Catalytic Subunit Of NADPH Oxidase Research Articles

Участь γ-аміномасляної кислоти у клітинних сигнальних процесах і в адаптації рослин до дії абіотичних стресорів

Adaptation of plants to stress factors occurs with the participation of stress phytohormones, signaling network and plant neurotransmitters. Among the latter, in particular, γ-aminobutyric acid (γ-aminobutyric acid – GABA) is a non-proteinogenic four-carbon amino acid found in many prokaryotic and eukaryotic organisms. Its functions in plants have been actively studied only in the past decade. During this period, a lot of information has been accumulated about the protective effect of exogenous GABA on plants of various taxonomic groups under the influence of stress factors of various nature. The first national review is devoted to the analysis and generalization of data on the mechanisms of stress-protective action of GABA in plants. The ways of synthesis and metabolism of GABA in plant cells and the mechanisms of activation of these processes under stressful conditions are described. It is noted that the main way of GABA formation in plants is decarboxylation of glutamate by means of glutamate decarboxy­lase. Possible mechanisms of GABA reception and signal transmission to the genetic apparatus are discussed. Special attention is paid to the analysis of new data on the role of calcium in the activation of GABA synthesis and the realization of its physiological effects. Possible mechanisms of GABA’s influence on the functioning of mitochondria, its role in maintaining redox homeostasis under stressful conditions are discussed. At the same time, data on the increase in the expression of genes encoding the catalytic subunit of NADPH oxidase under the influence of GABA are presented. Functional connections between GABA and nitric oxide as a signaling mediator are considered. The effect of exogenous GABA on the main protective reactions of plants is characte­rized: the state of the antioxidant system, the accumulation of multifunctional low-molecular protectors, the synthesis of dehydrins and chaperones. The data on the phenomeno­logy of the effects of GABA under the main abiotic stresses are presented: the effects of extreme temperatures, drought and salinity on plants. The prospects for the practical use of GABA as a compound that combines the functions of an energy metabolite and a signaling mediator are noted.

Naked Antibody-Based Conditioning Targeting CD47 and CD117 Plus Janus Kinase Inhibition Enables Toxin-Free, Radiation-Free, and Chemotherapy-Free Fully Mismatched Allogeneic Hematopoietic Stem Cell Transplantation

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for numerous malignant and non-malignant hematologic diseases. To enable engraftment of donor hematopoietic stem cells (HSCs), recipients undergo conditioning with chemotherapy and irradiation to make marrow space for incoming donor HSCs and suppress host immune responses that may lead to graft rejection. These cytotoxic conditioning regimens carry risks of severe toxicities which prevents many patients from accessing the lifesaving benefit of allo-HSCT. Previously, we developed fully ablative CD45- and cKit-targeted antibody-drug conjugates (ADC) which, when combined with Janus kinase 1/2 (JAK1/2) inhibitors, enabled fully mismatched allo-HSCT with full donor chimerism and minimal toxicity while providing durable antileukemia activity (Persaud et al (2022), ASH Abstract). However, even ADCs carry potential risks of acute and chronic toxicities that may preclude their common use as HSCT conditioning agents. Moreover, unlike malignant diseases, allo-HSCT for non-malignant diseases does not require the conditioning regimen to provide antitumor benefit and partial donor chimerism may be sufficient for cure, strongly favoring the use of less intense conditioning strategies with the fewest possible toxicities. We hypothesized that naked antibody-based conditioning strategies can achieve the therapeutic benefits of allo-HSCT for non-malignant hematologic diseases, obviating exposure to toxic ADC payloads. METHODS: We combined unconjugated antibodies targeting CD47 and cKit with JAK1/2 inhibition to perform minor histocompatibility antigen (miHA)-mismatched (129/Sv→B6-Ly5.1) and fully mismatched (BALB/c→B6) murine allo-HSCT. In some full mismatch HSCT experiments, recipients included B6-gp91phox -/- mice, which serve as a model of chronic granulomatous disease (CGD) due to their deficiency in a catalytic subunit of NADPH oxidase. Mice received 100 µg anti-CD47 (clone mIAP410; BioXcell) 8 days before HSCT (d-8) as a priming dose to reduce the severity of CD47 blockade-induced anemia. Next, mice received 500 µg anti-cKit (clone ACK2; BioLegend) on d-6 and daily 500 µg doses of anti-CD47 from d-6 through d-2. For JAK1/2 inhibition, mice received oral ruxolitinib (Incyte; 2 mg drug/g chow) starting d-3 or d-1 and continuing until d+28. On d0, recipients were transplanted with 20 x 10 6 whole bone marrow cells from 129/Sv (miHA) or BALB/c (fully mismatched) mice. Peripheral blood donor chimerism was followed monthly by flow cytometry for CD45.2 + (miHA) or H-2D d+ (fully mismatched) cells. Oxidative burst among granulocytes in B6-gp91phox -/- recipients was assessed by dihydrorhodamine 123 (DHR123) staining after phorbol 12-myristate 13-acetate (PMA) stimulation. RESULTS: CD47 and cKit antibodies plus ruxolitinib enabled multilineage engraftment in both miHA and fully mismatched allo-HSCT, including myeloid lineage donor chimerism of 95-99% (Figure 1A). As expected, CD47 blockade induced significant but transient morbidity from anemia during the peritransplant period; notably, a shorter duration of ruxolitinib treatment before HSCT (starting d-1 instead of d-3) improved the tolerability of the regimen without impacting engraftment success. Allo-HSCT of B6-gp91phox -/- mice led to near complete replacement of host granulocytes in peripheral blood with donor granulocytes, whose DHR123 positivity upon PMA stimulation indicated an intact oxidative burst capacity (Figure 1B). CONCLUSIONS: Combination of antibodies targeting CD47 and cKit with JAK1/2 inhibition provides a novel conditioning strategy for allo-HSCT in mouse models, achieving high-level, stable multilineage engraftment without radiation, chemotherapy, or exposure to toxic ADC payloads. The near complete donor chimerism among granulocytes made it particularly well suited to correcting phagocyte oxidase function in the gp91phox -/- model of CGD. Future studies seek to 1) mitigate the peritransplant anemia caused by CD47 blockade using Fc-silenced anti-CD47 reagents, 2) determine the proportions of donor- versus recipient-derived myeloid cells in non-hematopoietic tissues of allo-HSCT recipients, and 3) use murine infection models to evaluate the impact of allo-HSCT on host defense in transplanted B6-gp91phox -/- recipients.

Activation of neuronal NADPH oxidase NOX2 promotes inflammatory neurodegeneration.

Strong evidence indicates critical roles of NADPH oxidase (a key superoxide-producing enzyme complex during inflammation) in activated microglia for mediating neuroinflammation and neurodegeneration. However, little is known about roles of neuronal NADPH oxidase in neurodegenerative diseases. This study aimed to investigate expression patterns, regulatory mechanisms and pathological roles of neuronal NADPH oxidase in inflammation-associated neurodegeneration. The results showed persistent upregulation of NOX2 (gp91phox; the catalytic subunit of NADPH oxidase) in both microglia and neurons in a chronic mouse model of Parkinson's disease (PD) with intraperitoneal LPS injection and LPS-treated midbrain neuron-glia cultures (a cellular model of PD). Notably, NOX2 was found for the first time to exhibit a progressive and persistent upregulation in neurons during chronic neuroinflammation. While primary neurons and N27 neuronal cells displayed basal expression of NOX1, NOX2 and NOX4, significant upregulation only occurred in NOX2 but not NOX1 or NOX4 under inflammatory conditions. Persistent NOX2 upregulation was associated with functional outcomes of oxidative stress including increased ROS production and lipid peroxidation. Neuronal NOX2 activation displayed membrane translocation of cytosolic p47phox subunit and was inhibited by apocynin and diphenyleneiodonium chloride (two widely-used NADPH oxidase inhibitors). Importantly, neuronal ROS production, mitochondrial dysfunction and degeneration induced by inflammatory mediators in microglia-derived conditional medium were blocked by pharmacological inhibition of neuronal NOX2. Furthermore, specific deletion of neuronal NOX2 prevented LPS-elicited dopaminergic neurodegeneration in neuron-microglia co-cultures separately grown in the transwell system. The attenuation of inflammation-elicited upregulation of NOX2 in neuron-enriched and neuron-glia cultures by ROS scavenger N-acetylcysteine indicated a positive feedback mechanism between excessive ROS production and NOX2 upregulation. Collectively, our findings uncovered crucial contribution of neuronal NOX2 upregulation and activation to chronic neuroinflammation and inflammation-related neurodegeneration. This study reinforced the importance of developing NADPH oxidase-targeting therapeutics for neurodegenerative diseases.

Aircraft noise exposure drives the activation of white blood cells and induces microvascular dysfunction in mice

Epidemiological studies showed that traffic noise has a dose-dependent association with increased cardiovascular morbidity and mortality. Whether microvascular dysfunction contributes significantly to the cardiovascular health effects by noise exposure remains to be established. The connection of inflammation and immune cell interaction with microvascular damage and functional impairment is also not well characterized. Male C57BL/6J mice or gp91phox−/y mice with genetic deletion of the phagocytic NADPH oxidase catalytic subunit (gp91phox or NOX-2) were used at the age of 8 weeks, randomly instrumented with dorsal skinfold chambers and exposed or not exposed to aircraft noise for 4 days. Proteomic analysis (using mass spectrometry) revealed a pro-inflammatory phenotype induced by noise exposure that was less pronounced in noise-exposed gp91phox−/y mice. Using in vivo fluorescence microscopy, we found a higher number of adhesive leukocytes in noise-exposed wild type mice. Dorsal microvascular diameter (by trend), red blood cell velocity, and segmental blood flow were also decreased by noise exposure indicating microvascular constriction. All adverse effects on functional parameters were normalized or improved at least by trend in noise-exposed gp91phox−/y mice. Noise exposure also induced endothelial dysfunction in cerebral microvessels, which was associated with higher oxidative stress burden and inflammation, as measured using video microscopy. We here establish a link between a pro-inflammatory phenotype of plasma, activation of circulating leukocytes and microvascular dysfunction in mice exposed to aircraft noise. The phagocytic NADPH oxidase was identified as a central player in the underlying pathophysiological mechanisms.

Involvement of NADPH oxidase in patulin-induced oxidative damage and cytotoxicity in HEK293 cells

Patulin (PAT) is a kind of mycotoxins that commonly found in decayed fruits and their products. Our previous studies have shown that PAT induced cell apoptosis and the overproduction of reactive oxygen species (ROS) in human embryonic kidney (HEK293) cells. The present study aimed to further investigate the functional role of NADPH oxidase, one of the main cellular sources of ROS, in PAT-induced apoptosis and oxidative damage in HEK293 cells. We demonstrated that the protein and mRNA expression levels of NADPH oxidase catalytic subunit NOX2 and regulatory subunit p47phox were up-regulated under PAT stress. Inhibiting of NADPH oxidase with the specific antagonist diphenyleneiodonium (DPI) suppressed cytotoxicity and apoptosis induced by PAT as evidenced by the increase of cell viability, the decrease of LDH release and the inhibition of caspase activities. Furthermore, DPI re-established mitochondrial membrane potential (MMP) and enhanced cellular ATP content. Importantly, DPI supplementation elevated endogenous GSH contents as well as the ratio of GSH/GSSG. Meanwhile, the antioxidant-enzyme activities of GPx, GR, CAT and SOD were significantly promoted. Collectively, our results suggested that NADPH oxidase played a critical role in PAT-induced nephrotoxicity, and inhibition of NADPH oxidase by DPI attenuated cell injury and apoptosis via regulation of oxidative damage.

The Hypoxia Tolerance of the Goldfish (Carassius auratus) Heart: The NOS/NO System and Beyond.

The extraordinary capacity of the goldfish (Carassius auratus) to increase its cardiac performance under acute hypoxia is crucial in ensuring adequate oxygen supply to tissues and organs. However, the underlying physiological mechanisms are not yet completely elucidated. By employing an ex vivo working heart preparation, we observed that the time-dependent enhancement of contractility, distinctive of the hypoxic goldfish heart, is abolished by the Nitric Oxide Synthase (NOS) antagonist L-NMMA, the Nitric Oxide (NO) scavenger PTIO, as well as by the PI3-kinase (PI3-K) and sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) pumps’ inhibition by Wortmannin and Thapsigargin, respectively. In goldfish hearts exposed to hypoxia, an ELISA test revealed no changes in cGMP levels, while Western Blotting analysis showed an enhanced expression of the phosphorylated protein kinase B (pAkt) and of the NADPH oxidase catalytic subunit Nox2 (gp91phox). A significant decrease of protein S-nitrosylation was observed by Biotin Switch assay in hypoxic hearts. Results suggest a role for a PI3-K/Akt-mediated activation of the NOS-dependent NO production, and SERCA2a pumps in the mechanisms conferring benefits to the goldfish heart under hypoxia. They also propose protein denitrosylation, and the possibility of nitration, as parallel intracellular events.

NOX5 as a therapeutic target in cerebral ischemic injury.

In this issue of the JCI, Casas et al. define a previously unknown role of the NADPH oxidase catalytic subunit NOX5 in cerebral infarction. Using a mouse expressing human NOX5 in the endothelium, the investigators show that NOX5 is activated and plays a deleterious role in promoting edema, infarction, and ultimately, worsened neurological function following cerebral ischemia. They provide evidence that this is due to the breakdown of the blood-brain barrier (BBB) and that a unique pharmacological inhibitor of NOX5, ML090, if given early, around the time of reoxygenation, can maintain BBB integrity. Future studies of NOX5 inhibition in humans, particularly in the setting of thrombolysis, are warranted.

Abstract 17385: Endothelium Specific Reduction in Mitochondrial ROS Reverses the Deleterious Effects on Coronary Endothelium Caused by Prolonged Increase in NADPH Oxidase Derived ROS

Introduction: Recent findings from our lab demnstrated that prolonged increase in NADPH oxidase (NOX)-derived ROS results in increased mitochondrial (mito) ROS levels, reduced aspartate/dNTP synthesis, reduced coronary endothelial cell (EC) proliferation, and decreased recovery of post-MI cardiac function. Hypothesis: We hypothesized that endothelium-specific reduction in mitochondrial ROS will reverse the deleterious effects of prolonged NOX-ROS increase by improving mitochondrial function and aspartate/dNTP synthesis. Methods: Tetracycline (Tet)-ON/Tet-OFF binary transgenic mice, Tet-Nox2:VE-Cad-tTA (NOX-OE), were used as a model for endothelium-specific increase in the expression of the catalytic subunit of NADPH oxidase resulting in increased ROS. Mouse heart EC (MHEC) were isolated from Tet-ON and Tet-OFF Tg-Nox2 mice that were exposed to >16 weeks of increased EC-ROS. MHECs were then subject to mitochondrial antioxidants nitroxide (XJB-131) and nanoparticle (CeTiO2) followed by determination of mito-ROS, aspartate/dNTP synthesis, and EC proliferation. NOX-OE was crossbred with SOD-OE to generate a double-transgenic mice with EC-specific overexpression of mitochondrial antioxidant MnSOD (Sod2), Tet-Sod2:Tet-Nox2:VE-Cad-tTA ( SOD-OE ). Both NOX-OE and SOD-OE were subject to myocardial infarction (MI) LAD ligation experiments. Results: Mito-ROS levels were significantly reduced (by >2-fold) in MHEC from SOD-OE compared to NOX-OE. Both dNTP and proliferation were increased in SOD-OE, and also in NOX-EC treated with XJB-131 and CeTiO2. Together, these data suggest that reduction in mito-ROS abrogated negative effects of NOX-ROS in EC. Preliminary data demonstrate that there was an increase in capillary density (by 34± 5.8 %; p<0.05) and angiogenesis (by 2-fold) in SOD-OE post-MI myocardium compared to that of NOX-OE. Infarct sizes and cardiac function between NOX-OE and SOD-OE will be presented. Conclusion: These results suggest that reduction in mito-ROS can abrogate deleterious effects coronary endothelium caused by prolonged exposure to NOX-ROS. EC-specific modulation of mito-ROS may thus help develop therapeutic modalities to improve cardiovascular health and myocardial preservation in the post-MI heart.

In silico physicochemical characterization and topology analysis of Respiratory burst oxidase homolog (Rboh) proteins from Arabidopsis and rice.

NADPH oxidase (NOX) is a key enzyme involved in the production of apoplastic superoxide (O2-), a type of reactive oxygen species (ROS). Plant Noxes are the homologs of mammalian NADPH oxidase's catalytic subunit and are documented as respiratory burst oxidase homologs (Rbohs). A number of studies have reported their diverse functions in combating various stresses and in plant growth and development. In the present study, a total of 19 Rboh proteins (10 from Arabidopsis thaliana and 9 from Oryza sativa Japonica) were analyzed. We employed in silico approaches to compute the physiochemical properties (molecular weight, isoelectric point, total number of negatively and positively charged residues, extinction coefficient, half-life, instability and aliphatic index, grand average of hydropathicity, amino acid percentage). We observed a lot of variability in these parameters among the Rbohs accounting for their functional diversification. Their topological analysis, subcellular localization and signal peptide detection are also performed. To the best of our knowledge, the present study report on in silico physiochemical characterization, topology analysis, subcellular localization and signal peptide detection of Rboh proteins within two model plants. The study elucidates the variations in the key properties among Rbohs proteins, which may be responsible for their functional multiplicity.

Role of endoplasmic reticulum stress in 12/15-lipoxygenase-induced retinal microvascular dysfunction in a mouse model of diabetic retinopathy.

Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy. We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo-/-); ER stress was then evaluated after 12-14weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2-/- mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1μmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca2+) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leucocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO-derived metabolites both in vitro and in vivo. We also found that 15-HETE increases the intracellular calcium in HRECs. ER stress contributes to 12/15-LO-induced retinal inflammation in diabetic retinopathy via activation of NADPH oxidase and VEGFR2. Perturbation of calcium homeostasis in the retina might also play a role in linking 12/15-LO to retinal ER stress and subsequent microvascular dysfunction in diabetic retinopathy.

Abstract 21411: Coronary Endothelial ROS Protects Cardiac Function

Introduction: Recent findings from our lab demonstrated that modulation of NADPH oxidase-derived ROS for a short period (4 weeks), but not long period (20 weeks), improves coronary endothelial cell proliferation in vitro and aortic sprouting angiogenesis ex vivo . Hypothesis: We hypothesized that whereas short-term increase in endothelial ROS will improve post-MI cardiac function by increased coronary angiogenesis, long-term exposure to ROS will not. Methods: Tetracycline (Tet)-ON/Tet-OFF binary transgenic mice, Tet-Nox2:VE-Cad-tTA (Tg-Nox2), were used as a model for endothelium-specific increase in the expression of the catalytic subunit of NADPH oxidase resulting in increased ROS. Tet-ON and Tet-OFF Tg-Nox2 mice that were exposed to 4 weeks (short-term) and 16 weeks (long-term) of increased EC-ROS were subject to LAD ligation. Results: We demonstrate that there was an increase in capillary density (by 38± 6.45; p<0.05) and angiogenesis (by 2-fold) in short-term Tg-Nox2 post-MI myocardium but not in long-term Tg-Nox2. Short-term, but not long-term, Tg-Nox2 animals showed reduction in infarct size by Trichrome staining and improvement in cardiac function including EF, LVEDV and LVESV by echocardiography. Conclusion: These findings suggest that a short, but not long, exposure to increased EC-ROS play an important role in cardiovascular health and myocardial preservation in the post-MI heart.

Myeloid-specific deletion of NOX2 prevents the metabolic and neurologic consequences of high fat diet.

High fat diet-induced obesity is associated with inflammatory and oxidative signaling in macrophages that likely participates in metabolic and physiologic impairment. One key factor that could drive pathologic changes in macrophages is the pro-inflammatory, pro-oxidant enzyme NADPH oxidase. However, NADPH oxidase is a pleiotropic enzyme with both pathologic and physiologic functions, ruling out indiscriminant NADPH oxidase inhibition as a viable therapy. To determine if targeted inhibition of monocyte/macrophage NADPH oxidase could mitigate obesity pathology, we generated mice that lack the NADPH oxidase catalytic subunit NOX2 in myeloid lineage cells. C57Bl/6 control (NOX2-FL) and myeloid-deficient NOX2 (mNOX2-KO) mice were given high fat diet for 16 weeks, and subject to comprehensive metabolic, behavioral, and biochemical analyses. Data show that mNOX2-KO mice had lower body weight, delayed adiposity, attenuated visceral inflammation, and decreased macrophage infiltration and cell injury in visceral adipose relative to control NOX2-FL mice. Moreover, the effects of high fat diet on glucose regulation and circulating lipids were attenuated in mNOX2-KO mice. Finally, memory was impaired and markers of brain injury increased in NOX2-FL, but not mNOX2-KO mice. Collectively, these data indicate that NOX2 signaling in macrophages participates in the pathogenesis of obesity, and reinforce a key role for macrophage inflammation in diet-induced metabolic and neurologic decline. Development of macrophage/immune-specific NOX-based therapies could thus potentially be used to preserve metabolic and neurologic function in the context of obesity.

Nebivolol prevents ethanol-induced reactive oxygen species generation and lipoperoxidation in the rat kidney by regulating NADPH oxidase activation and expression

We studied whether the β1-adrenergic antagonist nebivolol would prevent ethanol-induced reactive oxygen species generation and lipoperoxidation in the rat renal cortex. Male Wistar rats were treated with ethanol (20% v/v) for 2 weeks. Nebivolol (10mg/kg/day; p.o. gavage) prevented both the increase in superoxide anion (O2-) generation and thiobarbituric acid reactive substances (TBARS) concentration induced by ethanol in the renal cortex. Ethanol decreased nitrate/nitrite (NOx) concentration in the renal cortex, and nebivolol prevented this response. Nebivolol did not affect the reduction of hydrogen peroxide (H2O2) concentration induced by ethanol. Nebivolol prevented the ethanol-induced increase of catalase (CAT) activity. Both SOD activity and the levels of reduced glutathione (GSH) were not affected by treatment with nebivolol or ethanol. Neither ethanol nor nebivolol affected the expression of Nox1, Nox4, eNOS, nNOS, CAT, Nox organizer 1 (Noxo1), c-Src, p47phox or superoxide dismutase (SOD) isoforms in the renal cortex. On the other hand, treatment with ethanol increased Nox2 expression, and nebivolol prevented this response. Finally, nebivolol reduced the expression of protein kinase (PK) Cδ and Rac1. The major finding of our study is that nebivolol prevented ethanol-induced reactive oxygen species generation and lipoperoxidation in the kidney by a mechanism that involves reduction on the expression of Nox2, a catalytic subunit of NADPH oxidase. Additionally, we demonstrated that nebivolol reduces NADPH oxidase-derived reactive oxygen species by decreasing the expression of PKCδ and Rac1, which are important activators of NADPH oxidase.

Constitutive NOS uncoupling and NADPH oxidase upregulation in the penis of type 2 diabetic men with erectile dysfunction.

Erectile dysfunction (ED) associated with type 2 diabetes mellitus (T2DM) involves dysfunctional nitric oxide (NO) signaling and increased oxidative stress in the penis. However, the mechanisms of endothelial NO synthase (eNOS) and neuronal NO synthase (nNOS) dysregulation, and the sources of oxidative stress, are not well defined, particularly at the human level. The objective of this study was to define whether uncoupled eNOS and nNOS, and NADPH oxidase upregulation, contribute to the pathogenesis of ED in T2DM men. Penile erectile tissue was obtained from 9 T2DM patients with ED who underwent penile prosthesis surgery for ED, and from six control patients without T2DM or ED who underwent penectomy for penile cancer. The dimer-to-monomer protein expression ratio, an indicator of uncoupling for both eNOS and nNOS, total protein expressions of eNOS and nNOS, as well as protein expressions of NADPH oxidase catalytic subunit gp91phox (an enzymatic source of oxidative stress) and 4-hydroxy-2-nonenal [4-HNE] and nitrotyrosine (markers of oxidative stress) were measured by western blot in this tissue. In the erectile tissue of T2DM men, eNOS and nNOS uncoupling and protein expressions of NADPH oxidase subunit gp91phox, 4-HNE- and nitrotyrosine-modified proteins were significantly (p<0.05) increased compared to control values. Total eNOS and nNOS protein expressions were not significantly different between the groups. In conclusion, mechanisms of T2DM-associated ED in the human penis may involve uncoupled eNOS and nNOS and NADPH oxidase upregulation. Our description of molecular factors contributing to the pathogenesis of T2DM-associated ED at the human level is relevant to advancing clinically therapeutic approaches to restore erectile function in T2DM patients.

The NADPH Oxidase Catalytic Subunit Nox2 Displays Differential Roles in Arterial Vs. Venous Thrombosis

NADPH oxidase is a major superoxide generating enzyme in vascular tissues. Deficiency of the Nox2 (gp91phox) catalytic subunit of NADPH oxidase is a genetic cause of X-linked chronic granulomatous disease. These patients are prone to infection due to loss of oxidant production by neutrophils, and recent evidence suggests they also have a defect in platelet adhesion to collagen. However, the role of Nox2 in thrombosis is controversial, with one study demonstrating no effect of murine Nox2 deficiency on platelet activation and another study implicating Nox2 in platelet aggregation as well as thrombosis in small vessels. Given that Nox2 may be important for both platelet activation as well as release of neutrophil extracellular traps (NETs, a mediator of venous thrombosis), we hypothesized that genetic deficiency in murine Nox2 leads to decreased susceptibility to experimental thrombosis in both arterial and venous systems. We studied male Nox2 deficient (Cybb-/y) mice and wild type (Cybb +/y) littermates at 10-14 weeks of age. There were no differences in platelet count (921.9±33.6 x103/µl vs. 978±85.2 x103/µl), hematocrit (36.5±0.6% vs. 38.7±0.8% ) or white blood cell count (5.5±0.3 x103/µl vs. 6.1±0.6 x103/µl) between Cybb+/y and Cybb-/y mice. We next, examined platelet activation in response to thrombin (0.05 and 0.2 U/ml) or collagen (80 and 320 ng/ml) using flow cytometry. We observed similar levels of integrin α2bβ3 activation, fibrinogen binding, and intra-platelet levels of H2O2 in platelets from Cybb+/y and Cybb-/y mice after activation with either agonist, which suggests no alteration in platelet inside-out signaling due to loss of Nox2. No significant difference in susceptibility to carotid artery thrombosis in a photochemical injury model was observed between Cybb+/y and Cybb-/- mice (time to stable occlusion 21.97±4.7 vs 27.6±4.2, P>0.4). In contrast, Cybb-/y mice demonstrated significant decreases in the weight and length of venous thrombi in the inferior vena cava after 48 hours of stenosis (P<0.05). Our findings suggest that Nox2 is not a major mediator of platelet activation or arterial thrombosis but contributes to the development of venous thrombi. Future studies are warranted to examine the role of NETs and the prothrombotic effects of Nox2 in association with other cardiovascular risk factors. DisclosuresLentz:Novo Nordisk: Consultancy, Research Funding; Celgene: Equity Ownership; Opko: Membership on an entity's Board of Directors or advisory committees.

β-eudesmol, a sesquiterpene from Teucrium ramosissimum, inhibits superoxide production, proliferation, adhesion and migration of human tumor cell.

Reactive oxygen species are well-known mediators of various biological responses. Recently, new homologues of the catalytic subunit of NADPH oxidase have been discovered in non phagocytic cells. These new homologues (Nox1-Nox5) produce low levels of superoxides compared to the phagocytic homologue Nox2/gp91phox. In this study we examined the effect of β-eudesmol, a sesquiterpenoid alcohol isolated from Teucrium ramosissimum leaves, on proliferation, superoxide anion production, adhesion and migration of human lung (A549) and colon (HT29 and Caco-2) cancer cell lines. Proliferation of tumor cells was inhibited by β-eudesmol. It also significantly inhibited superoxide production in A549 cells. Furthermore, β-eudesmol inhibited adhesion and migration of A549 and HT29 cell. These results demonstrate that β-eudesmol may be a novel anticancer agent for the treatment of lung and colon cancer by different ways: by inhibition of superoxide production or by blocking proliferation, adhesion and migration.

Reactive oxygen species derived from NADPH oxidase 1 and mitochondria mediate angiotensin II-induced smooth muscle cell senescence

Cellular senescence has emerged as an important player in both physiology and pathology. Excessive reactive oxygen species (ROS) is known to mediate cellular senescence. NADPH oxidases are major sources for ROS production in the vascular wall; the roles of different NADPH oxidase isoforms in cellular senescence remain unclear, however. We investigated the roles of two NADPH oxidase isoforms in mitochondrial dysfunction during angiotensin II (Ang II)-induced cellular senescence of human aortic vascular smooth muscle cells (VSMCs). Ang II (10−7M) stimulated ROS generation, exhibiting early increases between 30 and 60min and sustained increases between 24h and 72h, and induced VSMCs senescence after 48h or 72h treatment as assessed with senescence-associated β-galactosidase activity and the expression of two cell cycle inhibitors, p21 and p16. ROS scavengers and membrane-permeable catalase (catalase-PEG) reduced Ang II-stimulated cellular senescence. Furthermore, small interfering RNA (siRNA) of NADPH oxidase catalytic subunit Nox1, but not that of another isoform Nox4, inhibited Ang II-induced cellular senescence. Nox1 siRNA inhibited both early and sustained ROS increases induced by Ang II. In addition, a mitochondrial-specific antioxidant, mitoQ10, effectively inhibited Ang II-induced ROS increases and cellular senescence. Ang II decreased ATP synthesis and induced mitochondrial membrane depolarization, which were attenuated by pre-treating cells with Nox1 siRNA, mitoQ10 or catalase-PEG. The effect of Ang II on the mitochondrial regulator peroxisome-proliferator-activated receptor gamma coactivator-1α (PGC-1α) and its downstream genes was examined. Ang II stimulated S570 phosphorylation of PGC-1α with concomitant decreases in catalase and uncoupling protein-2 (UCP-2) levels between 12h and 72h, which were inhibited by Nox1 siRNA. Knockdown of both catalase and UCP-2 mimicked Ang II-induced VSMC senescence. These results suggested that Ang II-stimulated Nox1 activation mediates mitochondrial dysfunction, probably by decreasing PGC-1α activity and increasing mitochondrial oxidative stress, and leads to cellular senescence of VSMCs.

Change of concept about the regulation of angiotensin II-induced monocyte chemoattractant protein-1 production in human endothelial cells

AimsSome intriguing clinical observations about the anti-inflammatory effects of angiotensin type 1 (AT1) receptor blockers and angiotensin converting enzyme inhibitors in cardiovascular patients brought us to study the signalling pathways which lead to angiotensin II (ANG)-induced monocyte chemoattractant protein-1 (MCP-1) production in human endothelial cells. MethodsMCP-1 production in human umbilical vein endothelial cells (HUVECs) under treatments with ANG, AT1 and angiotensin type 2 (AT2) receptor blockers and pravastatin was measured by ELISA. The expression of AT1 and AT2 receptors and NADPH oxidase catalytic subunits (NOX 1–5) was analysed at mRNA and protein levels. Nuclear factor-kappa B (NF-κB) activation was studied by p65 subunit translocation to the cellular nucleus. Cell viability was tested by the MTT method. Nox4 subcellular distribution was analysed by subcellular protein fractionation and by immunoprecipitation followed by matrix-assisted laser desorption/ionization mass spectrometry analysis. ResultsANG-induced MCP-1 production was mediated by AT2 receptor, but not AT1 receptor in HUVECs in culture, which in turn activated NF-κB, promoting p65 subunit translocation to the nucleus. Reactive oxygen species produced by NADPH oxidase participated in this activation, mainly by the Nox4 subunit, ubiquitously expressed in all the compartments of HUVECs. Pravastatin inhibited ANG-induced MCP-1 production. ConclusionsOur results support that ANG-induced MCP-1 production in HUVECs is mediated by AT2 instead AT1 receptor activation, which in turn activates NF-κB involving reactive oxygen species produced by the NADPH oxidase complex. Statins can also block ANG-induced MCP-1 production, probably by their inhibitory effects on NADPH oxidase activity.

A lipidomic screen of hyperglycemia-treated HRECs links 12/15-Lipoxygenase to microvascular dysfunction during diabetic retinopathy via NADPH oxidase

Retinal hyperpermeability and subsequent macular edema is a cardinal feature of early diabetic retinopathy (DR). Here, we investigated the role of bioactive lipid metabolites, in particular 12/15-lipoxygenase (LOX)-derived metabolites, in this process. LC/MS lipidomic screen of human retinal endothelial cells (HRECs) demonstrated that 15-HETE was the only significantly increased metabolite (2.4 ± 0.4-fold, P = 0.0004) by high glucose (30 mM) treatment. In the presence of arachidonic acid, additional eicosanoids generated by 12/15-LOX, including 12- and 11-HETEs, were significantly increased. Fluorescein angiography and retinal albumin leakage showed a significant decrease in retinal hyperpermeability in streptozotocin-induced diabetic mice lacking 12/15-LOX compared with diabetic WT mice. Our previous studies demonstrated the potential role of NADPH oxidase in mediating the permeability effect of 12- and 15-HETEs, therefore we tested the impact of intraocular injection of 12-HETE in mice lacking the catalytic subunit of NADPH oxidase (NOX2). The permeability effect of 12-HETE was significantly reduced in NOX2(-/-) mice compared with the WT mice. In vitro experiments also showed that 15-HETE induced HREC migration and tube formation in a NOX-dependent manner. Taken together our data suggest that 12/15-LOX is implicated in DR via a NOX-dependent mechanism.

NADPH oxidase and the cardiovascular toxicity associated with smoking.

Smoking is one of the most serious but preventable causes of cardiovascular disease (CVD). Key aspects of pathological process associated with smoking include endothelial dysfunction, a prothrombotic state, inflammation, altered lipid metabolism, and hypoxia. Multiple molecular events are involved in smokinginduced CVD. However, the dysregulations of reactive oxygen species (ROS) generation and metabolism mainly contribute to the development of diverse CVDs, and NADPH oxidase (NOX) has been established as a source of ROS responsible for the pathogenesis of CVD. NOX activation and resultant ROS production by cigarette smoke (CS) treatment have been widely observed in isolated blood vessels and cultured vascular cells, including endothelial and smooth muscle cells. NOX-mediated oxidative stress has also been demonstrated in animal studies. Of the various NOX isoforms, NOX2 has been reported to mediate ROS generation by CS, but other isoforms were not tested thoroughly. Of the many CS constituents, nicotine, methyl vinyl ketone, and α,β-unsaturated aldehydes, such as, acrolein and crotonaldehyde, appear to be primarily responsible for NOX-mediated cytotoxicity, but additional validation will be needed. Human epidemiological studies have reported relationships between polymorphisms in the CYBA gene encoding p22phox, a catalytic subunit of NOX and susceptibility to smoking-related CVDs. In particular, G allele carriers of A640G and -930A/G polymorphisms were found to be vulnerable to smoking-induced cardiovascular toxicity, but results for C242T studies are conflicting. On the whole, evidence implicates the etiological role of NOX in smoking-induced CVD, but the clinical relevance of NOX activation by smoking and its contribution to CVD require further validation in human studies. A detailed understanding of the role of NOX would be helpful to assess the risk of smoking to human health, to define high-risk subgroups, and to develop strategies to prevent or treat smoking-induced CVD.