Role of endothelial Nox2 NADPH oxidase in angiotensin II-induced hypertension and vasomotor dysfunction

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are known to be involved in angiotensin II-induced hypertension and endothelial dysfunction. Several Nox isoforms are expressed in the vessel wall, among which Nox2 is especially abundant in the endothelium. Endothelial Nox2 levels rise during hypertension but little is known about the cell-specific role of endothelial Nox2 in vivo. To address this question, we generated transgenic mice with endothelial-specific overexpression of Nox2 (Tg) and studied the effects on endothelial function and blood pressure. Tg had an about twofold increase in endothelial Nox2 levels which was accompanied by an increase in p22phox levels but no change in levels of other Nox isoforms or endothelial nitric oxide synthase (eNOS). Basal NADPH oxidase activity, endothelial function and blood pressure were unaltered in Tg compared to wild-type littermates. Angiotensin II caused a greater increase in ROS production in Tg compared to wild-type aorta and attenuated acetylcholine-induced vasorelaxation. Both low and high dose chronic angiotensin II infusion increased telemetric ambulatory blood pressure more in Tg compared to wild-type, but with different patterns of BP change and aortic remodeling depending upon the dose of angiotensin II dose. These results indicate that an increase in endothelial Nox2 levels contributes to angiotensin II-induced endothelial dysfunction, vascular remodeling and hypertension. © 2011 The Author(s).

PPARα regulates endothelial progenitor cell maturation and myeloid lineage differentiation through a NADPH oxidase-dependent mechanism in mice

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Chronic intermittent hypoxia increases rat sternohyoid muscle NADPH oxidase expression with attendant modest oxidative stress

Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1α content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1α content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1α-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation-contraction coupling.

Cross-Talk Between Mitochondria and NADPH Oxidase: Effects of Mild Mitochondrial Dysfunction on Angiotensin II-Mediated Increase in Nox Isoform Expression and Activity in Vascular Smooth Muscle Cells

Mitochondria and NADPH oxidase activation are concomitantly involved in pathogenesis of many vascular diseases. However, possible cross-talk between those ROS-generating systems is unclear. We induced mild mitochondrial dysfunction due to mitochondrial DNA damage after 24 h incubation of rabbit aortic smooth muscle (VSMC) with 250 ng/mL ethidium bromide (EtBr). VSMC remained viable and had 29% less oxygen consumption, 16% greater baseline hydrogen peroxide, and unchanged glutathione levels. Serum-stimulated proliferation was unaltered at 24 h. Although PCR amplification of several mtDNA sequences was preserved, D-Loop mtDNA region showed distinct amplification of shorter products after EtBr. Such evidence for DNA damage was further enhanced after angiotensin-II (AngII) incubation. Remarkably, the normally observed increase in VSMC membrane fraction NADPH oxidase activity after AngII was completely abrogated after EtBr, together with failure to upregulate Nox1 mRNA expression. Conversely, basal Nox4 mRNA expression increased 1.6-fold, while being unresponsive to AngII. Similar loss in AngII redox response occurred after 24 h antimycin-A incubation. Enhanced Nox4 expression was unassociated with endoplasmic reticulum stress markers. Protein disulfide isomerase, an NADPH oxidase regulator, exhibited increased expression and inverted pattern of migration to membrane fraction after EtBr. These results unravel functionally relevant cross-talk between mitochondria and NADPH oxidase, which markedly affects redox responses to AngII. Antioxid Redox Signal 11, 1265-1278.

Tobacco Smoke Induces Ventricular Remodeling Associated with an Increase in NADPH Oxidase Activity

Background: Recent studies have assessed the direct effects of smoking on cardiac remodeling and function. However, the mechanisms of these alterations remain unknown. The aim of this study was to investigate de role of cardiac NADPH oxidase and antioxidant enzyme system on ventricular remodeling induced by tobacco smoke. Methods: Male Wistar rats that weighed 200-230 g were divided into a control group (C) and an experimental group that was exposed to tobacco smoke for a period of two months (ETS). After the two-month exposure period, morphological, biochemical and functional analyses were performed. Results: The myocyte cross-sectional area and left ventricle end-diastolic dimension was increased 16.2% and 33.7%, respectively, in the ETS group. The interstitial collagen volume fraction was also higher in ETS group compared to the controls. In addition to these morphological changes, the ejection fraction and fractional shortening were decreased in the ETS group. Importantly, these alterations were related to augmented heart oxidative stress, which was characterized by an increase in NADPH oxidase activity, increased levels of lipid hydroperoxide and depletion of antioxidant enzymes (e.g., catalase, superoxide dismutase and glutathione peroxidase). In addition, cardiac levels of IFN-gamma, TNF-alpha and IL-10 were not different between the groups. Conclusion: Cardiac alterations that are induced by smoking are associated with increased NADPH oxidase activity, suggesting that this pathway plays a role in the ventricular remodeling induced by exposure to tobacco smoke. Copyright (C) 2011 S. Karger AG, Basel

Protein disulfide isomerase overexpression in vascular smooth muscle cells induces spontaneous preemptive NADPH oxidase activation and Nox1 mRNA expression: Effects of nitrosothiol exposure

Mechanisms regulating NADPH oxidase remain open and include the redox chaperone protein disulfide isomerase (PDI). Here, we further investigated PDI effects on vascular NADPH oxidase. VSMC transfected with wild-type PDI (wt-PDI) OF PDI mutated in all four redox cysteines (mut-PDI) enhanced (2.5-fold) basal cellular ROS production and membrane NADPH oxidase activity, with 3-fold increase in Nox1, but not Nox4 mRNA. However, further ROS production, NADPH oxidase activity and Nox1 mRNA increase triggered by angiotensin-II (AngII) were totally lost with PDI overexpression, suggesting preemptive Nox1 activation in such cells. PDI overexpression increased Nox4 mRNA after AngII stimulus, although without parallel ROS increase. We also show that Nox inhibition by the nitric oxide donor GSNO is independent of PDI. PDI silencing decreased specifically Nox1 mRNA and protein, confirming that PDI may regulate Nox1 at transcriptional level in VSMC. Such data further strengthen the role of PDI as novel NADPH oxidase regulator. (C) 2009 Elsevier Inc. All rights reserved.

Desbalanço redox: NADPH oxidase como um alvo terapêutico no manejo cardiovascular

Vários estudos destacam as espécies reativas de oxigênio e nitrogênio (ERONs) como importantes contribuintes na patogênese de numerosas doenças cardiovasculares, incluindo hipertensão, aterosclerose e falência cardíaca. Tais espécies são moléculas altamente bioativas e com vida curta derivadas, principalmente, da redução do oxigênio molecular. O complexo enzimático da NADPH oxidase é a maior fonte dessas espécies reativas na vasculatura. Sob condições fisiológicas, a formação e eliminação destas substâncias aparecem balanceadas na parede vascular. Durante o desbalanço redox, entretanto, há um aumento na atividade da NADPH oxidase e predomínio de agentes pró-oxidantes, superando a capacidade de defesa orgânica antioxidante. Além disso, tal hiperatividade enzimática reduz a biodisponibilidade do óxido nítrico, crucial para a vasodilatação e a manutenção da função vascular normal. Apesar de a NADPH oxidase relacionar-se diretamente à disfunção endotelial, foi primeiramente descrita por sua expressão em fagócitos, onde sua atividade determina a eficácia dos mecanismos de defesa orgânica contra patógenos. As sutis diferenças existentes entre as unidades estruturais das NADPH oxidases, a depender do tipo celular que as expressa, podem ter implicações terapêuticas, permitindo a inibição seletiva do desequilíbrio redox induzido pela NADPH oxidase, sem comprometer, entretanto, sua participação nas vias fisiológicas de sinalização celular que garantem a proteção contra microorganismos.

Signal transduction and activation of the NADPH oxidase in eosinophils

Activation of the eosinophil NADPH oxidase and the subsequent release of toxic oxygen radicals has been implicated in the mechanism of parasite killing and inflammation. At present, little is known of the signal transduction pathway that govern agonist-induced activation of the respiratory burst and is the subject of this review. In particular, we focus on the ability of leukotrine B4 to activate the NADPH oxidase in guinea-pig peritoneal eosinophils which can be obtained in sufficient number and purity for detailed biochemical experiments to be performed.

Mechanisms for suppressing NADPH oxidase in the vascular wall

Oxidative stress underlies many forms of vascular disease as well as tissue injury following ischemia and reperfusion. The major source of oxidative stress in the artery wall is an NADPH oxidase. This enzyme complex as expressed in vascular cells differs from that in phagocytic leucocytes both in biochemical structure and functions. The crucial flavin-containing catalytic subunits, Nox1 and Nox4, are not found in leucocytes, but are highly expressed in vascular cells and upregulated with vascular remodeling, such as that found in hypertension and atherosclerosis. The difference in catalytic subunits offers the opportunity to develop "vascular specific" NADPH oxidase inhibitors that do not compromise the essential physiological signaling and phagocytic functions carried out by reactive oxygen and nitrogen species. Nitric oxide and targeted inhibitors of NADPH oxidase that block the source of oxidative stress in the vasculature are more likely to prevent the deterioration of vascular function that leads to stroke and heart attack, than are conventional antioxidants. The roles of Nox isoforms in other inflammatory conditions are yet to be explored.

Targeting vascular NADPH oxidase 1 blocks tumor angiogenesis through a PPAR 45; mediated mechanism.

Reactive oxygen species, ROS, are regulators of endothelial cell migration, proliferation and survival, events critically involved in angiogenesis. Different isoforms of ROS-generating NOX enzymes are expressed in the vasculature and provide distinct signaling cues through differential localization and activation. We show that mice deficient in NOX1, but not NOX2 or NOX4, have impaired angiogenesis. NOX1 expression and activity is increased in primary mouse and human endothelial cells upon angiogenic stimulation. NOX1 silencing decreases endothelial cell migration and tube-like structure formation, through the inhibition of PPAR 45;, a regulator of NF-_4;B. Administration of a novel NOX-specific inhibitor reduced angiogenesis and tumor growth in vivo in a PPAR 45; dependent manner. In conclusion, vascular NOX1 is a critical mediator of angiogenesis and an attractive target for anti-angiogenic therapies.

NADPH oxidase NOX4 mediates stellate cell activation and hepatocyte cell death during liver fibrosis development.

A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2−/−/p19ARF−/−, Stat3Δhc/Mdr2−/−) and a model of experimental induced fibrosis (CCl4) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes.

Restricted expression of NADPH oxidase/peroxidase gene (Duox) in zone VII of the ascidian endostyle

The ascidian Ciona intestinalis, a marine invertebrate chordate, is an emerging model system for developmental and evolutionary studies. The endostyle, one of the characteristic organs of ascidians, is a pharyngeal structure with iodine-concentrating and peroxidase activities and is therefore considered to be homologous to the follicular thyroid of higher vertebrates. We have previously reported that a limited part of the endostyle (zone VII) is marked by the expression of orthologs of the thyroid peroxidase (TPO) and thyroid transcription factor-2 (TTF-2/FoxE) genes. In this study, we have identified the Ciona homolog of NADPH oxidase/peroxidase (Duox), which provides hydrogen peroxide (H2O2) for iodine metabolism by TPO in the vertebrate thyroid. Expression patterns assessed by in situ hybridization have revealed that Ciona Duox (Ci-Duox) is predominantly expressed in the dorsal part of zone VII of the endostyle. Furthermore, two-color fluorescent in situ hybridization with Ci-Duox and Ciona TPO (CiTPO) has revealed that the ventral boundary of the Ci-Duox domain of expression is more dorsal than that of CiTPO. We have also characterized several genes, such as Ci-Fgf8/17/18, 5HT7, and Ci-NK4, which are predominantly expressed in the ventral part of zone VII, in a region complementary to the Ci-Duox expression domain. These observations suggest that, at the molecular level, zone VII has a complex organization that might have some impact on the specification of cell types and functions in this thyroid-equivalent element of the ascidian endostyle.

Molecular insights of p47phox phosphorylation dynamics in the regulation of NADPH oxidase activation and superoxide production

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox_/_ coronary microvascular cells. Compared with wild-type p47phoxcDNAtransfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2 . production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells.

Palmitate increases superoxide production through mitochondrial electron transport chain and NADPH oxidase activity in skeletal muscle cells

The effect of unbound palmitic acid (PA) at plasma physiological concentration range on reactive oxygen species (ROS) production by cultured rat skeletal muscle cells was investigated. The participation of the main sites of ROS production was also examined. Production of ROS was evaluated by cytochrome c reduction and dihydroethidium oxidation assays. PA increased ROS production after 1 h incubation. A xanthine oxidase inhibitor did not change PA-induced ROS production. However, the treatment with a mitochondrial uncoupler and mitochondrial complex III inhibitor decreased superoxide production induced by PA. The importance of mitochondria was also evaluated in 1 h incubated rat soleus and extensor digitorum longus (EDL) muscles. Soleus muscle, which has a greater number of mitochondria than EDL, showed a higher superoxide production induced by PA. These results indicate that mitochondrial electron transport chain is an important contributor for superoxide formation induced by PA in skeletal muscle. Results obtained with etomoxir and bromopalmitate treatment indicate that PA has to be oxidized to raise ROS production. A partial inhibition of superoxide formation induced by PA was observed by treatment with diphenylene iodonium, an inhibitor of NADPH oxidase. The participation of this enzyme complex was confirmed through an increase of p47(phox) phosphorylation after treatment with PA.