Nox2KO Mice Research Articles

Regulatory role of NADPH oxidase 2 in the polarization dynamics and neurotoxicity of microglia/macrophages after traumatic brain injury

Traumatic brain injury (TBI) is a leading cause of death and disability. Secondary injuries that develop after the initial trauma contribute to long-lasting neurophysiological deficits. Polarization of microglia/macrophages toward a pro-inflammatory (M1) phenotype may increase the progression of secondary injury following TBI; however, the regulatory and functional mechanisms underlying these changes remain poorly defined. In the present study, we showed elevated expression of NADPH oxidase 2 (NOX2) and activation of nuclear factor-kappa B (NF-κB) predominantly in microglia/macrophages at 4- and 7-days after controlled cortical impact in mice. Delayed inhibition of NOX2, beginning one day after TBI, reduced reactive oxygen species production of myeloid cells and protected neurons from oxidative damage. Moreover, delayed NOX inhibition or global genetic NOX2 knockout suppressed the M1 “pro-inflammatory” profile of microglia/macrophages and simultaneously increased the M2 “anti-inflammatory” profile in the injured brain. These changes were associated with marked down-regulation of the classical NF-κB pathway in microglia/macrophages and reduced production of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-1β, after TBI. Finally, we demonstrated that wild-type microglia/macrophages isolated from the ipsilateral cortex at 7 days post-TBI were neurotoxic to co-cultured primary neurons, whereas this neurotoxicity was largely attenuated in microglia/macrophages from NOX2-KO mice. Taken together, our study shows a direct link between NOX2 and the NF-κB pathway in microglia/macrophages after TBI, and it provides a novel mechanism by which NOX2 activation leads to the enhanced inflammatory response and neuronal damage after brain injury. Our data also supports the therapeutic potential of targeting NOX2, which may provide efficacy with an extended therapeutic window after TBI.

Insulin-dependent metabolic and inotropic responses in the heart are modulated by hydrogen peroxide from NADPH-oxidase isoforms NOX2 and NOX4

RationaleHydrogen peroxide (H2O2) is a stable reactive oxygen species (ROS) that has long been implicated in insulin signal transduction in adipocytes. However, H2O2's role in mediating insulin's effects on the heart are unknown. ObjectiveWe investigated the role of H2O2 in activating insulin-dependent changes in cardiac myocyte metabolic and inotropic pathways. The sources of insulin-dependent H2O2 generation were also studied. Methods and resultsIn addition to the canonical role of insulin in modulating cardiac metabolic pathways, we found that insulin also inhibited beta adrenergic-induced increases in cardiac contractility. Catalase and NADPH oxidase (NOX) inhibitors blunted activation of insulin-responsive kinases Akt and mTOR and attenuated beta adrenergic receptor-mediated responses. These insulin responses were lost in a mouse model of type 2 diabetes, suggesting a role for these H2O2-dependent pathways in the diabetic heart. The H2O2-sensitive fluorescent biosensor HyPer revealed rapid increases in cytosolic and caveolar H2O2 concentrations in response to insulin treatment, which were blocked by NOX inhibitors and attenuated in NOX2 KO and NOX4 KO mice. In NOX2 KO cardiac myocytes, insulin-mediated phosphorylation of Akt and mTOR was blocked, while these responses were unaffected in cardiac myocytes from NOX4 KO mice. In contrast, insulin's effects on contractility were lost in cardiac myocytes from NOX4 KO animals but were retained in NOX2 KO mice. ConclusionsThese studies identify a proximal point of bifurcation in cardiac insulin signaling through the simultaneous activation of both NOX2 and NOX4. Each NOX isoform generates H2O2 in cardiac myocytes with distinct time courses, with H2O2 derived from NOX2 augmenting Akt-dependent metabolic effects of insulin, while H2O2 from NOX4 blocks beta adrenergic increases in inotropy. These findings suggest that insulin resistance in the diabetic heart may lead to potentially deleterious potentiation of beta adrenergic responses.

Abstract 008: Leptin Treatment Attenuates Vascular Dysfunction and Inflammation in Mouse Model(s) of Acquired Lipodystrophy via Reducing Nox1-derived Ros

Although extremely efficient at suppressing HIV replication, highly active antiretroviral therapy (HAART) induces lipodystrophy, a metabolic disorder characterized by an abnormal adipose tissue distribution, reduced leptin levels and vascular dysfunction. Leptin replacement therapy (LRT) is currently used to improve metabolic function in patients suffering from congenital lipodystrophy. Here, we analyzed whether LRT restores vascular function and inflammation in mice treated with the antiretroviral agent, ritonavir (Rit). Four weeks of Rit reduced body weight [control (C): 28.4±0.5 vs. Rit: 24.4±0.2g*, *P<0.05] and fat mass (C: 10±1 vs. Rit: 6.5±1 %*) confirming that it induces lipodystrophy. Rit impaired aortic endothelial function [Relaxation to acetylcholine: C: 74±4 vs. Rit: 21±15%*), increased ROS producing enzymes (NOX1 and NOXA1), induced vascular inflammation (increased IL-1β, MCP-1, GATA Binding Protein 3 and INF-γ gene expression) and increased TBARS levels. ROS scavenging via tempol or GKT137117, (Nox1/4 inhibitor) pre-incubation blunted endothelial dysfunction. LRT (10μg/day/7 days, osmotic mini-pump), at the end of the 3-week Rit, restored endothelial function, reduced Nox1 and NOXA1 gene expression and vascular inflammation. NOX1 deficiency in Nox1 KO mice protected mice from Rit-induced endothelial dysfunction and vascular inflammation. Increasing endothelial leptin sensitivity via specific deletion of protein tyrosine phosphatase 1B (Ptp1b) in endothelial cells ( Ptp1b-/-EC mice) protected mice from Rit-induced endothelial dysfunction and reduced Nox1 and NOXA1 gene expression, and vascular inflammation. To address the relevance of these observations to other forms of acquired lipodystrophy, experiments were repeated in mice in which lipodystrophy was induced at 8 week of age, by the deletion of Bscl2, a gene involved in adipocyte maturation. Bscl2 deletion reduced fat mass, and induced endothelial dysfunction via ROS-mediated mechanisms. Again, LRT reverted endothelial dysfunction by downregulating Nox1 expression. All together, these data presents leptin as a key regulator of endothelial oxidative stress level and as a potential avenue for the treatment vascular disease.

Role of NOX2-Derived Reactive Oxygen Species in NK Cell-Mediated Control of Murine Melanoma Metastasis.

The NADPH oxidase of myeloid cells, NOX2, generates reactive oxygen species (ROS) to eliminate pathogens and malignant cells. NOX2-derived ROS have also been proposed to dampen functions of natural killer (NK) cells and other antineoplastic lymphocytes in the microenvironment of established tumors. The mechanisms by which NOX2 and ROS influence the process of distant metastasis have only been partially explored. Here, we utilized genetically NOX2-deficient mice and pharmacologic inhibition of NOX2 to elucidate the role of NOX2 for the hematogenous metastasis of melanoma cells. After intravenous inoculation of B16F1 or B16F10 cells, lung metastasis formation was reduced in B6.129S6-Cybbtm1DinK (Nox2-KO) versus Nox2-sufficient wild-type (WT) mice. Systemic treatment with the NOX2-inhibitor histamine dihydrochloride (HDC) reduced melanoma metastasis and enhanced the infiltration of IFNγ-producing NK cells into lungs of WT but not of Nox2-KO mice. IFNγ-deficient B6.129S7-Ifngtm1Ts /J mice were prone to develop melanoma metastases and did not respond to in vivo treatment with HDC. We propose that NOX2-derived ROS facilitate metastasis of melanoma cells by downmodulating NK-cell function and that inhibition of NOX2 may restore IFNγ-dependent, NK cell-mediated clearance of melanoma cells. Cancer Immunol Res; 5(9); 804-11. ©2017 AACR.

P 079 - Contribution of NADPH oxidase isoforms to angiotensin II-mediated oxidative stress and DNA damage in the mouse kidney

Hypertension caused by elevated levels of the blood pressure-regulating hormone angiotensin II (AngII) leads to oxidative stress and DNA damage in kidneys of mice. In vitro-studies showed a role of the free radical-generating NADPH oxidase (Nox) in this damage. Here, we studied the contribution of the isoforms Nox1, 2 and 4 to AngII-induced oxidative stress and DNA lesions in the kidney. Male wildtype (WT) C57BL/6, Nox1-, Nox2- and Nox4-knockout (KO) mice were infused with either vehicle or AngII during 28 days. Afterwards, urine and kidneys were analyzed for markers of oxidative stress and DNA damage. In WT mice, AngII induced hypertension, elevated urinary albumin levels and formation of ROS and DNA damage in the kidney. In Nox2- and Nox4-KO mice blood pressure was as high as in WT mice but lower in Nox1-KO mice. A rise in urinary albumin and systemic oxidative stress due to AngII was observed in all KO mice. In kidney tissue, only Nox1-KO mice showed no significant ROS formation. Basal values of genomic damage were higher in Nox2- and Nox4-KO mice. Only in Nox1-KO mice AngII did not cause significantly more DNA double strand breaks than in the controls. Nox1 seems to be mostly responsible for the AngII-induced generation of ROS in mouse kidney. Surprisingly, genomic damage in kidneys of Nox2- and Nox4-KO mice was already increased in the absence of AngII.

Distinct Regulatory Effects of Myeloid Cell and Endothelial Cell NAPDH Oxidase 2 on Blood Pressure

Hypertension caused by increased renin-angiotensin system activation is associated with elevated reactive oxygen species production. Previous studies implicate NADPH oxidase (Nox) proteins as important reactive oxygen species sources during renin-angiotensin system activation, with different Nox isoforms being potentially involved. Among these, Nox2 is expressed in multiple cell types, including endothelial cells, fibroblasts, immune cells, and microglia. Blood pressure (BP) is regulated at the central nervous system, renal, and vascular levels, but the cell-specific role of Nox2 in BP regulation is unknown. We generated a novel mouse model with a floxed Nox2 gene and used Tie2-Cre, LysM Cre, or Cdh5-CreERT2 driver lines to develop cell-specific models of Nox2 perturbation to investigate its role in BP regulation. Unexpectedly, Nox2 deletion in myeloid but not endothelial cells resulted in a significant reduction in basal BP. Both Tie2-CreNox2 knockout (KO) mice (in which Nox2 was deficient in both endothelial cells and myeloid cells) and LysM CreNox2KO mice (in which Nox2 was deficient in myeloid cells) had significantly lower BP than littermate controls, whereas basal BP was unaltered in Cdh5-CreERT2 Nox2KO mice (in which Nox2 is deficient only in endothelial cells). The lower BP was attributable to an increased NO bioavailability that dynamically dilated resistance vessels in vivo under basal conditions without a change in renal function. Myeloid-specific Nox2 deletion had no effect on angiotensin II-induced hypertension, which, however, was blunted in Tie2-CreNox2KO mice, along with preservation of endothelium-dependent relaxation during angiotensin II stimulation. We identify a hitherto unrecognized modulation of basal BP by myeloid cell Nox2, whereas endothelial cell Nox2 regulates angiotensin II-induced hypertension. These results identify distinct cell-specific roles for Nox2 in BP regulation.

Role of NADPH Oxidase as a Mediator of Oxidative Damage in Low-Dose Irradiated and Hindlimb-Unloaded Mice.

The purpose of this study was to determine whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived stress can account for unloading- and radiation-induced endothelial damage and neurovascular remodeling in a mouse model. Wild-type (WT, Nox2+/+) C57BL/6 mice or Nox2-/- (B6.129S6-CYBBM) knockout (KO) mice were placed into one of the following groups: age-matched control; hindlimb unloading (HLU); low-dose/low-dose-rate radiation (LDR); or HLU with LDR simultaneously for 21 days. The mice were then sacrificed one month later. Anti-orthostatic tail suspension was used to model the unloading, fluid shift and physiological stress aspects of microgravity. The LDR was delivered using 57Co plates (0.04 Gy at 0.01 cGy/h) to the simulate whole-body irradiation, similar to that experienced while in space. Brains were isolated for characterization of various oxidative stress markers and vascular topology. The level of 4-hydroxynonenal (4-HNE) protein, a specific marker for lipid peroxidation, was measured. Expression of aquaporin-4 (AQP4), a water channel protein expressed in astrocyte end-feet, was quantified. Thirty days after simulated spaceflight, KO mice showed decreased apoptosis (P < 0.05) in the brain compared to WT counterparts. The HLU-dependent increase in apoptosis in WT mice was not observed in KO mice. The level of 4-HNE protein was significantly elevated in the hippocampus of the LDR with HLU treatment group compared to WT controls (P < 0.05). However, there were no significant differences among groups of Nox2-KO mice at the one-month time point. In contrast to findings in the WT animals, superoxide dismutase (SOD) level and expression of AQP4 were similar among all KO groups. In summary, for most of the parameters, the oxidative response to HLU and LDR was suppressed in Nox2-KO mice. This suggests that Nox2-containing NADPH oxidase may contribute to spaceflight environment-induced oxidative stress.

NOX4 is an early initiator of neuropathic pain

Treatment of neuropathic pain remains challenging as the etiology is heterogeneous and pathomechanisms are incompletely understood. One possible mechanism is oxidative stress due to unphysiological reactive oxygen species (ROS) formation. The only know dedicated enzymatic source of ROS are NADPH oxidases of which the type 4 isoform (NOX4) has been suggested to be involved in the subacute and chronic phase of neuropathic pain. Here, we aim to translate this finding into a treatment strategy by examining the efficacy of the NOX1/4-specific inhibitor GKT136901 using the chronic constriction injury (CCI) mouse model of neuropathic pain. Unexpectedly, post-nerve lesion treatment using GKT136901 was ineffective to reduce pain-related behavior after CCI. We therefore re-investigated the role of NOX4 using an independent KO mouse model. Early after CCI we found an increase in pro-inflammatory cytokines, ROS formation and the oxidative stress marker nitrotyrosine in the lesioned nerve together with an upregulated Nox4 gene expression. In NOX4 KO mice, mechanical allodynia was markedly reduced from day 4 after nerve injury as were all ROS related and acute biomarkers. In addition, we observed a reduction in the CCI-induced upregulation of pro-inflammatory cytokines in the sciatic nerve and dorsal root ganglia along with NOX4-deficiency. Thus, we conclude that NOX4 is involved in the development of neuropathic pain states by producing oxidative stress and subsequent cytokine dysregulation at the lesion site. This appears at very early stages immediately after nerve injury explaining ineffectiveness of post-acute pharmacological NOX inhibition. We suggest that future target validation of NOX4 should now focus on defining the possible therapeutic window in human neuropathic pain.

NOX1/NADPH Oxidase Expressed in Colonic Macrophages Contributes to the Pathogenesis of Colonic Inflammation in Trinitrobenzene Sulfonic Acid-Induced Murine Colitis.

NOX1/NADPH oxidase, a nonphagocytic isoform of reactive oxygen species-producing enzymes, is highly expressed in the colon, but the physiologic and pathophysiologic roles of this isoform are not fully understood. The present study investigated the role of NOX1 in the development of colonic inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model. Intrarectal injection of TNBS caused severe colitis accompanied by body weight loss, diarrhea, and increased myeloperoxidase (MPO) activity in wild-type (WT) mice. In contrast, the severity of colitis was significantly attenuated in NOX1-deficient (NOX1KO) mice (the inhibitions of macroscopic damage score, body weight loss, diarrhea score, and MPO activity were 73.1%, 36.8%, 83.3%, and 98.4%, respectively). TNBS-induced upregulation of inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-1β), chemokines (CXCL1 and CXLC2), and inducible nitric oxide synthase (iNOS) was also significantly less in NOX1KO than in WT mice (the inhibitions were 100.8%, 89.0%, 63.5%, 96.7%, and 97.1%, respectively). Expression of NOX1 mRNA was detected not only in the lamina propria but also in peritoneal macrophages isolated from WT mice. Increased expression of TNF-α, IL-1β, and iNOS in peritoneal macrophages exposed to lipopolysaccharide was significantly attenuated in macrophages isolated from NOX1KO mice (68.1%, 67.0%, and 79.3% inhibition, respectively). These findings suggest that NOX1/NADPH oxidase plays an important role in the pathogenesis of TNBS-induced colonic inflammation via upregulation of inflammatory cytokines, chemokines, and iNOS. NOX1 in colonic macrophages may become a potential target in pharmacologic intervention for inflammatory bowel disease.

Abstract 097: Vascular Aging in Aldosterone Associated Hypertension: Role of NADPH Oxidase 1

The vascular phenotype in hypertension is characterised by features typically observed in the ageing vasculature. Pathophysiological processes underlying premature vascular aging in hypertension remains unclear but aldosterone (aldo) and oxidative stress may be important. We postulated that physiological aging is amplified in hypertension due to increased aldo-induced Nox activation and redox signalling. We used arteries from adult WKY (18 weeks), aged WKY (52 weeks) and adult stroke-prone spontaneously hypertensive (SHRSP) rats. Blood pressure was measured by tail-cuff. Vascular function/structure was analysed by myography. Gene level was assessed by qPCR and protein by immunoblotting. BP was increased in SHRSP (180.7±2.5 vs. 127±2.7 mmHg, p&lt;0.05). Endothelial dysfunction was observed in vessels from SHRSP. Increased vascular contraction in aged WKY rats was similar to SHRSP rats (p&lt;0.05 vs WKY). Increased vascular stiffness was observed in arteries from aged WKY and SHRSP compared to WKY rats. Nox2 (0.82±0.4/2.4±0.9 vs 0.22±0.2), NoxA1 (4.9±2/9.5±5 vs 1±0.3) and NoxO1 (1.9±0.6/4.1±1 vs 1±0.4) mRNA was increased (p&lt;0.05; SHRSP/aged WKY vs WKY). Nox1 mRNA (2.3±0.8 vs 1.1±0.4) was only increased in SHRSP rats (p&lt;0.05; vs WKY). Similarly, mRNA levels of MCP-1 (2.3±0.5/3.9±1.9 vs 0.3±0.1) and RANTES (7.4±2/6.3±1.7 vs 1.1±0.2), aging-related inflammatory markers, and cell cycle inhibitors, p21 (3.2±1.1/3.1±0.7 vs 1±0.1) and p27 (2.2±0.7/2±0.8 vs 0.4±0.1), were increased in SHRSP and aged WKY rats (p&lt;0.05; SHRSP/aged WKY vs WKY). ROS production (VSMC: 1.74±0.4 AU/protein), H2AX (DNA damage; 1.3±0.1) and aldosterone (plasma; 99.5±19 pg/mL) levels were increased in SHRSP rats (p&lt;0.05; vs WKY). Aldo-induced Nox1 mRNA expression and p66SHC activation was exacerbated in VSMCs from SHRSP rats; an effect blocked by ML171 (a Nox1 inhibitor) and blunted in VSMCs from Nox1 KO mice. In conclusion, endothelial dysfunction and vascular remodelling in hypertension are associated with increased aldo-mediated activation of pro-inflammatory and redox-sensitive pathways. These processes involve Nox1. Our findings identify an important role for aldo/Nox1/ROS in molecular processes underlying vascular changes of ageing in hypertension.

Abstract 279: Vascular Aging In Aldosterone Associated Hypertension: Role Of NADPH Oxidase 1

In hypertension, vascular aging is accelerated leading to heart failure, stroke and renal dysfunction. Cellular and molecular mechanisms of age-associated vascular changes are unclear, but enhanced ROS generation by NADPH oxidases (Nox) and aldosterone (aldo) have been suggested. Nox1 is of particular interest due to increased levels in hypertension. Here, we postulated that aldo plays an important role in vascular aging through Nox1-dependent mechanisms. We examined signalling molecules associated with aging in arteries from 2 experimental models: stroke-prone spontaneously hypertensive rats (SHRSP) rats and Nox1 knockout mice infused with aldo (300 ug/Kg/day). Gene expression was assessed by qPCR and protein levels by immunoblotting. Aldo was measured by ELISA. In SHRSP rats, aging-associated mRNA inflammatory markers, such as RANTES (5- fold), MCP-1 (6-fold) and IL-6 (2-fold); as well as aldo levels (6-fold) and H2AX (marker of aging-associated DNA damage - 1.5-fold) were increased compared with control rats, p&lt;0.05. In mice treated with aldo, JNK (pro-inflammatory - 69%) and p66SHC (pro-senescence - 92%) activation were increased in mesenteric arteries (p&lt;0.05); an effect blunted in vessels from Nox1 KO mice. In parallel studies, vascular smooth muscle cells (VSMCs) from adult and aged control mice, as well as, from adult Nox1 transgenic mice (Nox1 overexpression in VSMCs) were extracted and stimulated with H2O2 and aldo. Basal levels of p66SHC (39%) and OGG-1 (48%), markers of senescence, were increased in VSMCs from aged animals, p&lt;0.05. H2O2 (64%) and aldo (84%) increased p66SHC activation in VSMCs from adult mice to similar levels observed in cells from aged animals (p&lt;0.05). However, aldo effects on p66SHC activation were exacerbated in VSMCs from adult Nox1 transgenic mice. In conclusion, aldosterone may play an important role in the aging-like phenotype in vascular injury associated with hypertension. Such processes may involve Nox1 and redox-sensitive p66Shc signalling.

Nox1 upregulates the function of vascular T-type calcium channels following chronic nitric oxide deficit.

Cardiovascular disease is characterised by reduced nitric oxide bioavailability resulting from oxidative stress. Our previous studies have shown that nitric oxide deficit per se increases the contribution of T-type calcium channels to vascular tone through increased superoxide from NADPH oxidase (Nox). The aim of the present study was therefore to identify the Nox isoform responsible for modulating T-type channel function, as T-type channels are implicated in several pathophysiological conditions involving oxidative stress. We evaluated T-channel function in skeletal muscle arterioles in vivo, using a novel T-channel blocker, TTA-A2 (3 μmol/L), which demonstrated no cross reactivity with L-type channels. Wild-type and Nox2 knockout (Nox2ko) mice were treated with the nitric oxide synthase inhibitor L-NAME (40 mg/kg/day) for 2 weeks. L-NAME treatment significantly increased systolic blood pressure and the contribution of T-type calcium channels to arteriolar tone in wild-type mice, and this was not prevented by Nox2 deletion. In Nox2ko mice, pharmacological inhibition of Nox1 (10 μmol/L ML171), Nox4 (10 μmol/L VAS2870) and Nox4-derived hydrogen peroxide (500 U/mL catalase) significantly reduced the effect of chronic nitric oxide inhibition on T-type channel function. In contrast, in wild-type mice, ML171 and VAS2870, but not catalase, reduced the contribution of T-type channels to vascular tone, suggesting a role for Nox1 and non-selective actions of VAS2870. We conclude that Nox1, but not Nox2 or Nox4, is responsible for the upregulation of T-type calcium channels elicited by chronic nitric oxide deficit. These data point to an important role for this isoform in increasing T-type channel function during oxidative stress.

Tu1741 Involvement of IL-34 in Intestinal Inflammation of Crohn's Disease

PCR 6 h later. Results: Intrarectal injection of TNBS produced severe colitis accompanied by body weight loss and diarrhea as well as increased MPO activity and ROS production in WT mice. The severity of colitis, body weight loss, diarrhea, increased MPO activity and ROS production were significantly reduced in NOX1KO mice. The expression of iNOS, TNF-[alpha], IL-1[beta], KC, MIP-2, and MCP-1 expressions was markedly upregulated after the injection of TNBS in WT mice, but all these responses were significantly lower in NOX1KO mice. On the other hand, the apparent expression NOX1 mRNA in addition to NOX2 mRNA was detected in both lamina propria and peritoneal macrophages isolated fromWTmice. The exposure of LPS to peritoneal macrophages isolated fromWTmice caused marked upregulation of iNOS, TNF-[alpha], and IL-1[beta] mRNAs, but these increases were significantly mitigated in peritoneal macrophages isolated from NOX1KO mice. Conclusion: These findings suggest that NOX1 plays an important role in the pathogenesis of TNBSinduced colitis by upregulating inflammatory mediators, including iNOS, cytokines, and chemokines. It is likely that the NOX1 expressed in colonic macrophages may play an important role in these responses. Therefore, NOX1 expressed in colonic macrophages may be a novel target for the treatment and prevention of inflammatory bowel diseases.

Nox2‐derived superoxide does not contribute to oxidative stress‐induced vascular upregulation of T‐type channel function (893.6)

Cardiovascular disease is characterised by reduced nitric oxide (NO) and increased oxidative stress. We have shown that these conditions increase the expression and contribution of T‐type channels to vascular tone through increased bioavailability of reactive oxygen species produced from NADPH oxidase (Nox). As Nox2‐generated superoxide has been causally implicated in several vascular disease models, we investigated whether Nox2 was also responsible for upregulation of T‐type channel function in cremaster arterioles in vivo, using wildtype (WT) and Nox2‐deficient mice (Nox2KO). We first validated a novel T‐type channel antagonist, TTA‐A2 (3µM; provided by Merck and Co, Inc) which demonstrated no cross reactivity with L‐type channel function in these arterioles. In both WT and Nox2KO mice, acute inhibition of NO with L‐NAME (10µM) significantly increased the T‐type contribution to vascular tone. Chronic L‐NAME treatment (40mg/kg/day) for 2 weeks significantly increased blood pressure of both WT and Nox2KO mice, as well as the T‐type channel component of arteriolar tone. Pharmacological inhibition of Nox1 (10µM ML171) and Nox4 (10µM VAS2870) together reversed the effect of chronic L‐NAME on T‐type channel function in Nox2KO mice. We conclude that Nox2‐generated superoxide is not responsible for the upregulation of T‐type calcium channels elicited by oxidative stress.Grant Funding Source: Supported by the Heart Foundation of Australia

NOX2 deficiency attenuates markers of adiposopathy and brain injury induced by high-fat diet

The consumption of high-fat/calorie diets in modern societies is likely a major contributor to the obesity epidemic, which can increase the prevalence of cancer, cardiovascular disease, and neurological impairment. Obesity may precipitate decline via inflammatory and oxidative signaling, and one factor linking inflammation to oxidative stress is the proinflammatory, pro-oxidant enzyme NADPH oxidase. To reveal the role of NADPH oxidase in the metabolic and neurological consequences of obesity, the effects of high-fat diet were compared in wild-type C57Bl/6 (WT) mice and in mice deficient in the NAPDH oxidase subunit NOX2 (NOX2KO). While diet-induced weight gains in WT and NOX2KO mice were similar, NOX2KO mice had smaller visceral adipose deposits, attenuated visceral adipocyte hypertrophy, and diminished visceral adipose macrophage infiltration. Moreover, the detrimental effects of HFD on markers of adipocyte function and injury were attenuated in NOX2KO mice; NOX2KO mice had improved glucose regulation, and evaluation of NOX2 expression identified macrophages as the primary population of NOX2-positive cells in visceral adipose. Finally, brain injury was assessed using markers of cerebrovascular integrity, synaptic density, and reactive gliosis, and data show that high-fat diet disrupted marker expression in WT but not NOX2KO mice. Collectively, these data indicate that NOX2 is a significant contributor to the pathogenic effects of high-fat diet and reinforce a key role for visceral adipose inflammation in metabolic and neurological decline. Development of NOX-based therapies could accordingly preserve metabolic and neurological function in the context of metabolic syndrome.

177 The Roles of TNFR Signaling and NOX1 in Epithelial ß-Catenin Activation During Colitis

TNF blockade in Crohn's disease (CD) and ulcerative colitis (UC) patients reveals that TNF plays a central role in disease pathogenesis. Here we examine the potential that TNF activates epithelial stem and progenitor cell (ISC/PC) β-catenin through intestinal epithelial cell (IEC) TNFR1 and TNFR2 signaling. Tissue sections and purified IEC protein isolates were analyzed using Akt-phosphorylated β-catenin (P-β-catenin552 or P-β-cat) Ab. Results: IHC staining of biopsy tissue from control (uninflamed) and inflamed CD/UC patients revealed that Pβ-cat levels increased 2.8-fold in untreated colitis. However, in anti-TNF treated patients selected for ongoing active inflammation, P-β-cat levels were reduced by over 50% (p host) revealed that T cell activation increased numbers of P-βcat-stained IECs by 110% in WT->WT BMC with no increases seen in anti-CD3-treated WT-> TNFR1/2 KO mice. These findings suggest epithelial TNFR signaling regulates IEC β-catenin activation. In studies comparing colitic IL-10 KO (colitis score >3/4) mice to WT healthy controls (n=5 in each group) mRNA from CD44+-sorted IECs was used for genomewide expression array analysis revealing that levels of NADPH oxidase 1 (Nox1) were significantly induced (3.22-fold, p-value: 3 x106). We next tested whether Nox1 may participate in TNF-mediated activation of β-catenin in ISC. We confirmed Nox1 mRNA induction in IECs from IL-10 KO colitis mice (17-fold). Furthermore Nox1 mRNA was induced in purified IECs by anti-CD3 mAb treatment of WT mice (6-fold). Importantly, Nox1 mRNA induction was reduced by over 50% in T cell stimulated TNF-R1/R2 KO mice. Lastly, mRNA from purified CD44+-sorted IECs from Nox1 KO mice revealed that baseline levels of cMyc, cyclinD1, Ascl2 and Lrig1 expression were reduced compared to WT mice. Conclusion: Together the data suggest that epithelial TNF receptor signaling is required for optimal levels of IEC β-catenin activation in patients with active colitis. Data in IL-10 ko and WT mice suggest that epithelial TNFR signaling induces Nox1 which has recently been shown to play a role in Wnt/β-catenin signaling in the intestine (Coant et al. MCB 2010). Together these data are consistent with the hypothesis that Nox1 is a key mediator of TNFinduced epithelial β-catenin activation in UC and CD.

180 The Extracellular Calcium Sensing Receptor Mediates Local Inhibitory Reflexes Evoked by Aromatic L-Amino Acids in Guinea Pig Jejunum

TNF blockade in Crohn's disease (CD) and ulcerative colitis (UC) patients reveals that TNF plays a central role in disease pathogenesis. Here we examine the potential that TNF activates epithelial stem and progenitor cell (ISC/PC) β-catenin through intestinal epithelial cell (IEC) TNFR1 and TNFR2 signaling. Tissue sections and purified IEC protein isolates were analyzed using Akt-phosphorylated β-catenin (P-β-catenin552 or P-β-cat) Ab. Results: IHC staining of biopsy tissue from control (uninflamed) and inflamed CD/UC patients revealed that Pβ-cat levels increased 2.8-fold in untreated colitis. However, in anti-TNF treated patients selected for ongoing active inflammation, P-β-cat levels were reduced by over 50% (p host) revealed that T cell activation increased numbers of P-βcat-stained IECs by 110% in WT->WT BMC with no increases seen in anti-CD3-treated WT-> TNFR1/2 KO mice. These findings suggest epithelial TNFR signaling regulates IEC β-catenin activation. In studies comparing colitic IL-10 KO (colitis score >3/4) mice to WT healthy controls (n=5 in each group) mRNA from CD44+-sorted IECs was used for genomewide expression array analysis revealing that levels of NADPH oxidase 1 (Nox1) were significantly induced (3.22-fold, p-value: 3 x106). We next tested whether Nox1 may participate in TNF-mediated activation of β-catenin in ISC. We confirmed Nox1 mRNA induction in IECs from IL-10 KO colitis mice (17-fold). Furthermore Nox1 mRNA was induced in purified IECs by anti-CD3 mAb treatment of WT mice (6-fold). Importantly, Nox1 mRNA induction was reduced by over 50% in T cell stimulated TNF-R1/R2 KO mice. Lastly, mRNA from purified CD44+-sorted IECs from Nox1 KO mice revealed that baseline levels of cMyc, cyclinD1, Ascl2 and Lrig1 expression were reduced compared to WT mice. Conclusion: Together the data suggest that epithelial TNF receptor signaling is required for optimal levels of IEC β-catenin activation in patients with active colitis. Data in IL-10 ko and WT mice suggest that epithelial TNFR signaling induces Nox1 which has recently been shown to play a role in Wnt/β-catenin signaling in the intestine (Coant et al. MCB 2010). Together these data are consistent with the hypothesis that Nox1 is a key mediator of TNFinduced epithelial β-catenin activation in UC and CD.

Abstract P272: Physiological and Pathological Functions of Nox2 and Nox4 in Ischemia/Reperfusion Injury

NADPH oxidases (Noxes) represent an enzyme system whose primary function is to produce reactive oxygen species (ROS). Both Nox2 and Nox4 play an important role in regulating oxidative stress and growth/death of cardiomyocytes (CMs). However, the role of Nox2 and Nox4 during ischemia/reperfusion (I/R) is poorly understood. In order to elucidate the function of the Nox isoforms, wild type (WT), Nox2 KO (Nox2-/-), and cardiac specific Nox4 KO (cNox4-/-) mice were subjected to 30 minutes ischemia followed by 24 hours reperfusion. Myocardial infarction size/area at risk (MI/AAR) as evaluated by TTC staining was significantly smaller in Nox2-/- and cNox4 -/- than in WT mice (32 ± 3.1% and 28.0 ± 6.1% vs. 40.3 ± 8.3%, p &lt; 0.05). O 2 - production in Nox2 -/- and cNox4 -/- hearts was significantly lower than in WT hearts, as evaluated with the lucigenin-chemiluminescence assay (433 ± 52, 580 ± 106 vs. 1250 ± 236 RLU, p &lt; 0.05). The I/R experiment was also conducted with mice in which both Nox2 and Nox4 are deleted (double KO mice). O 2 - production in the double KO heart was significantly lower than in the single KO ones (257 ± 42 RLU, p &lt; 0.05). The MI/AAR in double KO mice was, however, significantly greater than that in WT mice (58.0 ± 6.3% vs. 40.3 ± 8.3%, p &lt; 0.05). These results raised the possibility that marked suppression of ROS by combined downregulation of Nox2 and Nox4 exacerbates I/R injury. To elucidate the underlying mechanism, we examined expression of hypoxia inducible factor-1alpha (HIF-1 α ), using DN-Nox transgenic (Tg) mice, which we have shown also exhibited marked suppression of O 2 - and significantly greater MI/AAR after I/R. HIF-1 α is lower in DN-Nox Tg mice than in WT mice in both the ischemic and non-ischemic areas. A genetic cross between DN-Nox Tg and mice lacking prolyl hydroxylase 2, an enzyme mediating hydroxylation and downregulation of HIF-1 α , partially rescued I/R injury in DN-Nox mice (35.8 ± 5.9% vs 65.2 ± 10.3%, p&lt;0.05). Taken together, these data show that both Nox2 and Nox4 mediate increases in oxidative stress and myocardial injury in response to I/R. However, combined downregulation of Nox2 and Nox4 induces marked downregulation of ROS, which in turn exacerbates I/R injury possibly through downregulation of HIF-1α and consequent impairment of hypoxic adaptation.

38 Does interaction between peroxisome proliferator-activated receptor-α and NADPH oxidases play an important role in regulating pressure-overload cardiac hypertrophy?

Activation of NADPH oxidases and deactivation of peroxisome proliferator-activated receptor-α (PPAR-α) are known individually to play key roles in the development of left ventricular hypertrophy (LVH). Interestingly, it appears that...

42 Identification of an inherent mechanism of NADPH oxidase signalling in doxorubicin cardiotoxicity

Anthracyclines used in cancer therapeutics, including doxorubicin (DOX), cause both short- and long-term cardiotoxicity associated with increased myocardial oxidative stress. Chronic DOX treatment increases superoxide anion production in vivo, which...