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The epigenetic landscape describes the chromatin structure of the eukaryotic genome and is therefore the major determinant of gene transcription and hence cellular phenotype. The molecular processes which act to shape the epigenetic landscape through cellular differentiation are thus central to cellular determination and specification. In addition, cellular adaptation to (patho)-physiological stress requires dynamic and reversible chromatin remodelling. It is becoming clear that redox-dependent molecular mechanisms are important determinants of this epigenetic regulation. NADPH oxidases generate reactive oxygen species (ROS) to activate redox-dependent signalling pathways in response to extracellular and intracellular environmental cues. This mini review aims to summarise the current knowledge of the role of NADPH oxidases in redox-dependent chromatin remodelling, and how epigenetic changes might feedback and impact upon the transcriptional expression of these ROS-producing enzymes themselves. The potential physiological significance of this relationship in the control of cellular differentiation and homeostasis by Nox4, specifically, is discussed.

An extensive body of research has demonstrated that pulmonary toxicity induced by fluoride is related to cell apoptosis. Although induction of death receptor-initiated extrinsic apoptosis by sodium fluoride (NaF) has been reported, its mechanism of action is still not clearly defined. Herein, we found that NaF treatment induced activation of caspase-8 in BEAS-2B cells, resulting in apoptosis, which was markedly reduced by blocking caspase-8 using small interfering RNA (siRNA). In this study, we report that death receptor 5 (DR5), a major component of the extrinsic apoptotic pathway, is markedly induced upon NaF stimulation. Enhanced DR5 induction was necessary for the apoptotic effects of NaF, inasmuch as transfected BEAS-2B cells with DR5 siRNA attenuated NaF-induced caspase-8 activation in lung cells. Mechanism investigation indicated that the induction of DR5, following NaF exposure, was mediated by tumor protein 53 (p53)-dependent transcriptional activation. Notably, we demonstrated that NaF could induce a significant increase in intracellular reactive oxygen species (ROS) level derived from nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4). Specifically, NOX4 knockdown inhibited NaF-induced the activation of p53/DR5 axis by reducing NOX4-derived ROS production. Further in vivo investigation demonstrated that NOX4 deficiency markedly attenuates NaF-induced lung injury, apoptosis, and ROS levels in the lung. Moreover, the expressions of p53 and DR5 were significantly reduced after NaF treatment in NOX4 knockout mice compared with the wild type mice. Taken together, our findings provide a novel insight into for the pulmonary apoptosis in response to NaF exposure.

Previous studies suggested that reactive oxygen species (ROS) produced by NADPH oxidase 4 (Nox4) affect the processing of neuropathic pain. However, mechanisms underlying Nox4-dependent pain signaling are incompletely understood. In this study, we aimed to identify novel Nox4 downstream interactors in the nociceptive system. Mice lacking Nox4 specifically in sensory neurons were generated by crossing Advillin-Cre mice with Nox4fl/fl mice. Tissue-specific deletion of Nox4 in sensory neurons considerably reduced mechanical hypersensitivity and neuronal action potential firing after peripheral nerve injury. Using a proteomic approach, we detected various proteins that are regulated in a Nox4-dependent manner after injury, including the small calcium-binding protein S100A4. Immunofluorescence staining and Western blot experiments confirmed that S100A4 expression is massively up-regulated in peripheral nerves and dorsal root ganglia after injury. Furthermore, mice lacking S100A4 showed increased mechanical hypersensitivity after peripheral nerve injury and after delivery of a ROS donor. Our findings suggest that S100A4 expression is up-regulated after peripheral nerve injury in a Nox4-dependent manner and that deletion of S100A4 leads to an increased neuropathic pain hypersensitivity.

Cardiac hypertrophy, an important cause of heart failure, is characterized by an increase in heart weight, the ventricular wall, and cardiomyocyte volume. The volume regulatory anion channel (VRAC) is an important regulator of cell volume. However, its role in cardiac hypertrophy remains unclear. The purpose of this study was to investigate the effect of leucine-rich repeat-containing 8A (LRRC8A), an essential component of the VRAC, on angiotensin II (AngII)-induced cardiac hypertrophy. Our results showed that LRRC8A expression, NADPH oxidase activity, and reactive oxygen species (ROS) production were increased in AngII-induced hypertrophic neonatal mouse cardiomyocytes and the myocardium of C57/BL/6 mice. In addition, AngII activated VRAC currents in cardiomyocytes. The delivery of adeno-associated viral (AAV9) bearing siRNA against mouse LRRC8A into the left ventricular wall inhibited AngII-induced cardiac hypertrophy and fibrosis. Accordingly, the knockdown of LRRC8A attenuated AngII-induced cardiomyocyte hypertrophy and VRAC currents in vitro. Furthermore, knockdown of LRRC8A suppressed AngII-induced ROS production, NADPH oxidase activity, the expression of NADPH oxidase membrane-bound subunits Nox2, Nox4, and p22phox, and the translocation of NADPH oxidase cytosolic subunits p47phox and p67phox. Immunofluorescent staining showed that LRRC8A co-localized with NADPH oxidase membrane subunits Nox2, Nox4, and p22phox. Co-immunoprecipitation and analysis of a C-terminal leucine-rich repeat domain (LRRD) mutant showed that LRRC8A physically interacts with Nox2, Nox4, and p22phox via the LRRD. Taken together, the results of this study suggested that LRRC8A might play an important role in promoting AngII-induced cardiac hypertrophy by interacting with NADPH oxidases via the LRRD.

Background and aims Our previous findings have demonstrated the protective effect of endothelial Nox4-based NADPH oxidase on atherosclerosis. One of the possible mechanisms is the inhibition of soluble epoxide hydrolase (sEH), a proinflammatory and atherogenic factor. Our goal was to investigate whether in vivo inhibition of sEH by 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) alleviates endothelial Nox4 dysfunction caused atherosclerosis and the regulatory mechanism of endothelial Nox4 on sEH. Methods & results: We used endothelial human Nox4 dominant-negative (EDN) transgenic mice in ApoE deficient background to mimic the dysfunction of endothelial Nox4 in atherosclerosis-prone conditions. In EDN aortic endothelium, sEH and the inflammatory marker vascular cell adhesion molecule 1 (VCAM1) were upregulated. TPPU reduced atherosclerotic lesions in EDN mice. In EDN endothelial cells (ECs), the endoplasmic reticulum (ER) stress markers (BIP, IRE1α, phosphorylation of PERK, ATF6) were upregulated, and they can be suppressed by ER stress inhibitor 4-phenyl butyric acid (4-PBA). In EDN ECs, 4-PBA downregulated the expression of sEH and VCAM1, suppressed inflammation, and its application in vivo reduced atherosclerotic lesions of EDN mice. Conclusions Endothelial Nox4 dysfunction upregulated sEH to enhance inflammation, probably by its induction of ER stress. Inhibition of ER stress or sEH is beneficial to alleviate atherosclerosis caused by endothelial Nox4 dysfunction.

Changes in vascular smooth muscle cell (VSMC) phenotype underlie disease pathophysiology and are strongly regulated by NOX NADPH oxidases, with NOX1 favoring synthetic proliferative phenotype and NOX4 supporting differentiation. Growth factor-triggered NOX1 expression/activity strictly depends on the chaperone oxidoreductase protein disulfide isomerase-A1 (PDIA1). Intracellular PDIA1 is required for VSMC migration and cytoskeleton organization, while extracellular PDIA1 fine-tunes cytoskeletal mechanoadaptation and vascular remodeling. We hypothesized that PDIA1 orchestrates NOX1/NOX4 balance and VSMC phenotype. Using an inducible PDIA1 overexpression model in VSMC, we showed that early PDIA1 overexpression (for 24–48 h) increased NOX1 expression, hydrogen peroxide steady-state levels and spontaneous VSMC migration distances. Sustained PDIA1 overexpression for 72 h and 96 h supported high NOX1 levels while also increasing NOX4 expression and, remarkably, switched VSMC phenotype to differentiation. Differentiation was preceded by increased nuclear myocardin and serum response factor-response element activation, with no change in cell viability. Both NOX1 and hydrogen peroxide were necessary for later PDIA1-induced VSMC differentiation. In primary VSMC, PDIA1 knockdown decreased nuclear myocardin and increased the proliferating cell nuclear antigen expression. Newly-developed PDIA1-overexpressing mice (TgPDIA1) exhibited normal general and cardiovascular baseline phenotypes. However, in TgPDIA1 carotids, NOX1 was decreased while NOX4 and calponin expressions were enhanced, indicating overdifferentiation vs. normal carotids. Moreover, in a rabbit overdistension injury model during late vascular repair, PDIA1 silencing impaired VSMC redifferentiation and NOX1/NOX4 balance. Our results suggest a model in which PDIA1 acts as an upstream organizer of NOX1/NOX4 balance and related VSMC phenotype, accounting for baseline differentiation setpoint.

Dysfunctional reactive oxygen species (ROS) signaling is considered an important disease mechanism. Therapeutically, non-selective scavenging of ROS by antioxidants, however, has failed in multiple clinical trials to provide patient benefit. Instead, pharmacological modulation of disease-relevant, enzymatic sources of ROS appears to be an alternative, more promising and meanwhile successfully validated approach. With respect to targets, the family of NADPH oxidases (NOX) stands out as main and dedicated ROS sources. Validation of the different NOX isoforms has been mainly through genetically modified rodent models and is lagging behind in other species. It is unclear whether the different NOX isoforms are sufficiently distinct to allow selective pharmacological modulation. Here we show for five widely used NOX inhibitors that isoform selectivity can be achieved, although individual compound specificity is as yet insufficient. NOX1 was most potently (IC50) targeted by ML171 (0.1 mu M); NOX2, by VAS2870 (0.7 mu M); NOX4, by M13 (0.01 mu M) and NOX5, by ML090 (0.01 mu M). In addition, some non-specific antioxidant and assay artefacts may limit the interpretation of data, which included, surprisingly, the clinically advanced NOX inhibitor, GKT136901. In a human ischemic blood-brain barrier hyperpermeability model where genetic target validation is not an option, we provide proof-of-principle that pharmacological target validation for different NOX isoforms is possible by applying an inhibitor panel at IC50 concentrations. Moreover, our findings encourage further lead optimization and development efforts for isoform-selective NOX inhibitors in different indications.

International audience; Reactive oxygen species (ROS) produced in hematopoietic stem cells (HSCs) are involved in the balance between quiescence, self-renewal, proliferation and differentiation processes. However the role of NOX enzymes on the early stages of hematopoietic differentiation is poorly investigated. For that, we used induced pluripotent stem cells (iPSCs) derived from X-linked Chronic Granulomatous Disease (X0CGD) patients with deficiency in NOX2, and AR220CGD patients with deficiency in p22phox subunit which decreases NOX1, NOX2, NOX3 and NOX4 activities. CD34+ hematopoietic progenitors were obtained after 7, 10 and 13 days of iPS/OP9 co-culture differentiation system. Neither NOX expression nor activity was found in Wild-type (WT), X0CGD and AR220CGD iPSCs. Although NOX2 and NOX4 mRNA were found in WT, X0CGD and AR220CGD iPSC-derived CD34+ cells at day 10 and 13 of differentiation, NOX4 protein was the only NOX enzyme expressed in these cells. A NADPH oxidase activity was measured in WT and X0CGD iPSC-derived CD34+ cells but not in AR220CGD iPSC-derived CD34+ cells because of the absence of p22phox, which is essential for the NOX4 activity. The absence of NOX4 activity and the poor NOX-independent ROS production in AR220CGD iPSC-derived CD34+ cells favored the CD34+ cells production but lowered their hematopoietic potential compared to WT and X0CGD iPSC-derived CD34+ cells. In addition we found a large production of primitive AR220CGD iPSC-derived progenitors at day 7 compared to the WT and X0CGD cell types. In conclusion NOX4 is the major NOX enzyme involved in the early stages of hematopoietic differentiation from iPSCs and its activity can modulate the production, the hematopoietic potential and the phenotype of iPSC-derived CD34+.

Reactive oxygen species derived from NADPH oxidase contribute to a wide variety of renal diseases. Nox4, the major NADPH isoform in kidney, produces mainly H2O2 that regulates physiological functions. Nox4 contributes to redox processes involved in diabetic nephropathy, acute kidney injury, obstructive nephropathy, hypertensive nephropathy, renal cell carcinoma and other renal diseases by activating multiple signaling pathways. Although Nox4 is found in a variety of cell types, including epithelial cells, podocytes, mesangial cells, endothelial cells and fibroblasts, its role is not clear and even controversial. In some conditions, Nox4 protects cells by promoting cell survival in response to harmful stimuli. In other scenarios it induces cell apoptosis, inflammation or fibrogenesis. This functional variability may be attributed to distinct cell types, subcellular localization, molecular concentrations, disease type or stage, and other factors yet unexplored. In this setting, we reviewed the function and mechanism of Nox4 in renal diseases, highlighted the contradictions in Nox4 literature, and discussed promising therapeutic strategies targeting Nox4 in the treatment of certain types of renal diseases.

Background Insulin resistance in endothelial cells contributes to the development of cardiovascular disease in type 2 diabetes mellitus (T2DM). Therefore, there are great potential clinical implications in developing pharmacological interventions targeting endothelial insulin resistance. Our previous studies indicated that argirein which was developed by combining rhein with L -arginine by a hydrogen bond, could substantially relieved stress related exacerbation of cardiac failure and alleviated cardiac dysfunction in T2DM, which was associated with suppressing NADPH oxidase activity. However, it is unclear whether argirein treatment attenuates the vascular lesion and dysfunction in T2DM and its underlying mechanisms. Methods and results The rat aortic endothelial cells (RAECs) were used to treat with palmitic acid (PA), a most common saturated free fatty acid, which could induce insulin resistance. It was showed that argirein increased glucose uptake and glucose transporter-4 (Glut4) expression and reversed the phosphorylation of IRS-1-ser307 and AKT-ser473, consequently resulting in the increase of the production of eNOS and NO in PA-induced RAECs. We further found that argirein blocked the Nox4-dependent superoxide (O2-.) generation, which regulated glucose metabolism in RAECs during PA stimulation. In vitro, argirein increased the release of endothelial NO to relieve the vasodilatory response to acetylcholine and insulin, and restored the expression of Nox4 and pIRS-1-ser307 in the aorta endothelium of high-fat diet (HFD)-fed rats following an injection of streptozocin (STZ). Conclusion These results suggested that argirein could improve endothelial insulin resistance which was attributed to inhibiting Nox4-dependent redox signaling in RAECs. These studies thus revealed the novel effect of argirein to prevent the vascular complication in T2DM.

The p22phox protein is an essential component of the phagocytic- and inner ear NADPH oxidases but its relationship to other Nox proteins is less clear. We have studied the role of p22phox in the TGF-β1-stimulated H2O2 production of primary human and murine fibroblasts. TGF-β1 induced H2O2 release of the examined cells, and the response was dependent on the expression of both Nox4 and p22phox. Interestingly, the p22phox protein was present in the absence of any detectable Nox/Duox expression, and the p22phox level was unaffected by TGF-β1. On the other hand, Nox4 expression was dependent on the presence of p22phox, establishing an asymmetrical relationship between the two proteins. Nox4 and p22phox proteins localized to the endoplasmic reticulum and their distribution was unaffected by TGF-β1. We used a chemically induced protein dimerization method to study the orientation of p22phox and Nox4 in the endoplasmic reticulum membrane. This technique is based on the rapamycin-mediated heterodimerization of the mammalian FRB domain with the FK506 binding protein. The results of these experiments suggest that the enzyme complex produces H2O2 into the lumen of the endoplasmic reticulum, indicating that Nox4 contributes to the development of the oxidative milieu within this organelle.

Obesity is associated with an increased risk of chronic kidney diseases and the conventional treatment with renin-angiotensin-aldosterone system (RAAS) inhibitors is not enough to prevent renal injury and prolong the progression of disease. Recently, silymarin has shown protective effects on renal tissue injury, but the underlying mechanisms remain elusive. The goal of this study was to investigate the potential capacity of silymarin to prevent renal injury during obesity induced by high fat diet (HFD) in mice. In vivo, male C57BL/6 mice received HFD (60% of total calories) for 12 weeks, randomized and treated orally with vehicle saline or silymarin (30mg/kg body weight/d) for 4 weeks. In vitro, human proximal tubular epithelial cells (HK2) were exposed to 300μM palmitic acid (PA) for 36h followed by silymarin administration at different concentrations. The administration of silymarin significantly ameliorated HFD induced glucose metabolic disorders, oxidative stress and pathological alterations in the kidney. Silymarin significantly mitigated renal lipid accumulation, fatty acid β-oxidation and mitochondrial biogenesis in HFD mice and PA treated HK2 cells. Furthermore, silymarin partly restored mitochondrial membrane potential of HK2 cells after PA exposure. In conclusion, silymarin can improve oxidative stress and preserve mitochondrial dysfunction in the kidney, potentially via preventing accumulation of renal lipids and fatty acid β-oxidation.

Aims The role of oxidative stress and inflammation in the development and progression of cardiovascular diseases (CVD) is well established. Increases in oxidative stress can further exacerbate the inflammatory response and lead to cellular senescence. We previously reported that angiotensin II (Ang II) and zinc increase reactive oxygen species (ROS) and cause senescence of vascular smooth muscle cells (VSMCs) and that senescence induced by Ang II is a zinc-dependent process. Zinc stimulated NADPH oxidase (Nox) activity; however, the role of Nox isoforms in zinc effects was not determined. Results Here, we show that downregulation of Nox1, but not Nox4, by siRNA prevented both Ang II- and zinc-induced senescence in VSMCs. On the other hand, overexpression of Nox1 induced senescence, which was associated with reduced proliferation, reduced expression of telomerase and increased DNA damage. Zinc increased Nox1 protein expression, which was inhibited by chelation of zinc with TPEN and by overexpression of the zinc exporters ZnT3 and ZnT10. These transporters work to reduce cytosolic zinc, suggesting that increased cytosolic zinc mediates Nox1 upregulation. Other metals including copper, iron, cobalt and manganese failed to upregulate Nox1, suggesting that this pathway is zinc specific. Nox1 upregulation was inhibited by actinomycin D (ACD), an inhibitor of transcription, by inhibition of NF-κB, a known Nox1 transcriptional regulator and by N-acetyl cysteine (NAC) and MitoTEMPO, suggesting that NF-κB and mitochondrial ROS mediate zinc effects. Supporting this idea, we found that zinc increased NF-κB activation in the cytosol, stimulated the translocation of the p65 subunit to the nucleus, and that zinc accumulated in mitochondria increasing mitochondrial ROS, measured using MitoSox. Further, zinc-induced senescence was reduced by inhibition of NF-κB or reduction of mitochondrial ROS with MitoTEMPO. NF-κB activity was also reduced by MitoTEMPO, suggesting that mitochondrial ROS is upstream of NF-κB. Innovation and conclusion Our data demonstrate that altered zinc distribution leading to accumulation of zinc in the mitochondria increases mitochondrial ROS production causing NF-κB activation which in turn upregulates Nox1 expression inducing senescence of VSMCs.

Excessive levels of reactive oxygen species (ROS) and increased expression of NADPH oxidases (Nox) have been proposed to contribute to pulmonary artery hypertension (PAH) and other cardiovascular diseases (CVD). Nox enzymes are major sources of ROS but the mechanisms regulating changes in Nox expression in disease states remain poorly understood. Epigenetics encompasses a number of mechanisms that cells employ to regulate the ability to read and transcribe DNA. Histone acetylation is a prominent example of an epigenetic mechanism regulating the expression of numerous genes by altering chromatin accessibility. The goal of this study was to determine whether inhibition of histone deacetylases (HDAC) affects the expression of Nox isoforms and reduces pulmonary hypertension. In immune cells, we found that multiple HDAC inhibitors robustly decreased Nox2 mRNA and protein expression in a dose-dependent manner concomitant with reduced superoxide production. This effect was not restricted to Nox2 as expression of Nox1, Nox4 and Nox5 was also reduced by HDAC inhibition. Surprisingly, Nox promoter-luciferase activity was unchanged in the presence of HDAC inhibitors. In macrophages and lung fibroblasts, ChIP experiments revealed that HDAC inhibitors block the binding of RNA polymerase II and the histone acetyltransferase p300 to the Nox2, Nox4 and Nox5 promoter regions and decrease histones activation marks (H3K4me3 and H3K9ac) at these promoter sites. We further show that the ability of CRISPR-ON to drive transcription of Nox1, Nox2, Nox4 and Nox5 genes is blocked by HDAC inhibitors. In a monocrotaline (MCT) rat model of PAH, multiple HDAC isoforms are upregulated in isolated pulmonary arteries, and HDAC inhibitors attenuate Nox expression in isolated pulmonary arteries and reduce indices of PAH. In conclusion, HDAC inhibitors potently suppress Nox gene expression both in vitro and in vivo via epigenetically regulating chromatin accessibility.

We have reported that hematopoietic system injury induced by total body irradiation (TBI) leads to generation of intracellular reactive oxygen species (ROS) and DNA damage, which are ameliorated by antioxidant agents. In the present study, we reported that administration of DIM, a potent antioxidant agent, not only protected mice against TBI-induced lethality, also ameliorated TBI-induced hematopoietic injury. The latter effect was probably attributable to DIM's inhibition of TBI-induced increases in ROS production in hematopoietic stem cells (HSCs) and the phosphorylation of histone H2AX (γ-H2AX). In particular, DIM led to significant improvements in bone marrow (BM) HSC frequency, hematopoietic progenitor cell (HPC) clonogenic function, and multilineage engraftment after transplantation. A downregulation of NADPH oxidase 4 (NOX4) and an upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression were observed following DIM treatment. Notably, the anti-apoptotic potential of DIM was correlated with increased expression of the anti-apoptotic protein Bcl-2 and decreased expression of the pro-apoptotic protein Bax. These findings suggest that DIM attenuates TBI-induced hematopoietic injury through the inhibition of both oxidative stress in HSCs and hematopoietic cell apoptosis. Furthermore, we demonstrated that DIM protected BM hematopoietic cells against ionizing radiation and led to increased clonogenicity in vitro. Therefore, DIM has the potential to be used as an effective radioprotectant to ameliorate TBI-induced hematopoietic injury.

Fibrosis represents a common pathway to end-stage renal disease. Transforming growth factor-β (TGF-β) plays a critical role in the progression of kidney fibrosis. In the present study, we explored the effect of carnosic acid (CA) against TGF-β-induced fibroblast activation in vitro and unilateral ureteral obstruction (UUO)-induced kidney fibrosis in vivo. CA attenuated TGF-β-induced up-regulation of profibrogenic proteins, α-smooth muscle actin (α-SMA), collagen I (COLI), fibronectin (FN), and plasminogen activator inhibitor-1 (PAI-1) in kidney fibroblast cells (NRK-49F). CA inhibited TGF-β-induced hydrogen peroxide generation via inhibition of NADPH oxidase 4 (Nox4) expressions. In mice, CA-administration markedly mitigated the UUO-induced interstitial extension, collagen deposition, superoxide anion formation, hydrogen peroxide production, and lipid peroxidation. In addition, CA significantly attenuated the expression of α-SMA, COLI, FN, PAI-1, and Nox4 in UUO-induced kidneys. These results indicated that CA attenuated oxidative stress via inhibition of Nox4 expression in TGF-β-stimulated fibroblasts and UUO operated-kidneys, suggesting that CA may be useful for the treatment of fibrosis-related diseases.

Liver is a unique organ in displaying a reparative and regenerative response after acute/chronic damage or partial hepatectomy, when all the cell types must proliferate to re-establish the liver mass. The NADPH oxidase NOX4 mediates Transforming Growth Factor-beta (TGF-β) actions, including apoptosis in hepatocytes and activation of stellate cells to myofibroblasts. Aim of this work was to analyze the impact of NOX4 in liver regeneration by using two mouse models where Nox4 was deleted: 1) general deletion of Nox4 (NOX4−/−) and 2) hepatocyte-specific deletion of Nox4 (NOX4hepKO). Liver regeneration was analyzed after 2/3 partial hepatectomy (PH). Results indicated an earlier recovery of the liver-to-body weight ratio in both NOX4−/− and NOX4hepKO mice and an increased survival, when compared to corresponding WT mice. The regenerative hepatocellular fat accumulation and the parenchyma organization recovered faster in NOX4 deleted livers. Hepatocyte proliferation, analyzed by Ki67 and phospho-Histone3 immunohistochemistry, was accelerated and increased in NOX4 deleted mice, coincident with an earlier and increased Myc expression. Primary hepatocytes isolated from NOX4 deleted mice showed higher proliferative capacity and increased expression of Myc and different cyclins in response to serum. Transcriptomic analysis through RNA-seq revealed significant changes after PH in NOX4−/− mice and support a relevant role for Myc in a node of regulation of proliferation-related genes. Interestingly, RNA-seq also revealed changes in the expression of genes related to activation of the TGF-β pathway. In fact, levels of active TGF-β1, phosphorylation of Smads and levels of its target p21 were lower at 24 h in NOX4 deleted mice. Nox4 did not appear to be essential for the termination of liver regeneration in vivo, neither for the in vitro hepatocyte response to TGF-β1 in terms of growth inhibition, which suggest its potential as therapeutic target to improve liver regeneration, without adverse effects., Graphical abstract Image 1, Highlights • General or hepatocyte-specific Nox4 deletion accelerates mice liver regeneration. • Increased hepatocyte proliferation is observed, coincident with higher Myc expression. • RNA-seq analysis reveals a role for Myc in a node of regulation of gene expression. • Transcriptional and functional attenuation of the TGF-β1 pathway is observed in vivo. • In vitro, Nox4 deleted hepatocytes maintain the growth inhibitory response to TGF-β1.

Glutathione is the major intracellular redox buffer in the liver and is critical for hepatic detoxification of xenobiotics and other environmental toxins. Hepatic glutathione is also a major systemic store for other organs and thus impacts on pathologies such as Alzheimer's disease, Sickle Cell Anaemia and chronic diseases associated with aging. Glutathione levels are determined in part by the availability of cysteine, generated from homocysteine through the transsulfuration pathway. The partitioning of homocysteine between remethylation and transsulfuration pathways is known to be subject to redox-dependent regulation, but the underlying mechanisms are not known. An association between plasma Hcy and a single nucleotide polymorphism within the NADPH oxidase 4 locus led us to investigate the involvement of this reactive oxygen species- generating enzyme in homocysteine metabolism. Here we demonstrate that NADPH oxidase 4 ablation in mice results in increased flux of homocysteine through the betaine-dependent remethylation pathway to methionine, catalysed by betaine-homocysteine-methyltransferase within the liver. As a consequence NADPH oxidase 4-null mice display significantly lowered plasma homocysteine and the flux of homocysteine through the transsulfuration pathway is reduced, resulting in lower hepatic cysteine and glutathione levels. Mice deficient in NADPH oxidase 4 had markedly increased susceptibility to acetaminophen-induced hepatic injury which could be corrected by administration of N-acetyl cysteine. We thus conclude that under physiological conditions, NADPH oxidase 4-derived reactive oxygen species is a regulator of the partitioning of the metabolic flux of homocysteine, which impacts upon hepatic cysteine and glutathione levels and thereby upon defence against environmental toxins., Highlights • Ablation of Nox4 results in lowered plasma homocysteine levels in mice. • This results from increased folate-independent remethylation of homocysteine. • As a consequence mice display reduced hepatic cysteine and glutathione. • Reduced glutathione levels render the mice susceptible to hepatotoxic agents. • Nox4 is a physiological regulator of partitioning of homocysteine metabolic flux.

Pulmonary hypertension (PH) is characterized by increased pulmonary vascular remodeling, resistance, and pressures. Reactive oxygen species (ROS) contribute to PH-associated vascular dysfunction. NADPH oxidases (Nox) and mitochondria are major sources of superoxide (O(2)(•-)) and hydrogen peroxide (H(2)O(2)) in pulmonary vascular cells. Hypoxia, a common stimulus of PH, increases Nox expression and mitochondrial ROS (mtROS) production. The interactions between these two sources of ROS generation continue to be defined. We hypothesized that mitochondria-derived O(2)(•-) (mtO(2)(•-)) and H(2)O(2) (mtH(2)O(2)) increase Nox expression to promote PH pathogenesis and that mitochondria-targeted antioxidants can reduce mtROS, Nox expression, and hypoxia-induced PH. Exposure of human pulmonary artery endothelial cells to hypoxia for 72 h increased mtO(2)(•-) and mtH(2)O(2). To assess the contribution of mtO(2)(•-) and mtH(2)O(2) to hypoxia-induced PH, mice that overexpress superoxide dismutase 2 (Tg(hSOD2)) or mitochondria-targeted catalase (MCAT) were exposed to normoxia (21% O(2)) or hypoxia (10% O(2)) for three weeks. Compared with hypoxic control mice, MCAT mice developed smaller hypoxia-induced increases in RVSP, α-SMA staining, extracellular H(2)O(2) (Amplex Red), Nox2 and Nox4 (qRT-PCR and Western blot), or cyclinD1 and PCNA (Western blot). In contrast, Tg(hSOD2) mice experienced exacerbated responses to hypoxia. These studies demonstrate that hypoxia increases mtO(2)(•-) and mtH(2)O(2). Targeting mtH(2)O(2) attenuates PH pathogenesis, whereas targeting mtO(2)(•-) exacerbates PH. These differences in PH pathogenesis were mirrored by RVSP, vessel muscularization, levels of Nox2 and Nox4, proliferation, and H(2)O(2) release. These studies suggest that targeted reductions in mtH(2)O(2) generation may be particularly effective in preventing hypoxia-induced PH.

Hypoxia stimulates pulmonary hypertension (PH) in part by increasing the proliferation of pulmonary vascular wall cells. Recent evidence suggests that signaling events involved in hypoxia-induced cell proliferation include sustained nuclear factor-kappaB (NF-κB) activation, increased NADPH oxidase 4 (Nox4) expression, and downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) levels. To further understand the role of reduced PPARγ levels associated with PH pathobiology, siRNA was employed to reduce PPARγ levels in human pulmonary artery smooth muscle cells (HPASMC) in vitro under normoxic conditions. PPARγ protein levels were reduced to levels comparable to those observed under hypoxic conditions. Depletion of PPARγ for 24-72 h activated mitogen-activated protein kinase, ERK 1/2, and NF-κB. Inhibition of ERK 1/2 prevented NF-κB activation caused by PPARγ depletion, indicating that ERK 1/2 lies upstream of NF-κB activation. Depletion of PPARγ for 72 h increased NF-κB-dependent Nox4 expression and H2O2 production. Inhibition of NF-κB or Nox4 attenuated PPARγ depletion-induced HPASMC proliferation. Degradation of PPARγ depletion-induced H2O2 by PEG-catalase prevented HPASMC proliferation and also ERK 1/2 and NF-κB activation and Nox4 expression, indicating that H2O2 participates in feed-forward activation of the above signaling events. Contrary to the effects of PPARγ depletion, HPASMC PPARγ overexpression reduced ERK 1/2 and NF-κB activation, Nox4 expression, and cell proliferation. Taken together these findings provide novel evidence that PPARγ plays a central role in the regulation of the ERK1/2-NF-κB-Nox4-H2O2 signaling axis in HPASMC. These results indicate that reductions in PPARγ caused by pathophysiological stimuli such as prolonged hypoxia exposure are sufficient to promote the proliferation of pulmonary vascular smooth muscle cells observed in PH pathobiology.

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

The endocannabinoid system (CS) has been implicated in the development of hepatic fibrosis such as schistosomiasis-associated liver fibrosis (SSLF). However, the mechanisms mediating the action of the CS in hepatic fibrosis are unclear. The present study hypothesized that Schistosoma J. infection upregulates cannabinoid receptor 1 (CB1) due to activation of NADPH oxidase leading to a fibrotic phenotype in hepatic stellate cells (HSCs). The SSLF model was developed by infecting mice with Schistosoma J. cercariae in the skin, and HSCs from control and infected mice were then isolated, cultured, and confirmed by analysis of HSC markers α-SMA and desmin. CB1 significantly increased in HSCs isolated from mice with SSLF, which was accompanied by a greater expression of fibrotic markers α-SMA, collagen I, and TIMP-1. CB1 upregulation and enhanced fibrotic changes were also observed in normal HSCs treated with soluble egg antigen (SEA) from Schistosoma J. Electron spin resonance (ESR) analysis further demonstrated that superoxide [Formula: see text] production was increased in infected HSCs or normal HSCs stimulated with SEA. Both Nox4 and Nox1 siRNA prevented SEA-induced upregulation of CB1, α-SMA, collagen I, and TIMP-1 by inhibition of [Formula: see text] production, while CB1 siRNA blocked SEA-induced fibrotic changes without effect on [Formula: see text] production in these HSCs. Taken together, these data suggest that the fibrotic activation of HSCs on Schistosoma J. infection or SEA stimulation is associated with NADPH oxidase-mediated redox regulation of CB1 expression, which may be a triggering mechanism for SSLF.

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.

Increased oxidative stress exacerbates the condition of Non-Alcoholic Fatty Liver Disease (NAFLD) which is a global pandemic, leading to its progression to the state of chronic inflammation and fibrosis (NASH). Protein phosphatase 2A (PP2A) is an enzyme that plays an important role in fibrosis, but its role in controlling the production of reactive oxygen species is unclear. Our studies show that the environmental toxin microcystin, an inhibitor of PP2A, specifically inhibits the enzyme and generates higher oxidative stress. The above process activated TGF-β fibrotic signaling pathway in the intestines that was mediated by NOX2. Results using mouse NAFLD models and transformed rat intestinal epithelial cells showed that inhibiting PP2A by administration of microcystin led to increased NOX2 activation, increased SMAD2/3-SMAD4 co-localization and increased alpha smooth muscle actin immuno-reactivity. This process was greatly attenuated in p47 phox knockout mice signifying an important role of NOX2. Mechanistically, rat intestinal epithelial cells were treated with apocynin, DMPO and phenylboronic acid which showed significantly decreased NOX2 activation, SMAD co-localization and α-SMA immuno-reactivity. A significant decrease of NOX2 and TGF-β signaling in NOX4 inhibitor treated samples indicated that there might be a possibility of dependency of NOX isoforms on each other. This study gives us hope to explore novel therapeutic strategies by increasing PP2A in situ by administration of PP2A agonists for the treatment of NASH.

NADPH oxidases (Nox) are expressed in all layers of the vessel wall and are integral to vascular physiology. Of particular interest is Nox4, which regulates vascular smooth muscle differentiation and vessel relaxation, but also has pathophysiological roles. Our laboratory has shown that polymerase delta interacting protein 2 (Poldip2) can increase the activity of Nox4. The Nox4/Poldip2 axis plays a role in cell proliferation and migration via its ability to regulate focal adhesion turnover and actin oxidation. However, Poldip2 has multiple binding partners and many additional effects on vascular pathology and physiology. Heterozygous Poldip2 mice have stiffer arteries, a reduced capacity to generate arterial force and increased collagen deposition. In addition, Poldip2 heterozygous mice have a reduced inflammatory response after ischemia in the hindlimb or following transient middle cerebral artery occlusion, which results in defective collateral formation in the hindlimb, but protection from aneurysm formation and reduced edema following stroke. The inhibition of edema results from a lack of disruption of the endothelial barrier and confers some neuroprotection. Overall, Poldip2, in part via Nox4 activity, plays a critical role in vascular physiology and pathology.

Non-toxic level of reactive oxygen species (ROS) has been reported to be involved in transducing intracellular signals and thereby regulating fundamental cell behaviors such as proliferation, differentiation and survival. Previous study showed that ROS is highly generated in pre-adipocytes and be involved in modulating pre-adipocyte differentiation. In this study, the suppressive effect of CAPE on 3T3-L1 differentiation to adipocyte through the inhibition of ROS generation was investigated. Cells were stained with Oil-Red-O to detect oil droplets in adipocytes. Also, the expression of adipogenic transcription factors such as CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator activated receptor γ (PPAR γ) was observed by western blot. Lipid accumulation in the mature adipocyte was significantly suppressed by CAPE treatment between 10 and 50 μM in a dose-dependent manner. PPARγ and C/EBPα known to be associated with adipogenic transcription factor in adipocytes were significantly attenuated by CAPE treatment. Intracellular ROS level increased to its highest level within 2 days and was dose-dependently reduced by CAPE treatment. In addition, the expression of NADPH oxidase 4 (NOX4), p22phox was suppressed and the translocation of p47phox to membrane by CAPE treatment.

Glioblastoma multiforme is a common primary brain tumor in adults and one of the most devastating human cancers. Reactive oxygen species (ROS) generated by NADPH oxidase (Nox) 4 have recently been a focus of attention in the study of glioblastomas, but the molecular mechanisms underlying the actions of Nox4 remain elusive. In this study, we demonstrated that silencing of Nox4 expression by Nox4-targeted siRNA suppressed cell growth and motility of glioblastoma U87 cells, indicating the involvement of Nox4. Furthermore, Nox4-derived ROS oxidized and inactivated protein tyrosine phosphatase (PTP):1B: PTP1B in its active form downregulates cell proliferation and migration. By affinity purification with the substrate-trapping mutant of PTP1B, tyrosine-phosphorylated coronin-1C was identified as a substrate of PTP1B. Its tyrosine phosphorylation level was suppressed by Nox4 inhibition, suggesting that tyrosine phosphorylation of coronin-1C is regulated by the Nox4-PTP1B pathway. Finally, ablation of coronin-1C attenuated the proliferative and migratory activity of the cells. Collectively, these findings reveal that Nox4-mediated redox regulation of PTP1B serves as a modulator, in part through coronin-1C, of the growth and migration of glioblastoma cells, and provide new insight into the mechanistic aspect of glioblastoma malignancy.

The prevalence of metabolic syndrome persistently increases and affects over 30% of U.S. adults. To study how metabolic syndrome may induce tubulointerstitial injury and whether acetaminophen has renal-protective properties, 4-week-old obese Zucker rats were randomly assigned into three groups, control (OC), vehicle dimethyl sulfoxide (OV), and acetaminophen treatment (30mg/kg/day for 26 weeks), and lean Zucker rats served as healthy controls. Significant tubulointerstitial injuries were observed in both OC and OV animals, evidenced by increased tubular cell death, tubular atrophy/dilation, inflammatory cell infiltration, and fibrosis. These tubulointerstitial alterations were significantly reduced by treatment with a chronic but low dose of acetaminophen, which acted to diminish NADPH oxidase isoforms Nox2 and Nox4 and decrease tubulointerstitial oxidative stress (reduced tissue superoxide and macromolecular oxidation). Decreased oxidative stress by acetaminophen was paralleled by the reduction of tubular proapoptotic signaling (diminished Bax/Bcl-2 ratio and caspase 3 activation) and the alleviation of tubular epithelial-to-mesenchymal transition (decreased transforming growth factor β, connective tissue growth factor, α-smooth muscle actin, and laminin). These data suggest that increased oxidative stress plays a critical role in mediating metabolic syndrome-induced tubulointerstitial injury and provide the first evidence suggesting that acetaminophen may be of therapeutic benefit for the prevention of tubulointerstitial injury.

Peroxynitrite, the reaction product of nitric oxide and superoxide, contributes to the pathogenesis of chronic pulmonary hypertension in immature animals by stimulating proliferation of pulmonary arterial smooth muscle cells (PASMCs). Pulmonary vasoconstriction, secondary to hypoxia and other stimuli, leads to enhanced pulsatile stretch of cells in the vascular wall, particularly in smooth muscle, which we hypothesized would cause increased peroxynitrite generation. Our objectives in this study were to determine whether cyclic mechanical stretch, at supraphysiologic levels, would cause increased production of reactive oxygen species (ROS), nitric oxide, and peroxynitrite in vitro. Early passage neonatal rat PASMCs were seeded and grown to subconfluence on collagen-coated elastomer-bottom plates and subjected to cyclic mechanical stretch (10% (“physiologic”) or 20% (“supraphysiologic”) at 0.5 Hz) for up to 24 h. Compared to nonstretched controls and to cells subjected to 10% stretch, 20% stretch increased H 2 O 2 (stable marker of ROS) and nitrate/nitrite (stable marker of nitric oxide) in conditioned medium. These effects were accompanied by increased peroxynitrite, as evidenced by increased in situ dihydroethidium fluorescence and immunoreactive nitrotyrosine and by increased expression of nitric oxide synthase (NOS)-1 and NADPH oxidase 4 (NOX4), but not NOS-2. Stretch-induced H 2 O 2 release and increased dihydroethidium fluorescence were prevented by pretreatment with a superoxide scavenger, nonspecific inhibitors of NADPH oxidase or NOS, or a peroxynitrite decomposition catalyst. Short-interfering RNA-mediated knockdown of NOS-1 or NOX4 attenuated increased nitric oxide and H 2 O 2 content, respectively, in stretched-cell-conditioned medium. Knockdown of NOS-1 also attenuated increased immunoreactive nitrotyrosine content and stretch-induced proliferation, whereas knockdown of NOS-2 had no effect. We conclude that increased peroxynitrite generation by neonatal rat PASMCs subjected to supraphysiologic levels of cyclic stretch is NOS-1-dependent and that increased ROS production is predominantly mediated by NADPH oxidase, specifically NOX4.

Endothelial cell (EC) migration in response to vascular endothelial growth factor (VEGF) is a critical step in both physiological and pathological angiogenesis. Although VEGF signaling has been extensively studied, the mechanisms by which VEGF-dependent reactive oxygen species (ROS) production affects EC signaling are not well understood. The aim of this study was to elucidate the involvement of Nox2- and Nox4-dependent ROS in VEGF-mediated EC Ca(2+) regulation and migration. VEGF induced migration of human aortic ECs into a scratch wound over 6 h, which was inhibited by overexpression of either catalase or superoxide dismutase (SOD). EC stimulation by micromolar concentrations of H2O2 was inhibited by catalase, but also unexpectedly by SOD. Both VEGF and H2O2 increased S-glutathiolation of SERCA2b and increased Ca(2+) influx into EC, and these events could be blocked by overexpression of catalase or overexpression of SERCA2b in which the reactive cysteine-674 was mutated to a serine. In determining the source of VEGF-mediated ROS production, our studies show that specific knockdown of either Nox2 or Nox4 inhibited VEGF-induced S-glutathiolation of SERCA, Ca(2+) influx, and EC migration. Treatment with H2O2 induced S-glutathiolation of SERCA and EC Ca(2+) influx, overcoming the knockdown of Nox4, but not Nox2, and Amplex red measurements indicated that Nox4 is the source of H2O2. These results demonstrate that VEGF stimulates EC migration through increased S-glutathiolation of SERCA and Ca(2+) influx in a Nox4- and H2O2-dependent manner, requiring Nox2 downstream.

The dopamine D(2) receptor (D(2)R) regulates renal reactive oxygen species (ROS) production, and impaired D(2)R function results in ROS-dependent hypertension. Paraoxonase 2 (PON2), which belongs to the paraoxonase gene family, is expressed in various tissues, acting to protect against cellular oxidative stress. We hypothesized that PON2 may be involved in preventing excessive renal ROS production and thus may contribute to maintenance of normal blood pressure. Moreover, D(2)R may decrease ROS production, in part, through regulation of PON2. D(2)R colocalized with PON2 in the brush border of mouse renal proximal tubules. Renal PON2 protein was decreased (-33±6%) in D(2)(-/-) relative to D(2)(+/+) mice. Renal subcapsular infusion of PON2 siRNA decreased PON2 protein expression (-55%), increased renal oxidative stress (2.2-fold), associated with increased renal NADPH oxidase expression (Nox1, 1.9-fold; Nox2, 2.9-fold; and Nox4, 1.6-fold) and activity (1.9-fold), and elevated arterial blood pressure (systolic, 134±5 vs 93±6mmHg; diastolic, 97±4 vs 65±7mmHg; mean 113±4 vs 75±7mmHg). To determine the relevance of the PON2 and D(2)R interaction in humans, we studied human renal proximal tubule cells. Both D(2)R and PON2 were found in nonlipid and lipid rafts and physically interacted with each other. Treatment of these cells with the D(2)R/D(3)R agonist quinpirole (1μM, 24h) decreased ROS production (-35±6%), associated with decreased NADPH oxidase activity (-32±3%) and expression of Nox2 (-41±7%) and Nox4 (-47±8%) protein, and increased expression of PON2 mRNA (2.1-fold) and protein (1.6-fold) at 24h. Silencing PON2 (siRNA, 10nM, 48h) not only partially prevented the quinpirole-induced decrease in ROS production by 36%, but also increased basal ROS production (1.3-fold), which was associated with an increase in NADPH oxidase activity (1.4-fold) and expression of Nox2 (2.1-fold) and Nox4 (1.8-fold) protein. Inhibition of NADPH oxidase with diphenylene iodonium (10μM/30 min) inhibited the increase in ROS production caused by PON2 silencing. Our results suggest that renal PON2 is involved in the inhibition of renal NADPH oxidase activity and ROS production and contributes to the maintenance of normal blood pressure. PON2 is positively regulated by D(2)R and may, in part, mediate the inhibitory effect of renal D(2)R on NADPH oxidase activity and ROS production.

Specific inhibitors of the production of reactive oxygen species (ROS) by the NADPH oxidases (Nox's) are potentially important therapeutic agents in the wide range of human diseases that are characterized by excessive ROS production. It has been proposed that VAS2870 (3-benzyl-7-(2-benzoxazolyl)thio-1,2,3- triazolo[4,5-d]pyrimidine), identified as an inhibitor of Nox2 by small-molecule screening, may serve as an example of such an agent. Here we show that VAS2870 inhibits ROS production in the sarcoplasmic reticulum (SR) of mammalian skeletal muscle, previously identified with Nox4, and thereby abrogates O(2)-coupled redox regulation of the ryanodine receptor-Ca(2+) channel (RyR1). However, we also find that VAS2870 modifies directly identified cysteine thiols within RyR1. Mass spectrometric analysis of RyR1 exposed in situ to VAS2870 and of VAS2870-treated glutathione indicated that thiol modification is through alkylation by the benzyltriazolopyrimidine moiety of VAS2870. Thus, VAS2870 exerts significant off-target effects, and thiol alkylation by VAS2870 (and closely related Nox inhibitors) may in fact replicate some of the effects of ROS on cellular thiol redox status. In addition, we show that SR-localized Nox4 is inhibited by other thiol-alkylating agents, consistent with a causal role for cysteine modification in the inhibition of ROS production by VAS2870.

There is current discussion whether reactive oxygen species are up- or downregulated in the pulmonary circulation during hypoxia, from which sources (i.e., mitochondria or NADPH oxidases) they are derived, and what the downstream targets of ROS are. We recently showed that the NADPH oxidase homolog NOX4 is upregulated in hypoxia-induced pulmonary hypertension in mice and contributes to the vascular remodeling in pulmonary hypertension. We here tested the hypothesis that NOX4 regulates K(v) channels via an increased ROS formation after prolonged hypoxia. We showed that (1) NOX4 is upregulated in hypoxia-induced pulmonary hypertension in rats and isolated rat pulmonary arterial smooth muscle cells (PASMC) after 3days of hypoxia, and (2) that NOX4 is a major contributor to increased reactive oxygen species (ROS) after hypoxia. Our data indicate colocalization of K(v)1.5 and NOX4 in isolated PASMC. The NADPH oxidase inhibitor and ROS scavenger apocynin as well as NOX4 siRNA reversed the hypoxia-induced decrease in K(v) current density whereas the protein levels of the channels remain unaffected by siNOX4 treatment. Determination of cysteine oxidation revealed increased NOX4-mediated K(v)1.5 channel oxidation. We conclude that sustained hypoxia decreases K(v) channel currents by a direct effect of a NOX4-derived increase in ROS.

Redox signaling contributes to the regulation of cancer cell proliferation, survival and invasion, and participates in the adaptation of cancer cells to their microenvironment. NADPH oxidases are important mediators of redox signaling in normal and cancer cells. Redox signal specificity in normal cells is in part achieved by targeting enzymes that generate reactive oxygen species to specific subcellular microdomains such as focal adhesions, dorsal ruffles, lipid rafts or caveolae. In a similar fashion, redox signal specificity during cancer cell invasion can be regulated by targeting reactive oxygen generation to invasive microdomains such as invadopodia. Here we summarize recent advances in the understanding of the redox signaling processes that control the cancer cell pro-invasive program by modulating cell adhesion, migration and proteolysis as well as the interaction of cancer cells with the tumor microenvironment. We will focus on redox signaling events mediated by invadopodia NADPH oxidase complexes and their contribution to cancer cell invasion.

Introduction NADPH oxidase (Nox)-mediated generation of reactive oxygen species (ROS) and Rho GTPase signaling play a vital role in vascular smooth muscle cell (VSMC) physiology and their aberrant signaling contributes to vascular disease. Polymerase δ-interacting protein 2 (Poldip2) modulates both Nox and Rho activity, yet the mechanisms are unknown. Here we explore how Poldip2 enhances Rho GTPase activation. Methods A nucleotide-free-RhoA pulldown followed by mass spectrometry was used to identify Rho activators, guanine nucleotide exchange factors (GEFs), that are stimulated upon Poldip2 overexpression. These pulldowns followed by western blot were subsequently used to examine the activity of the identified GEF. Co-immunoprecipitation experiments were performed to look for direct interaction and immuno-fluorescence microscopy was used to examine subcellular localization and cytokinesis defects (via multinucleated cells). Finally, proliferation was examined by counting cells post-treatment with siRNA. Results The RhoGEF, Epithelial cell transforming sequence 2 (Ect2), is activated by overexpression of Poldip2 (>2-fold, p=2e-7) in VSMCs. However, no interaction was observed between Poldip2 and Ect2, nor did Poldip2 regulate Ect2 subcellular localization. Nox4 is not required for Poldip2-driven Ect2 activation, as Ect2 activation in Nox4 knockout VSMCs is even more dramatic (>5 fold, p=0.001) than in wild type cells. Instead, depletion of Nox1 inhibits the activation of Ect2 by Poldip2. Consistent with ROS derived from Nox1 activating Ect2, the ROS scavenger N-acetyl-cysteine blocks Poldip2-driven Ect2 activation. Depletion of Ect2 or Poldip2 inhibits VSMC proliferation (p Conclusions Poldip2 activates Ect2, via ROS generated by Nox1, to regulate VSMC cytokinesis and promote proliferation.

Previously, we have shown that TGF-beta induced NADPH oxidase 4 (NOX4) expression is involved in the epithelial-to-mesenchymal transition (EMT), a process critical for cancer metastasis. Further, we found that wild-type and tumor-associated mutated forms of p53 cause divergent effects on TGF-beta induction of NOX4: wild-type (WT-p53) suppresses whereas mutant p53 (mut-p53) augments NOX4 expression in several established tumor cell models. We sought to confirm and extend our findings using publically-available sequencing data from primary tumor samples in the Cancer Genome Atlas (TCGA). Here, we report that NOX4 is differentially expressed depending on tumor type and histopathological stages. To investigate the broad significance of our previous model in many cancers, we created a pan-cancer cohort of over 23 types of primary tumors. NOX4 strongly correlates with EMT gene signatures relative to other isoforms in the NOX family in this pan-cancer cohort. We found that the strength of correlation is dependent on specific p53 hot-spot mutations. Interestingly, increased NOX4 expression is associated with poor survival outcome in patients with mut-p53, whereas increased NOX4 is correlated with better survival in patients with WT-p53. We uncovered other divergent effects of p53 mutation status on apoptosis and proliferation marker mRNA levels as a function of NOX4 that may further support roles for NOX4 in cancer progression.

The objective of this work was to analyze the possible association between cyclooxygenase-2 (COX-2) and NADPH oxidases (NOX) in liver cells, in response to various proinflammatory and toxic insults. First, we observed that treatment of Chang liver (CHL) cells with various COX-2 inducers increased reactive oxygen species (ROS) production concomitant with GSH depletion, phorbol 12-myristate 13-acetate (PMA) being the most effective treatment. Moreover, early changes in the oxidative status induced by PMA were inhibited by glutathione ethyl ester, which also impeded COX-2 induction. In fact, CHL cells expressed NOX1 and NOX4, although only NOX4 expression was up-regulated in the presence of PMA. Knock-down experiments suggested that PMA initiated a pathway in which NOX1 activation controlled COX-2 expression and activity, which, in turn, induced NOX4 expression by activation of the prostaglandin receptor EP4. In addition, CHL cells overexpressing COX-2 showed higher NOX4 expression and ROS content, which were decreased in the presence of the COX-2 inhibitor DFU. Interestingly, we found that addition of prostaglandin E2 (PGE2) also induced NOX4 expression and ROS production, which might promote cell adhesion. Finally, we determined that NOX4 induction by PGE2 was dependent on ERK1/2 signaling. Taken together, these results indicate that NOX proteins and COX-2 are reciprocally regulated in liver cells., This work was supported by grants from the Ministerio de Ciencia e Innovación, Spain (BFU2009-07219 and ISCIII-RTICC RD06/0020 to I.F.; SAF2007-60551 and SAF2010-16037 to P.M.-S), and AGAUR–Generalitat de Catalunya (2009SGR-312, to I.F.). P.S. was the recipient of a postdoctoral contract from the Instituto de Salud Carlos III.

In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2, and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In this study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B-loop of Nox2, would inhibit ROS production by the Nox2-, but not the Nox1- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1-, or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O(2)(•-)) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance confirmed that Nox2ds inhibits O(2)(•-) production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production by either Nox1- or Nox4-oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2-oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent.

Ascorbic acid (AA) increases cardiomyogenesis of embryonic stem (ES) cells. Herein we show that treatment of mouse ES cells with AA enhanced cardiac differentiation accompanied by an upregulation of the NADPH oxidase isoforms NOX2 and NOX4, phosphorylation of endothelial nitric oxide synthase (eNOS), and cyclic GMP (cGMP) formation, indicating that reactive oxygen species (ROS) as well as nitric oxide (NO) may be involved in cardiomyogenesis. In whole mount embryoid bodies as well as isolated Flk-1-positive (Flk-1(+)) cardiovascular progenitor cells ROS elevation by AA was observed in early stages of differentiation (Days 4-7), and absent at Day 10. In contrast NO generation following incubation with AA was absent at Day 4 and increased at Days 7 and 10. AA-mediated cardiomyogenesis was blunted by the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin, the free radical scavengers N-(2-mercaptopropionyl)-glycine (NMPG) and ebselen, and the NOS inhibitor L-NAME. Downregulation of NOX4 by short hairpin RNA (shRNA) resulted in significant inhibition of cardiomyogenesis and abolished the stimulation of MHC-ß and MLC2v gene expression observed on AA treatment. Our data demonstrate that AA stimulates cardiomyocyte differentiation from ES cells by signaling pathways that involve ROS generated at early stages and NO at late stages of cardiomyogenesis.

Reactive oxygen species (ROS) generated by vascular endothelial and smooth muscle cells contribute to the development and progression of vascular diseases. We have recently shown that hyperoxia enhances NADPH oxidase 4 (Nox4) expression, which regulates lung endothelial cell migration and angiogenesis. Regulation of Nox4 in the vasculature is poorly understood. The objective of this study was to identify the transcriptional factor(s) involved in regulation of endothelial Nox4. We found that hyperoxia-induced Nox4 expression was markedly reduced in Nrf2(-/-) mice, compared to Nrf2(+/+) mice. Exposure of human lung microvascular endothelial cells (HLMVECs) to hyperoxia stimulated Nrf2 translocation from the cytoplasm to the nucleus and increased Nox4 expression. Knockdown of Nrf2 expression using an siRNA approach attenuated basal Nox4 expression; however, it enhanced superoxide/ROS generation under both normoxia and hyperoxia. In silico analysis revealed the presence of at least three consensus sequences for the antioxidant response element (ARE) in the promoter region of Nox4. In transient transfections, hyperoxia stimulated Nox4 promoter activity in HLMVECs, and deletion of the -438 to -458 and -619 to -636 sequences markedly reduced hyperoxia-stimulated Nox4 promoter activation. ChIP analysis revealed an enhanced recruitment of Nrf2 to the endogenous Nox4 promoter spanning these two AREs after hyperoxic insult. Collectively, these results demonstrate, for the first time, a novel role for Nrf2 in regulating hyperoxia-induced Nox4 transcription via AREs in lung endothelium.

In contrast to other cell types, vascular smooth muscle cells modify their phenotype in response to external signals. NADPH oxidase 4 (Nox4) is critical for maintenance of smooth muscle gene expression; however, the underlying mechanisms are incompletely characterized. Using smooth muscle α-actin (SMA) as a prototypical smooth muscle gene and transforming growth factor-β (TGF-β) as a differentiating agent, we examined Nox4-dependent signaling. TGF-β increases Nox4 expression and activity in human aortic smooth muscle cells (HASMC). Transfection of HASMC with siRNA against Nox4 (siNox4) abolishes TGF-β-induced SMA expression and stress fiber formation. siNox4 also significantly inhibits TGF-β-stimulated p38MAPK phosphorylation, as well as that of its substrate, mitogen-activated protein kinase-activated protein kinase-2. Moreover, the p38MAPK inhibitor SB-203580 nearly completely blocks the SMA increase induced by TGF-β. Inhibition of either p38MAPK or NADPH oxidase-derived reactive oxygen species impairs the TGF-β-induced phosphorylation of Ser103 on serum response factor (SRF) and reduces its transcriptional activity. Binding of SRF to myocardin-related transcription factor (MRTF) is also necessary, because downregulation of MRTF by siRNA abolishes TGF-β-induced SMA expression. Taken together, these data suggest that Nox4 regulates SMA expression via activation of a p38MAPK/SRF/MRTF pathway in response to TGF-β.

There is ambiguity as to the biologic function of the Nox4 NADPH oxidase with evidence that it is both protective and deleterious in the cardiovascular system. The constitutive activity of Nox4 suggests that the reactive oxygen species (ROS) generated do not function primarily as second messengers. Furthermore, regulation of Nox4 activity is mediated primarily by changes in expression levels. Regulation of alternative splicing and functional effects of Nox4 isoforms are not known. We hypothesized that changes in Nox4 isoform expression and subcellular distribution regulate subcellular redox state and specific cellular processes. We found that Nox4 is differentially spliced in atherosclerotic monkey aortic smooth muscle cells (SMC) with an increase in the shorter, non-transmembrane Nox4D and a decrease in the transmembrane form Nox4A. Analysis of the Nox4 3’UTR identified putative binding sites for the microRNAs miR-9 and miR-25 and these miRNAs were elevated in the SMCs isolated from aorta of atherosclerotic monkeys. Similarly, the overexpression of miR-9 or miR-25 in cultured SMCs increased Nox4D expression and decreased Nox4A expression. Using subcellular-targeted HyPer probes, the overexpression of Nox4A increased ROS in the mitochondria, cytosol and nucleus, whereas overexpression of Nox4D predominately showed increased ROS in the nucleus. Changes in Nox4 expression differentially regulate transcriptional activation of the antioxidant response element and expression of HIF-1α. Knockdown of Nox4 decreased NADP+/NADPH and GSSG/2GSH couples while overexpression increased these redox couples. In addition, changes in Nox4 levels resulted in protein-specific changes in thiol oxidation. These data suggest that Nox4 splicing is in part regulated by miR-9 and miR-25 and the dynamic regulation of subcellular redox status by Nox4 isoforms is a mechanism by which the cell integrates information and coordinates complex signaling pathways.

Gap junctions (Gjs), formed by specific protein termed connexins (Cxs), are intercellular channels that regulate many cell responses, including oxidation, inflammation, and cell damage. Currently, the mechanisms involved are still poorly understood. Given that NLRP3 inflammasome activation is a pivotal cellular event linking oxidative stress, inflammatory responses, and cell injury, we tested the possible regulation of inflammasome by Gjs and explored the potential mechanisms. Exposure of mouse peritoneal macrophages (PMs) to LPS plus ATP caused NLRP3 inflammasome activation, together with an increased Cx43. Inhibition of Cx channels blunted inflammasome activation. Consistently, PMs from Cx43 heterozygous mouse (Cx43+/-) exhibited a weak inflammasome activation, in comparison with those from Cx43+/+ mouse. Further analysis revealed that inflammasome activation was associated with an early increase in NADPH oxidase 2 (NOX2), protein carbonylation and MAPK activation. Suppression of ROS with antioxidant, or inhibition of NADPH oxidase or MAPK kinases with inhibitors blocked Cx43 elevation and inflammasome activation. Intriguingly, the suppression of Cx43 also blunted NOX2 expression, protein carbonylation, p38 phosphorylation and inflammasome activation. It also inhibited TXNIP expression. In a model of acute renal injury induced by LPS, Cx43+/- mouse exhibited a significantly lower level of blood IL-1β, blood urea nitrogen, and urinary protein, together with a milder renal pathological change and renal expression of NLRP3 and NOX4, as compared with Cx43+/+ mouse. Moreover, inhibition of Cx channels suppressed IL-1β- and TNF-α-induced expression of NOX4 in glomerular podocytes and tubular epithelial cells. Collectively, our study characterized Cx43 as a novel determinant of inflammasome activation and inflammatory cell injury through a mechanism involving its regulation on cellular redox status. Our study thus provides novel mechanistic insight into the actions of Cx43 and suggests that it could be a promising therapeutic target for the treatment of certain inflammatory diseases.

NADPH oxidase (Nox) variants Nox1, Nox2 and Nox4 have been implicated in the progression of liver fibrosis. However, the role of Nox5 is unknown, mainly due to the lack of this enzyme in rat and mouse genomes. Here we describe the expression and functional relevance of Nox5 in the human cell line of hepatic stellate cells (HSC), LX-2. Under basal conditions, these cells expressed a long (Nox5L) and a short (Nox5S) variant which were silenced with specific siRNAs for Nox5. Overexpression of Nox5L generated ROS in the presence of calcium, as judged by the production of extracellular hydrogen peroxide, L-012 luminescence and cytochrome c reduction, while Nox5S did not generated ROS under these conditions. In contrast, dihydroethidium oxidation was increased when either Nox5L or Nox5S were overexpressed. Functional studies revealed that both Nox5L and Nox5S stimulated the proliferation of LX-2 cells and the synthesis of type I collagen, while Nox5 siRNAs inhibited these effects. Interestingly, TGF-beta and angiotensin II induced Nox5, and silencing Nox5 reduced collagen production stimulated by TGF-beta. Collectively, these results suggest for the first time that Nox5 can play a relevant role in HSC proliferation and fibrogenesis.

Reactive oxygen species (ROS) derived from NADPH oxidase (NOX) as well as mitochondria play a critical role in promoting angiogenic switch from a quiescent endothelial cells (ECs). However, the mechanism by which ROS produced from different sources can organize to enhance VEGF signaling and angiogenic process remains unknown. Using the cytosol- and mitochondria-targed redox-sensitive RoGFP biosensors with real-time imaging, we found that VEGF stimulation in human ECs rapidly increases Nox4-derived H2O2 that activates Nox2 to produce ROS in the cytosol, which in turn induce mitochondrial ROS (mtROS) production via phosphorylation of p66Shc at Ser36. This ROS-induced ROS release orchestrated by the Nox4/Nox2/pS36-p66Shc/mtROS axis promotes activation and sustained angiogenesis signaling program. Moreover, emerging evidence suggest that EC metabolism and mitochondria dynamics regulated by fission and fusion also regulate angiogenesis; however, their relationships with redox signaling remains unclear. Here we provide the novel mechanism for redox regulation of mitochondria fission protein Drp1 and for how VEGF-induced ROS are sensed via Cysteine oxidation to promote glycolysis driving formation of new blood vessels.

Cerebral Cavernous Malformation (CCM) is a neurovascular disease characterized by abnormal vascular structures associated with defects on vascular lumen formation and vascular fragility, mostly localized in the brain. Lack of KRIT1 protein associated to CCM drives the stable and quiescent endothelium toward an active state characterized by loss of barrier function, increased vascular permeability, loss of adherens junction integrity, increased β-catenin nuclear localization and vascular endothelial growth factor (VEGF) expression and angiogenesis. Multiple pathways are responsible for changes in endothelial phenotype and we contributed to demonstrate that the dysregulation of reactive oxygen species (ROS) production following loss of KRIT1 expression play a key role in increasing vascular permeability and is linked to NADPH oxidase (NOX) up-regulation. By using wt and KRIT1-/- mouse embryonic fibroblasts (MEFs) we showed that loss of KRIT1 induced NOX4 overexpression that is associated with alteration of angiogenic growth factors expression and with the angiogenic switch of endothelial cells. Our data contribute to demonstrate the pivotal role of oxidative stress in the progression of CCM disease, indicating NOX4 and ROS as potential targets for pharmaceutical approaches.