NADPH Oxidase-derived Reactive Oxygen Species Research Articles

Urotensin II-induced insulin resistance is mediated by NADPH oxidase-derived reactive oxygen species in HepG2 cells.

To investigated the effects of urotensin II (UII) on hepatic insulin resistance in HepG2 cells and the potential mechanisms involved. Human hepatoma HepG2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucose-oxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species (ROS) levels were detected with a multimode reader using a 2',7'-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase (JNK), insulin signal essential molecules such as insulin receptor substrate -1 (IRS-1), protein kinase B (Akt), glycogen synthase kinase-3β (GSK-3β), and glucose transporter-2 (Glut 2), and NADPH oxidase subunits such as gp91(phox), p67(phox), p47(phox), p40(phox), and p22(phox) were evaluated by Western blot. Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption (P < 0.05) and glycogen content (P < 0.01) in HepG2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression (P < 0.01) and phosphorylation of IRS-1 (P < 0.05), associated with down-regulation of Akt (P < 0.05) and GSK-3β (P < 0.05) phosphorylation levels, and the expression of Glut 2 (P < 0.001), indicating an insulin-resistance state in HepG2 cells. Furthermore, UII enhanced the phosphorylation of JNK (P < 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1 (P < 0.001), phosphorylation of IRS-1 (P < 0.001) and GSK-3β (P < 0.05), and glycogen synthesis (P < 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation (P < 0.05) and NADPH oxidase subunit expression (P < 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production (P < 0.05), JNK phosphorylation (P < 0.05), and insulin resistance (P < 0.05) in HepG2 cells. UII induces insulin resistance, and this can be reversed by JNK inhibitor SP600125 and antioxidant/NADPH oxidase inhibitor apocynin targeting the insulin signaling pathway in HepG2 cells.

Abstract 13097: Novel Interaction of Antioxidant-1 With NADPH Oxidase p47phox and TRAF4: Role in TNFα-Induced Inflammation and Atherosclerosis

Tumor necrosis factor-TNFα plays a key role in atherosclerosis via NADPH oxidase-derived reactive oxygen species (ROS) and its downstream redox-sensitive inflammatory responses. Copper (Cu) has been implicated in atherosclerosis and inflammation with unknown mechanism. Bioavailability of Cu is controlled by transport-proteins including Cu-chaperone Atox1 which obtains Cu via Cu-importer CTR1 localized at plasma membrane. We recently found that Atox1 also functions as a Cu-dependent transcription factor for NADPH oxidase p47phox. However, role of Atox1 in atherosclerosis is unknown. Here we show that ApoE-/-Atox1-/- mice with 16 weeks high-fat-diet (HFD) showed decreased atherosclerotic lesion (40%, Oil-O-red staining). inflammatory cell recruitment (50% decrease, Mac3+) and extracellular matrix deposition (Masson’s Trichrome) compared to ApoE-/-HFD. Mechanistically, in cultured endothelial cells (ECs), Atox1 knockdown with siRNA, or Cu-chelator BCS inhibited TNFα-induced ROS production (70%) at later phase (16hrs) and its downstream VCAM1/ICAM1 expression and monocyte adhesion by reducing transcription of p47phox. Unexpectedly, TNFα stimulation rapidly promoted Atox1/p47phox binding in a PKCδ-dependent and Cu-“independent” manner, which is required for p47phox membrane translocation involved in early phase of (5min) ROS production and subsequent Cys oxidation of Cu-importer CTR1 detected by a biotin-labeled Cys-OH trapping reagent. This was followed by CTR1/Cu-dependent Atox1 binding to TNFα receptor associated factor (TRAF)4, which promoted Atox1 nuclear translocation to increase p47phox expression leading to late-phase of ROS production, facilitating further monocyte adhesion to ECs. In summary, Atox1 plays an important role in activating p47phox-based NADPH oxidase and inflammatory responses in ECs by binding to p47phox that induces early phase of ROS to promote CTR1 oxidation in Cu-independent manner. This is followed by Cu-dependent Atox1/TRAF4 binding required for Atox1 nuclear translocation to function as a transcription factor for p47phox, leading to late-phase of ROS production, which contributes to acceleration of atherosclerosis in vivo.

Tyrosine kinases as key modulators of smooth muscle function in health and disease

This issue contains two review articles based on talks given as part of a symposium entitled ‘Tyrosine kinases and smooth muscle function’ that took place in London, July 2014, hosted by the Physiological Society and supported by The Journal of Physiology. The symposium examined evidence for the involvement of non-receptor tyrosine kinases in multiple aspects of smooth muscle function, including their role in normal contractile responses to various stimuli and potentially important contributions to hyper-contractile states and vascular remodelling in hypertension. Senior speakers at the symposium were Professor Gervaise Loirand (University of Nantes, France), Professor Ralph Schermuly (Max-Plank Institute for Heart and Lung Research, Germany), and Dr Jacqueline Ohanian (Institute of Cardiovascular Sciences, University of Manchester, UK).

Functional genomics identifies negative regulatory nodes controlling phagocyte oxidative burst.

The phagocyte oxidative burst, mediated by Nox2 NADPH oxidase-derived reactive oxygen species, confers host defense against a broad spectrum of bacterial and fungal pathogens. Loss-of-function mutations that impair function of the Nox2 complex result in a life-threatening immunodeficiency, and genetic variants of Nox2 subunits have been implicated in pathogenesis of inflammatory bowel disease (IBD). Thus, alterations in the oxidative burst can profoundly impact host defense, yet little is known about regulatory mechanisms that fine-tune this response. Here we report the discovery of regulatory nodes controlling oxidative burst by functional screening of genes within loci linked to human inflammatory disease. Implementing a multi-omics approach, we define transcriptional, metabolic and ubiquitin-cycling nodes controlled by Rbpj, Pfkl and Rnf145, respectively. Furthermore, we implicate Rnf145 in proteostasis of the Nox2 complex by endoplasmic reticulum-associated degradation. Consequently, ablation of Rnf145 in murine macrophages enhances bacterial clearance, and rescues the oxidative burst defects associated with Ncf4 haploinsufficiency.

Resveratrol attenuates high glucose-induced oxidative stress and cardiomyocyte apoptosis through AMPK

BackgroundDiabetic cardiomyopathy (DCM) suggests a direct cellular insult to myocardium. Hyperglycemia-induced oxidative stress and apoptosis have been implicated in the pathogenesis of DCM. NADPH oxidase is a major source of reactive oxygen species (ROS) generation in cardiomyocytes. Resveratrol, a naturally occurring polyphenol, has shown beneficial effects on some cardiovascular complications associated with diabetes. ObjectivesWe aimed to examine the role of resveratrol on high glucose-induced NADPH oxidase-derived ROS production and cardiac apoptosis, together with modulation of protein signaling pathways in cardiomyocytes. MethodsPrimary cultures of neonatal rat cardiomyocytes were exposed to 30 mmol/L high glucose with or without resveratrol. Cell viability, apoptosis, superoxide formation, NADPH oxidase activity and its subunits expression, antioxidant enzymes activities, as well as the potential regulatory molecules AMPK, Akt and GSK-3β were assessed in cardiac cells. ResultsElevated ROS production induced by 30 mmol/L high glucose was inhibited with the addition of resveratrol in primary cultured neonatal rat cardiomyocytes. Consistently, resveratrol markedly suppressed the increased activity of NADPH oxidase and Rac1, as well as the enhanced expression of p67phox, p47phox, and gp91phox induced by high glucose. Lipid peroxidation, SOD, catalase, GSH-px activity and GSH content was reversed in the presence of resveratrol. Moreover, the expression of pro-apoptotic protein Bax was down regulated while anti-apoptotic protein Bcl-2 was up regulated. And cardiac cell injury and apoptosis were markedly rescued by resveratrol. In addition, resveratrol significantly increased phosphorylation of AMP-activated protein kinase (AMPK) at Thr172 in cardiomyocytes exposed to high glucose. Compound C, the pharmacologic inhibitor of AMPK, could mostly abrogate roles of resveratrol on cardiomyocytes in high glucose. In contrast, Akt and GSK-3β were little influenced by resveratrol. ConclusionsOur data demonstrated that resveratrol protected cardiomyocytes against high glucose-induced apoptosis through suppression NADPH oxidase-derived ROS generation and maintenance endogenous antioxidant defenses. And the protective effects of resveratrol are mostly mediated by AMPK related pathway.

Mutual enhancement between high-mobility group box-1 and NADPH oxidase-derived reactive oxygen species mediates diabetes-induced upregulation of retinal apoptotic markers.

The expression of the proinflammatory cytokine high-mobility group box-1 (HMGB1) is upregulated in epiretinal membranes and vitreous fluid from patients with proliferative diabetic retinopathy (PDR) and in the diabetic retina. We hypothesized that a novel mechanism exists where HMGB1 and NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are mutually enhanced in the diabetic retina, which may be a novel mechanism for promoting upregulation of retinal apoptotic markers induced by diabetes. Vitreous samples from 48 PDR and 34 nondiabetic patients, retinas from 1-month diabetic rats and from normal rats intravitreally injected with HMGB1 and human retinal microvascular endothelial cells (HRMEC) stimulated with HMGB1 were studied by enzyme-linked immunosorbent and spectrophotometric assays, Western blot analysis, RT-PCR, and immunofluorescence. We also studied the effect of the HMGB1 inhibitor glycyrrhizin and apocynin on diabetes-induced biochemical changes in the retinas of rats (n = 5-7 in each groups). HMGB1 and the oxidative stress marker protein carbonyl content levels in the vitreous fluid from PDR patients were significantly higher than in controls (p = 0.021; p = 0.005, respectively). There was a significant positive correlation between vitreous fluid levels of HMGB1 and the levels of protein carbonyl content (r = 0.62, p = 0.001). HMGB1 enhanced interleukin-1β, ROS, Nox2, poly (ADP-ribose) polymerase (PARP)-1, and cleaved caspase-3 production by HRMEC. Diabetes and intravitreal injection of HMGB1 in normal rats induced significant upregulation of ROS, Nox2, PARP-1, and cleaved caspase-3 in the retina. Constant glycyrrhizin and apocynin intake from onset of diabetes did not affect the metabolic status of the diabetic rats, but restored these increased mediators to control values. The results of this study suggest that there is a mutual enhancement between HMGB1 and Nox-derived ROS in the diabetic retina, which may promote diabetes-induced upregulation of retinal apoptotic markers.

The Role of Reactive Oxygen Species (ROS) in the Formation of Extracellular Traps (ETs) in Humans.

Extracellular traps (ETs) are reticulate structures of extracellular DNA associated with antimicrobial molecules. Their formation by phagocytes (mainly by neutrophils: NETs) has been identified as an essential element of vertebrate innate immune defense. However, as ETs are also toxic to host cells and potent triggers of autoimmunity, their role between pathogen defense and human pathogenesis is ambiguous, and they contribute to a variety of acute and chronic inflammatory diseases. Since the discovery of ET formation (ETosis) a decade ago, evidence has accumulated that most reaction cascades leading to ET release involve ROS. An important new facet was added when it became apparent that ETosis might be directly linked to, or be a variant of, the autophagy cell death pathway. The present review analyzes the evidence to date on the interplay between ROS, autophagy and ETosis, and highlights and discusses several further aspects of the ROS-ET relationship that are incompletely understood. These aspects include the role of NADPH oxidase-derived ROS, the molecular requirements of NADPH oxidase-dependent ETosis, the roles of NADPH oxidase subtypes, extracellular ROS and of ROS from sources other than NADPH oxidase, and the present evidence for ROS-independent ETosis. We conclude that ROS interact with ETosis in a multidimensional manner, with influence on whether ETosis shows beneficial or detrimental effects.

A Central Role for H2O2 in Insulin Signal Transduction in Cardiac Myocytes

Molecular mechanisms of insulin action have been extensively characterized in the classic insulin target tissues: fat, liver, and skeletal muscle. But in cardiac myocytes (CM), understanding of insulin‐modulated signaling pathways has lagged far behind. Early studies of insulin action implicated a physiological role for H2O2, but the relationships between insulin and H2O2 in CM signal transduction are almost entirely unexplored. In these studies, we identified a role for H2O2 in insulin signaling pathways in adult mouse CM. Insulin promoted the rapid (maximal at 5 min) robust (10‐ to 18‐fold) and reversible phosphorylation of insulin receptorβTyr1146/1150; IRS‐1 Tyr895; and Akt Ser473/308; these responses all had an EC50 of 1 nM insulin (n=3‐7). Insulin‐dependent phosphorylation of IRS‐1 Ser636/639, mTOR Ser2448, p70S6 Thr389/421, and S6 Ser235/Ser236 showed a slower time course (maximal at 30 min), again with an EC50 of 1 nM insulin and with robust phosphorylation responses (6‐ to 40‐fold increases from baseline; n=3‐7). All of these phosphorylation responses were blocked by the NADPH oxidase inhibitors apocynin and VAS2870, implicating NADPH oxidase‐derived reactive oxygen species in modulating insulin‐dependent phosphorylation responses. Importantly, the cell‐permeant derivatized enzyme PEG‐catalase (which degrades H2O2) attenuated all these insulin‐dependent phosphorylation responses. Our findings implicate NADPH oxidase‐generated H2O2 as a critical determinant of insulin‐dependent signaling responses in CM, and may have important implications for the abnormalities in cardiac function seen in disease states associated with oxidative stress. Research support: NIH and AHA.

Contribution of reactive oxygen species to cerebral amyloid angiopathy, vasomotor dysfunction, and microhemorrhage in aged Tg2576 mice

Cerebral amyloid angiopathy (CAA) is characterized by deposition of amyloid β peptide (Aβ) within walls of cerebral arteries and is an important cause of intracerebral hemorrhage, ischemic stroke, and cognitive dysfunction in elderly patients with and without Alzheimer's Disease (AD). NADPH oxidase-derived oxidative stress plays a key role in soluble Aβ-induced vessel dysfunction, but the mechanisms by which insoluble Aβ in the form of CAA causes cerebrovascular (CV) dysfunction are not clear. Here, we demonstrate evidence that reactive oxygen species (ROS) and, in particular, NADPH oxidase-derived ROS are a key mediator of CAA-induced CV deficits. First, the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, are shown to reduce oxidative stress and improve CV reactivity in aged Tg2576 mice. Second, the observed improvement in CV function is attributed both to a reduction in CAA formation and a decrease in CAA-induced vasomotor impairment. Third, anti-ROS therapy attenuates CAA-related microhemorrhage. A potential mechanism by which ROS contribute to CAA pathogenesis is also identified because apocynin substantially reduces expression levels of ApoE-a factor known to promote CAA formation. In total, these data indicate that ROS are a key contributor to CAA formation, CAA-induced vessel dysfunction, and CAA-related microhemorrhage. Thus, ROS and, in particular, NADPH oxidase-derived ROS are a promising therapeutic target for patients with CAA and AD.

NADPH Oxidase Limits Sterile Inflammation By Modulating IL-1/G-CSF-Driven Neutrophilic Inflammation

The phagocyte NADPH oxidase is a multi-subunit enzyme that generates superoxide in response to multiple agonists. Besides their role in microbial killing, NADPH oxidase-derived reactive oxygen species (ROS) are important in regulating inflammation and have emerging links to autoimmunity. Patients with chronic granulomatous disease (CGD) have null mutations in NADPH oxidase subunit genes and absent enzyme activity, presenting not only with recurrent bacterial and fungal infections but also inflammatory conditions and an association with autoimmune disorders. How the NADPH oxidase regulates inflammatory processes independent of its role in microbial infection is incompletely characterized. We hypothesized that NADPH oxidase deficiency influences the magnitude of inflammatory mediators released in response to Danger Associated Molecular Patterns (DAMPs) during the acute phase of sterile tissue injury. Of note, prior studies (Chen et al Nature Medicine 2007; Kono et al, J Immunol 2010) identified IL-1α released by sentinel macrophages as a key mediator of the initial response to sterile cell death.We compared acute inflammation between WT and CGD mice lacking NADPH oxidase due to inactivation of X-linked Cybb (Cybb KO), using peritoneal instillation of periodate to induce sterile tissue injury. We found that early recruitment of neutrophils and local production of IL-1 α,IL-1β and IL-1 receptor (IL-1-R)-driven secondary cytokines IL-6 and G-CSF were significantly elevated in Cybb KO mice compared to WT. Results were similar following peritoneal injection of uric acid crystals, a canonical DAMP. Studies in transplantation chimeras indicated that NADPH oxidase-deficient hematopoietic cells were required for the enhanced neutrophilic inflammation in response to periodate-induced tissue injury. In vivo neutralization of IL-1α but not IL-β abrogated peritoneal neutrophilic influx and the increase in peritoneal G-CSF in both WT and Cybb KO mice. G-CSF is a major regulator of neutrophil production, survival, and under inflammatory conditions, augments effector functions of immune cells. Cybb KO mice exhibited relatively greater acute phase peripheral blood neutrophilia following peritoneal injury and maintained a steady low-level recruitment of neutrophils into the tissue even in later stages of inflammation. However, steady state granulopoiesis was similar in WT and Cybb KO mice.Our studies provide the first evidence that early IL-1α release elicited by sterile tissue injury is exacerbated by NADPH oxidase deficiency and this is central to the exaggerated acute inflammatory response by CGD mice in this setting. These findings complement recent work demonstrating elevated IL-1β release by NADPH oxidase-deficient myeloid cells elicited by microbial products or other inflammasome-activating agents ( de Luca et al PNAS 2014; Meisener et al Blood 2009). Targeting the IL-1/IL-R axis may help ameliorate some of the autoinflammatory complications seen in CGD patients. However, as non-redundant roles for IL-1α and IL-1β in driving inflammation have been recently shown in a number of models, further studies are required to examine how dysregulated production of each IL-1 family member contributes to disease manifestations in NADPH oxidase deficiency. DisclosuresNo relevant conflicts of interest to declare.

Smad-independent pathway involved in transforming growth factor β1-induced Nox4 expression and proliferation of endothelial cells.

NADPH oxidase-derived reactive oxygen species are important for various cellular functions, including proliferation. Endothelial cells predominantly express the Nox4 isoform of NADPH oxidase, but it is not entirely clear how it is regulated. In this study, we investigated the signalling pathways involved in transforming growth factor-β1 (TGF-β1)-induced Nox4 expression and the proliferation of human microvascular endothelial cells (HMECs). TGF-β1 stimulated Nox4 messenger RNA and protein expression in HMECs. TGF-β1-induced Nox4 also increased hydrogen peroxide production, which was inhibited by diphenyleneiodonium and EUK134. The acute treatment of HMECs with TGF-β1 enhanced the phosphorylation of Smad2 and extracellular signal-regulated kinase (ERK) 1/2, without affecting p38MAPK, Akt, or Jun N-terminal kinase 1/2 (JNK1/2) pathways. Further, inhibition of Smad2 signalling using an inhibitor of activin receptor-linked kinase 5 SB431542 reduced TGF-β1-induced Nox4 expression, while inhibition of ERK1/2 with the inhibitor of mitogen-activated protein kinase kinase 1/2 U0126 decreased both basal and TGF-β1-induced Nox4 expression. Inhibition of ERK1/2 phosphorylation with U0126 did not affect Smad2 phosphorylation. Finally, TGF-β1 enhanced endothelial cell proliferation, which was reduced by U0126 but not by SB431542. These findings suggest that the non-canonical pathway ERK1/2 regulates Nox4 expression and may be involved in TGF-β1-induced proliferation of endothelial cells, which is vital during angiogenesis and vascular development.

Nod-like Receptor Protein 3 (NLRP3) Inflammasome Activation and Podocyte Injury via Thioredoxin-Interacting Protein (TXNIP) during Hyperhomocysteinemia

NADPH oxidase-derived reactive oxygen species (ROS) have been reported to activate NLRP3 inflammasomes resulting in podocyte and glomerular injury during hyperhomocysteinemia (hHcys). However, the mechanism by which the inflammasome senses ROS is still unknown in podocytes upon hHcys stimulation. The current study explored whether thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of the antioxidant thioredoxin and ROS sensor, mediates hHcys-induced NLRP3 inflammasome activation and consequent glomerular injury. In cultured podocytes, size exclusion chromatography and confocal microscopy showed that inhibition of TXNIP by siRNA or verapamil prevented Hcys-induced TXNIP protein recruitment to form NLRP3 inflammasomes and abolished Hcys-induced increases in caspase-1 activity and IL-1β production. TXNIP inhibition protected podocytes from injury as shown by normal expression levels of podocyte markers, podocin and desmin. In vivo, adult C57BL/6J male mice were fed a folate-free diet for 4 weeks to induce hHcys, and TXNIP was inhibited by verapamil (1 mg/ml in drinking water) or by local microbubble-ultrasound TXNIP shRNA transfection. Evidenced by immunofluorescence and co-immunoprecipitation studies, glomerular inflammasome formation and TXNIP binding to NLRP3 were markedly increased in mice with hHcys but not in TXNIP shRNA-transfected mice or those receiving verapamil. Furthermore, TXNIP inhibition significantly reduced caspase-1 activity and IL-1β production in glomeruli of mice with hHcys. Correspondingly, TXNIP shRNA transfection and verapamil attenuated hHcys-induced proteinuria, albuminuria, glomerular damage, and podocyte injury. In conclusion, our results demonstrate that TXNIP binding to NLRP3 is a key signaling mechanism necessary for hHcys-induced NLRP3 inflammasome formation and activation and subsequent glomerular injury.

Redox modulation of FAK controls melanoma survival--role of NOX4.

Studies have demonstrated that reactive oxygen species (ROS) generated by NADPH oxidase are essential for melanoma proliferation and survival. However, the mechanisms by which NADPH oxidase regulates these effects are still unclear. In this work, we investigate the role of NADPH oxidase-derived ROS in the signaling events that coordinate melanoma cell survival. Using the highly metastatic human melanoma cell line MV3, we observed that pharmacological NADPH oxidase inhibition reduced melanoma viability and induced dramatic cellular shape changes. These effects were accompanied by actin cytoskeleton rearrangement, diminished FAKY397 phosphorylation, and decrease of FAK-actin and FAK-cSrc association, indicating disassembly of focal adhesion processes, a phenomenon that often results in anoikis. Accordingly, NADPH oxidase inhibition also enhanced hypodiploid DNA content, and caspase-3 activation, suggesting activation of the apoptotic machinery. NOX4 is likely to be involved in these effects, since silencing of NOX4 significantly inhibited basal ROS production, reduced FAKY397 phosphorylation and decreased tumor cell viability. Altogether, the results suggest that intracellular ROS generated by the NADPH oxidase, most likely NOX4, transmits cell survival signals on melanoma cells through the FAK pathway, maintaining adhesion contacts and cell viability.

Caveolin-1 is a negative regulator of NADPH oxidase-derived reactive oxygen species

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

Abstract 161: Differential Effects of Short- and Long-Term Increase in Endothelial ROS on Coronary Vascular Function

Introduction: Increase in reactive oxygen species (ROS) is often associated with vascular pathophysiological conditions. Our recent findings demonstrated that transient increase in ROS improved coronary endothelial function. However, long-term effects of endothelium-specific increase in ROS on endothelial function are not known. Hypothesis: The short-term (four to eight weeks) versus the long-term (16 to 20 weeks) increase in NADPH oxidase-derived ROS will have differential effects on vascular endothelium. Methods: Our binary (Tet-ON/OFF) conditional transgenic mouse (Tet-Nox2:VE-Cad-tTA) induces 1.8±0.42-fold increase in NADPH oxidase-derived ROS in endothelium. Using these animals, we have examined the effects of short-term vs. the long-term effects of ROS on endothelial cell (EC) signaling and coronary endothelial function. Results: We demonstrate that whereas EC-dependent coronary vasodilation was significantly increased after short-term increase in ROS, coronary vasodilation was drastically reduced after long-term increase in EC-ROS in Tet-OFF Nox2 mice compared to their Tet-ON (control) littermates. Using EC isolated from mouse heart, we demonstrate that, whereas Nox2-derived both short-term and long-term increase in ROS increased activation of AMPK and eNOS, and nitric oxide (NO) synthesis, long-term increase in ROS abrogated these positive effects due to increased peroxynitrite and nitrotyrosylation of MnSOD. We also show that long-term ROS increase resulted in abrogation of EC proliferation and reduction in EC mitochondrial content compared to short-term ROS exposure. Conclusion: This study demonstrates that whereas short-term increase in ROS is beneficial for EC function, long-term increase in ROS results in loss of EC mitochondria and inhibition of endothelial function.

NADPH oxidase-derived reactive oxygen species contribute to impaired cutaneous microvascular function in chronic kidney disease.

Oxidative stress promotes vascular dysfunction in chronic kidney disease (CKD). We utilized the cutaneous circulation to test the hypothesis that reactive oxygen species derived from NADPH oxidase and xanthine oxidase impair nitric oxide (NO)-dependent cutaneous vasodilation in CKD. Twenty subjects, 10 stage 3 and 4 patients with CKD (61 ± 4 yr; 5 men/5 women; eGFR: 39 ± 4 ml·min(-1)·1.73 m(-2)) and 10 healthy controls (55 ± 2 yr; 4 men/6 women; eGFR: >60 ml·min(-1)·1.73 m(-2)) were instrumented with 4 intradermal microdialysis fibers for the delivery of 1) Ringer solution (Control), 2) 10 μM tempol (scavenge superoxide), 3) 100 μM apocynin (NAD(P)H oxidase inhibition), and 4) 10 μM allopurinol (xanthine oxidase inhibition). Skin blood flow was measured via laser-Doppler flowmetry during standardized local heating (42°C). N(g)-nitro-l-arginine methyl ester (L-NAME; 10 mM) was infused to quantify the NO-dependent portion of the response. Cutaneous vascular conductance (CVC) was calculated as a percentage of the maximum CVC achieved during sodium nitroprusside infusion at 43°C. Cutaneous vasodilation was attenuated in patients with CKD (77 ± 3 vs. 88 ± 3%, P = 0.01), but augmented with tempol and apocynin (tempol: 88 ± 2 (P = 0.03), apocynin: 91 ± 2% (P = 0.001). The NO-dependent portion of the response was reduced in patients with CKD (41 ± 4 vs. 58 ± 2%, P = 0.04), but improved with tempol and apocynin (tempol: 58 ± 3 (P = 0.03), apocynin: 58 ± 4% (P = 0.03). Inhibition of xanthine oxidase did not alter cutaneous vasodilation in either group (P > 0.05). These data suggest that NAD(P)H oxidase is a source of reactive oxygen species and contributes to microvascular dysfunction in patients with CKD.

Contribution of guanine nucleotide exchange factor Vav2 to homocysteine‐induced NLRP3 inflammasome activation in mouse podocytes (1063.6)

NADPH oxidase‐derived reactive oxygen species (ROS) have been demonstrated to mediate the activation of podocyte NLRP3 inflammasomes in response to elevated levels of homocysteine (Hcys). We have also previously provided evidence that the guanine nucleotide exchange factor Vav2 exhibits high specificity to Rac1‐mediated NADPH oxidase activation by elevated Hcys. The precise role of Vav2 in NLRP3 inflammasome activation remains unknown. The present study was designed to test whether Vav2 contributes to Hcys‐induced NLRP3 inflammasome activation through activation of NADPH oxidase. In our experiments, podocytes were transfected with either a scramble, constitutively active form of Vav2 (oncoVav2) or Vav2 shRNA plasmid directly to the nucleus via nucleofection. Confocal microscopic analysis showed Hcys treatment as well as oncoVav2 transfection alone increased colocalization between inflammasome proteins NLRP3 with ASC and NLRP3 with caspase‐1, suggesting NLRP3 inflammasome formation. Additionally, both Hcys treatment as well as oncoVav2 transfection alone resulted in increased caspase‐1 activation and IL‐1β production, signifying NLRP3 inflammasome activation. These Hcys‐induced measures of inflammasome formation and activation were all inhibited by Vav2 shRNA transfection, observed in decreased colocalization of inflammasome components, caspase‐1 activity, and IL‐1β production in podocytes. Furthermore, Vav2 shRNA transfection also prevented podocyte damage by restoring impaired VEGF secretion as well as decreased podocyte marker podocin expression seen in Hcys injured podocytes. Together, these findings suggest that NADPH oxidase activation by Vav2 overexpression by oncoVav2 was sufficient to activate NLRP3 inflammasomes, independent of Hcys treatment. However, Vav2 inhibition was able to inhibit Hcys‐induced NLRP3 inflammasome activation and podocyte injury, further substantiating the role of Vav2 in this mechanism.Grant Funding Source: Supported by NIH grants DK54927 and 1F31AG043289

Protective effects of puerarin against Aß40-induced vascular dysfunction in zebrafish and human endothelial cells

Aß40-induced vascular dysfunction has been implicated in the pathogenesis of Alzheimer׳s disease (AD). In the present study, we investigated the possible protective effects of puerarin against Aß40-induced vascular damage and impairment to angiogenesis in transgenic TG (fli1:EGFP) zebrafish and human endothelial cells. Aß40 peptides at 5μM caused an obvious reduction of vessel branches in the subintestinal vein basket, induced NADPH oxidase-derived reactive oxygen species and impaired vascular endothelial growth factor (VEGF)-dependent angiogenesis. Pretreatment with puerarin attenuated Aβ40-induced vessel reduction and impairment to angiogenesis in a dose-dependent manner. In addition, Aß40 decreased VEGF-dependent phosphorylation of Akt and eNOS, whereas puerarin treatment attenuated these detrimental effects. Furthermore, the restoration of Aß40-induced-angiogenesis impairment by puerarin was abolished by either the PI3 kinase inhibitor LY294002 (10μM) or eNOS inhibitor L-NAME. The present study suggests that puerarin exerts its protective action probably through reduction of NADPH oxidase-derived reactive oxygen species overproduction and activation of the PI3K/Akt/eNOS pathways.

DUOX2 and DUOXA2 Form the Predominant Enzyme System Capable of Producing the Reactive Oxygen Species H2O2 in Active Ulcerative Colitis and are Modulated by 5-Aminosalicylic Acid

NADPH oxidase-derived reactive oxygen species, such as H2O2, are part of the intestinal innate immune system but may drive carcinogenesis through DNA damage. We sought to identify the predominant enzyme system capable of producing H2O2 in active ulcerative colitis and assess whether it is affected by 5-aminosalicylic acid (5-ASA). We studied human mucosal biopsies by expression arrays, quantitative real-time polymerase chain reaction for NADPH oxidase family members, in situ hybridization (DUOX2 and DUOXA2) and immunofluorescence for DUOX, 8-OHdG (DNA damage), and γH2AX (DNA damage response) and sought effects of 5-ASA on ex vivo cultured biopsies and cultured rectal cancer cells. DUOX2 with maturation partner DUOXA2 forms the predominant system for H2O2 production in human colon and is upregulated in active colitis. DUOX2 in situ is exclusively epithelial, varies between and within individual crypts, and increases near inflammation. 8-OHdG and γH2AX were observed in damaged crypt epithelium. 5-ASA upregulated DUOX2 and DUOXA2 levels in the setting of active versus quiescent disease and altered DUOX2 expression in cultured biopsies. Ingenuity pathway analysis confirmed that inflammation status and 5-ASA increase expression of DUOX2 and DUOXA2. An epithelial cell model confirmed that cultured cancer cells expressed DUOX protein and produced H2O2 in response to hypoxia and 5-ASA exposure. Both DUOX2 and DUOXA2 expression are involved specifically in inflammation and are regulated on a crypt-by-crypt basis in ulcerative colitis tissues. Synergy between inflammation, hypoxia, and 5-ASA to increase H2O2 production could explain how 5-ASA supports innate defense, although potentially increasing the burden of DNA damage.

Resveratrol treatment rescues neurovascular coupling in aged mice: role of improved cerebromicrovascular endothelial function and downregulation of NADPH oxidase

Moment-to-moment adjustment of cerebral blood flow (CBF) to neuronal activity via neurovascular coupling is essential for the maintenance of normal neuronal function. Increased oxidative stress that occurs with aging was shown to impair neurovascular coupling, which likely contributes to a significant age-related decline in higher cortical function, increasing the risk for vascular cognitive impairment. Resveratrol is a polyphenolic compound that exerts significant antiaging protective effects in large vessels, but its effects on the cerebromicrovasculature remain poorly defined. The present study was undertaken to investigate the capacity of resveratrol to improve neurovascular coupling in aging. In aged (24-mo-old) C57BL/6 mice Nω-nitro-l-arginine methyl ester-sensitive, nitric oxide-mediated CBF responses to whisker stimulation and to the endothelium-dependent dilator acethylcholine (ACh) were impaired compared with those in young (3-mo-old) mice. Treatment of aged mice with resveratrol rescued neurovascular coupling and ACh-induced responses, which was associated with downregulation of cortical expression of NADPH oxidase and decreased levels of biomarkers of oxidative/nitrative stress (3-nitrotyrosine, 8-isoprostanes). Resveratrol also attenuated age-related increases in reactive oxygen species (ROS) production in cultured cerebromicrovascular endothelial cells (DCF fluorescence, flow cytometry). In conclusion, treatment with resveratrol rescues cortical neurovascular coupling responses to increased neuronal activity in aged mice, likely by restoring cerebromicrovascular endothelial function via downregulation of NADPH oxidase-derived ROS production. Beneficial cerebromicrovascular effects of resveratrol may contribute to its protective effects on cognitive function in aging.