Nox4 Protein Levels Research Articles

Amyloid β‐induced astrogliosis is mediated by β1‐integrin via NADPH oxidase 2 in Alzheimer's disease

SummaryAstrogliosis is a hallmark of Alzheimer′s disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid‐β modulates β1‐integrin activity and triggers NADPH oxidase (NOX)‐dependent astrogliosis in vitro and in vivo. Amyloid‐β oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by β1‐integrin in cultured astrocytes. This mechanism promotes β1‐integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple‐transgenic AD mouse model shows increased levels of GFAP, NOX2, and β1‐integrin in reactive astrocytes which correlates with the amyloid β‐oligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and β1‐integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and β1‐integrin were significantly associated with increased amyloid‐β load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid β oligomers with β1‐integrin which in turn leads to enhancing β1‐integrin and NOX2 activity via NOX‐dependent mechanisms. These observations may be relevant to AD pathophysiology.

Intermedin1-53 Attenuates Abdominal Aortic Aneurysm by Inhibiting Oxidative Stress.

Oxidative stress plays a critical role in the development of abdominal aortic aneurysm (AAA). Intermedin (IMD) is a regulator of oxidative stress. Here, we investigated whether IMD reduces AAA by inhibiting oxidative stress. In angiotensin II-induced ApoE-/- mouse and CaCl2-induced C57BL/6J mouse model of AAA, IMD1-53 significantly reduced the incidence of AAA and maximal aortic diameter. Ultrasonography, hematoxylin, and eosin staining and Verhoeff-van Gieson staining showed that IMD1-53 significantly decreased the enlarged aortas and elastic lamina degradation induced by angiotensin II or CaCl2. Mechanistically, IMD1-53 attenuated oxidative stress, inflammation, vascular smooth muscle cell apoptosis, and matrix metalloproteinase activation. IMD1-53 inhibited the activation of redox-sensitive signaling pathways, decreased the mRNA and protein expression of nicotinamide adenine dinucleotide phosphate oxidase subunits, and reduced the activity of nicotinamide adenine dinucleotide phosphate oxidase in AAA mice. Expression of Nox4 was upregulated in human AAA segments and in angiotensin II-treated mouse aortas and was markedly decreased by IMD1-53. In vitro, vascular smooth muscle cells with small-interfering RNA knockdown of IMD showed significantly increased angiotensin II-induced reactive oxygen species, and small-interfering RNA knockdown of Nox4 markedly inhibited the reactive oxygen species. IMD knockdown further increased the apoptosis of vascular smooth muscle cells and inflammation, which was reversed by Nox4 knockdown. Preincubation with IMD17-47 and protein kinase A inhibitor H89 inhibited the effect of IMD1-53, reducing Nox4 protein levels. IMD1-53 could have a protective effect on AAA by inhibiting oxidative stress.

Salvianolic acid B improves vascular endothelial function in diabetic rats with blood glucose fluctuations via suppression of endothelial cell apoptosis

Vascular endothelial cell injury is an initial event in atherosclerosis. Salvianolic acid B (Sal B), a main bioactive component in the root of Salvia miltiorrhiza, has vascular protective effect in diabetes, but the underlying mechanisms remain unclear. The present study investigated the effect of Sal B on vascular endothelial function in diabetic rats with blood glucose fluctuations and the possible mechanisms implicated. The results showed that diabetic rats developed marked endothelial dysfunction as exhibited by impaired acetylcholine induced vasodilation. Supplementation with Sal B resulted in an evident improvement of endothelial function. Phosphorylation (Ser 1177) of endothelial nitric oxide synthase (eNOS) was significantly restored in Sal B treated diabetic rats, accompanied by an evident recovery of NO metabolites. Sal B effectively reduced vascular endothelial cell apoptosis, with Bcl-2 protein up-regulated and Bax protein down-regulated markedly. Treatment with Sal B led to an evident amelioration of oxidative stress in diabetic rats as manifested by enhanced antioxidant capacity and decreased contents of malondialdehyde in aortas. Protein levels of NOX2 and NOX4, two main isoforms of NADPH oxidase known as the major source of reactive oxygen species in the vasculature, were markedly decreased in Sal B treated groups. In addition, treatment with Sal B led to an evident decrease of serum lipids. Taken together, this study indicates that Sal B is capable of improving endothelial function in diabetic rats with blood glucose fluctuations, of which the underlying mechanisms might be related to suppression of endothelial cell apoptosis and stimulation of eNOS phosphorylation (Ser 1177).

NADPH Oxidase Accounts for Changes in Cerebrovascular Redox Status in Hindlimb Unweighting Rats.

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 microgravit.

Angiotensin-(1-7) Improves Liver Fibrosis by Regulating the NLRP3 Inflammasome via Redox Balance Modulation.

Angiotensin II (Ang II) aggravates hepatic fibrosis by inducing NADPH oxidase (NOX)-dependent oxidative stress. Angiotensin-(1-7) [Ang-(1-7)], which counter-regulates Ang II, has been evidenced to protect against hepatic fibrosis. The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, being activated by reactive oxygen species (ROS), is identified as a novel mechanism of liver fibrosis. However, whether the NLRP3 inflammasome involves in regulation of Ang II-induced hepatic fibrosis remains unclear. This study investigates the different effects of the Ang II and Ang-(1-7) on collagen synthesis by regulating the NLRP3 inflammasome/Smad pathway via redox balance modulation. In vivo, Ang-(1-7) improved bile duct ligation-induced hepatic fibrosis, reduced H2O2 content, protein levels of NOX4, and the NLRP3 inflammasome, whereas it increased glutathione (GSH) and nuclear erythroid 2-related factor 2 (Nrf2) antioxidant response element (ARE). In vitro, Ang II treatment elevated NOX4 protein expression and ROS production in hepatic stellate cells (HSCs), whereas it inhibited GSH and Nrf2-ARE, resulting in the activation of the NLRP3 inflammasome in the mitochondria of HSCs. NLRP3 depletion inhibited Ang II-induced collagen synthesis. Furthermore, Ang II increased NLRP3 and pro-IL-1β levels by activating the Toll-like receptor 4 (TLR4)/MyD88/NF-κB pathway. Treatment with antioxidants, NOX4 small interference RNA (siRNA), or Nrf2 activator inhibited Ang II-induced NLRP3 inflammasome activation and collagen synthesis. In contrast, the action of Ang-(1-7) opposed the effects of Ang II. Ang-(1-7) improved liver fibrosis by regulating NLRP3 inflammasome activation induced by Ang II-mediated ROS via redox balance modulation. Antioxid. Redox Signal. 24, 795-812.

Hu antigen R is required for NOX-1 but not NOX-4 regulation by inflammatory stimuli in vascular smooth muscle cells.

NOX-1 and NOX-4 are key enzymes responsible for reactive oxygen species (ROS) generation in vascular smooth muscle cells (VSMC). The RNA-binding protein Hu antigen R (HuR) is implicated in posttranscriptional regulation of gene expression; however, its role regulating NOX is unknown. We investigated transcriptional and posttranscriptional mechanisms underlying angiotensin II (AngII) and IL-1β regulation of NOX-1 and NOX-4 in VSMC and their implications in cell migration. Rat and human VSMC were stimulated with AngII (0.1 μmol/l) and/or IL-1β (10 ng/ml). NOX-1 and NOX-4 mRNA and protein levels, NOX-1 and NOX-4 promoter and 3'UTR activities, NADPH oxidase activity, ROS production, and cell migration were studied. IL-1β increased NOX-1 expression, NADPH oxidase activity and ROS production, and decreased NOX-4 expression and H2O2 production in VSMC. AngII potentiated the IL-1β-mediated induction of NOX-1 expression, NADPH oxidase activity, ROS production, and cell migration. However, AngII did not influence IL-1β-induced NOX-4 downregulation. AngII + IL-1β interfered with the decay of NOX-1 mRNA and promoted HuR binding to NOX-1 mRNA. Moreover, HuR blockade reduced NOX-1 mRNA stability and AngII + IL-1β-induced NOX-1 mRNA levels. IL-1β decreased NOX-4 expression through a transcriptional mechanism that involved response elements situated in the proximal promoter. AngII and/or IL-1β-induced cell migration were prevented by NOX-1 and HuR blockade and were augmented by NOX-4 overexpression. In VSMC HuR-mediated mRNA stabilization is partially responsible for AngII + IL-1β-dependent NOX-1 expression, whereas transcriptional mechanisms are involved in decreased NOX-4 expression induced by IL-1β. NOX4 and HuR regulation of NOX-1 contributes to VSMC migration, important in vascular inflammation and remodeling.

Abstract 19752: Nox4 Expression Regulates Inflammation in Tgfβ-treated Vascular Smooth Muscle Cells

NADPH oxidases (NOX) are a major source of reactive oxygen species in the cardiovascular system, contributing to the pathogenesis of disease. We recently reported that increased NOX4 expression in aging results in enhanced cellular and mitochondrial oxidative stress, vascular inflammation, dysfunction, and atherosclerosis. However, the role of NOX4 in the activation of inflammatory molecular pathways in vascular smooth muscle cells (VSMC) under pathophysiological conditions has not been elucidated. In this study, we investigated whether inhibition of NOX4 expression modulates proinflammatory gene expression in mouse VSMC by suppressing its expression with a novel peptide-mediated siRNA transfection approach. Using melittin derived peptide p5RHH, we achieved significantly higher transfection efficiency (92% vs 85% with Lipofectamine) and decreased toxicity (p<0.01 vs Lipofectamine in MTT assay). TGFβ, a proinflammatory cytokine, known to induce NOX4 expression significantly upregulated NOX4 mRNA (p<0.01) and protein expression (p<0.01) in VSMC. p5RHH mediated NOX4 siRNA transfection attenuated TGFβ induced upregulation of NOX4 mRNA (p<0.01) and protein (p<0.01) levels. Analysis of inflammatory marker genes, CCL2, CCL5, IL6, and VCAM-1, showed that NOX4 knockdown significantly attenuated the expression of these genes in TGFβ-treated VSMC, with the highest inhibition seen in the expression of IL6. Congruent with this, inflammatory marker gene expression, which increases with aging, was significantly lower in VSMC isolated from aged (16 months old) NOX4 knockout mice compared with the aged wild-type. TGFβ-activated kinase 1 (TAK1, also known as MAP3K7) is an upstream regulator of inflammatory signaling in cells treated with cytokines. p5RHH mediated TAK1 siRNA transfection downregulated AP1 and NFκB activation and inhibited NOX4 expression in wild-type VSMC, indicating that TAK1 regulates NOX4 expression via regulation of AP1 and NFκB. Because plasma TGFβ levels increase with aging, it may be of one of the factors contributing to increased NOX4 expression with aging. Our data indicate that NOX4 is a potential therapeutic target for modulation of inflammation in aging and aging-associated cardiovascular disease.

NADPH Oxidase Accounts for Changes in Cerebrovascular Redox Status in Hindlimb Unweighting Rats

ObjectiveThe 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. MethodsWe 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. ResultsROS 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. ConclusionThese results suggest that vascular NADPH oxidases regulate cerebrovascular redox status and participate in vascular oxidative stress injury during simulated microgravity.

Mechanisms of bradykinin-induced expression of connective tissue growth factor and nephrin in podocytes.

Diabetic nephropathy (DN) is the main cause of morbidity and mortality in diabetes and is characterized by mesangial matrix deposition and podocytopathy, including podocyte loss. The risk factors and mechanisms involved in the pathogenesis of DN are still not completely defined. In the present study, we aimed to understand the cellular mechanisms through which activation of B2 kinin receptors contribute to the initiation and progression of DN. Stimulation of cultured rat podocytes with bradykinin (BK) resulted in a significant increase in ROS generation, and this was associated with a significant increase in NADPH oxidase (NOX)1 and NOX4 protein and mRNA levels. BK stimulation also resulted in a signicant increase in the phosphorylation of ERK1/2 and Akt, and this effect was inhibited in the presence of NOX1 and Nox4 small interfering (si)RNA. Furthermore, podocytes stimulated with BK resulted in a significant increase in protein and mRNA levels of connective tissue growth factor (CTGF) and, at the same time, a significant decrease in protein and mRNA levels of nephrin. siRNA targeted against NOX1 and NOX4 significantly inhibited the BK-induced increase in CTGF. Nephrin expression was increased in response to BK in the presence of NOX1 and NOX4 siRNA, thus implicating a role for NOXs in modulating the BK response in podocytes. Moreover, nephrin expression in response to BK was also significantly increased in the presence of siRNA targeted against CTGF. These findings provide novel aspects of BK signal transduction pathways in pathogenesis of DN and identify novel targets for interventional strategies.

Protective effect of allicin on high glucose/hypoxia-induced aortic endothelial cells via reduction of oxidative stress

This study was designed to explore the protective effect of allicin on aortic endothelial cell injury induced by high glucose/hypoxia and to investigate the corresponding mechanisms. The primary-cultured murine aortic endothelial cells were subcultured. The third passage of cells was adopted and randomly divided into five groups: The normal group (NG), the mannitol group (MG), the high-glucose/hypoxia group (HG), the allicin group (AG) and the protein kinase C (PKC) inhibitor group (GG). The general morphology was observed under an inverted phase-contrast microscope and cell viability was assessed using the MTT assay. Intracellular reactive oxygen species (ROS) levels in the endothelial cells were quantified using dihydroethidium staining. The levels of 8-hydroxydeoxyguanosine (8-OHdG), nuclear factor-κB (NF-κB), NADPH oxidase 4 (Nox4) and hypoxia-inducible factor-1α (HIF-1α) and the activity of PKC were measured using ELISA. A quantitative polymerase chain reaction (qPCR) was adopted to evaluate the mRNA expression of Nox4, HIF-1α and NF-κB. The altered cell morphology observed in HG was notably ameliorated in the AG and GG. The protein levels of 8-OHdG, NF-κB, Nox4, HIF-1α and PKC in the HG were higher than those in the other groups. Furthermore, the cell viability in the AG was significantly increased and the protein levels of 8-OHdG, NF-κB, Nox4, HIF-1α and PKC were significantly decreased compared with those in the HG. The ROS production was found to be increased in the HG cells, while there was a significant decrease in the AG cells. These data indicate that allicin exerts a protective effect against high glucose/hypoxia-induced injury in aortic endothelial cells through its antioxidative action, which may involve the inhibition of the PKC pathway and regulation of HIF-1α.

Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation

Reactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate oxidase (NOX) play a key role in liver injury and fibrosis. Previous studies demonstrated that GKT137831, a dual NOX1/4 inhibitor, attenuated liver fibrosis in mice as well as pro-fibrotic genes in hepatic stellate cells (HSCs) as well as hepatocyte apoptosis. The effect of NOX1 and NOX4 deficiency in liver fibrosis is unclear, and has never been directly compared. HSCs are the primary myofibroblasts in the pathogenesis of liver fibrosis. Therefore, we aimed to determine the role of NOX1 and NOX4 in liver fibrosis, and investigated whether NOX1 and NOX4 signaling mediates liver fibrosis by regulating HSC activation. Mice were treated with carbon tetrachloride (CCl4) to induce liver fibrosis. Deficiency of either NOX1 or NOX4 attenuates liver injury, inflammation, and fibrosis after CCl4 compared to wild-type mice. NOX1 or NOX4 deficiency reduced lipid peroxidation and ROS production in mice with liver fibrosis. NOX1 and NOX4 deficiency are approximately equally effective in preventing liver injury in the mice. The NOX1/4 dual inhibitor GKT137831 suppressed ROS production as well as inflammatory and proliferative genes induced by lipopolysaccharide (LPS), platelet-derived growth factor (PDGF), or sonic hedgehog (Shh) in primary mouse HSCs. Furthermore, the mRNAs of proliferative and pro-fibrotic genes were downregulated in NOX1 and NOX4 knock-out activated HSCs (cultured on plastic for 5 days). Finally, NOX1 and NOX4 protein levels were increased in human livers with cirrhosis compared with normal controls. Thus, NOX1 and NOX4 signaling mediates the pathogenesis of liver fibrosis, including the direct activation of HSC.

MAPK pathway activation by chronic lead-exposure increases vascular reactivity through oxidative stress/cyclooxygenase-2-dependent pathways

Chronic exposure to low lead concentration produces hypertension; however, the underlying mechanisms remain unclear. We analyzed the role of oxidative stress, cyclooxygenase-2-dependent pathways and MAPK in the vascular alterations induced by chronic lead exposure. Aortas from lead-treated Wistar rats (1st dose: 10 μg/100g; subsequent doses: 0.125μg/100g, intramuscular, 30days) and cultured aortic vascular smooth muscle cells (VSMCs) from Sprague Dawley rats stimulated with lead (20μg/dL) were used. Lead blood levels of treated rats attained 21.7±2.38μg/dL. Lead exposure increased systolic blood pressure and aortic ring contractile response to phenylephrine, reduced acetylcholine-induced relaxation and did not affect sodium nitroprusside relaxation. Endothelium removal and L-NAME left-shifted the response to phenylephrine more in untreated than in lead-treated rats. Apocynin and indomethacin decreased more the response to phenylephrine in treated than in untreated rats. Aortic protein expression of gp91(phox), Cu/Zn-SOD, Mn-SOD and COX-2 increased after lead exposure. In cultured VSMCs lead 1) increased superoxide anion production, NADPH oxidase activity and gene and/or protein levels of NOX-1, NOX-4, Mn-SOD, EC-SOD and COX-2 and 2) activated ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized superoxide anion production, NADPH oxidase activity and mRNA levels of NOX-1, NOX-4 and COX-2. Blockade of the ERK1/2 and p38 signaling pathways abolished lead-induced NOX-1, NOX-4 and COX-2 expression. Results show that lead activation of the MAPK signaling pathways activates inflammatory proteins such as NADPH oxidase and COX-2, suggesting a reciprocal interplay and contribution to vascular dysfunction as an underlying mechanisms for lead-induced hypertension.

Heat Shock Protein 70 and CHIP Promote Nox4 Ubiquitination and Degradation within the Losartan Antioxidative Effect in Proximal Tubule Cells

Background: Angiotensin II/Angiotensin II type 1 receptor (AT₁R) effects are dependent on ROS production stimulated by NADPH oxidase activation. Hsp70 regulates a diverse set of signaling pathways through their interactions with proteins. CHIP is a E3 ubiquitin ligase that targets proteins for polyubiquitination and degradation. Aim: We study whether Hsp70/CHIP contribute to the negative regulation of Nox4 after AT₁R blockage. Methods/Results: Primary culture of proximal tubule epithelial cells (PTCs) from SHR and WKY were stimulated with Angiotensin II (AII) or treated with Losartan (L) or Losartan plus Angiotensin II (L+AII). Losartan decreased AT₁R and Nox4 while enhancing caveolin-1 and Hsp70 protein expression in SHR PTCs. Immunoprecipitation and immunofluorescence proved interaction and colocalization of increased Hsp70/CHIP with decreased Nox4 in SHR PTCs (L) vs (All). Hsp72 knockdown resulted in enhanced Nox4 protein levels, NADPH oxidase activity and ROS generation in (L+AII) revealing that Losartan was unable to abrogate AII effects on Nox4 expression and oxidative activity. Moreover, MG132 exposed PTCs (L) demostrated blocked ubiquitinated Nox4 degradation and increased colocalization of Nox4/Ubiquitin by inmunofluorescence. Conversely, Hsp72 depletion reduced Nox4/Ubiquitin colocalization causing Nox4 upregulation due to proteosomal degradation inhibition, although Losartan treatment. Conclusion: Our study demonstrates that Hsp70 and CHIP mediates the ubiquitination and proteasomal degradation of Nox4 as part of the antioxidative effect of Losartan in SHR.

Netrin-1 abrogates ischemia/reperfusion-induced cardiac mitochondrial dysfunction via nitric oxide-dependent attenuation of NOX4 activation and recoupling of NOS

Despite an established role of mitochondrial dysfunction in cardiac ischemia/reperfusion (I/R) injury, the upstream activators have remained incompletely defined. We have recently identified an innovative role of exogenously applied netrin-1 in cardioprotection, which is mediated by increased nitric oxide (NO) bioavailability. Here, we tested the hypothesis that this “pharmacological” treatment of netrin-1 preserves mitochondrial function via novel mechanisms that are NO dependent. Freshly isolated C57BL6 mouse hearts were perfused using a Langendorff system, and subjected to a 20min global ischemia/60min reperfusion, in the presence or absence of netrin-1. I/R induced marked increases in infarct size, total superoxide and hydrogen peroxide production, activity and protein abundance of NADPH oxidase (NOX) isoform 4 (NOX4), as well as impaired mitochondrial integrity and function, all of which were attenuated by netrin-1. This protective effect of netrin-1 is attributed to cGMP, a downstream effector of NO. The protein levels of NOX1 and NOX2 were however unaffected, and infarct size from NOX1 and NOX2 knockouts was not different from wild type animals. Scavenging of NO with PTIO reversed inhibitory effects of netrin-1 on NOX4, while NO donor attenuated NOX4 protein abundance. In vivo NOX4 RNAi, or sepiapterin perfusion, resulted in recoupling of NOS, decreased infarct size, and blockade of dysfunctional mitochondrial swelling and mitochondrial superoxide production. These data demonstrate that netrin-1 induces cardioprotection through inhibition of NOX4 activity, which leads to recoupling of NOS, augmented NO bioavailability, reduction in oxidative stress, and ultimately preservation of mitochondrial function. The NO-dependent NOX4 inhibition connects with our previously established pathway of DCC/ERK1/2/eNOS/NO/DCC feed-forward mechanism, to maintain NOS in the coupling state to attenuate oxidative stress to preserve mitochondrial function. These findings may promote development of novel therapeutics for cardiac I/R injury. This article is part of a Special Issue entitled “Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease”.

Alterations in the time course of expression of the Nox family in the brain in a rat experimental cerebral ischemia and reperfusion model: effects of melatonin.

Ischemia-reperfusion (I/R) injury induces the generation of reactive oxygen species (ROS), which results in a poor prognosis for ischemic stroke patients. This study was designed to evaluate the time course of expression of the Nox family, a major source of ROS, and whether melatonin, a potent scavenger of ROS, influences these parameters in a rat model of cerebral I/R caused by middle cerebral artery occlusion (MCAO). After 2-hr occlusion, the filament was withdrawn to allow reperfusion. At 0, 3, 6, 12, 24, and 48hr after reperfusion, brain tissue samples were obtained for assays. Among the Nox family, the mRNA and protein levels of Nox2 and Nox4 were increased both in the ischemic hemisphere and contralateral counterpart in the experimental I/R rats at 0hr after reperfusion, peaked at 3hr, and then returned to the basal level at 24hr. Double-immunofluorescence staining further confirmed the expressions of Nox2 and Nox4 in three major types of brain cells, including neurons, astrocytes, and endothelial cells. In addition, melatonin (5mg/kg) or its vehicle was injected intraperitoneally at 0.5hr before MCAO. Compared with I/R + vehicle group, melatonin pretreatment diminished the increased expression of Nox2 and Nox4, reduced ROS levels, and inhibited cell apoptosis. Our findings suggested that the inhibition of Nox2 and Nox4 expressions by melatonin may essentially contribute to its antioxidant and anti-apoptotic effects during brain I/R.

Mitochondrial regulation of NADPH oxidase in hindlimb unweighting rat cerebral arteries.

Exposure to microgravity results in post-flight cardiovascular deconditioning and orthostatic intolerance in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been indicated in this process. To elucidate the mechanism for this condition, we investigated whether mitochondria regulated NADPH oxidase in hindlimb unweighting (HU) rat cerebral and mesenteric arteries. Four-week HU was used to simulate microgravity in rats. Vascular superoxide generation, protein and mRNA levels of Nox2/Nox4, and the activity of NADPH oxidase were examined in the present study. Compared with control rats, the levels of superoxide increased in cerebral (P<0.001) but not in mesenteric vascular smooth muscle cells. The protein and mRNA levels of Nox2 and Nox4 were upregulated significantly (P<0.001 and P<0.001 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly by HU (P<0.001) in cerebral arteries but not in mesenteric arteries. Chronic treatment with mitochondria-targeted antioxidant mitoTEMPO attenuated superoxide levels (P<0.001), decreased the protein and mRNA expression levels of Nox2/Nox4 (P<0.01 and P<0.05 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) and the activity of NADPH oxidase (P<0.001) in HU rat cerebral arteries, but exerted no effects on HU rat mesenteric arteries. Therefore, mitochondria regulated the expression and activity of NADPH oxidases during simulated microgravity. Both mitochondria and NADPH oxidase participated in vascular redox status regulation.

NADPH oxidase 4 represents a potential target for the treatment of osteoporosis

NADPH oxidase 4 represents a potential target for the treatment of osteoporosis

The NADPH oxidase NOX4 inhibits hepatocyte proliferation and liver cancer progression

The NADPH oxidase NOX4 has emerged as an important source of reactive oxygen species in signal transduction, playing roles in physiological and pathological processes. NOX4 mediates transforming growth factor-β-induced intracellular signals that provoke liver fibrosis, and preclinical assays have suggested NOX4 inhibitors as useful tools to ameliorate this process. However, the potential consequences of sustained treatment of liver cells with NOX4 inhibitors are yet unknown. The aim of this work was to analyze whether NOX4 plays a role in regulating liver cell growth either under physiological conditions or during tumorigenesis. In vitro assays proved that stable knockdown of NOX4 expression in human liver tumor cells increased cell proliferation, which correlated with a higher percentage of cells in S/G2/M phases of the cell cycle, downregulation of p21(CIP1/WAF1), increase in cyclin D1 protein levels, and nuclear localization of β-catenin. Silencing of NOX4 in untransformed human and mouse hepatocytes also increased their in vitro proliferative capacity. In vivo analysis in mice revealed that NOX4 expression was downregulated under physiological proliferative situations of the liver, such as regeneration after partial hepatectomy, as well as during pathological proliferative conditions, such as diethylnitrosamine-induced hepatocarcinogenesis. Xenograft experiments in athymic mice indicated that NOX4 silencing conferred an advantage to human hepatocarcinoma cells, resulting in earlier onset of tumor formation and increase in tumor size. Interestingly, immunochemical analyses of NOX4 expression in human liver tumor cell lines and tissues revealed decreased NOX4 protein levels in liver tumorigenesis. Overall, results described here strongly suggest that NOX4 would play a growth-inhibitory role in liver cells.

Mammalian Target of Rapamycin Complex 2 (mTORC2) Coordinates Pulmonary Artery Smooth Muscle Cell Metabolism, Proliferation, and Survival in Pulmonary Arterial Hypertension

Enhanced proliferation, resistance to apoptosis, and metabolic shift to glycolysis of pulmonary arterial vascular smooth muscle cells (PAVSMCs) are key pathophysiological components of pulmonary vascular remodeling in idiopathic pulmonary arterial hypertension (PAH). The role of the distinct mammalian target of rapamycin (mTOR) complexes mTORC1 (mTOR-Raptor) and mTORC2 (mTOR-Rictor) in PAVSMC proliferation and survival in PAH and their therapeutic relevance are unknown. Immunohistochemical and immunoblot analyses revealed that mTORC1 and mTORC2 pathways are markedly upregulated in small remodeled pulmonary arteries and isolated distal PAVSMCs from subjects with idiopathic PAH that have increased ATP levels, proliferation, and survival that depend on glycolytic metabolism. Small interfering RNA- and pharmacology-based analysis showed that although both mTORC1 and mTORC2 contribute to proliferation, only mTORC2 is required for ATP generation and survival of idiopathic PAH PAVSMCs. mTORC2 downregulated the energy sensor AMP-activated protein kinase, which led to activation of mTORC1-S6 and increased proliferation, as well as a deficiency of the proapoptotic protein Bim and idiopathic PAH PAVSMC survival. NADPH oxidase 4 (Nox4) protein levels were increased in idiopathic PAH PAVSMCs, which was necessary for mTORC2 activation, proliferation, and survival. Nox4 levels and mTORC2 signaling were significantly upregulated in small pulmonary arteries from hypoxia-exposed rats at days 2 to 28 of hypoxia. Treatment with the mTOR kinase inhibitor PP242 at days 15 to 28 suppressed mTORC2 but not Nox4, induced smooth muscle-specific apoptosis in small pulmonary arteries, and reversed hypoxia-induced pulmonary vascular remodeling in rats. These data provide a novel mechanistic link of Nox4-dependent activation of mTORC2 via the energy sensor AMP-activated protein kinase to increased proliferation and survival of PAVSMCs in PAH, which suggests a new potential pathway for therapeutic interventions.

Palmitate induces reactive oxygen species production and β-cell dysfunction by activating nicotinamide adenine dinucleotide phosphate oxidase through Src signaling.

Chronic hyperlipidemia impairs pancreatic β-cell function, referred to as lipotoxicity. We have reported an important role of endogenous reactive oxygen species (ROS) overproduction by activation of Src, a non-receptor tyrosine kinase, in impaired glucose-induced insulin secretion (GIIS) from diabetic rat islets. In the present study, we investigated the role of ROS production by Src signaling in palmitate-induced dysfunction of β-cells. After rat insulinoma INS-1D cells were exposed to 0.6mmol/L palmitate for 24h (palmitate exposure); GIIS, ROS production and nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity were examined with or without exposure to10μmol/L 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), a Src inhibitior, for 30 or 60min. Exposure to PP2 recovered impaired GIIS and decreased ROS overproduction as a result of palmitate exposure. Palmitate exposure increased activity of NOX and protein levels of NOX2, a pathological ROS source in β-cells. Palmitate exposure increased the protein level of p47 (phox) , a regulatory protein of NOX2, in membrane fraction compared with control, which was reduced by PP2. Transfection of small interfering ribonucleic acid of p47 (phox) suppressed the augmented p47 (phox) protein level in membrane fraction, decreased augmented ROS production and increased impaired GΙIS by palmitate exposure. In addition, exposure to PP2 ameliorated impaired GIIS and decreased ROS production in isolated islets of KK-A(y) mice, an obese diabetic model with hyperlipidemia. Activation of NOX through Src signaling plays an important role in ROS overproduction and impaired GΙIS caused by chronic exposure to palmitate, suggesting a lipotoxic mechanism of β-cell dysfunction of obese mice.