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Harald H H W Schmidt - One of the best experts on this subject based on the ideXlab platform.
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Isoform-specific NADPH oxidase inhibition for pharmacological target validation
bioRxiv, 2018Co-Authors: Sebastian Altenhöfer, Mahmoud H. Elbatreek, Ana I. Casas, Peter Lijnen, Merlijn J. Meens, Ulla G. Knaus, Harald H H W SchmidtAbstract:Abstract Unphysiological reactive oxygen species (ROS) formation is considered an important pathomechanism for several diseasephenotypes with high unmet medical need. After the clinical failure of antioxidants, inhibition of disease-relevant enzymatic sources of ROS appears to be the most promising alternative approach. With respect to most promising drug target, NADPH oxidases (NOXs) stand out, however, validation has been restricted mainly to genetically modified mice. Validation in other species including human is lacking and it is unclear whether the different NOX isoforms are sufficiently distinct for selective pharmacological inhibition. Here we show for the five most advanced NOX inhibitors that pharmacological isoform selectivity can be achieved. NOX1 was most potently (IC50) targeted by ML171 (0.1 μM); NOX2, by VAS2870 (0.7 μM); NOX4, by M13 (0.01 μM) and NOX5, by ML090 (0.01 μM). Of note, previously unrecognised non-specific antioxidant and assay artefacts may limit interpretations in some systems, which included, surprisingly, the clinically most advanced compound, GKT136901. As proof-of-principle for our pharmacological target validation approach, we used a human blood-brain barrier model and our NOX inhibitor panel at IC50 concentrations. Indeed, the protective efficacy pattern of this compound panel pointed towards a functional role NOX4 confirming previous genetic targeting. These findings strongly encourage further lead optimisation efforts for isoform-selective NOX inhibitors and their clinical development and provide already now an experimental alternative when genetic targeting of NOXs is not an option.
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differential effects of NOX4 and nox1 on immune cell mediated inflammation in the aortic sinus of diabetic apoe mice
Clinical Science, 2016Co-Authors: Elyse Di Marco, Stephen P Gray, Phyllis Chew, Kit Kennedy, Karin Jandeleitdahm, Harald H H W Schmidt, Mark E. CooperAbstract:Oxidative stress and inflammation are central mediators of atherosclerosis particularly in the context of diabetes. The potential interactions between the major producers of vascular reactive oxygen species (ROS), NADPH oxidase (NOX) enzymes and immune-inflammatory processes remain to be fully elucidated. In the present study we investigated the roles of the NADPH oxidase subunit isoforms, NOX4 and NOX1, in immune cell activation and recruitment to the aortic sinus atherosclerotic plaque in diabetic ApoE −/− mice. Plaque area analysis showed that NOX4- and NOX1-derived ROS contribute to atherosclerosis in the aortic sinus following 10 weeks of diabetes. Immunohistochemical staining of the plaques revealed that NOX4-derived ROS regulate T-cell recruitment. In addition, NOX4-deficient mice showed a reduction in activated CD4 + T-cells in the draining lymph nodes of the aortic sinus coupled with reduced pro-inflammatory gene expression in the aortic sinus. Conversely, NOX1-derived ROS appeared to play a more important role in macrophage accumulation. These findings demonstrate distinct roles for NOX4 and NOX1 in immune-inflammatory responses that drive atherosclerosis in the aortic sinus of diabetic mice.
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neuroprotection after stroke by targeting NOX4 as a source of oxidative stress
Antioxidants & Redox Signaling, 2013Co-Authors: Kim A. Radermacher, Sebastian Altenhöfer, Kirstin Wingler, Pamela W. M. Kleikers, Christoph Kleinschnitz, Friederike Langhauser, J Rob J Hermans, Martin Hrabě De Angelis, Harald H H W SchmidtAbstract:Significance: Stroke, a leading cause of death and disability, poses a substantial burden for patients, relatives, and our healthcare systems. Only one drug is approved for treating stroke, and more than 30 contraindications exclude its use in 90% of all patients. Thus, new treatments are urgently needed. In this review, we discuss oxidative stress as a pathomechanism of poststroke neurodegeneration and the inhibition of its source, type 4 nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX4), as a conceptual breakthrough in stroke therapy. Recent Advances: Among potential sources of reactive oxygen species (ROS), the NOXes stand out as the only enzyme family that is solely dedicated to forming ROS. In rodents, three cerebrovascular NOXes exist: the superoxide-forming NOX1 and 2 and the hydrogen peroxide-forming NOX4. Studies using NOX1 knockout mice gave conflicting results, which overall do not point to a role for this isoform. Several reports find NOX2 to be relevant in stroke, albeit to variable and moderate degrees. In our hands, NOX4 is, by far, the major source of oxidative stress and neurodegeneration on ischemic stroke. Critical Issues: We critically discuss the tools that have been used to validate the roles of NOX in stroke. We also highlight the relevance of different animal models and the need for advanced quality control in preclinical stroke research. Future Directions: The development of isoform-specific NOX inhibitors presents a precious tool for further clarifying the role and drugability of NOX homologues. This could pave the avenue for the first clinically effective neuroprotectant applied poststroke, and even beyond this, stroke could provide a proof of principle for antioxidative stress therapy. Antioxid. Redox Signal. 18, 1418–1427.
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VAS2870 is a pan-NADPH oxidase inhibitor
Cellular and Molecular Life Sciences, 2012Co-Authors: Kirstin Wingler, Sebastian A. Altenhoefer, Pamela W. M. Kleikers, Kim A. Radermacher, Christoph Kleinschnitz, Harald H H W SchmidtAbstract:In their detailed review ‘‘NADPH oxidases as therapeutic targets in ischemic stroke’’ [1], Kahles and Brandes discuss the pathological roles of NADPH oxidases in ischemic brain injury and the therapeutic implications. In agreement with the authors, we consider inhibition of NADPH oxidases as a promising strategy to treat ischemic stroke. As described in the review, we recently reported that NOX4-deficient mice are largely protected from brain damage caused by ischemic stroke, whereas we did not observe any effects by deleting NOX1 or NOX2. Thus, we believe that NOX4 is a highly promising target for stroke therapy. To further support our findings, we treated wildtype and NOX4 knockout mice with the NADPH oxidase inhibitor VAS2870 in a therapeutically relevant time window, i.e., post-stroke. Indeed, in wild-type mice, inhibition of NADPH oxidases by VAS2870 resulted in a similar degree of protection as did deletion of NOX4. In contrast, in NOX4 knockout mice, VAS2870 did not have any additional effects in reducing ischemic brain damage. This further supports our statement that NOX4, and not other NOX isoforms, is the likely detrimental NOX isoform in ischemic stroke in mice. Unfortunately, to the best of our knowledge, there was and is no NOX4-selective inhibitor that could have been used to further support our findings. Kahles and Brandes correctly describe that VAS2870 inhibits NOX1 and NOX2 and cite our relevant publications [2–4]. In the same issue of this journal, we provided evidence that VAS2870 also inhibits NOX4 [5]. In conclusion, we believe that VAS2870 is a pan-NOX inhibitor and not selective for any NOX isoform. However, in their review, Kahles and Brandes [1] state that we concluded that ‘‘VAS2870 was a NOX4-specific inhibitor based on the fact that the compound had no effect on the small infarcts they produced in NOX4 knockout mice’’. This is not true. We have never published such a statement on the NOX isoform-specificity of VAS2870. Both in the respective paper [6] and our other publications we describe VAS2870 as an NADPH oxidase inhibitor with no relevant specificity for any NOX isoform (data on NOX3 are not available) [5]. We have published similar data on the closely related derivative of VAS2870, VAS3947 [7]. Thus, we would kindly ask the authors to revoke their statement.
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oxidative stress and endothelial dysfunction in aortas of aged spontaneously hypertensive rats by nox1 2 is reversed by nadph oxidase inhibition
Hypertension, 2010Co-Authors: Sven Wind, Daniel Janowitz, Christina Neff, Arun H. S. Kumar, Knut Beuerlein, Melanie E Armitage, Ashraf Taye, Kirstin Wingler, Ajay M. Shah, Harald H H W SchmidtAbstract:Arterial hypertension is associated with increased levels of reactive oxygen species, which may scavenge endothelium-derived NO and thereby diminish its vasorelaxant effects. However, the quantitatively relevant source of reactive oxygen species is unclear. Thus, this potential pathomechanism is not yet pharmacologically targetable. Several enzymatic sources of reactive oxygen species have been suggested: uncoupled endothelial NO synthase, xanthine oxidase, and NADPH oxidases. Here we show that increased reactive oxygen species formation in aortas of 12- to 14-month–old spontaneously hypertensive rats versus age-matched Wistar Kyoto rats is inhibited by the specific NADPH oxidase inhibitor VAS2870 but neither by the xanthine oxidase inhibitor oxypurinol nor the NO synthase inhibitor N G -nitro-l-arginine methyl ester. NADPH oxidase activity, as well as protein expression of its catalytic subunits, NOX1 and NOX2, was increased in the aortas of spontaneously hypertensive rats, whereas the expression of NOX4 protein, the most abundant NOX isoform, was not significantly changed. Impaired acetylcholine-induced relaxation of spontaneously hypertensive rat aortas was significantly improved by VAS2870. In conclusion, NOX1 and NOX2 but not NOX4 proteins are increased in aged spontaneously hypertensive rat aortas. Importantly, these NOX isoforms, in particular, ectopic expression of NOX1 in endothelial cells, appear to affect vascular function in an NADPH oxidase inhibitor-reversible manner. NADPH oxidases may, thus, be a novel target for the treatment of systemic hypertension.
Rhian M Touyz - One of the best experts on this subject based on the ideXlab platform.
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angiotensin ii nadph oxidase and redox signaling in the vasculature
Antioxidants & Redox Signaling, 2013Co-Authors: Augusto C Montezano, Dylan Burger, Rhian M TouyzAbstract:Significance: Angiotensin II (Ang II) influences the function of many cell types and regulates many organ systems, in large part through redox-sensitive processes. In the vascular system, Ang II is a potent vasoconstrictor and also promotes inflammation, hypertrophy, and fibrosis, which are important in vascular damage and remodeling in cardiovascular diseases. The diverse actions of Ang II are mediated via Ang II type 1 and Ang II type 2 receptors, which couple to various signaling molecules, including NADPH oxidase (Nox), which generates reactive oxygen species (ROS). ROS are now recognized as signaling molecules, critically placed in pathways activated by Ang II. Mechanisms linking Nox and Ang II are complex and not fully understood. Recent Advances: Ang II regulates vascular cell production of ROS through various recently characterized Noxs, including Nox1, Nox2, NOX4, and Nox5. Activation of these Noxs leads to ROS generation, which in turn influences many downstream signaling targets of Ang II, including MAP kinases, RhoA/Rho kinase, transcription factors, protein tyrosine phosphatases, and tyrosine kinases. Activation of these redox-sensitive pathways regulates vascular cell growth, inflammation, contraction, and senescence. Critical Issues: Although there is much evidence indicating a role for Nox/ROS in Ang II function, there is still a paucity of information on how Ang II exerts cell-specific effects through ROS and how Nox isoforms are differentially regulated by Ang II. Moreover, exact mechanisms whereby ROS induce oxidative modifications of signaling molecules mediating Ang II actions remain elusive. Future Directions: Future research should elucidate these issues to better understand the significance of Ang II and ROS in vascular (patho) biology. Antioxid. Redox Signal. 19, 1110–1120.
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reactive oxygen species and endothelial function role of nitric oxide synthase uncoupling and nox family nicotinamide adenine dinucleotide phosphate oxidases
Basic & Clinical Pharmacology & Toxicology, 2012Co-Authors: Augusto C Montezano, Rhian M TouyzAbstract:Abstract: The healthy endothelium prevents platelet aggregation and leucocyte adhesion, controls permeability to plasma components and maintains vascular integrity. Damage to the endothelium promotes endothelial dysfunction characterized by: altered endothelium-mediated vasodilation, increased vascular reactivity, platelet aggregation, thrombus formation, increased permeability, leucocyte adhesion and monocyte migration. Molecular processes contributing to these phenomena include increased expression of adhesion molecules, synthesis of pro-inflammatory and pro-thrombotic factors and increased endothelin-1 secretion. Decreased nitric oxide bioavailability and increased generation of reactive oxygen species (ROS) are among the major molecular changes associated with endothelial dysfunction. A critical source of endothelial ROS is a family of non-phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, including the prototypic Nox2-based NADPH oxidases, Nox1, NOX4 and Nox5. Other possible sources include mitochondrial electron transport enzymes, xanthine oxidase, cyclooxygenase, lipoxygenase and uncoupled nitric oxide synthase (NOS). Cross-talk between ROS-generating enzymes, such as mitochondrial oxidases and Noxs, is increasingly implicated in cellular ROS production. The present review discusses the importance of endothelial ROS in health and disease and focuses on the major ROS-generating systems in the endothelium, namely uncoupled endothelial nitric oxide synthase and NADPH oxidases.
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differential regulation of nox1 nox2 and NOX4 in vascular smooth muscle cells from wky and shr
Journal of The American Society of Hypertension, 2011Co-Authors: Mercedes Salaices, Glaucia E. Callera, Alvaro Yogi, Fatiha Tabet, Augusto C Montezano, Alejandro M. Briones, Mark T Quinn, Ying He, Rhian M TouyzAbstract:The functional significance and regulation of NAD(P)H oxidase (Nox) isoforms by angiotensin II (Ang II) and endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs) from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) was studied. Expression of Nox1, Nox2, and NOX4 (gene and protein) and NAD(P)H oxidase activity were increased in SHR. Basal NAD(P)H oxidase activity was blocked by GKT136901 (Nox1/4 inhibitor) and by Nox1 siRNA in WKY cells and by siNOX1 and siNOX2 in SHR. Whereas Ang II increased expression of all Noxes in WKY, only Nox1 was influenced in SHR. Ang II–induced NAD(P)H activity was inhibited by siNOX1 in WKY and by siNOX1 and siNOX2 in SHR. ET-1 upregulated Nox expression only in WKY and increased NAD(P)H oxidase activity, an effect inhibited by siNOX1 and siNOX2. Nox1 co-localized with Nox2 but not with NOX4, implicating association between Nox1 and Nox2 but not between Nox1 and NOX4. These data highlight the complexity of Nox biology in VSMCs, emphasising that more than one Nox member, alone or in association, may be involved in NAD(P)H oxidase-mediated •O2− production. Nox1 regulation by Ang II, but not by ET-1, may be important in •O2− formation in VSMCs from SHR.
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critical role of NOX4 based nadph oxidase in glucose induced oxidative stress in the kidney implications in type 2 diabetic nephropathy
American Journal of Physiology-renal Physiology, 2010Co-Authors: Mona Sedeek, Glaucia E. Callera, Augusto C Montezano, Cedric Szyndralewiez, Freddy Heitz, Alexey Gutsol, Patrick Page, Christopher R J Kennedy, Kevin D Burns, Rhian M TouyzAbstract:Molecular mechanisms underlying renal complications of diabetes remain unclear. We tested whether renal NADPH oxidase (Nox) 4 contributes to increased reactive oxygen species (ROS) generation and hyperactivation of redox-sensitive signaling pathways in diabetic nephropathy. Diabetic mice (db/db) (20 wk) and cultured mouse proximal tubule (MPT) cells exposed to high glucose (25 mmol/l, D-glucose) were studied. Expression (gene and protein) of NOX4, p22(phox), and p47(phox), but not Nox1 or Nox2, was increased in kidney cortex, but not medulla, from db/db vs. control mice (db/m) (P < 0.05). ROS generation, p38 mitogen-activated protein (MAP) kinase phosphorylation, and content of fibronectin and transforming growth factor (TGF)-β1/2 were increased in db/db vs. db/m (P < 0.01). High glucose increased expression of NOX4, but not other Noxes vs. normal glucose (P < 0.05). This was associated with increased NADPH oxidase activation and enhanced ROS production. NOX4 downregulation by small-interfering RNA and inhibition of NOX4 activity by GK-136901 (Nox1/4 inhibitor) attenuated d-glucose-induced NADPH oxidase-derived ROS generation. High d-glucose, but not l-glucose, stimulated phosphorylation of p38MAP kinase and increased expression of TGF-β1/2 and fibronectin, effects that were inhibited by SB-203580 (p38MAP kinase inhibitor). GK-136901 inhibited d-glucose-induced actions. Our data indicate that, in diabetic conditions: 1) renal NOX4 is upregulated in a cortex-specific manner, 2) MPT cells possess functionally active NOX4-based NADPH, 3) NOX4 is a major source of renal ROS, and 4) activation of profibrotic processes is mediated via NOX4-sensitive, p38MAP kinase-dependent pathways. These findings implicate NOX4-based NADPH oxidase in molecular mechanisms underlying fibrosis in type 2 diabetic nephropathy.
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Novel Nox homologues in the vasculature: focusing on NOX4 and Nox5.
Clinical Science, 2010Co-Authors: Augusto C Montezano, Dylan Burger, Graziela S. Ceravolo, Hiba Yusuf, María J. Montero, Rhian M TouyzAbstract:The Noxes (NADPH oxidases) are a family of ROS (reactive oxygen species)-generating enzymes. Of the seven family members, four have been identified as important sources of ROS in the vasculature: Nox1, Nox2, NOX4 and Nox5. Although Nox isoforms can be influenced by the same stimulus and co-localize in cellular compartments, their tissue distribution, subcellular regulation, requirement for cofactors and NADPH oxidase subunits and ability to generate specific ROS differ, which may help to understand the multiplicity of biological functions of these oxidases. NOX4 and Nox5 are the newest isoforms identified in the vasculature. NOX4 is the major isoform expressed in renal cells and appear to produce primarily H2O2. The Nox5 isoform produces ROS in response to increased levels of intracellular Ca 2+ and does not require the other NADPH oxidase subunits for its activation. The present review focuses on these unique Noxes, NOX4 and Nox5, and provides novel concepts related to the regulation and interaction in the vasculature, and discusses new potential roles for these isoforms in vascular biology.
David J Lambeth - One of the best experts on this subject based on the ideXlab platform.
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NOX4 a hydrogen peroxide generating oxygen sensor
Biochemistry, 2014Co-Authors: Yukio Nisimoto, Becky A Diebold, Daniela Cosentinogomes, David J LambethAbstract:NOX4 is an oddity among members of the Nox family of NADPH oxidases [seven isoenzymes that generate reactive oxygen species (ROS) from molecular oxygen] in that it is constitutively active. All other Nox enzymes except for NOX4 require upstream activators, either calcium or organizer/activator subunits (p47phox, NOXO1/p67phox, and NOXA1). NOX4 may also be unusual as it reportedly releases hydrogen peroxide (H2O2) in contrast to Nox1–Nox3 and Nox5, which release superoxide, although this result is controversial in part because of possible membrane compartmentalization of superoxide, which may prevent detection. Our studies were undertaken (1) to identify the NOX4 ROS product using a membrane-free, partially purified preparation of NOX4 and (2) to test the hypothesis that NOX4 activity is acutely regulated not by activator proteins or calcium, but by cellular pO2, allowing it to function as an O2 sensor, the output of which is signaling H2O2. We find that approximately 90% of the electron flux through isola...
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role of NOX4 and nox2 in hyperoxia induced reactive oxygen species generation and migration of human lung endothelial cells
Antioxidants & Redox Signaling, 2009Co-Authors: Srikanth Pendyala, David J Lambeth, Irina Gorshkova, Peter V Usatyuk, Arjun Pennathur, Victor J Thannickal, Viswanathan NatarajanAbstract:Abstract In vascular endothelium, the major research focus has been on reactive oxygen species (ROS) derived from Nox2. The role of NOX4 in endothelial signal transduction, ROS production, and cytoskeletal reorganization is not well defined. In this study, we show that human pulmonary artery endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) express higher levels of NOX4 and p22phox compared to Nox1, Nox2, Nox3, or Nox5. Immunofluorescence microscopy and Western blot analysis revealed that NOX4 and p22phox, but not Nox2 or p47phox, are localized in nuclei of HPAECs. Further, knockdown of NOX4 with siRNA decreased NOX4 nuclear expression significantly. Exposure of HPAECs to hyperoxia (3–24 h) enhanced mRNA and protein expression of NOX4, and NOX4 siRNA decreased hyperoxia-induced ROS production. Interestingly, NOX4 or Nox2 knockdown with siRNA upregulated the mRNA and protein expression of the other, suggesting activation of compensatory mechanisms. A similar upregulation o...
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redox dependent expression of cyclin d1 and cell proliferation by nox1 in mouse lung epithelial cells
Antioxidants & Redox Signaling, 2006Co-Authors: Priya Ranjan, Vikas Anathy, Peter M Burch, Kelly Weirather, David J Lambeth, Nicholas H. HeintzAbstract:NADPH oxidases produce reactive oxygen species (ROS) that serve as co-stimulatory signals for cell proliferation. In mouse lung epithelial cells that express Nox1, Nox2, NOX4, p22phox, p47phox, p67phox, and Noxo1, overexpression of Nox1 delayed cell cycle withdrawal by maintaining AP-1-dependent expression of cyclin D1 in low serum conditions. In cycling cells, the effects of Nox1 were dose dependent: levels of Nox1 that induced 3- to 10-fold increases in ROS promoted phosphorylation of ERK1/2 and expression of cyclin D1, whereas expression of Nox1 with Noxo1 and Noxa1 (or expression of NOX4 alone) that induced substantial increases in intracellular ROS inhibited cyclin D1 and proliferation. Catalase reversed the effects of Nox1 on cyclin D1 and cell proliferation. Diphenylene iodonium, an inhibitor of NADPH oxidase activity, did not affect dosedependent responses of ERK1/2 or Akt to serum, but markedly inhibited the sequential expression of c-Fos and Fra-1 required for induction of cyclin D1 during cell ...
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point mutations in the proline rich region of p22phox are dominant inhibitors of nox1 and nox2 dependent reactive oxygen generation
Journal of Biological Chemistry, 2005Co-Authors: Tsukasa Kawahara, Guangjie Cheng, Darren R. Ritsick, David J LambethAbstract:Abstract The integral membrane protein p22phox is an indispensable component of the superoxide-generating phagocyte NADPH oxidase, whose catalytic core is the membrane-associated gp91phox (also known as Nox2). p22phox associates with gp91phox and, through its proline-rich C terminus, provides a binding site for the tandem Src homology 3 domains of the activating subunit p47phox. Whereas p22phox is expressed ubiquitously, its participation in regulating the activity of other Nox enzymes is less clear. This study investigates the requirement of p22phox for Nox enzyme activity and explores the role of its proline-rich region (PRR) for regulating activity. Coexpression of specific Nox catalytic subunits (Nox1, Nox2, Nox3, NOX4, or Nox5) along with their corresponding regulatory subunits (NOXO1/NOXA1 for Nox1; p47phox/p67phox/Rac for Nox2; NOXO1 for Nox3; no subunits for NOX4 or Nox5) resulted in marked production of reactive oxygen. Small interfering RNAs decreased endogenous p22phox expression and inhibited reactive oxygen generation from Nox1, Nox2, Nox3, and NOX4 but not Nox5. Truncated forms of p22phox that disrupted the PRR-inhibited reactive oxygen generation from Nox1, Nox2, and Nox3 but not from NOX4 and Nox5. Similarly, p22phox (P156Q), a mutation that disrupts Src homology 3 binding by the PRR, potently inhibited reactive oxygen production from Nox1 and Nox2 but not from NOX4 and Nox5. Expression of p22phox (P156Q) inhibited NOXO1-stimulated Nox3 activity, but co-expression of NOXA1 overcame the inhibitory effect. The P157Q and P160Q mutations of p22phox showed selective inhibition of Nox2/p47phox/p67phox, and selectivity was specific for the organizing subunit (p47phox or NOXO1) rather than the Nox catalytic subunit. These studies stress the importance of p22phox for the function of Nox1, Nox2, Nox3, and NOX4, and emphasize the key role of the PRR for regulating Nox proteins whose activity is dependent upon p47phox or NOXO1.
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distinct subcellular localizations of nox1 and NOX4 in vascular smooth muscle cells
Arteriosclerosis Thrombosis and Vascular Biology, 2004Co-Authors: Lula Hilenski, Roza E Clempus, David J Lambeth, Mark T Quinn, Kathy K. GriendlingAbstract:Objective— Reactive oxygen species (ROS) that act as signaling molecules in vascular smooth muscle cells (VSMC) and contribute to growth, hypertrophy, and migration in atherogenesis are produced by multi-subunit NAD(P)H oxidases. Nox1 and NOX4, two homologues to the phagocytic NAD(P)H subunit gp91 phox , both generate ROS in VSMC but differ in their response to growth factors. We hypothesize that the opposing functions of Nox1 and NOX4 are reflected in their differential subcellular locations. Methods and Results— We used immunofluorescence to visualize the NAD(P)H subunits Nox1, NOX4, and p22 phox in cultured rat and human VSMC. Optical sectioning using confocal microscopy showed that Nox1 is co-localized with caveolin in punctate patches on the surface and along the cellular margins, whereas NOX4 is co-localized with vinculin in focal adhesions. These immunocytochemical distributions are supported by membrane fractionation experiments. Interestingly, p22 phox , a membrane subunit that interacts with the Nox proteins, is found in surface labeling and in focal adhesions in patterns similar to Nox1 and NOX4, respectively. Conclusions— The differential roles of Nox1 and NOX4 in VSMC may be correlated with their differential compartmentalization in specific signaling domains in the membrane and focal adhesions.
Ralf P Brandes - One of the best experts on this subject based on the ideXlab platform.
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The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein.
Journal of Biological Chemistry, 2016Co-Authors: Kim-kristin Prior, Ajay M. Shah, Ilka Wittig, Matthias S. Leisegang, Jody Groenendyk, Norbert Weissmann, Marek Michalak, Pidder Jansen-dürr, Ralf P BrandesAbstract:Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin(-/-)mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.
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the e loop is involved in hydrogen peroxide formation by the nadph oxidase NOX4
Journal of Biological Chemistry, 2011Co-Authors: Ina Takac, Bernard Lardy, Françoise Morel, Ajay M. Shah, Katrin Schroder, Leilei Zhang, Narayana Anilkumar, James David Lambeth, Ralf P BrandesAbstract:Abstract In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O2), NOX4 produces hydrogen peroxide (H2O2). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H2O2 versus L-012 and cytochrome c for O2. The third extracytosolic loop (E-loop) of NOX4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in NOX4 or exchange of the two cysteines in these stretches switched NOX4 from H2O2 to O2 generation while preserving expression and intracellular localization. In the presence of an NO donor, the O2-producing NOX4 mutants, but not wild-type NOX4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O2. In Cos7 cells, in which NOX4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H2O2 but increased O2 formation by NOX4 without affecting Nox1-dependent O2 formation. The E-loop of NOX4 but not Nox1 and Nox2 contains a highly conserved histidine that could serve as a source for protons to accelerate spontaneous dismutation of superoxide to form H2O2. Mutation of this but not of four other conserved histidines also switched NOX4 from H2O2 to O2 formation. Thus, H2O2 formation is an intrinsic property of NOX4 that involves its E-loop. The structure of the E-loop may hinder O2 egress and/or provide a source for protons, allowing dismutation to form H2O2.
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No Superoxide—No Stress? NOX4, the Good NADPH Oxidase!
Arteriosclerosis Thrombosis and Vascular Biology, 2011Co-Authors: Ralf P Brandes, Ina Takac, Katrin SchroderAbstract:NADPH oxidases of the Nox family are enzymes whose only known function is the production of reactive oxygen species (ROS). Since the first report on a vascular NADPH oxidase by Griendling et al in 1994,1 several thousand publications have been devoted to this topic. The general impression is that under physiological conditions, Nox proteins contribute to vascular signaling, whereas the overactivation and induction of NADPH oxidases promote vascular disease.2 See accompanying article on page 1368 In the vascular system, the NADPH oxidases Nox1, Nox2, NOX4, and Nox5 are expressed. Unlike the case with the other vascular Nox homologues, the activity of NOX4 appears to be predominantly controlled by its expression level, and proinflammatory mediators that induce Nox1 or Nox2 instead appear to suppress NOX4 expression.3 NOX4 is also the only vascular homolog that directly produces hydrogen peroxide (H2O2) and thus is incapable of scavenging nitric oxide (NO) or producing peroxynitrite (ONOO−).4 As a consequence of these unique properties, so far little consensus regarding the physiological function of NOX4 has been reached.3 In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Ray et al report the generation and characterization of a transgenic mouse with endothelial-specific NOX4 overexpression.5 Vascular segments and endothelial cells of these animals had a significant increase in H2O2 generation that was sufficient to result in a substantial increase in the oxidation of peroxiredoxin 1. Despite these signs of increased protein oxidation, the blood pressure of the animals was lower under basal conditions and after angiotensin II treatment. Furthermore, endothelium-dependent relaxation was enhanced compared with wild-type animals. The latter effects were sensitive to catalase ex vivo and N -acetylcysteine in vivo, suggesting that they were mediated by H2 …
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identification of structural elements in nox1 and NOX4 controlling localization and activity
Antioxidants & Redox Signaling, 2009Co-Authors: Ina Helmcke, Sabine Heumuller, Ritva Tikkanen, Katrin Schroder, Ralf P BrandesAbstract:Abstract Nox NADPH oxidases differ in their mode of activation, subcellular localization, and physiological function. Nox1 releases superoxide anions (O2−) and depends on cytosolic activator proteins, whereas NOX4 extracellularly releases hydrogen peroxide (H2O2), and its activity does not require cotransfection of additional proteins. We constructed chimeric proteins consisting of Nox1 and NOX4 expressed in HEK293 cells. When the cytosolic tail of NOX4 was fused with the transmembrane part of Nox1, Nox1 became constitutively active. The reciprocal construct was inactive, suggesting that cytosolic subunit–dependent activation requires elements in the transmembrane loops. By TIRF-microscopy, Nox1 was observed in the plasma membrane, whereas NOX4 colocalized with proteins of the endoplasmic reticulum. Fusion proteins of Myc and Nox revealed that the N-terminal part of Nox1 but not NOX4 is cleaved. When the potential signal peptide of NOX4 was inserted into Nox1, plasma-membrane localization was lost, and th...
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NOX4 Acts as a Switch Between Differentiation and Proliferation in Preadipocytes
Arteriosclerosis Thrombosis and Vascular Biology, 2009Co-Authors: Katrin Schroder, Ina Helmcke, Katalin Wandzioch, Ralf P BrandesAbstract:Objective— Insulin promotes differentiation of preadipocytes into adipocytes. Insulin also stimulates reactive oxygen species (ROS) production, and the NADPH oxidases Nox1 and NOX4 are important sources of ROS. We determined in human and mouse preadipocytes whether Nox proteins contribute to ROS formation and differentiation in response to insulin. Methods and Results— The expression of Nox1 and NOX4 was increased during insulin-induced differentiation, and insulin increased ROS production. SiRNA against NOX4 but not Nox1 inhibited insulin-induced differentiation and ROS production but promoted proliferation. NOX4 overexpression yielded the opposite effect. As observed by siRNA and overexpression, NOX4 controlled the expression of MAP kinase phosphatase-1 (MKP-1), which reduces insulin-induced ERK1/2 activation. Consequently, downregulation of NOX4 promoted ERK1/2 signaling: Proliferation was increased and through phosphorylation of the inhibitory site serine612, ERK1/2 inhibited the activation of the insulin-receptor substrate-1 (IRS-1) and thereby prevented differentiation in response to insulin. Inhibition of ERK1/2 or overexpression of MPK-1 promoted insulin-induced differentiation. Accordingly, insulin-induced proliferation was enhanced by siRNA against MKP-1, whereas inhibition of ERK1/2 or overexpression of MKP-1 attenuated proliferation. Conclusions— NOX4 acts as a switch from insulin-induced proliferation to differentiation by controlling MKP-1 expression, which limits ERK1/2 signaling.
Kirstin Wingler - One of the best experts on this subject based on the ideXlab platform.
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genetic targeting or pharmacologic inhibition of nadph oxidase NOX4 provides renoprotection in long term diabetic nephropathy
Journal of The American Society of Nephrology, 2014Co-Authors: Stephen P Gray, Kirstin Wingler, David Barit, Jun Okabe, Assam Elosta, Tamehachi Namikoshi, Vicki Thallasbonke, Cedric Szyndralewiez, Freddy HeitzAbstract:Diabetic nephropathy may occur, in part, as a result of intrarenal oxidative stress. NADPH oxidases comprise the only known dedicated reactive oxygen species (ROS)–forming enzyme family. In the rodent kidney, three isoforms of the catalytic subunit of NADPH oxidase are expressed (Nox1, Nox2, and NOX4). Here we show that NOX4 is the main source of renal ROS in a mouse model of diabetic nephropathy induced by streptozotocin administration in ApoE−/− mice. Deletion of NOX4, but not of Nox1, resulted in renal protection from glomerular injury as evidenced by attenuated albuminuria, preserved structure, reduced glomerular accumulation of extracellular matrix proteins, attenuated glomerular macrophage infiltration, and reduced renal expression of monocyte chemoattractant protein-1 and NF-κB in streptozotocin-induced diabetic ApoE−/− mice. Importantly, administration of the most specific Nox1/4 inhibitor, GKT137831, replicated these renoprotective effects of NOX4 deletion. In human podocytes, silencing of the NOX4 gene resulted in reduced production of ROS and downregulation of proinflammatory and profibrotic markers that are implicated in diabetic nephropathy. Collectively, these results identify NOX4 as a key source of ROS responsible for kidney injury in diabetes and provide proof of principle for an innovative small molecule approach to treat and/or prevent chronic kidney failure.
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neuroprotection after stroke by targeting NOX4 as a source of oxidative stress
Antioxidants & Redox Signaling, 2013Co-Authors: Kim A. Radermacher, Sebastian Altenhöfer, Kirstin Wingler, Pamela W. M. Kleikers, Christoph Kleinschnitz, Friederike Langhauser, J Rob J Hermans, Martin Hrabě De Angelis, Harald H H W SchmidtAbstract:Significance: Stroke, a leading cause of death and disability, poses a substantial burden for patients, relatives, and our healthcare systems. Only one drug is approved for treating stroke, and more than 30 contraindications exclude its use in 90% of all patients. Thus, new treatments are urgently needed. In this review, we discuss oxidative stress as a pathomechanism of poststroke neurodegeneration and the inhibition of its source, type 4 nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX4), as a conceptual breakthrough in stroke therapy. Recent Advances: Among potential sources of reactive oxygen species (ROS), the NOXes stand out as the only enzyme family that is solely dedicated to forming ROS. In rodents, three cerebrovascular NOXes exist: the superoxide-forming NOX1 and 2 and the hydrogen peroxide-forming NOX4. Studies using NOX1 knockout mice gave conflicting results, which overall do not point to a role for this isoform. Several reports find NOX2 to be relevant in stroke, albeit to variable and moderate degrees. In our hands, NOX4 is, by far, the major source of oxidative stress and neurodegeneration on ischemic stroke. Critical Issues: We critically discuss the tools that have been used to validate the roles of NOX in stroke. We also highlight the relevance of different animal models and the need for advanced quality control in preclinical stroke research. Future Directions: The development of isoform-specific NOX inhibitors presents a precious tool for further clarifying the role and drugability of NOX homologues. This could pave the avenue for the first clinically effective neuroprotectant applied poststroke, and even beyond this, stroke could provide a proof of principle for antioxidative stress therapy. Antioxid. Redox Signal. 18, 1418–1427.
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VAS2870 is a pan-NADPH oxidase inhibitor
Cellular and Molecular Life Sciences, 2012Co-Authors: Kirstin Wingler, Sebastian A. Altenhoefer, Pamela W. M. Kleikers, Kim A. Radermacher, Christoph Kleinschnitz, Harald H H W SchmidtAbstract:In their detailed review ‘‘NADPH oxidases as therapeutic targets in ischemic stroke’’ [1], Kahles and Brandes discuss the pathological roles of NADPH oxidases in ischemic brain injury and the therapeutic implications. In agreement with the authors, we consider inhibition of NADPH oxidases as a promising strategy to treat ischemic stroke. As described in the review, we recently reported that NOX4-deficient mice are largely protected from brain damage caused by ischemic stroke, whereas we did not observe any effects by deleting NOX1 or NOX2. Thus, we believe that NOX4 is a highly promising target for stroke therapy. To further support our findings, we treated wildtype and NOX4 knockout mice with the NADPH oxidase inhibitor VAS2870 in a therapeutically relevant time window, i.e., post-stroke. Indeed, in wild-type mice, inhibition of NADPH oxidases by VAS2870 resulted in a similar degree of protection as did deletion of NOX4. In contrast, in NOX4 knockout mice, VAS2870 did not have any additional effects in reducing ischemic brain damage. This further supports our statement that NOX4, and not other NOX isoforms, is the likely detrimental NOX isoform in ischemic stroke in mice. Unfortunately, to the best of our knowledge, there was and is no NOX4-selective inhibitor that could have been used to further support our findings. Kahles and Brandes correctly describe that VAS2870 inhibits NOX1 and NOX2 and cite our relevant publications [2–4]. In the same issue of this journal, we provided evidence that VAS2870 also inhibits NOX4 [5]. In conclusion, we believe that VAS2870 is a pan-NOX inhibitor and not selective for any NOX isoform. However, in their review, Kahles and Brandes [1] state that we concluded that ‘‘VAS2870 was a NOX4-specific inhibitor based on the fact that the compound had no effect on the small infarcts they produced in NOX4 knockout mice’’. This is not true. We have never published such a statement on the NOX isoform-specificity of VAS2870. Both in the respective paper [6] and our other publications we describe VAS2870 as an NADPH oxidase inhibitor with no relevant specificity for any NOX isoform (data on NOX3 are not available) [5]. We have published similar data on the closely related derivative of VAS2870, VAS3947 [7]. Thus, we would kindly ask the authors to revoke their statement.
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post stroke inhibition of induced nadph oxidase type 4 prevents oxidative stress and neurodegeneration
PLOS Biology, 2010Co-Authors: Christoph Kleinschnitz, Melanie E Armitage, Kirstin Wingler, David Barit, Manish Mittal, Henrike Grund, Emma S Jones, Tobias Schwarz, Christian Geis, Peter KraftAbstract:Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (NOX4 2/2 )o f either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in NOX4 2/2 mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy.
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oxidative stress and endothelial dysfunction in aortas of aged spontaneously hypertensive rats by nox1 2 is reversed by nadph oxidase inhibition
Hypertension, 2010Co-Authors: Sven Wind, Daniel Janowitz, Christina Neff, Arun H. S. Kumar, Knut Beuerlein, Melanie E Armitage, Ashraf Taye, Kirstin Wingler, Ajay M. Shah, Harald H H W SchmidtAbstract:Arterial hypertension is associated with increased levels of reactive oxygen species, which may scavenge endothelium-derived NO and thereby diminish its vasorelaxant effects. However, the quantitatively relevant source of reactive oxygen species is unclear. Thus, this potential pathomechanism is not yet pharmacologically targetable. Several enzymatic sources of reactive oxygen species have been suggested: uncoupled endothelial NO synthase, xanthine oxidase, and NADPH oxidases. Here we show that increased reactive oxygen species formation in aortas of 12- to 14-month–old spontaneously hypertensive rats versus age-matched Wistar Kyoto rats is inhibited by the specific NADPH oxidase inhibitor VAS2870 but neither by the xanthine oxidase inhibitor oxypurinol nor the NO synthase inhibitor N G -nitro-l-arginine methyl ester. NADPH oxidase activity, as well as protein expression of its catalytic subunits, NOX1 and NOX2, was increased in the aortas of spontaneously hypertensive rats, whereas the expression of NOX4 protein, the most abundant NOX isoform, was not significantly changed. Impaired acetylcholine-induced relaxation of spontaneously hypertensive rat aortas was significantly improved by VAS2870. In conclusion, NOX1 and NOX2 but not NOX4 proteins are increased in aged spontaneously hypertensive rat aortas. Importantly, these NOX isoforms, in particular, ectopic expression of NOX1 in endothelial cells, appear to affect vascular function in an NADPH oxidase inhibitor-reversible manner. NADPH oxidases may, thus, be a novel target for the treatment of systemic hypertension.
