NADPH Oxidase Isoforms Research Articles

Divergent effects of genetic and pharmacological inhibition of Nox2 NADPH oxidase on insulin resistance-related vascular damage

Insulin resistance leads to excessive endothelial cell (EC) superoxide generation and accelerated atherosclerosis. The principal source of superoxide from the insulin-resistant endothelium is the Nox2 isoform of NADPH oxidase. Here we examine the therapeutic potential of Nox2 inhibition on superoxide generation in saphenous vein ECs (SVECs) from patients with advanced atherosclerosis and type 2 diabetes and on vascular function, vascular damage, and lipid deposition in apolipoprotein E-deficient (ApoE−/−) mice with EC-specific insulin resistance (ESMIRO). To examine the effect of genetic inhibition of Nox2, ESMIRO mice deficient in ApoE−/− and Nox2 (ESMIRO/ApoE−/−/Nox2−/y) were generated and compared with ESMIRO/ApoE−/−/Nox2+/y littermates. To examine the effect of pharmacological inhibition of Nox2, we administered gp91dstat or scrambled peptide to ESMIRO/ApoE−/− mice. SVECs from diabetic patients had increased expression of Nox2 protein with concomitant increase in superoxide generation, which could be reduced by the Nox2 inhibitor gp91dstat. After 12 wk Western diet, ESMIRO/ApoE−/−/Nox2−/y mice had reduced EC superoxide generation and greater aortic relaxation to acetylcholine. ESMIRO/ApoE−/−/Nox2−/y mice developed more lipid deposition in the thoraco-abdominal aorta with multiple foci of elastin fragmentation at the level of the aortic sinus and greater expression of intercellular adhesion molecule-1 (ICAM-1). Gp91dstat reduced EC superoxide and lipid deposition in the thoraco-abdominal aorta of ESMIRO/ApoE−/− mice without causing elastin fragmentation or increased ICAM-1 expression. These results demonstrate that insulin resistance is characterized by increased Nox2-derived vascular superoxide. Complete deletion of Nox2 in mice with EC insulin resistance exacerbates, whereas partial pharmacological Nox2 inhibition protects against, insulin resistance-induced vascular damage.

Glucose-Stimulated Insulin Secretion Fundamentally Requires H2O2 Signaling by NADPH Oxidase 4.

NADPH facilitates glucose-stimulated insulin secretion (GSIS) in pancreatic islets (PIs) of β-cells through an as yet unknown mechanism. We found NADPH oxidase isoform 4 (NOX4) to be the main producer of cytosolic H2O2, which is essential for GSIS; an increase in ATP alone was insufficient for GSIS. The fast GSIS phase was absent from PIs from NOX4-null, β-cell-specific knockout mice (NOX4βKO) (though not from NOX2 knockout mice) and from NOX4-silenced or catalase-overexpressing INS-1E cells. Lentiviral NOX4 overexpression or H2O2 rescued GSIS in PIs from NOX4βKO mice. NOX4 silencing suppressed Ca2+ oscillations, and the patch-clamped KATP channel opened more frequently when glucose was high. Mitochondrial H2O2, decreasing upon GSIS, provided alternative redox signaling when 2-oxo-isocaproate or fatty acid oxidation formed superoxides through electron-transfer flavoprotein:Q-oxidoreductase. Unlike GSIS, such insulin secretion was blocked with mitochondrial antioxidant SkQ1. Both NOX4 knockout and NOX4βKO mice exhibited impaired glucose tolerance and peripheral insulin resistance. Thus, the redox signaling previously suggested to cause β-cells to self-check hypothetically induces insulin resistance when it is absent. In conclusion, increases in ATP and H2O2 constitute an essential signal that switches on insulin exocytosis for glucose and branched-chain oxoacids as secretagogues (it does so partially for fatty acids). Redox signaling could be impaired by cytosolic antioxidants; hence, those targeting mitochondria should be preferred for clinical applications to treat (pre)diabetes at any stage.

Canagliflozin provides cardioprotection and renoprotection against isoprenaline‐induced oxidative stress by stimulating AMPK‐Akt‐eNOS‐NO signaling axis

The antidiabetic drug canagliflozin, a SGLT2 inhibitor, has been reported to possess protective effects against ischemia‐reperfusion and chemically‐induced cardiac and renal injuries. However, to our knowledge, no studies have investigated the protective effects of canagliflozin in isoprenaline (ISO)‐induced cardiac and renal oxidative damage – a model mimicking sympathetic nervous system (SNS) overstimulation‐evoked organ injuries in humans. Therefore, we investigated effects of canagliflozin in ISO‐induced cardiac and renal oxidative stress, and their underlying molecular mechanisms. Our tissue biochemistry, histology, and Western blotting data showed that ISO administration inflicted pro‐oxidative, pro‐inflammatory and pro‐apoptotic changes in the heart and kidney tissues. In contrast, treatment with canagliflozin at 5 mg/kg body weight attenuated levels of cardiac and renal oxidative stress markers including malondialdehyde (MDA), advanced protein oxidation product (APOP), myeloperoxidase (MPO) and nitric oxide (NO), and restored levels of endogenous antioxidant molecules such as catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH) that were depleted in ISO‐treated rats. Western blot analysis revealed that canagliflozin increased activity of anti‐oxidant/anti‐inflammatory proteins such as AMP‐activated protein kinase (AMPK), Akt and eNOS, and decreased pro‐oxidative/pro‐inflammatory proteins including iNOS and NADPH oxidase isoform 4 (NOX4). Protein expression analysis further revealed that ISO increased the ratio of pro‐apoptotic/anti‐apoptotic protein (Bax:Bcl‐2) expression, which was significantly reduced by canagliflozin treatment. Our histological examination demonstrated that in the heart and kidney tissue sections, canagliflozin attenuated ISO‐induced histopathological changes, including inflammatory cell infiltration, collagen deposition, and fibrosis. Consistent with our biochemical data, canagliflozin augmented heart and kidney functions in ISO rats, further supporting a protective role of this drug in ISO‐induced oxidative stress. In summary, we show that canagliflozin produces cardioprotective and renoprotective actions via suppression of iNOS, NOX4 and Bax, and activation of AMPK‐Akt‐eNOS‐NO signaling axis.Support or Funding InformationThis work was supported by a start‐up grant from the Mercer University College of Pharmacy to Dr. Raquibul Hasan and a research grant from North South University to Dr. Md Ashraful Alam

Noise‐induced vascular dysfunction, oxidative stress and inflammationare improved by pharmacological heme oxygenase‐1 induction

Endothelial dysfunction, vascular oxidative stress and inflammation are consequences of traditional cardiovascular risk factors and triggers of cardiovascular disease. However, emerging environmental stressors such as traffic noise exposure may facilitate the development of cardiovascular and metabolic diseases.C57BL/6J mice were exposed to aircraft noise (maximum sound level of 85 dB(A), average sound pressure level of 72 dB(A)) applied for 1, 2 and 4 days caused an increase in systolic blood pressure, stress hormones and induced endothelial dysfunction, oxidative stress and inflammation (Eur Heart J 2017). We also observed reduced vascular NO levels due to eNOS uncoupling and increased levels of nitrotyrosine, interleukin‐6, NADPH oxidase isoform Nox2 and endothelin‐1 in the mice exposed to aircraft noise. More recently, we have shown that aircraft noise induces also cerebral oxidative stress and a neuroinflammatory phenotype that is up‐stream of the observed adverse cardiovascular effects, all of which was normalized by genetic deletion of NADPH oxidase isoform Nox2 (Eur Heart J 2018).The role of heme oxygenase‐1 (HO‐1) as an antioxidant response is currently being investigated. C57BL/6J mice were treated with the HO‐1 activator hemin (25 mg/kg i.p.). Our preliminary data show a protective effect of hemin infusion on aircraft noise exposure (4 days) induced increase of systolic blood pressure and vascular dysfunction. There was also a trend of normalization of noise‐triggered oxidative stress ny hemin infusion. Within the ongoing studies, we will address the impact of HO‐1 induction in noise‐exposed mice on expression of genes and proteins involved in pathways of oxidative stress and systemic inflammation. In a later stage of the study, we will also test whether genetic deficiency of HO‐1 (HO‐1+/− mice) aggravates and the Nrf2 activator dimethyl fumarate (DMF) ameliorates noise‐triggered cardiovascular complications. The present study aims at identifying new targets for mitigation strategies against adverse health effects of environmental noise exposure of the general population. Since HO‐1 and Nrf2 induction can be achieved by natural dietary constituents, these pathways represent promising targets for preventive measures.Support or Funding InformationOur experimental studies on noise effects in mice were supported by vascular biology research grants from the Else‐Kröner‐Fresenius Foundation for “Noise and arterial hypertension” (2017_A106) and the Boehringer Ingelheim Foundation for the collaborative research group „Novel and neglected cardiovascular risk factors: molecular mechanisms and therapeutic implications”.

Integrated Computational Modeling of the Kinetics and Regulation of NADPH Oxidase 2 Assembly, Activation, Electron Flow, and Superoxide Production

IntroductionReactive oxygen species (ROS) play a crucial role in key physiological processes, including cell signaling. However, ROS overproduction leads to oxidative stress, which plays a critical role in cell injury/death and pathogenesis of many diseases, including cardiovascular disease. Members of the NADPH oxidase (NOX) family that comprise of membrane and cytosolic components are known to be the major non‐mitochondrial sources of ROS in most mammalian cells. NOX2 is the most complex, widely expressed, and well‐studied of the NOX isoforms. NOX2 is activated upon assembly of its membrane‐bound subunits gp91phox and p22phox with the cytosolic subunits p40phox, p47phox, p67phox, and Rac facilitating electron flow and ROS production. Specifically, upon NOX2 assembly and activation, electrons are transferred from substrate NADPH to molecular O2 through different redox centers of NOX2 complex (further regulated by pH) resulting in oxidized NADP+ and reduced O2−. (superoxide). Yet, there is a lack of mechanistic and quantitative understanding of the kinetics and regulation of NOX2 assembly and the relative contributions of NOX2 subunits towards NOX2 activation, electron flow, and ROS production.MethodWe developed an integrated computational model to quantitatively describe the kinetics and regulation of NOX2 assembly, activation, electron flow, and ROS production. The model incorporates our hypothesized “random rapid equilibrium binding mechanism” for NOX2 assembly and activation, regulations by guanine nucleotides (GTP, GDP), and mutual binding enhancements between individual cytosolic subunits (p40phox, p47phox, p67phox, Rac). The model also considers the thermodynamics of electron transfer through different redox centers of NOX2 complex and biphasic inhibition by pH. The model uses diverse published experimental data to modularly estimate the unknown model parameters using the built‐in “fmincon” optimization algorithm in MATLAB.ResultsThe modeling and analysis show that NOX2 is differentially activated and regulated by p40phox, p47phox, p67phox, and Rac subunits. In addition, the assembly of NOX2 subunits is regulated by GTP, GDP, and other cytosolic subunits. The model can quantify enhancements in the binding affinities of p47phox, p67phox and Rac subunits by GTP, reductions in the binding affinities of the same cytosolic subunits by GDP, mutual binding enhancement between p47phox, p67phox and Rac subunits, and enhancement in the binding affinity of p47phox by p40phox. The model can also quantify the binding affinities of NADPH and O2 for NOX2 complex and the maximal NOX2 activities as functions of pH.ConclusionThe proposed model provides a quantitative and integrated understanding of the kinetics and regulation of NOX2 assembly, activation, and the importance of these processes in facilitating electron transfer and ROS production under different conditions. The model also serves as a mechanistic and quantitative framework for investigating the critical role of NOX2 complex‐mediated ROS production in regulating diverse cellular mechanisms under physiological and pathophysiological conditions.Support or Funding InformationNIH P01‐GM066730, P01‐HL116264, U01‐HL122199

Kidney mitochondrial redox state 3 days following uninephrectomy in rats determined by cyro‐fluorescence optical imaging.

Uninephrectomy (UNX) is known to result in structural and metabolic changes to the remaining kidney although it is uncertain if this results in changes in the mitochondrial redox state and how soon such changes may occur. A custom designed fluorescence cryo‐imaging technique was used to quantitatively assess the effect of UNX on the mitochondrial bioenergetics of the remaining kidney. Responses were obtained in kidneys of normal Sprague Dawley (SD) rats, Dahl salt‐ sensitive (SS) rats, and SS rats in which the gene for NADPH‐oxidase isoform 4 (NOX4) was knocked out (SSNOX4−/−). Kidneys were snap frozen and the intrinsic fluorescence of two coenzymes, Nicotinamide Adenine Dinucleotide (NADH) and oxidized Flavin Adenine Dinucleotide (FAD) were optically acquired and 3D images created to characterize the NADH/FAD redox ratios (RR) of each kidney 3 days following UNX. Each of the strains exhibited similar increases of kidney weights averaging 17% after 3 days of UNX. It was especially notable that following UNX, SD and SSNOX4−/− rats exhibited global reductions of the RR ratios (P<0.05) with a similar tendency observed in SS rats (P<0.08) indicative of increased reactive oxygen species (ROS) production. The unexpected reduction of the mitochondrial RR in the remaining kidneys of SSNOX4−/− rats indicates that mechanisms independent of H2O2 produced from NOX4 may be largely response for this global increase of renal oxidative stress. We propose that enhanced mitochondrial bioenergetics due to increased tubular workload of the remaining kidney becomes the dominant source of increased ROS following UNX and could represent an important hypertrophic signaling mechanism.Support or Funding InformationHL‐116264

NADPH Oxidase Inhibition: Preclinical and Clinical Studies in Diabetic Complications.

Significance: Oxidative stress plays a critical role in the development and progression of serious micro- and macrovascular complications of diabetes. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)-derived reactive oxygen species (ROS) significantly contribute to oxidative stress-associated inflammatory pathways that lead to tissue damage of different organs, including the kidneys, retina, brain, nerves, and the cardiovascular system. Recent Advances: Preclinical studies, including genetic-modified mouse models or cell culture models, have revealed the role of specific NOX isoforms in different diabetic complications, and suggested them as a promising target for the treatment of these diseases. Critical Issues: In this review, we provide an overview of the role of ROS and oxidative stress in macrovascular complications, such as stroke, myocardial infarction, coronary artery disease, and peripheral vascular disease that are all mainly driven by atherosclerosis, as well as microvascular complications, such as diabetic retinopathy, nephropathy, and neuropathy. We summarize conducted genetic deletion studies of different Nox isoforms as well as pharmacological intervention studies using NOX inhibitors in the context of preclinical as well as clinical research on diabetic complications. Future Directions: We outline the isoforms that are most promising for future clinical trials in the context of micro- and macrovascular complications of diabetes.

Antioxidant Blueberry Anthocyanins Induce Vasodilation via PI3K/Akt Signaling Pathway in High-Glucose-Induced Human Umbilical Vein Endothelial Cells.

Blueberries are rich in antioxidant anthocyanins. The hypotensive effects of blueberry anthocyanins in endothelial cells was investigated here. Pretreatment with blueberry anthocyanin extract, malvidin, malvidin-3-glucoside, and malvidin-3-galactoside significantly ameliorated high-glucose-induced damage by enhancing endogenous antioxidant superoxide dismutase (SOD) and heme oxygenase-1 (HO-1), lowering reactive oxygen species (ROS) generation and NADPH oxidase isoform 4 (NOX4) expression, and increasing the cell vitalities. They also effectively induced a vasodilatory effect by increasing the vasodilator nitric oxide (NO) and its promoters endothelial NO synthase (eNOS) and peroxisome proliferator-activated receptor-γ (PPARγ) levels as well as by decreasing the vasoconstrictor angiotensin-converting enzyme (ACE), xanthine oxidase-1 (XO-1), and low-density lipoprotein (LDL) levels. The activation of phosphoinositide 3-kinase (PI3K)/Akt signaling pathway and the breakdown of protein kinase C zeta (PKCζ) pathway were involved in the bioactivities. The results indicated blueberry anthocyanins protected endothelial function against high-glucose (HG) injury via antioxidant and vasodilatory mechanisms, which could be promising molecules as a hypotensive nutraceutical for diabetes patients.

Inhibiting NOXs with extra-virgin olive oil polyphenols as a strategy to prevent hepatic fibrogenesis

Introduction: An exaggerated activation of NADPH oxidases (NOXs), key enzymes in the production of reactive oxygen species (ROS), is implicated in inflammatory and fibrotic disorders. Although polyphenols display antioxidant activity, only few in vitro results have been obtained about their effect on the inhibition of specific NOX isoforms (e.g. NOX1 and 4) and nothing is known about the consequences of NOX inhibition on immune cell activation and/or recruitment in the liver, a process playing a pivotal role in fibrogenesis. Aim: to describe the in vivo role of NOXs in fibrogenesis and evaluate the antifibrotic and antioxidant effects of 4 polyphenols (oleacein, oleocanthal, tyrosol and hydroxytyrosol) extracted from extra-virgin olive oil.

KRIT1 loss-mediated upregulation of NOX1 in stromal cells promotes paracrine pro-angiogenic responses

Cerebral cavernous malformation (CCM) is a cerebrovascular disorder of proven genetic origin characterized by abnormally dilated and leaky capillaries occurring mainly in the central nervous system, with a prevalence of 0.3–0.5% in the general population. Genetic studies have identified causative mutations in three genes, CCM1/KRIT1, CCM2 and CCM3, which are involved in the maintenance of vascular homeostasis. However, distinct studies in animal models have clearly shown that CCM gene mutations alone are not sufficient to cause CCM disease, but require additional contributing factors, including stochastic events of increased oxidative stress and inflammation. Consistently, previous studies have shown that up-regulation of NADPH oxidase-mediated production of reactive oxygen species (ROS) in KRIT1 deficient endothelium contributes to the loss of microvessel barrier function.In this study, we demonstrate that KRIT1 loss-of-function in stromal cells, such as fibroblasts, causes the up-regulation of NADPH oxidase isoform 1 (NOX1) and the activation of inflammatory pathways, which in turn promote an enhanced production of proangiogenic factors, including vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2). Furthermore and importantly, we show that conditioned media from KRIT1 null fibroblasts induce proliferation, migration, matrix metalloproteinase 2 (MMP2) activation and VE-cadherin redistribution in wild type human endothelial cells.Taken together, our results demonstrate that KRIT1 loss-of-function in stromal cells affects the surrounding microenvironment through a NOX1-mediated induction and release of angiogenic factors that are able to promote paracrine proangiogenic responses in human endothelial cells, thus pointing to a novel role for endothelial cell-nonautonomous effects of KRIT1 mutations in CCM pathogenesis, and opening new perspectives for disease prevention and treatment.

NOX Inhibitors: From Bench to Naxibs to Bedside.

Reactive oxygen species (ROS) are ubiquitous metabolic products and important cellular signaling molecules that contribute to several biological functions. Pathophysiology arises when ROS are generated either in excess or in cell types or subcellular locations that normally do not produce ROS or when non-physiological types of ROS (e.g., superoxide instead of hydrogen peroxide) are formed. In the latter scenario, antioxidants were considered as the apparent remedy but, clinically, have consistently failed and even sometimes induced harm. The obvious reason for that is the non-selective ROS scavenging effects of antioxidants which interfere with both qualities of ROS, physiological and pathological. Therefore, it is essential to overcome this "antidote or neutralizer" strategy. We here review the most promising alternative approach by identifying the disease-relevant enzymatic sources of ROS, target these selectively, but leave physiological ROS signaling through other sources intact. Among all ROS sources, NADPH oxidases (NOX1-5 and DUOX1-2) stand out as their sole function is to produce ROS, whereas most other enzymatic sources only produce ROS as a by-product or upon biochemical uncoupling or damage. This qualifies NOXs as the main potential drug-target candidates in diseases associated with dysfunction in ROS signaling. As a reflection of this, the development of several NOX inhibitors has taken place. Recently, the WHO approved a new stem, "naxib," which refers to NADPH oxidase inhibitors, and thereby recognized NOX inhibitors as a new therapeutic class. This has been announced while clinical trials with the first-in-class compound, setanaxib (initially known as GKT137831) had been initiated. We also review the differences between the seven NOX family members in terms of structure and function in health and disease and then focus on the most advanced NOX inhibitors with an exclusive focus on clinically relevant validations and applications. Therapeutically relevant NADPH oxidase isoforms type 1, 2, 4, and 5 (NOX1, NOX2, NOX4, NOX5). Of note, NOX5 is not present in mice and rats and thus pre-clinically less studied. NOX2, formerly termed gp91phox, has been correlated with many, too many, diseases and is rather relevant as genetic deficiency in chronic granulomatous disease (CGD), treated by gene therapy. Overproduction of ROS through NOX1, NOX4, and NOX5 leads to the indicated diseases states including atherosclerosis (red), a condition where NOX4 is surprisingly protective.

Elevated Intraocular Pressure Causes Abnormal Reactivity of Mouse Retinal Arterioles.

Objective Glaucoma is a leading cause of severe visual impairment and blindness. Although high intraocular pressure (IOP) is an established risk factor for the disease, the role of abnormal ocular vessel function in the pathophysiology of glaucoma gains more and more attention. We tested the hypothesis that elevated intraocular pressure (IOP) causes vascular dysfunction in the retina. Methods High IOP was induced in one group of mice by unilateral cauterization of three episcleral veins. The other group received sham surgery only. Two weeks later, retinal vascular preparations were studied by video microscopy in vitro. Reactive oxygen species (ROS) levels and expression of hypoxia markers and of prooxidant and antioxidant redox genes as well as of inflammatory cytokines were determined. Results Strikingly, responses of retinal arterioles to stepwise elevation of perfusion pressure were impaired in the high-IOP group. Moreover, vasodilation responses to the endothelium-dependent vasodilator, acetylcholine, were markedly reduced in mice with elevated IOP, while no differences were seen in response to the endothelium-independent nitric oxide donor, sodium nitroprusside. Remarkably, ROS levels were increased in the retinal ganglion cell layer including blood vessels. Expression of the NADPH oxidase isoform, NOX2, and of the inflammatory cytokine, TNF-α, was increased at the mRNA level in retinal explants. Expression of NOX2, but not of the hypoxic markers, HIF-1α and VEGF-A, was increased in the retinal ganglion cell layer and in retinal blood vessels at the protein level. Conclusion Our data provide first-time evidence that IOP elevation impairs autoregulation and induces endothelial dysfunction in mouse retinal arterioles. Oxidative stress and inflammation, but not hypoxia, appear to be involved in this process.

Angiotensin II and aldosterone activate retinal microglia

Microglial cells are important contributors to the neuroinflammation and blood vessel damage that occurs in ischemic retinopathies. We hypothesized that key effectors of the renin-angiotensin aldosterone system, angiotensin II (Ang II) and aldosterone, increase the density of microglia in the retina and stimulate their production of reactive oxygen species (ROS) as well as pro-angiogenic and pro-inflammatory factors. Two animal models were studied that featured up-regulation of Ang II or aldosterone and included transgenic Ren-2 rats which overexpress renin and Ang II in tissues including the retina, and Sprague Dawley rats with ischemic retinopathy and infused with aldosterone. Complementary studies were performed in primary cultures of retinal microglia from neonatal Sprague Dawley rats exposed to hypoxia (0.5% O2) and inhibitors of the angiotensin type 1 receptor (valsartan), the mineralocorticoid receptor (spironolactone) or aldosterone synthase (FAD286). In both in vivo models, the density of ionized calcium-binding adaptor protein-1 labelled microglia/macrophages was increased in retina compared to genetic or vehicle controls. In primary cultures of retinal microglia, hypoxia increased ROS (superoxide) levels as well as the expression of the NADPH oxidase (NOX) isoforms, NOX1, NOX2 and NOX4. The elevated levels of ROS as well as NOX2 and NOX4 were reduced by all of the treatments, and valsartan and FAD286 also reduced NOX1 mRNA levels. A protein cytokine array of retinal microglia revealed that valsartan, spironolactone and FAD286 reduced the hypoxia-induced increase in the potent pro-angiogenic and pro-inflammatory agent, vascular endothelial growth factor as well as the inflammatory factors, CCL5 and interferon γ. Valsartan also reduced the hypoxia-induced increase in IL-6 and TIMP-1 as well as the chemoattractants, CXCL2, CXCL3, CXCL5 and CXCL10. Spironolactone and FAD286 reduced the levels of CXCL2 and CXCL10, respectively. In conclusion, our findings that both Ang II and aldosterone influence the activation of retinal microglia implicates the renin-angiotensin aldosterone system in the pathogenesis of ischemic retinopathies.

Isoform-selective NADPH oxidase inhibitor panel for pharmacological target validation

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 μM); NOX2, by VAS2870 (0.7 μM); NOX4, by M13 (0.01 μM) and NOX5, by ML090 (0.01 μM). 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.

Connexin32 ameliorates renal fibrosis in diabetic mice by promoting K48-linked NADPH oxidase 4 polyubiquitination and degradation.

Nox4 is the major isoform of NADPH oxidase found in the kidney and contributes to the pathogenesis of diabetic nephropathy. However, the molecular mechanisms of increased Nox4 expression induced by hyperglycaemia remain to be elucidated. Here, the role of the connexin32-Nox4 signalling axis in diabetic nephropathy and its related mechanisms were investigated. Diabetes was induced in mice by low-dose streptozotocin (STZ) combined with a high-fat diet. Effects of connexin32 on Nox4 expression and on renal function and fibrosis in STZ-induced diabetic mice were investigated using adenovirus-overexpressing connexin32 and connexin32-deficient mice. Interactions between connexin32 and Nox4 were analysed by co-immunoprecipitation and immunofluorescence assays. Connexin32 was down-regulated in the kidneys of STZ-induced diabetic mice. Overexpression of connexin32 reduced expression of Nox4 and improved renal function and fibrosis in diabetic mice, whereas connexin32 deficiency had opposite effects. Down-regulation of fibronectin expression by connexin32 was not dependent on gap junctional intercellular communication involving connexin32. Connexin32 interacted with Nox4 and reduced the generation of hydrogen peroxide, leading to the down-regulation of fibronectin expression. Mechanistically, connexin32 decreased Nox4 expression by promoting its K48-linked polyubiquitination. Interestingly, Smurf1 overexpression inhibited K48-linked polyubiquitination of Nox4. Furthermore, connexin32 interacted with Smurf1 and inhibited its expression. Connexin32 ameliorated renal fibrosis in diabetic mice by promoting K48-linked Nox4 polyubiquitination and degradation via inhibition of Smurf1 expression. Targeting the connexin32-Nox4 signalling axis may contribute to the development of novel treatments for diabetic nephropathy.

NADPH oxidase 2 as a potential therapeutic target for protection against cognitive deficits following systemic inflammation in mice

BackgroundResearch indicates that sepsis increases the risk of developing cognitive impairment. After systemic inflammation, a corresponding activation of microglia is rapidly induced in the brain, and multiple neurotoxic factors, including inflammatory mediators (e.g., cytokines) and reactive oxygen species (e.g., superoxide), are also released that contribute to neuronal injury. NADPH oxidase (NOX) enzymes play a vital role in microglial activation through the generation of superoxide anions. We hypothesized that NOX isoforms, particularly NOX2, could exhibit remarkable abilities in developing cognitive deficits induced by systemic inflammation. MethodsMice with deficits of NOX2 organizer p47phox (p47phox−/−) and wild-type (WT) mice treated with the NOX inhibitor diphenyleneiodonium (DPI) were used in this study. Intraperitoneal lipopolysaccharide (LPS) injection was used to induce systemic inflammation. Spatial learning and memory were compared among treatment groups using the radial arm maze task. Brain tissues were collected for evaluating the transcript levels of proinflammatory cytokines, whereas immunofluorescence staining and immunoblotting were conducted to determine the percentage of activated glia (microglia and astroglia) and damaged neurons and the expression of synaptic proteins and BDNF. ResultsCognitive impairment induced by systemic inflammation was significantly attenuated in the p47phox−/− mice compared to that in the WT mice. The p47phox−/− mice exhibited reduced microglial and astroglial activation and neuronal damage and attenuated the induction of multiple proinflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and CCL2. Similar to that observed in the p47phox−/− mice, the administration of DPI significantly attenuated the cognitive impairment, reduced the glial activation and brain cytokine concentrations, and restored the expression of postsynaptic proteins (PSD-95) and BDNF in neurons and astrocytes, compared to those in the vehicle-treated controls within 10 days after LPS injection. ConclusionsThis study clearly demonstrates that NOX2 contributes to glial activation with subsequent reduction in the expression of BDNF, synaptic dysfunction, and cognitive deficits after systemic inflammation in an LPS-injected mouse model. Our results provide evidence that NOX2 might be a promising pharmacological target that could be used to protect against synaptic dysregulation and cognitive impairment following systemic inflammation.

NADPH oxidase 4 mediates the protective effects of physical activity against obesity-induced vascular dysfunction.

Physical activity is one of the most potent strategies to prevent endothelial dysfunction. Recent evidence suggests vaso-protective properties of hydrogen peroxide (H2O2) produced by main endothelial NADPH oxidase isoform 4 (Nox4) in the vasculature. Therefore, we hypothesized that Nox4 connects physical activity with vaso-protective effects. Analysis of the endothelial function using Mulvany Myograph showed endothelial dysfunction in wild-type (WT) as well as in C57BL/6J/ Nox4-/- (Nox4-/-) mice after 20 weeks on high-fat diet (HFD). Access to running wheels during the HFD prevented endothelial dysfunction in WT but not in Nox4-/- mice. Mechanistically, exercise led to an increased H2O2 release in the aorta of WT mice with increased phosphorylation of eNOS pathway member AKT serine/threonine kinase 1 (AKT1). Both H2O2 release and phosphorylation of AKT1 were diminished in aortas of Nox4-/- mice. Deletion of Nox4 also resulted in lower intracellular calcium release proven by reduced phenylephrine-mediated contraction, whilst potassium-induced contraction was not affected. H2O2 scavenger catalase reduced phenylephrine-induced contraction in WT mice. Supplementing H2O2 increased phenylephrine-induced contraction in Nox4-/- mice. Exercise-induced peroxisome proliferative-activated receptor gamma, coactivator 1 alpha (Ppargc1a), as key regulator of mitochondria biogenesis in WT but not Nox4-/- mice. Furthermore, exercise-induced citrate synthase activity and mitochondria mass were reduced in the absence of Nox4. Thus, Nox4-/- mice became less active and ran less compared with WT mice. Nox4 derived H2O2 plays a key role in exercise-induced adaptations of eNOS and Ppargc1a pathway and intracellular calcium release. Hence, loss of Nox4 diminished physical activity performance and vascular protective effects of exercise.

Apolipoprotein E Deficiency Causes Endothelial Dysfunction in the Mouse Retina.

Objective Atherogenic lipoproteins may impair vascular reactivity consecutively causing tissue damage in multiple organs via abnormal perfusion and excessive reactive oxygen species generation. We tested the hypothesis that chronic hypercholesterolemia causes endothelial dysfunction and cell loss in the retina. Methods Twelve-month-old apolipoprotein E-deficient (ApoE-/-) mice and age-matched wild-type controls were used in this study (n = 8 per genotype for each experiment). Intraocular pressure, blood pressure, and ocular perfusion pressure were determined. Retinal arteriole responses were studied in vitro, and reactive oxygen and nitrogen species were quantified in the retinal and optic nerve cryosections. The expression of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and the NADPH oxidase isoforms, NOX1, NOX2, and NOX4, were determined in retinal cryosections by immunofluorescence microscopy. Pro- and antioxidant redox genes were quantified in retinal explants by PCR. Moreover, cell number in the retinal ganglion cell layer and axon number in the optic nerve was calculated. Results Responses to the endothelium-dependent vasodilator, acetylcholine, were markedly impaired in retinal arterioles of ApoE-/- mice (P < 0.01). LOX-1 (P = 0.0007) and NOX2 (P = 0.0027) expressions as well as levels of reactive oxygen species (P = 0.0022) were increased in blood vessels but not in other retinal structures. In contrast, reactive nitrogen species were barely detectable in both mouse genotypes. Messenger RNA for HIF-1α, VEGF-A, NOX1, and NOX2, but also for various antioxidant redox genes was elevated in the retina of ApoE-/- mice. Total cell number in the retinal ganglion cell layer did not differ between ApoE-/- and wild-type mice (P = 0.2171). Also, axon number in the optic nerve did not differ between ApoE-/- and wild-type mice (P = 0.6435). Conclusion Apolipoprotein E deficiency induces oxidative stress and endothelial dysfunction in retinal arterioles, which may trigger hypoxia in the retinal tissue. Oxidative stress in nonvascular retinal tissue appears to be prevented by the upregulation of antioxidant redox enzymes, resulting in neuron preservation.

NOX4 activation is involved in ROS-dependent Jurkat T-cell death induced by Entamoeba histolytica.

Entamoeba histolytica can induce host cell death through induction of various intracellular signalling pathways. The responses triggered by E.histolytica are closely associated with tissue pathogenesis and immune evasion. Although E.histolytica can induce reactive oxygen species (ROS) in host cells, which NADPH oxidase (NOX) isoform contributes to amoeba-triggered Jurkat T-cell death is unclear. In this study, we investigated the signalling role of NOX4-derived ROS in E.histolytica-induced Jurkat T-cell death process. In resting-state Jurkat T cells, NOX4 is strongly expressed. When Jurkat T cells were incubated with live E.histolytica trophozoites, intracellular ROS was significantly increased compared to cells incubated with medium alone. E.histolytica-induced ROS production was inhibited by pretreating Jurkat T cells with a NOX inhibitor. In addition, pretreating Jurkat T cells with a NOX inhibitor (Diphenyleneiodonium chloride) effectively blocked E.histolytica-induced phosphatidylserine (PS) exposure and DNA fragmentation of host cells. Moreover, siRNA-mediated knockdown of NOX4 protein expression in Jurkat T cells prevented E.histolytica-induced ROS generation and DNA fragmentation. These results suggest that NOX4 has a critical role in ROS-dependent cell death process in Jurkat T cells induced by E. histolytica.

Пути поиска новых мишеней для фармакологической коррекции эндотелиальной дисфункции

Сердечно-сосудистые заболевания остаются ведущей причиной смертности и инвалидизации, что диктует необходимость углубленного изучения их патогенеза и разработки мер лечения и профилактики. Основой современной терапии артериальной гипертензии и других сердечно-сосудистых заболеваний является постулат о необходимости коррекции дисфункции эндотелия как показателя адекватности антигипертензивного и других видов лечения. Фактически это означает, что снижение артериального давления (АД) без нормализации функции эндотелия не может считаться успешно решенной клинической задачей. Однако в настоящее время не существует препаратов для специфической фармакологической коррекции метаболического пути L-аргинин — eNOS — оксид азота, нарушения которого являются универсальным и основополагающим триггерным механизмом инициализации каскада метаболических нарушений, ведущих к артериальной гипертензии и другим заболеваниям сердечно-сосудистой системы. Аргиназы 1 и 2, NOX1 и NOX2 оксидазы как изоформы НАДФ·H-оксидазы, экспрессируемые эндотелиальными клетками, ангиотензинпревращающий фермент 2, а также GSNO-редуктазы могут представлять интерес в направленном поиске лекарственных средств для фармакологической коррекции эндотелиальной дисфункции.