. Peroxisomes in these tissues differ in their enzyme activity and protein concentration not only in adjacent cells but even within the same one. Moreover, the enzyme appears preferentially concentrated in the central region of the peroxisomal matrix compartment. Urate oxidase, a cuproprotein catalyzing the oxidation of urate to allantoin, is confined to the peroxisomal core, yet is lacking in human PO. Recent experiments revealed that cores in rat hepatocytes appear in close association with the peroxisomal membrane releasing H₂O₂ generated by urate oxidase to the surrounding cytoplasma. Xanthine oxidase is exclusively located to cores, oxidizes xanthine thereby generating H₂O₂ and O₂ ^– radicals. The latter are converted to O₂ and H₂O₂ by CuZn superoxide dismutase, which has been shown recently to be a bona fide peroxisomal protein. Presented at the 50th Anniversary Symposium of the Society for Histochemistry, Interlaken, Switzerland, October 1-4, 2008.     

Suppression of Mycobacterium tuberculosis induced reactive oxygen species (ROS) and TNF-alpha mRNA expression in human monocytes by allicin

Chronic inflammation associated with tumor necrosis factor (TNF)-alpha and reactive oxygen species (ROS) is the hallmark of tuberculosis. Mycobacterium tuberculosis (MTB) directly stimulates human monocytes to secrete TNF-alpha. We show the augmented expression of TNF-alpha mRNA in MTB-infected monocytes by cellular activation and ROS was suppressed by allicin in a dose-dependent manner. Also, allicin enhanced the glutathione peroxidase activity, which correlated inversely with the downregulation of ROS and TNF-alpha in MTB-infected monocytes. Hence, allicin may prove to be a valuable natural antioxidant in combating tuberculosis.     

Decomposition of reactive oxygen species by copper(II) bis(1-pyrazolyl)methane complexes

Two bis(1-pyrazolyl)alkane ligands, bis(3,5-dimethyl-1-pyrazolyl)methane and bis(4-iodo-3,5-dimethyl-1-pyrazolyl)methane, and their copper(II) complexes, bis(3,5-dimethyl-1-pyrazolyl)methanedinitratocopper(II) [CuL₁(NO₃)₂] and bis(4-iodo-3,5-dimethyl-1-pyrazolyl)methanedinitratocopper(II) [CuL₂(NO₃)₂]·2H₂O, were prepared. Physiochemical properties of the copper(II) complexes were studied by spectroscopic (UV–vis, IR, EPR) techniques and cyclic voltammetry. Spectroscopic analysis revealed a 1:1 stoichiometry of ligand:copper(II) ion and a bindentate coordination mode for the nitrate ions in both of the complexes. According to experimental and theoretical ab initio data, the copper(II) ion is located in an octahedral hexacoordinated environment. Both complexes were able to catalyze the dismutation of superoxide anion () (pH 7.5) and decomposition of H₂O₂ (pH 7.5) and peroxynitrite (pH 10.9). In addition, both complexes exhibited superoxide dismutase (SOD) like activity toward extracellular and intracellular reactive oxygen species produced by activated human neutrophils in whole blood. Thus, these complexes represent useful SOD mimetics with a broad range of antioxidant activity toward a variety of reactive oxidants.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-l-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the OH generation. UV and O₂ consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H₂O₂ did not generate any significant amount of OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H₂O₂ → Co(II) + OH + OH⁻] seems responsible for OH generation. H₂O₂ is produced from O₂⁻ via dismutation. O₂⁻ is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or β-ananyl-3-methyl- -histidine alters, its oxidation–reduction potential and makes Co(II) capable of generating OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H₂O₂ → Co(III) + OH + OH⁻]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and β-ananyl-3-methyl- -histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.     

The effect of reactive oxygen species on ethylene production induced by osmotic stress in etiolated mungbean seedling

After 10 h osmotic stress in 25% polyethylene glycol (PEG6000) solution (–1.8 MPa) at 25 °C in darkness, the etiolated mungbean seedlings were transferred to pure water for recovery. The ethylene release rate and the level of reactive oxygen species (ROS), including superoxide radical (O₂^–) and hydrogen peroxide (H₂O₂), were investigated during the recovery process. The results showed that ethylene production rate and amount of ROS increased dramatically after osmotic stress, and a close correlation was observed between ethylene release rate and concentrations of ROS. Inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) or aminooxyacetic acid (AOA), could reduce the ethylene release rate, but had no significant influence to the content of O₂^– and H₂O₂. As well as, silver thiosulfate (STS), an inhibitor of ethylene action, exhibited no obvious effect to the concentration of ROS, showing stress-inducible ethylene was not the cause for the increase of stress-inducible ROS. On the other hand, exogenous generator of superoxide radical (methylviologen, MV, or sodium dithionite, Na₂S₂O₄) could enhance the ethylene production evidently, which could be inhibited by exogenous scavenger of superoxide radical (superoxide dismutase, SOD, or 1, 4-diazabicyclo (2,2,2) octane, DABCO). However, either exogenous H₂O₂ or catalase (CAT) had no significant influence on ethylene production. The results suggested that it was superoxide radical but not H₂O₂which was involved directly in osmotic stress-inducible ethylene biosynthesis. The dual-role of superoxide radical on stress ethylene biosynthesis was also discussed.