Knockdown Of Nox4 Research Articles

쥐의 뇌실 하 영역(SVZ) 신경 줄기 세포의 신경 세포로의 분화 과정에서 Nox4의 역할

Reactive oxygen species (ROS), at appropriate concentrations, mediate various normal cellular functions, including defense against pathogens, signal transduction, cellular growth, and gene expression. A recent study demonstrated that ROS and ROS-generating NADPH oxidase (Nox) are important in self-renewal and neuronal differentiation of subventricular zone (SVZ) neural stem cells in adult mouse brains. In this study, we found that endogenous ROS were detected in SVZ neural stem cells cultured from postnatal mouse brains. Nox4 was predominantly expressed in cultured cells, while the levels of the Nox1 and Nox2 transcripts were very low. In addition, the Nox4 gene was highly upregulated (by up to 10-fold) during neuronal differentiation. Immunocytochemical analysis detected the Nox4 protein mainly in neurons positive for the neuronal specific tubulin Tuj1. After differentiation, endogenous ROS were detected exclusively in neuron-like cells with processes. In addition, perturbation of the cellular redox state with N-acetyl cysteine, a ROS scavenger, during neuronal differentiation greatly inhibited neurogenesis. Lastly, knockdown of Nox4 using short hairpin RNA decreased neurogenesis. These findings suggest that Nox4 may be a major ROS-generating enzyme in postnatal SVZ neural stem cells, and Nox4-mediated ROS generation may be important in their neuronal differentiation.

Myocardin-related Transcription Factor Regulates Nox4 Protein Expression: LINKING CYTOSKELETAL ORGANIZATION TO REDOX STATE

TGFβ-induced expression of the NADPH oxidase Nox4 is essential for fibroblast-myofibroblast transition. Rho has been implicated in Nox4 regulation, but the underlying mechanisms are largely unknown. Myocardin-related transcription factor (MRTF), a Rho/actin polymerization-controlled coactivator of serum response factor, drives myofibroblast transition from various precursors. We have shown that TGFβ is necessary but insufficient for epithelial-myofibroblast transition in intact epithelia; the other prerequisite is the uncoupling of intercellular contacts, which induces Rho-dependent nuclear translocation of MRTF. Because the Nox4 promoter harbors a serum response factor/MRTF cis-element (CC(A/T)6GG box), we asked if MRTF (and thus cytoskeleton organization) could regulate Nox4 expression. We show that Nox4 protein is robustly induced in kidney tubular cells exclusively by combined application of contact uncoupling and TGFβ. Nox4 knockdown abrogates epithelial-myofibroblast transition-associated reactive oxygen species production. Laser capture microdissection reveals increased Nox4 expression in the tubular epithelium also during obstructive nephropathy. MRTF down-regulation/inhibition suppresses TGFβ/contact disruption-provoked Nox4 protein and mRNA expression, Nox4 promoter activation, and reactive oxygen species production. Mutation of the CC(A/T)6GG box eliminates the synergistic activation of the Nox4 promoter. Jasplakinolide-induced actin polymerization synergizes with TGFβ to facilitate MRTF-dependent Nox4 mRNA expression/promoter activation. Moreover, MRTF inhibition prevents Nox4 expression during TGFβ-induced fibroblast-myofibroblast transition as well. Although necessary, MRTF is insufficient; Nox4 expression also requires TGFβ-activated Smad3 and TAZ/YAP, two contact- and cytoskeleton-regulated Smad3-interacting coactivators. Down-regulation/inhibition of TAZ/YAP mitigates injury-induced epithelial Nox4 expression in vitro and in vivo. These findings uncover new MRTF- and TAZ/YAP-dependent mechanisms, which link cytoskeleton remodeling and redox state and impact epithelial plasticity and myofibroblast transition.

Activation of ENaC in collecting duct cells by prorenin and its receptor PRR: involvement of Nox4-derived hydrogen peroxide.

The collecting duct (CD) has been recognized as an important source of prorenin/renin, and it also expresses (pro)renin receptor (PRR). The goal of this study was to examine the hypothesis that prorenin or renin via PRR regulates epithelial Na(+) channel (ENaC) activity in mpkCCD cells. Transepithelial Na(+) transport was measured by using a conventional epithelial volt-ohmmeter and was expressed as the calculated equivalent current (Ieq). Amiloride-inhibitable Ieq was used as a reflection of ENaC activity. Administration of prorenin in the nanomolar range induced a significant increase in Ieq that was detectable as early as 1 min, peaked at 5 min, and gradually returned to baseline within 15 min. These changes in Ieq were completely prevented by a newly developed PRR decoy inhibitor, PRO20. Prorenin-induced Ieq was inhibitable by amiloride. Compared with prorenin, renin was less effective in stimulating Ieq Prorenin-induced Ieq was attenuated by apocynin but enhanced by tempol, the latter effect being prevented by catalase. In response to prorenin treatment, the levels of total reactive oxygen species and H2O2 were both increased, as detected by spin-trap analysis and reactive oxygen species (ROS)-Glo H2O2 assay, respectively. Both siRNA-mediated Nox4 knockdown and the dual Nox1/4 inhibitor GKT137892 attenuated prorenin-induced Ieq Overall, our results demonstrate that activation of PRR by prorenin stimulates ENaC activity in CD cells via Nox4-derived H2O2.

Involvement of NADPH oxidases and non-muscle myosin light chain in senescence of endothelial progenitor cells in hyperlipidemia.

NADPH oxidase (NOX)-derived reactive oxygen species (ROS) is involved in endothelial dysfunction of hyperlipidemia, and non-muscle myosin regulatory light chain (nmMLC20) is reported to have a transcriptional function in regulation of gene expression. The purposes of this study are to determine whether NOX-derived ROS can promote endothelial progenitor cell (EPC) senescence and whether nmMLC20 can regulate NOX expression through a phosphorylation-dependent manner. The rats were subjected to 8 weeks of high-fat diet feeding to establish a hyperlipidemic model, which showed an increase in plasma lipids and the accelerated senescence and reduced number of circulating EPCs, accompanied by an increase in myosin light chain kinase (MLCK) and NOX activities, p-nmMLC20 level, NOX (NOX2, NOX4) expression, and H2O2 content. Next, EPCs isolated from normal rats were incubated with ox-LDL (100 μg/mL) for 24 h to establish a senescent model in vitro. Consistent with our in vivo findings, ox-LDL treatment increased the senescence of EPCs concomitant with an increase in MLCK and NOX activities, p-nmMLC20 level (in total or nuclear proteins), NOX expression, and H2O2 content; these phenomena were reversed by MLCK inhibitor. NOX inhibitor achieved similar results to that of MLCK inhibitor except that there is no effect on MLCK activity and p-nmMLC20 level. Furthermore, knockdown of nmMLC20, NOX2, or NOX4 led to a down-regulation in NOX and a reduction in ox-LDL-induced EPC senescence. These results suggest that NOX-derived ROS promotes the senescence of circulating EPCs in hyperlipidemia and nmMLC20 may play a transcriptional role in the upregulation of NOX through a phosphorylation-dependent manner.

Regulation of cellular oxidative stress and apoptosis by G protein-coupled receptor kinase-2; The role of NADPH oxidase 4

Cardiac myocyte oxidative stress and apoptosis are considered important mechanisms for the development of heart failure (HF). Chronic HF is characterized by increased circulating catecholamines to augment cardiac output. Long-term stimulation of myocardial β-adrenergic receptors (β-ARs) is deleterious in cardiac myocytes, however, the potential mechanisms underlying increased cell death are unclear. We hypothesize that GRK2, a critical regulator of myocardial β-AR signaling, plays an important role in mediating cellular oxidative stress and apoptotic cell death in response to β-agonist stimulation. Stimulation of H9c2 cells with a non-selective β-agonist, isoproterenol (Iso) caused increased oxidative stress and apoptosis. There was also increased Nox4 expression, but no change in Nox2, the primary NADPH isoforms and major sources of ROS generation in cardiac myocytes. Adenoviral-mediated overexpression of GRK2 led to similar increases in ROS production and apoptosis as seen with Iso stimulation. These increases in oxidative stress were abolished by pre-treatment with the non-specific Nox inhibitor, apocynin, or siRNA knockdown of Nox4. Adenoviral-mediated expression of a GRK2 inhibitor prevented ROS production and apoptosis in response to Iso stimulation. β-Arrestins are signaling proteins that function downstream of GRK2 in β-AR uncoupling. Adenoviral-mediated overexpression of β-arrestins increased ROS production and Nox4 expression. Chronic β-agonist stimulation in mice increased Nox4 expression and apoptosis compared to PBS or AngII treatment. These data demonstrate that GRK2 may play an important role in regulating oxidative stress and apoptosis in cardiac myocytes and provides an additional novel mechanism for the beneficial effects of cardiac-targeted GRK2 inhibition to prevent the development of HF.

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.

30 NADPH oxidases influence the angiogenic function of endothelial colony-forming cells

Introduction Endothelial colony-forming cells (ECFCs) hold great cytotherapeutic potential for ischaemic disease. Interestingly, recent evidence supports a key role for NADPH oxidases in underlying angiogenic processes of these and other endothelial cells. Aims To study the effects of Nox NADPH oxidases on pro-angiogenic function of ECFCs. Methods Human ECFCs isolated from umbilical cord blood were treated with pro-oxidant PMA and assessed in vitro , both basally and after siRNA knockdown of Nox4, a key endothelial isoform, alongside primary mature human aortic endothelial cells (HAoECs). Results PMA (500 nM for 8 h) increased cell migration (control 18.6 ± 2.8, PMA 32.7 ± 6.6% closure; n = 6, P Conclusion ECFC migration is enhanced by low concentrations of superoxide, and to a greater extent as compared to mature endothelial cells, and appears to be at least partly dependent upon Nox4 NADPH oxidase. These findings may have significant implications for potential ECFC-based therapies for ischaemic disease, which is associated with an oxidative microenvironment.

The Nox1/4 Dual Inhibitor GKT137831 or Nox4 Knockdown Inhibits Angiotensin-II-Induced Adult Mouse Cardiac Fibroblast Proliferation and Migration. AT1 Physically Associates With Nox4

Both oxidative stress and inflammation contribute to chronic hypertension-induced myocardial fibrosis and adverse cardiac remodeling. Here we investigated whether angiotensin (Ang)-II-induced fibroblast proliferation and migration are NADPH oxidase (Nox) 4/ROS and IL-18 dependent. Our results show that the potent induction of mouse cardiac fibroblast (CF) proliferation and migration by Ang-II is markedly attenuated by Nox4 knockdown and the Nox inhibitor DPI. Further, Nox4 knockdown and DPI pre-treatment attenuated Ang-II-induced IL-18, IL-18Rα and collagen expression, and MMP9 and LOX activation. While neutralization of IL-18 blunted Ang-II-induced CF proliferation and migration, knockdown of MMP9 attenuated CF migration. The antioxidant NAC and the cell-permeable SOD mimetics Tempol, MnTBAP, and MnTMPyP attenuated oxidative stress and inhibited CF proliferation and migration. The Nox1/Nox4 dual inhibitor GKT137831 also blunted Ang-II-induced H2 O2 production and CF proliferation and migration. Further, AT1 bound Nox4, and Ang-II enhanced their physical association. Notably, GKT137831 attenuated the AT1/Nox4 interaction. These results indicate that Ang-II induces CF proliferation and migration in part via Nox4/ROS-dependent IL-18 induction and MMP9 activation, and may involve AT1/Nox4 physical association. Thus, either (i) neutralizing IL-18, (ii) blocking AT1/Nox4 interaction or (iii) use of the Nox1/Nox4 inhibitor GKT137831 may have therapeutic potential in chronic hypertension-induced adverse cardiac remodeling.

NADPH OXIDASE 2, 4 AND MITOCHONDRIA-DERIVED REACTIVE OXYGEN SPECIES CONTRIBUTE TO ANGIOPOIETIN-1 INDUCED ANGIOGENESIS

The NOX proteins are reactive oxygen species (ROS) generating enzymes and together with Mitochondrial-derived ROS, they play an important role in pathological as well as physiological processes of the vasculature. Recent evidences indicate that ROS generated by NOX and the mitochondria may play important roles in growth factors downstream signaling. Our own study have pointed out that Angiopoietin-1 (Ang-1) induced ROS production is important for Ang-1/Tie-2 receptor signaling in endothelial cells, however, we did not identify the molecular sources of ROS generation in this respect. In this study, we have identified the origins of Ang-1 induced ROS production in endothelial cells and their involvement in Ang-1 induced angiogenesis. The most abundant isoforms of NOX in human umbilical endothelial cells were knocked down using adenoviruses expressing shRNA against NOX2 and NOX4. Mitochondrial-derived ROS were inhibited using SS31 peptide or Mito-Tempol. Cellular ROS levels were measured upon Ang-1 treatment using Amplex Red and Mitochondrial ROS level were measured with MitoSox. To evaluate the involvement of NOX or Mitochondrial-derived ROS in Ang-1 induced angiogenesis we evaluated cell survival by cell count and Caspase-3 cleavage, tubulogenesis by matrigel assay and migration by scratch wound assay. Activation level of the MAPK and PI3K/AKT pathways downstream of Ang-1/Tie-2 were measured by immunoblotting. Ang-1treatment leads to NOX-derived and Mitochondrial ROS production. NOX4 and the mitochondrial ROS are required for Ang-1 induced ROS production. NOX4 knockdown and Mitochondrial ROS blockage lead to impaired Ang-1 induced migration. NOX2 minimally contributes to Ang-1 induced ROS production and appears to be required for Ang-1 induced cell survival. In addition, NOX4 and mitochondrial-derived ROS are required for optimal activation of p38 and inactivation of ERK and AKT. On the other hand NOX2 appears to be required for optimal AKT activation and ERK and p38 inactivation. We conclude that NOX4 and mitochondrial ROS are required for Ang-1 induced migration through p38 activation. We also concluded that NOX2-derived ROS regulates Ang-1 induced cell survival by activation of AKT. This study provides new insight into cellular signaling mechanism downstream of Ang-1/Tie-2 signaling axis.

Increased expression of NAPDH oxidase 4 in systemic sclerosis dermal fibroblasts: regulation by transforming growth factor β.

Systemic sclerosis (SSc) is characterized by severe and often progressive fibrosis of the skin and multiple internal organs. The mechanisms responsible for these alterations remain obscure, although excessive reactive oxygen species (ROS)-mediated oxidative stress has been implicated. NOX-4 is 1 of 7 isoforms of NADPH oxidase responsible for the generation of ROS. The purpose of this study was to examine NOX-4 expression in skin and cultured dermal fibroblasts from SSc patients and to examine its regulation by transforming growth factor β1 (TGFβ1). NOX-4 was assessed in normal and SSc skin by immunohistologic analysis and in normal and SSc cultured dermal fibroblasts by quantitative polymerase chain reaction analysis, fluorescence microscopy, and Western blotting. ROS levels were assessed by fluorescence measurement of H2 O2 production. Specific kinase inhibitors were used to study the TGFβ1 signaling involved in NOX-4 stimulation. NOX-4 inhibition/down-regulation was induced with a selective NOX-4 small-molecule inhibitor and NOX-4 small interfering RNA (siRNA). In contrast with normal skin fibroblasts, those from SSc skin showed intense NOX-4 staining. Cultured SSc fibroblasts displayed increased NOX-4 expression. TGFβ1 caused potent NOX-4 protein and messenger RNA stimulation in normal and SSc fibroblasts, which was mediated by the protein kinase Cδ (PKCδ) and Smad2/3 pathways. NOX-4 knockdown in SSc fibroblasts reduced the production of ROS and lowered the expression of type I collagen. NOX-4 expression and production were found to be constitutively elevated in SSc skin and cultured SSc dermal fibroblasts. TGFβ1 stimulated NOX-4 expression in normal and SSc fibroblasts through PKCδ and Smad2/3 signaling pathways. A small-molecule NOX-4 inhibitor decreased collagen and fibronectin production by normal and SSc fibroblasts, and NOX-4 siRNA knockdown reduced ROS and collagen production by SSc fibroblasts. These results demonstrate the involvement of NOX-4 in SSc-associated fibrosis and indicate NOX-4 inhibitors as novel therapeutic approaches for SSc.

Inhibition of NADPH oxidase 4 induces apoptosis in malignant mesothelioma: Role of reactive oxygen species.

Malignant pleural mesothelioma (MPM) is an aggressive tumor that is characterized by dysregulated growth and resistance to apoptosis. Reactive oxygen species (ROS)-generating NADPH oxidase (Nox) family enzymes have been suggested to be involved in neoplastic proliferation. Both the antioxidant N-acetylcysteine (NAC) and the inhibitor of flavoprotein-dependent oxidase, diphenylene iodonium (DPI), inhibited the cell viability of MPM cells in a dose-dependent manner. To examine whether Nox-mediated ROS generation confers antiapoptotic activity and thus a growth advantage to MPM cells, we analyzed the mRNA expression of Nox family members using quantitative RT-PCR in 7 MPM cell lines and a normal mesothelial cell line. Nox4 mRNA was expressed in all of the examined MPM cell lines, whereas little or no Nox2, Nox3 and Nox5 mRNA expression was detected. In 2 MPM cell lines, Nox4 mRNA expression was significantly higher than that in a normal mesothelial cell line. siRNAs targeting Nox4 suppressed ROS generation and cell viability in the MPM cell lines. In addition, DPI treatment and knockdown of Nox4 attenuated phosphorylation of AKT and ERK. Taken together, our results indicate that Nox4-mediated ROS, at least in part, transmit cell survival signals and their depletion leads to apoptosis, thus highlighting the Nox4-ROS-AKT signaling pathway as a potential therapeutic target for MPM treatment.

IFNγ induces oxidative stress, DNA damage and tumor cell senescence via TGFβ/SMAD signaling-dependent induction of Nox4 and suppression of ANT2.

Cellular senescence provides a biological barrier against tumor progression, often associated with oncogene-induced replication and/or oxidative stress, cytokine production and DNA damage response (DDR), leading to persistent cell-cycle arrest. While cytokines such as tumor necrosis factor-alpha (TNFα) and interferon gamma (IFNγ) are important components of senescence-associated secretome and induce senescence in, for example, mouse pancreatic β-cancer cell model, their downstream signaling pathway(s) and links with oxidative stress and DDR are mechanistically unclear. Using human and mouse normal and cancer cell models, we now show that TNFα and IFNγ induce NADPH oxidases Nox4 and Nox1, reactive oxygen species (ROS), DDR signaling and premature senescence. Unlike mouse tumor cells that required concomitant presence of IFNγ and TNFα, short exposure to IFNγ alone was sufficient to induce Nox4, Nox1 and DDR in human cells. siRNA-mediated knockdown of Nox4 but not Nox1 decreased IFNγ-induced DDR. The expression of Nox4/Nox1 required Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling and the effect was mediated by downstream activation of transforming growth factor-beta (TGFβ) secretion and consequent autocrine/paracrine activation of the TGFβ/Smad pathway. Furthermore, the expression of adenine nucleotide translocase 2 (ANT2) was suppressed by IFNγ contributing to elevation of ROS and DNA damage. In contrast to mouse B16 cells, inability of TC-1 cells to respond to IFNγ/TNFα by DDR and senescence correlated with the lack of TGFβ and Nox4 response, supporting the role of ROS induced by NADPH oxidases in cytokine-induced senescence. Overall, our data reveal differences between cytokine effects in mouse and human cells, and mechanistically implicate the TGFβ/SMAD pathway, via induction of NADPH oxidases and suppression of ANT2, as key mediators of IFNγ/TNFα-evoked genotoxicity and cellular senescence.

Cardiac-targeted NADPH oxidase 4 in the adaptive cardiac remodelling of the murine heart

BackgroundThe mechanisms that determine whether the heart adapts to overload stress, or fails, are poorly understood. NADPH oxidase (NOX) proteins produce reactive oxygen species (ROS) involved in redox signalling, and our recent studies have found that an increase in Nox4 during pressure overload protects the heart against failure. We aimed to identify novel Nox4-driven cardioprotective mechanisms that promote adaptive cardiac remodelling. MethodsWe first undertook a proteomic comparison of heart tissue from cardiac-targeted Nox4-overexpressing mice and controls. The Nox4 cardiac metabolome was then investigated by 1H nuclear magnetic resonance (NMR) spectroscopy. Effects on cardiac metabolism were assessed by ex-vivo working heart perfusions and isolated mitochondrial respiration studies. Ex-vivo cardiac energetics were assessed by 31P NMR. Alterations to cardiac fatty acid oxidation were explored in primary cardiomyocytes (extracellular flux analysis). FindingsCardiac-targeted Nox4 overexpression profoundly remodelled the cardiac proteome in an isoform-specific manner, both in the unstressed and stressed heart. Glycolysis and fatty acid oxidation were identified as the most enriched pathways that were altered by Nox4. Metabolomic analysis showed a 2·2 times increase in acetylcarnitine concentrations (p=0·002). Ex-vivo heart perfusions demonstrated a profound increase in palmitate oxidation relative to wild-type hearts (3·6 times increase, p=0·01), with opposite findings observed in primary cardiomyocytes with a knockdown of Nox4. A preference for fatty acid oxidation in Nox4 hearts correlated with a better energetic state (phosphocreatine:ATP ratio) when subjected to increasing doses of isoprenaline stress under baseline and pressure-overload. InterpretationIn this study we identified a novel role for Nox4 in the regulation of cardiac fatty acid oxidation. Cardiomyocyte-targeted Nox4 hearts preferentially oxidised fatty acids for energy provision, improving myocardial energetics under stress. Enhancing fatty acid oxidation might have an adaptive role in the setting of pressure-overload hypertrophy. These data provide novel insights into ROS-dependent metabolic programming. FundingUK Medical Research Council, British Heart Foundation.

Role of neuronal NADPH oxidase 1 in the peri-infarct regions after stroke.

The molecular mechanism underlying the selective vulnerability of neurons to oxidative damage caused by ischemia—reperfusion (I/R) injury remains unknown. We sought to determine the role of NADPH oxidase 1 (Nox1) in cerebral I/R-induced brain injury and survival of newborn cells in the ischemic injured region. Male Wistar rats were subjected to 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion. After reperfusion, infarction size, level of superoxide and 8-hydroxy-2′-deoxyguanosine (8-oxo-2dG), and Nox1 immunoreactivity were determined. RNAi-mediated knockdown of Nox1 was used to investigate the role of Nox1 in I/R-induced oxidative damage, neuronal death, motor function recovery, and ischemic neurogenesis. After I/R, Nox1 expression and 8-oxo-2dG immunoreactivity was increased in cortical neurons of the peri-infarct regions. Both infarction size and neuronal death in I/R injury were significantly reduced by adeno-associated virus (AAV)-mediated transduction of Nox1 short hairpin RNA (shRNA). AAV-mediated Nox1 knockdown enhanced functional recovery after MCAO. The level of survival and differentiation of newborn cells in the peri-infarct regions were increased by Nox1 inhibition. Our data suggest that Nox-1 may be responsible for oxidative damage to DNA, subsequent cortical neuronal degeneration, functional recovery, and regulation of ischemic neurogenesis in the peri-infarct regions after stroke.

NADPH oxidase 4-derived H2O2 promotes aberrant retinal neovascularization via activation of VEGF receptor 2 pathway in oxygen-induced retinopathy.

NADPH oxidase 4 (Nox4) is a major isoform of NADPH oxidase in retinal endothelial cells. Our previous study suggests that upregulation of Nox4 in retinal endothelial cells contributes to retinal vascular leakage in diabetes. In the current study, we investigated the role and mechanism of Nox4 in regulation of retinal neovascularization (NV), a hallmark of proliferative diabetic retinopathy (PDR), using a mouse model of oxygen-induced retinopathy (OIR). Our results confirmed that Nox4 was expressed predominantly in retinal vasculature of mouse retina. Retinal expression of Nox4 was markedly increased in OIR, in parallel with enhanced phosphorylation of ERK. In human retinal microvascular endothelial cells (HRECs), overexpression of Nox4 by adenovirus significantly increased extracellular H2O2 generation, resulting in intensified VEGFR2 activation and exacerbated angiogenesis upon VEGF stimulation. In contrast, silencing Nox4 expression or scavenging H2O2 by polyethylene glycol- (PEG-) conjugated catalase inhibited endothelial migration, tube formation, and VEGF-induced activation of VEGFR2 signaling. Importantly, knockdown of retinal Nox4 by adenovirus-delivered siRNA significantly reduced ERK activation and attenuated retinal NV formation in OIR. Taken together, our data indicate that Nox4 promotes retinal NV formation through H2O2/VEGFR2/ERK signaling pathway. Reducing retinal Nox4 expression may represent a promising therapeutic approach for neovascular retinal diseases such as PDR.

Regulation of mitochondrial oxidative stress by β-arrestins in cultured human cardiac fibroblasts.

ABSTRACTOxidative stress in cardiac fibroblasts (CFs) promotes transformation to myofibroblasts and collagen synthesis leading to myocardial fibrosis, a precursor to heart failure (HF). NADPH oxidase 4 (Nox4) is a major source of cardiac reactive oxygen species (ROS); however, mechanisms of Nox4 regulation are unclear. β-arrestins are scaffold proteins that signal in G-protein-dependent and -independent pathways; for example, in ERK activation. We hypothesize that β-arrestins regulate oxidative stress in a Nox4-dependent manner and increase fibrosis in HF. CFs were isolated from normal and failing adult human left ventricles. Mitochondrial ROS/superoxide production was quantitated using MitoSox. β-arrestin and Nox4 expressions were manipulated using adenoviral overexpression or short interfering RNA (siRNA)-mediated knockdown. Mitochondrial oxidative stress and Nox4 expression in CFs were significantly increased in HF. Nox4 knockdown resulted in inhibition of mitochondrial superoxide production and decreased basal and TGF-β-stimulated collagen and α-SMA expression. CF β-arrestin expression was upregulated fourfold in HF. β-arrestin knockdown in failing CFs decreased ROS and Nox4 expression by 50%. β-arrestin overexpression in normal CFs increased mitochondrial superoxide production twofold. These effects were prevented by inhibition of either Nox or ERK. Upregulation of Nox4 seemed to be a primary mechanism for increased ROS production in failing CFs, which stimulates collagen deposition. β-arrestin expression was upregulated in HF and plays an important and newly identified role in regulating mitochondrial superoxide production via Nox4. The mechanism for this effect seems to be ERK-mediated. Targeted inhibition of β-arrestins in CFs might decrease oxidative stress as well as pathological cardiac fibrosis.

Hyperketonemia (Acetoacetate) Upregulates NADPH Oxidase 4 and Elevates Oxidative Stress, ICAM-1, and Monocyte Adhesivity in Endothelial Cells

Background/Aims: The incidence of developing microvascular dysfunction is significantly higher in type 1 diabetic (T1D) patients. Hyperketonemia (acetoacetate, β-hydroxybutyrate) is frequently found along with hyperglycemia in T1D. Whether hyperketonemia per se contributes to the excess oxidative stress and cellular injury observed in T1D is not known. Methods: HUVEC were treated with ketones in the presence or absence of high glucose for 24 h. NOX4 siRNA was used to specifically knockdown NOX4 expression in HUVEC. Results: Ketones alone or in combination with high glucose treatment cause a significant increase in oxidative stress, ICAM-1, and monocyte adhesivity to HUVEC. Using an antisense approach, we show that ketone induced increases in ROS, ICAM-1 expression, and monocyte adhesion in endothelial cells were prevented in NOX4 knockdown cells. Conclusion: This study reports that elevated levels of ketones upregulate NOX, contributing to increased oxidative stress, ICAM-1 levels, and cellular dysfunction. This provides a novel biochemical mechanism that elucidates the role of hyperketonemia in the excess cellular injury in T1D. New drugs targeting inhibition of NOX seems promising in preventing higher risk of complications associated with T1D.

Matrix metalloproteinase-3 causes dopaminergic neuronal death through Nox1-regenerated oxidative stress.

In the present study we investigated the interplay between matrix metalloproteinase 3 (MMP3) and NADPH oxidase 1 (Nox1) in the process of dopamine (DA) neuronal death. We found that MMP3 activation causes the induction of Nox1 via mitochondrial reactive oxygen species (ROS) production and subsequently Rac1 activation, eventually leading to Nox1-derived superoxide generation in a rat DA neuronal N27 cells exposed to 6-OHDA. While a MMP3 inhibitor, NNGH, largely attenuated mitochondrial ROS and subsequent Nox1 induction, both apocynin, a putative Nox inhibitor and GKT137831, a Nox1 selective inhibitor failed to reduce 6-OHDA-induced mitochondrial ROS. However, both inhibitors for MMP3 and Nox1 similarly attenuated 6-OHDA-induced N27 cell death. RNAi-mediated selective inhibition of MMP3 or Nox1 showed that knockdown of either MMP3 or Nox1 significantly reduced 6-OHDA-induced ROS generation in N27 cells. While 6-OHDA-induced Nox1 was abolished by MMP3 knockdown, Nox1 knockdown did not alter MMP3 expression. Direct overexpression of autoactivated MMP3 (actMMP3) in N27 cells or in rat substantia nigra (SN) increased expression of Nox1. Selective knockdown of Nox1 in the SN achieved by adeno-associated virus-mediated overexpression of Nox1-specific shRNA largely attenuated the actMMP3-mediated dopaminergic neuronal loss. Furthermore, Nox1 expression was significantly attenuated in Mmp3 null mice treated with N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Together we established novel molecular mechanisms underlying oxidative stress-mediated dopaminergic neuronal death in which MMP3 activation is a key upstream event that leads to mitochondrial ROS, Nox1 induction and eventual dopaminergic neuronal death. Our findings may lead to the development of novel therapeutic approach.

P-Cresyl sulfate, a uremic toxin, causes vascular endothelial and smooth muscle cell damages by inducing oxidative stress.

The major cause of death in patients with chronic kidney disease (CKD) is cardiovascular disease. Here, p-Cresyl sulfate (PCS), a uremic toxin, is considered to be a risk factor for cardiovascular disease in CKD. However, our understanding of the vascular toxicity induced by PCS and its mechanism is incomplete. The purpose of this study was to determine whether PCS enhances the production of reactive oxygen species (ROS) in vascular endothelial and smooth muscle cells, resulting in cytotoxicity. PCS exhibited pro-oxidant properties in human umbilical vein endothelial cells (HUVEC) and aortic smooth muscle cells (HASMC) by enhancing NADPH oxidase expression. PCS also up-regulates the mRNA levels and the protein secretion of monocyte chemotactic protein-1 (MCP-1) in HUVEC. In HASMC, PCS increased the mRNA levels of alkaline phosphatase (ALP), osteopontin (OPN), core-binding factor alpha 1, and ALP activity. The knockdown of Nox4, a subunit of NADPH oxidase, suppressed the cell toxicity induced by PCS. The vascular damage induced by PCS was largely suppressed in the presence of probenecid, an inhibitor of organic anion transporters (OAT). In PCS-overloaded 5/6-nephrectomized rats, plasma MCP-1 levels, OPN expression, and ALP activity of the aortic arch were increased, accompanied by the induction of Nox4 expression. Collectively, the vascular toxicity of PCS can be attributed to its intracellular accumulation via OAT, which results in an enhanced NADPH oxidase expression and increased ROS production. In conclusion, we found for the first time that PCS could play an important role in the development of cardiovascular disease by inducing vascular toxicity in the CKD condition.

Astrocytic phospholipase A2 contributes to neuronal glutamate toxicity

The role of astrocytes in glutamate toxicity has been controversial. Here, we show that astrocytes in neuron–astrocyte co-cultures increased neuronal sensitivity to chronic glutamate exposure but not to acute exposure. Enhanced neuronal toxicity by chronic exposure was dependent on astrocyte cell numbers. A reduced generation of extracellular H2O2 induced by glutamate was observed in co-cultures. Further, neuronal glutamate toxicity was not suppressed by NADPH oxidase (Nox) inhibitors, catalase or Nox4 knockdown in co-cultures, whereas these compounds effectively reduced the toxicity in pure neuron cultures. Instead, the intracellular scavenger of reactive oxygen species, N-acetylcysteine (NAC), reduced neuronal cytotoxicity in co-cultures, whereas catalase worked in pure neuron cultures. Lipoxygenase (LOX) inhibitors attenuated neuronal glutamate toxicity in co-cultures but not in pure neuron cultures. Neuronal 5-LOX activity was increased only in co-cultures, whereas 12-LOX activity was increased in both types of cultures. The cyclooxygenase (COX) inhibitors, indomethacin and NS-398, and the phospholipase A2 (PLA2) inhibitors, LY311727 and MAFP, more effectively reduced neuronal glutamate toxicity in co-cultures than in pure neuron cultures. However, in co-cultures, pre-treating neurons and astrocytes with the same inhibitors generated opposite results. COX inhibitors suppressed neuronal glutamate toxicity in pre-treated neurons rather than astrocytes, whereas PLA2 inhibitors reduced the toxicity in pre-treated astrocytes rather than neurons. Gene-specific knockdown of PLA2 confirmed these results. Knockdown of cPLA2α and/or sPLA2-V in astrocytes rather than in neurons more effectively reduced glutamate toxicity in co-cultures. These findings suggest that astrocytic PLA2 activity increases neuronal sensitivity to chronic glutamate exposure in neuron–astrocyte co-cultures.