Nox4 KO Research Articles

Blockade of Activin Receptor IIB Protects Arthritis Pathogenesis by Non-Amplification of Activin A-ACVR2B-NOX4 Axis Pathway.

Although activin receptor IIB (ACVR2B) is emerging as a novel pathogenic receptor, its ligand and assembled components (or assembly) are totally unknown in the context of osteoarthritis (OA) pathogenesis. The present results suggest that upregulation of ACVR2B and its assembly could affect osteoarthritic cartilage destruction. It is shown that the ACVR2B ligand, activin A, regulates catabolic factor expression through ACVR2B in OA development. Activin A Tg mice (Col2a1-Inhba) exhibit enhanced cartilage destruction, whereas heterozygous activin A KO mice (Inhba+/- ) show protection from cartilage destruction. In silico analysis suggests that the Activin A-ACVR2B axis is involved in Nox4-dependent ROS production. Activin A Tg:Nox4 KO (Col2a1-Inhba:Nox4-/- ) mice show inhibition of experimental OA pathogenesis. NOX4 directly binds to the C-terminal binding site on ACVR2B-ACVR1B and amplifies the pathogenic signal for cartilage destruction through SMAD2/3 signaling. Together, the findings reveal that the ACVR2B assembly, which comprises Activin A, ACVR2B, ACVR1B, Nox4, and AP-1-induced HIF-2α, accelerates OA development. Furthermore, it is shown that shRNA-mediated ACVR2B knockdown or trapping ligands of ACVR2B abrogate OA development by competitively disrupting the ACVR2B-Activin A interaction. These results suggest that the ACVR2B assembly is required to amplify osteoarthritic cartilage destruction and could be a potential therapeutic target in efforts to treat OA.

PS-BPB01-6: PROTECTIVE NOX4 EFFECT IN ENDOTHELIAL CELLS INVOLVES ACTIVATION OF TRANSIENT RECEPTOR POTENTIAL MELASTATIN 2 (TRPM2) CATION CHANNEL

Objective: In endothelial cells, Nox4 is the most prevalent isoform and, in contrast to other Noxes, which generate superoxide O2, Nox4 produces mainly H2O2. Low H2O2 levels have been described to lower blood pressure, which suggests that a certain level of reactive oxygen species (ROS) can have protective effects on the cardiovascular system. We demonstrated that TRPM2 is a target of H2O2 in vascular cells. TRPM2 is a redox-sensitive Ca2+ channel and once activated leads to Ca2 influx. Endothelial dysfunction involves a reduction in eNOS activation by Ca2+ and is potentiated in the absence of Nox4. As TRPM2 channel is a H2O2 sensor and regulates Ca2+ entry, we questioned whether protective Nox4 effect in endothelial cells involves the H2O2-TRPM2-Ca2+ axis, with consequent regulation of NO signalling. Design and Methods: WT and TTRhRen hypertensive mice were crossed with Nox4 KO mice. Vascular function was measured by wire myograph in resistance arteries. ROS and H2O2 generation were assessed by lucigenin and amplex red, and Ca2+ influx by fluorescence microscopy in rat aortic endothelial cells (RAEC). eNOS activation was assessed by immunoblotting. Results: Blood pressure was significantly increased in TTRhRen (130.3 ± 7.0 mmHg) and TTRhRen/Nox4 KO mice (141.3 ± 18 mmHg) versus control mice (98.1 ± 8.0 mmHg). TTRhRen mice had reduced endothelium-dependent relaxation (Emax: WT 83.5 ± 4.03 vs TTRhRen 59.1 ± 3.5); an effect worsened by Nox4 KO (37.9 ± 5.4), p < 0.05. The TRPM2 activator, ADPR, improved vascular relaxation in TTRhRen/Nox4 KO mice (75.9 ± 7.7); an effect recapitulated by H2O2 (74.2 ± 15.4), p < 0.05. Ang II-induced ROS and H2O2 generation in RAEC, effect reversed by Nox4 siRNA and followed by increased Ca2+ influx (AUC - Ca2+: 20440.3333 ± 1870.9), important in eNOS activation. Ang II-induced Ca2+ influx in endothelial cells was reduced by TRPM2 siRNA, TRPM2 inhibitors (AUC - Ca2+: 8-br-cADPR 15232.2 ± 1052.1; Olaparib 14952.6 ± 843.2 and 2-APB 13270.8333 ± 277.4, p < 0.05) and by Nox4 siRNA and Nox4 inhibitor GKT137831 (AUC - Ca2+: GKT 15067.5 ± 255.6). Ang II-induced eNOS activation (Ser1177) (%: 72.3 ± 24.7) and NO release were blocked by Nox4 and TRPM2 sirNA, GKT137831, PEG-catalase and the TRPM2 inhibitor 8-br-cADPR (p < 0.05). Conclusions: We defined a novel molecular pathway in endothelial cells, where Nox4-induced H2O2 production activates PARP/TRPM2 signalling followed by Ca2+ influx, eNOS activation and NO release. The absence of Nox4 impairs Ca2+ homeostasis leading to endothelial dysfunction, an effect exacerbated in hypertension. The protective role of Nox4 corroborates the idea that the use of antioxidants based on the sole evidence that decreased oxidative stress is cardioprotective, it is not effective.

Nox2 underpins microvascular inflammation and vascular contributions to cognitive decline

Chronic microvascular inflammation and oxidative stress are inter-related mechanisms underpinning white matter disease and vascular cognitive impairment (VCI). A proposed mediator is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (Nox2), a major source of reactive oxygen species (ROS) in the brain. To assess the role of Nox2 in VCI, we studied a tractable model with white matter pathology and cognitive impairment induced by bilateral carotid artery stenosis (BCAS). Mice with genetic deletion of Nox2 (Nox2 KO) were compared to wild-type (WT) following BCAS. Sustained BCAS over 12 weeks in WT mice induced Nox2 expression, indices of microvascular inflammation and oxidative damage, along with white matter pathology culminating in a marked cognitive impairment, which were all protected by Nox2 genetic deletion. Neurovascular coupling was impaired in WT mice post-BCAS and restored in Nox2 KO mice. Increased vascular expression of chemoattractant mediators, cell-adhesion molecules and endothelial activation factors in WT mice post-BCAS were ameliorated by Nox2 deficiency. The clinical relevance was confirmed by increased vascular Nox2 and indices of microvascular inflammation in human post-mortem subjects with cerebral vascular disease. Our results support Nox2 activity as a critical determinant of VCI, whose targeting may be of therapeutic benefit in cerebral vascular disease.

Early Cytokine-Induced Transient NOX2 Activity Is ER Stress-Dependent and Impacts β-Cell Function and Survival

In type 1 diabetes (T1D) development, proinflammatory cytokines (PIC) released by immune cells lead to increased reactive oxygen species (ROS) production in β-cells. Nonetheless, the temporality of the events triggered and the role of different ROS sources remain unclear. Isolated islets from C57BL/6J wild-type (WT), NOX1 KO and NOX2 KO mice were exposed to a PIC combination. We show that cytokines increase O2•− production after 2 h in WT and NOX1 KO but not in NOX2 KO islets. Using transgenic mice constitutively expressing a genetically encoded compartment specific H2O2 sensor, we show, for the first time, a transient increase of cytosolic/nuclear H2O2 in islet cells between 4 and 5 h during cytokine exposure. The H2O2 increase coincides with the intracellular NAD(P)H decrease and is absent in NOX2 KO islets. NOX2 KO confers better glucose tolerance and protects against cytokine-induced islet secretory dysfunction and death. However, NOX2 absence does not counteract the cytokine effects in ER Ca2+ depletion, Store-Operated Calcium Entry (SOCE) increase and ER stress. Instead, the activation of ER stress precedes H2O2 production. As early NOX2-driven ROS production impacts β-cells’ function and survival during insulitis, NOX2 might be a potential target for designing therapies against early β-cell dysfunction in the context of T1D onset.

NOX4 PROTECTIVE EFFECT IN ENDOTHELIAL FUNCTION INVOLVES ACTIVATION OF TRANSIENT RECEPTOR POTENTIAL MELASTATIN 2 (TRPM2) CATION CHANNEL

Objective: Nox4 regulates endothelium-dependent relaxation through production of H2O2, which in turn acts as an EDHF. We demonstrated that TRPM2 is also a target of H2O2 in vascular cells. TRPM2 is a redox-sensitive Ca2+ channel and once activated leads to Ca2 influx. Hypertension-associated endothelial dysfunction involves reduction in eNOS activation and is potentiated in the absence of NOX4. As TRPM2 channel is a H2O2 sensor and regulates Ca2+ entry, we questioned whether impaired vascular relaxation in hypertensive NOX4 KO mice involves the H2O2-TRPM2-Ca2+ axis in endothelial cells. Design and method: WT and TTRhRen hypertensive mice were crossed with Nox4 KO mice. Vascular function was measured by wire myograph in resistance arteries. Ca2+ influx was assessed by fluorescence microscopy in aortic endothelial cells. eNOS activation was assessed by immunoblotting. Results: Blood pressure was significantly increased in TTRhRen (130.3 ± 7.0 mmHg) and TTRhRen/NOX4 KO mice (141.3 ± 18 mmHg) versus control mice (98.1 ± 8.0 mmHg). TTRhRen mice had reduced endothelium-dependent relaxation (Emax: WT 83.5 ± 4.03 vs TTRhRen 59.1 ± 3.5); an effect worsened by NOX4 KO (37.9 ± 5.4), p<0.05. The TRPM2 activator, ADPR, improved vascular relaxation in TTRhRen/NOX4 KO mice (75.9 ± 7.7); an effect recapitulated by H2O2 (74.2 ± 15.4), p < 0.05. Ang II-induced Ca2+ influx in endothelial cells, important in eNOS activation, was reduced by TRPM2 inhibitors (AUC - Ca2+: 8-br-cADPR 76161 ± 1052.0; Olaparib 74763 ± 843.2 and 2-APB 79625 ± 277.4, p < 0.05) and the NOX1/4 inhibitor GKT137831 (AUC - Ca2+: Ang II 107357 ± 1940.2 vs GKT 60270 ± 255.6, p < 0.05). Ang II-induced activation of eNOS (Ser1177) (%: 72.3 ± 24.7) was blocked by GKT137831, PEG-catalase and the TRPM2 inhibitor 8-br-cADPR (p < 0.05). Conclusions: Endothelial dysfunction in TTRhRen mice involves impaired TRPM2 activation. Inhibition/downregulation of Nox4 and reduced bioavailability of H2O2 exacerbates these processes. We identify a novel protective effect of NOX4 in endothelial cells involving H2O2-mediated activation of TRPM2-Ca2+-eNOS signalling.

Tuberculous Fibrosis Enhances Tumorigenic Potential via the NOX4-Autophagy Axis.

Simple SummaryAlthough previous studies have reported coexistence of pulmonary TB and carcinoma, the underlying mechanism of tuberculous fibrosis-induced tumorigenicity remains to be investigated. We previously reported that NOX4 signaling mediates tuberculous pleural fibrosis by activating ERK–ROS–EMT pathways. We were interested in the role of NOX4 in the tumor microenvironment changed by tuberculosis fibrosis. Our results showed that lung cancer cells enhanced the NOX4 expression and invasive potential after exposure to the conditioned medium of heat-killed Mycobacterium tuberculosis stimulated mesothelial cells or tuberculous pleural effusion. NOX4–autophagy signaling axis contributes to the interaction between tuberculosis fibrosis and lung cancer. Silencing of NOX4 signaling in tuberculous fibrosis reduced the metastatic potential by enhancing autophagy in both in vivoand in vitro studies. This result suggests that NOX4-P62 might serve as a therapeutic target for tuberculous fibrosis-associated lung cancer.While a higher incidence of lung cancer in subjects with previous tuberculous infection has been reported in epidemiologic data, the mechanism by which previous tuberculosis affects lung cancer remains unclear. We investigated the role of NOX4 in tuberculous pleurisy-assisted tumorigenicity both in vitro and in vivo.Heat-killed Mycobacterium tuberculosis-stimulated mesothelial cells augmented the migrationand invasive potential of lung cancer cells in a NOX4-dependent manner. Mice with Mycobacterium bovis bacillus Calmette–Guérin (BCG) pleural infection exhibited increased expression of NOX4 and enhanced malignant potential of lung cancer compared to mice with intrathoracic injection of phosphate-buffered saline. The BCG+ KLN205 (KLN205 cancer cell injection after BCG treatment) NOX4 KO mice group showed reduced tuberculous fibrosis-promoted metastatic potential of lung cancer, increased autophagy, and decreased expression of TGF-β, IL-6, and TNF-α compared to the BCG+KLN205 WT mice group. Finally, NOX4 silencing mitigated the malignant potential of A549 cells that was enhanced by tuberculous pleural effusion and restored autophagy signaling. Our results suggest that the NOX4–autophagy axis regulated by tuberculous fibrosis could result in enhanced tumorigenic potential and that NOX4-P62 might serve as a target for tuberculous fibrosis-induced lung cancer.

Abstract P083: Fetuin-a Induces Vascular Dysfunction Through Toll-like Receptor 4 And Nox1/4 Activation

Although studies demonstrate an important role for fetuin-A (FetA) in the inhibition of vascular calcification, convincing evidence suggests that fetuin-A is also involved in insulin resistance, inflammation and cardiovascular damage. The present study seeks to unravel FetA vascular effects and associated molecular mechanisms, focusing on oxidative stress and toll-like receptor 4 (TLR4). Vascular function studies were performed in mesenteric resistance arteries from WKY rats, wild-type, Nox1 KO, Nox4 KO and Ang II-dependent hypertensive mice (LinA3) and rat aortic endothelial cells (RAEC). ROS production (chemiluminescence, Amplex Red, ELISA) and pro-inflammatory markers expression (RT-PCR) were measured in VSMCs from WKY rats and RAEC. FetA impaired endothelium-dependent (LogEC50 7.320±0.08 M vs control 8.025±0.06) and endothelium-independent vasorelaxation (LogEC50 6.48±0.19 M vs control 7.38±0.12), p<0.05; effects blocked by tempol (superoxide dismutase mimetic), Nox1 inhibitor, ML171, and TLR4 inhibitor, CLI095. We did not observe any changes in contraction. FetA increased ROS production (62%) and peroxynitrite levels (158%) in VSMCs; while in RAEC, FetA increased ROS production (105%) followed by a decrease in H2O2 (62%) levels (p<0.05 vs control). FetA-induced effects on ROS were inhibited by ML171 and GKT137831 (Nox1/Nox4 inhibitor), as well as CLI095. Vascular dysfunction in arteries from Nox1 and Nox4 KO mice was unaffected by FetA. Activation of the FetA/TLR4/Nox axis led to an increase in IL-1β (190%), Il-6 (124%) and RANTES mRNA expression(116%) in RAEC, p<0.05 vs control. FetA enhanced vascular dysfunctionin LinA3 mice. Together, these results suggest that FetA through TLR4/Nox1 and 4-derived ROS leads to vascular dysfunction and inflammation, which may play an important role in the development of vascular injury during hypertension.

Abstract MP29: Activation Of Transient Receptor Potential Melastatin 2 (trpm2) Cation Channel Contributes To Nox4-induced Protective Effects In Endothelial Cells.

NOX4-induced H 2 O 2 production promotes vasodilation and is cardiovascular protective. H 2 O 2 also regulates TRPM2, a redox-sensitive channel that regulates Ca 2 influx. eNOS is a Ca 2+ -dependent enzyme, and hypertension-associated endothelial dysfunction involves eNOS inactivation. As NOX4-derived H 2 O 2 regulates TRPM2 and consequently Ca 2+ influx, we questioned whether downregulation of the H 2 O 2 -TRPM2-Ca 2+ axis in endothelial cells may contributes to impaired vascular relaxation in hypertension. WT and TTRhRen hypertensive mice were crossed with Nox4 KO mice. Vascular function was studied in mesenteric resistance arteries by wire myography. Ca 2+ influx was assessed by fluorescence microscopy in aortic endothelial cells, eNOS activation and TRPM2 expression were assessed by immunoblotting and immunohistochemistry, respectively. Blood pressure in TTRhRen (130.3±7.0 mmHg) and TTRhRen/NOX4 KO mice (141.3±18 mmHg) was significantly increased compared to control mice (98.1±8.0 mmHg). Endothelium-dependent relaxation was impaired in TTRhRen mice (Emax: WT 83.5±4.03 vs TTRhRen 59.1±3.5), effects worsened by NOX4 KO (37.9±5.4), p<0.05. Activation of TRPM2 with ADPR, improved vascular relaxation in TTRhRen/NOX4 KO mice (75.9±7.7); an effect also achieved with H 2 O 2 incubation (74.2±15.4), p<0.05. Ang II stimulated H 2 O 2 generation (% of control: 138.23±9.04) followed by Ca 2+ influx (AUC - Ca 2+ : 19401.25±1940.21), an important regulator of eNOS. These processes were reduced by TRPM2 inhibition (AUC - Ca 2+ : 8-br-cADPR 15232.2±1052.0; Olaparib 14260±843.2 and 2-APB 13095.2±277.4, p<0.05) and by the NOX1/4 inhibitor GKT137831 (AUC - Ca 2+ : Ang II 107357±1940.2 vs GKT 15067.5±255.6, p<0.05). Activation of eNOS (Ser1177) by Ang II in endothelial cells was blocked by PEG-catalase, GKT137831, and the TRPM2 inhibitor 8-br-cADPR. TRPM2 inhibitors also increased MAPK expression in endothelial cells. In conclusion, endothelial dysfunction in TTRhRen/NOX4 KO mice involves impaired TRPM2 activation. Reduced bioavailability of H 2 O 2 due to Nox4 downregulation is a major driver of this process. We identify a new axis in endothelial cells involving Nox4-H 2 O 2 -mediated activation of TRPM2-Ca 2+ -eNOS signalling which is vasoprotective.

Abstract 1247: NOX4 P62 regulates tuberculous fibrosis promoted tumorigenic potential

Abstract Background Increased incidence of lung cancer in subjects with previous tuberculous infection was reported in epidemiologic data. NOX4 signaling contributes to tuberculous pleural fibrosis and lung cancer proliferation, respectively. The objective of this study was to investigate the role of NOX4 in tuberculous pleurisy-assisted tumorigenicity both in vitro and in vivo. Methods After pleural mesothelial cells from WT and NOX4 KO C57BL/6 mice were stimulated with Heat-Killed mycobacterium tuberculosis (HKMT) for 24 hour, cellular interactions between mesothelial cell and cancer cell were evaluated by using transwell invasion assay and wound healing assay. A murine model injected with cancer at 4 weeks after Mycobacterium bovis bacillus Calmette-Guérin (BCG) pleural infection were used to validate the contribution of tuberculous fibrosis to tumor invasion. Results HKMT -stimulated mesothelial cells induced enhanced invasion and migration potential of lung cancer cells in a NOX4-dependent manner. The inhibition of NOX4 signaling exerted a negative effect on the metastatic potential of lung cancer by tuberculous fibrosis in mice model. The expression of inflammatory cytokines like TNF-a, TGF-β and IL-6 was enhanced in the WT-BCG+CA than WT+PBS+CA and NOX4 KO+BCG+CA. The expression of P62 in lung tissue was higher in the WT-BCG+CA than WT+PBS+CA and NOX4 KO+BCG+CA. Discussions Our results suggest that NOX4 and autophagy signaling changed by tuberculous fibrosis could result enhanced tumorigenic potential. NOX4-P62 might seve as a target for the development of anti-tuberculous fibrosis and anti-cancer drugs. Citation Format: Yoonki Hong, Seong-Ji Woo, Youngmi Kim, Ji Young Hong. NOX4 P62 regulates tuberculous fibrosis promoted tumorigenic potential [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1247.

Nox4 Expression Is Not Required for OVX-Induced Osteoblast Senescence and Bone Loss in Mice.

ABSTRACTEstrogen deficiency and aging play critical roles in the pathophysiology of bone as a result of increased oxidative stress. It has been suggested that prevention of NADPH oxidase‐ (Nox‐) dependent accumulation of ROS may be an approach to potentially minimize bone loss caused by these conditions. Using ovariectomized (OVX) and Nox4 gene‐deletion mouse models, we investigated the role of Nox4 in OVX‐induced bone loss and osteoblast senescence signaling. Six‐month‐old WT C57Bl6 mice were allocated to a sham control group, OVX, and OVX plus E2 treatment group for 8 weeks. Decreased bone mass including BMD and BMC were found in the OVX group compared with the sham control (p < 0.05); E2 treatment completely reversed OVX‐induced bone loss. Interestingly, the prevention of OVX‐induced bone loss by E2 was associated with the elimination of increased senescence signaling in bone osteoblastic cells from the OVX group. E2 blunted OVX‐induced p53 and p21 overexpression, but not p16 and Nox4 in bone. In addition, 8‐ and 11‐month‐old Nox4 KO female mice were OVX for 8 weeks. Significant bone loss and increased bone osteoblastic cell senescence signaling occurred not only in Nox4 KO OVX mice compared with sham‐operated animals, but also in 11‐month‐old Nox4 KO sham mice compared with 8‐month‐old Nox4 KO sham mice (p < 0.05). These data suggest that Nox4‐mediated ROS in bone osteoblastic cells may be dispensable for sex steroid deficiency‐induced bone loss and senescence. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

478-P: Nox5 Aggravates Renal Injury Independent of Nox4 Expression in Diabetes

Background: Oxidative stress plays an important role in the pathogenesis of diabetic kidney disease (DKD). Evidences suggest roles for pro-oxidant enzymes Nox4 and Nox5 in animal models of DKD. Nox5 is present in mans but not in mice/rats. It appears that Nox5 could be a main culprit in human DKD. Therefore, we aimed to examine the roles of Nox5 vs. Nox4 and their relative contribution to renal pathology in DKD. Methods: We examined the expression and interaction of Nox5 and Nox4 and ROS formation in human kidney biopsies. In vitro, certain human renal cells being knockdown for Nox4 and Nox5 were exposed to high glucose. In vivo, we examined the effect of Nox5 expression in the absence of Nox4 in STZ- diabetic mice. We developed rabbit models of DKD and Nox5KO rabbits. Results: Increased Nox5 expression and enhanced ROS level was seen in human diabetic kidney biopsies compared to nondiabetic kidney. Nox5 shows the highest upregulation in human renal cells exposed to high glucose in comparison to other Nox isoforms. Nox5 silencing attenuated high glucose induced increased expression of markers of fibrosis, inflammation and putative elements via reduction in ROS formation. Nox5 appears to be upstream of Nox4 and that Nox5 inhibition downregulates Nox4, but not vice versa. In vivo, cell specific expression of Nox5 in both Nox4 KO and GKT137831 (a renal Nox4 inhibitor) treated diabetic mice demonstrated a 30-40% increase in albuminuria, mesangial expansion, renal fibrosis and inflammation as well as enhanced ROS production in comparison to diabetic mice not expressing Nox5. In addition, both high fat feeding and alloxan induced diabetic rabbits showed increased renal Nox5 expression in association with increased renal injury along with upregulation of CTGF, fibronectin and MCP-1 as well as enhanced renal ROS production. Conclusions: These findings provide evidence that Nox5 plays a superior pathogenic role in DKD in comparison to Nox4. Therefore, targeting Nox5 may represent a better approach to treat and prevent DKD in human. Disclosure J.C. Jha: None. S. Urner: None. A. Dai: None. M.E. Cooper: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Mundipharma International. Research Support; Self; Boehringer Ingelheim International GmbH, Novo Nordisk A/S. Speaker’s Bureau; Self; AstraZeneca, Merck Sharp &amp; Dohme Corp., Novartis AG, Sanofi-Aventis, Servier. M. Roden: Advisory Panel; Self; Servier. Board Member; Self; Poxel SA. Consultant; Self; Eli Lilly and Company, Gilead Sciences, Inc., ProSciento, TARGET PharmaSolutions. Research Support; Self; Boehringer Ingelheim International GmbH, Novartis Pharma K.K., Sanofi US. Speaker’s Bureau; Self; Novo Nordisk A/S. C. Kennedy: None. K. Jandeleit-Dahm: None.

Evidence for NADPH oxidase activation by GPR40 in pancreatic β-cells

ABSTRACT Objective: Investigate the involvement of the fatty acids receptor GPR40 in the assembly and activation of NADPH oxidase and the implications on pancreatic β-cell function. Methods: BRIN-BD11 β-cells were exposed to GPR40 agonist (GW9508) or linoleic acid in different glucose concentrations. Superoxide and H2O2 were analyzed, respectively, by DHE fluorescence and by fluorescence of the H2O2 sensor, roGFP2-Orp1. Protein contents of p47phox in plasma membrane and cytosol were analyzed by western blot. NADPH oxidase role was evaluated by p22phox siRNA or by pharmacological inhibition with VAS2870. NOX2 KO islets were used to measure total cytosolic calcium and insulin secretion. Results: GW9508 and linoleic acid increased superoxide and H2O2 contents at 5.6 and 8.3 mM of glucose. In addition, in 5.6 mM, but not at 16.7 mM of glucose, activation of GPR40 led to the translocation of p47phox to the plasma membrane. Knockdown of p22phox abolished the increase in superoxide after GW9508 and linoleic acid. No differences in insulin secretion were found between wild type and NOX2 KO islets treated with GW9508 or linoleic acid. Discussion: We report for the first time that acute activation of GPR40 leads to NADPH oxidase activation in pancreatic β-cells, without impact on insulin secretion.

SUN-302 The Relative Roles of Pro-oxidant Ezymes Nox4 versus Nox5 in Diabetic Kidney Disease

Renal oxidative stress plays an important role in mediating kidney injury in diabetes. There is increasing evidences that recently discovered pro-oxidant enzyme, Nox5 plays a significant role in human diabetic kidney disease (DKD). Nox5 is present in humans and rabbits but not in mice or rats. Thus, there is a paucity of information about Nox5 in conventional mice/rats models of DKD. We examined the role of Nox5 in human diabetic nephropathy, in human renal cell populations as well as in a high fat fed rabbit model of kidney disease. In addition, in vivo, we also examined the contribution of Nox5 in the pathogenesis of DN in the absence of Nox4 expression. Protein expression of Nox4 and Nox5 and their localization in glomerular (podocytes and mesangial cells) and tubular cells were examined by immunostaining in human kidney biopsies obtained from non-diabetic and diabetic individuals. In vitro, human mesangial cells, podocytes and proximal tubules were exposed to high glucose, TGF-β and AngII and Nox4 and Nox5 were knockdown in these renal cells. Cell morphology, gene and protein expression of markers of fibrosis and inflammation as well as putative signalling pathways and the level of ROS were assessed in these human renal cells. In addition, we have developed a novel mode of diabetic complications which is more similar to the human context, the New Zealand White rabbit which expresses Nox5 and the other Nox isoforms as in humans. We have exposed the rabbits to high fat feeding (as a model of type 2 diabetes) as well as inducing insulin deficient diabetes (type 1 diabetes) using alloxan. As a world first, we have generated a Nox5 knockout rabbit using CRISP/Cas9.We have also generated a unique humanised Nox5 transgenic mouse with concomitant Nox4 KO with the aim to definitively delineate the relative effects of Nox4 versus Nox5 in diabetic complications. Expression of Nox5 was increased in both glomerular and tubular compartments of kidney biopsies obtained from diabetic individuals when compared to non-diabetic individuals. Nox5 is the Nox isoform which shows the highest upregulation in response to high glucose. Silencing of Nox5 reduces ROS formation and expression of proinflammatory and profibrotic cytokines and growth factors as well as putative elements that are implicated in DKD. Our data also suggest that Nox5 is upstream of Nox5 and that Nox5 inhibition also downregulates Nox4, but not vice versa. High fat feeding and alloxan induced diabetes rabbits develop increased mesangial area, increased Nox5 expression in renal cortex and upregulation of a range of pro-inflammatory (MCP-1) and pro-fibrotic genes (CTGF) as analysed by RNAseq and confirmed by RT-PCR. In addition, over expression of Nox5 in mesangial cells of Nox4 KO diabetic mice demonstrated 30% increase in albuminuria, mesangial expansion, increased renal injury and inflammation via enhanced ROS production. These studies will for the first time explore in a comprehensive manner the role of Nox5 in recently generated rabbit models of diabetes (type 1 and type 2) as well as delineate the relative role of Nox4 versus Nox5 in diabetic complications. These studies will provide evidence if Nox5 blockade alone or combined with Nox4/Nox5 blockade is the optimal approach to treat and prevent diabetic complications, in particular nephropathy.

The flagellin-TLR5-Nox4 axis promotes the migration of smooth muscle cells in atherosclerosis

We hypothesized that NADPH oxidase 4 (Nox4) is involved in the formation of neointimal atherosclerotic plaques through the migration of smooth muscle cells (SMCs) in response to flagellin. Here, we demonstrate that TLR5-mediated Nox4 activation regulates the migration of SMCs, leading to neointimal plaque formation in atherosclerosis. To investigate the molecular mechanism by which the TLR5-Nox4 cascade mediates SMC migration, we analyzed the signaling cascade in primary vascular SMCs (VSMCs) from wild-type (WT) or Nox4 KO mice. Stimulation of VSMCs from Nox4 KO mice with flagellin failed to induce H2O2 production and Rac activation compared with stimulation of VSMCs from WT mice. Moreover, the migration of Nox4-deficient VSMCs was attenuated in response to flagellin in transwell migration and wound healing assays. Finally, we performed partial carotid artery ligation in ApoE KO and Nox4ApoE DKO mice fed a high-fat diet (HFD) with or without recombinant FliC (rFliC) injection. Injection of rFliC into ApoE KO mice fed a HFD resulted in significantly increased SMC migration into the intimal layer, whereas SMC accumulation was not detected in Nox4ApoE DKO mice. We conclude that activation of the TLR5-Nox4 cascade plays an important role in the formation of neointimal atherosclerotic plaques.

499-P: The Relative Roles of Pro-oxidant Enzymes Nox4 and Nox5 in Diabetic Kidney Disease

Background: Renal oxidative stress plays an important role in mediating kidney injury in diabetes. There is increasing evidences that recently discovered pro-oxidant enzyme, Nox5 plays a significant role in human diabetic kidney disease (DKD). We examined the role of Nox5 in human DKD, in human renal cell populations as well as in a high fat fed and alloxan rabbit model of DKD. In addition, we also examined the contribution of Nox5 in the in DKD in the absence of Nox4 expression using renal cell specific Nox5Tg mice. Results: Expression of Nox5 was increased in both glomerular and tubular compartments of kidney biopsies obtained from diabetic individuals. Nox5 is the Nox isoform which shows the highest upregulation in response to high glucose. Silencing of Nox5 reduces ROS formation and expression of proinflammatory and profibrotic cytokines and growth factors as well as putative elements that are implicated in DKD. Our data also suggest that Nox5 is upstream of Nox4 and that Nox5 inhibition downregulates Nox4, but not vice versa. High fat feeding and alloxan induced diabetes rabbits showed upregulation of Nox5 and increased ROS in association with increased mesangial area, ECM protein accumulation and upregulation of a range of pro-inflammatory (MCP-1) and pro-fibrotic genes (CTGF) as analysed by RNAseq and confirmed by RT-PCR. In addition, over expression of Nox5 in mesangial cells of Nox4 KO diabetic mice demonstrated 30% increase in albuminuria, mesangial expansion, increased renal injury and inflammation via enhanced ROS production. Conclusions: These studies will for the first time explore in a comprehensive manner the role of Nox5 in recently generated Nox5KO rabbit models in the context of type 1 and type 2 diabetes as well as delineate the relative role of Nox4 versus Nox5 in diabetic complications. These studies will provide evidence if Nox5 blockade alone or combined with Nox4/Nox5 blockade is the optimal approach to treat and prevent diabetic complications, in particular nephropathy. Disclosure J.C. Jha: None. M.E. Cooper: Advisory Panel; Self; AstraZeneca, Lilly Diabetes. Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Servier. Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Novo Nordisk A/S. Speaker's Bureau; Self; Bayer AG, Merck Sharp &amp; Dohme Corp., Novartis AG. C. Kennedy: Research Support; Self; Prometic Life Sciences Inc. K. Jandeleit-Dahm: Advisory Panel; Self; Australian Diabetes Society, Genkyotex. Consultant; Self; Metavention.

NADPH oxidase 2 deletion enhances neurogenesis following traumatic brain injury

The NADPH oxidase (NOX) enzyme family is a major source of reactive oxygen species (ROS) and contributor to the secondary pathology underlying traumatic brain injury (TBI). However, little is known about how NOX-derived ROS influences the proliferation and cell-fate determination of neural stem/progenitor cells (NSCs/NPCs) following TBI. In the current study, we found that deletion of NOX2 (NOX2-KO) significantly decreases the population of radial glia-like NSCs and neuroblasts but maintains the population of non-radial Sox2 expressing stem cells under physiological (non-injury) conditions. Surprisingly, the brains of NOX2-KO mice demonstrated a robust increase in the number of neuroblasts during the first week after TBI, as compared to the wild-type group. This increase may result from an enhanced proliferation of NPCs in a lower ROS environment after brain injury, as further examination revealed a significant increase of dividing neuroblasts in both NOX2-KO and NOX inhibitor-treated mouse brain during the first week following TBI. Finally, 5-Bromo-2′-deoxyuridine (BrdU) lineage tracing demonstrated a significantly increased number of newborn neurons were present in the perilesional cortex of NOX2-KO mice at 5 weeks post TBI, indicating that deletion of NOX2 promotes long-term neurogenesis in the injured brain following TBI. Altogether, these findings suggest that targeting NOX through genetic deletion or inhibition enhances post-injury neurogenesis, which may be beneficial for recovery following 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 P510: Nox4 Deficiency Leads to Hypertension and Vascular Damage With Enhanced Effects in Ang II-dependent Hypertension

We previously showed that Nox1 and Nox2 are not involved in chronic Ang II-dependent hypertension, which recapitulates human hypertension. Here we questioned the role of Nox4 by studying transgenic mice expressing human renin (LinA3) crossed with Nox4-/- mice. Four groups were used: wildtype (WT), LinA3, Nox4 KO (Nox4), and LinA3/Nox4 KO (LinA3/Nox4). Blood pressure was measured by tail cuff. Aorta was collected to assess wall thickness, collagen and glycosaminoglycans (GAGs) deposition, and TNFα expression. Mesenteric arteries were used to access vascular function by myography. Blood pressure was increased in LinA3, Nox4 and LinA3/Nox4 mice vs WT (p&lt;0.05). All three experimental groups exhibited vascular remodeling with evidence of increased fibrosis. Although LinA3 had increased aortic wall thickness (+31%), there was no significant change in collagen (10.3±3 vs. 8.5±2% in WT) and GAGs (6.6±3 vs 2.8±2% in WT) deposition (p&lt;0.05). Nox4 mice, which presented a similar increase in wall thickness to LinA3 (+31%), had significant increase in collagen (20.6±6%) and GAGs (22.3±4%) in aorta (p&lt;0.05). In LinA3/Nox4 mice, collagen (24.6±7%) and GAGs (37.1±10%) deposition were increased vs LinA3. TNFα was increased in LinA3 (130.4±6 a.u.) and LinA3/Nox4 mice (129±5 a.u.) vs WT (116.8±9 a.u.) (p&lt;0.05). Mesenteric arteries from LinA3, Nox4 and LinA3/Nox4 mice, exhibit increased Phenylephrine-induced vasoconstriction vs WT (Emax: WT 6.79±0.29 vs LinA3 9.37±0.51; Nox4 9.87±1.59; LinA3/Nox4 9.12±1.63, p&lt;0.05). Endothelium-dependent vasodilation was not reduced in Nox4 but impaired in LinA3 and LinA3/Nox4 (Emax: WT 86.48±0.01 vs LinA3 59.70±0.03; LinA3/Nox4 33.57±0.26, p&lt;0.05). In conclusion, Nox4 deficiency was associated with increased blood pressure, vascular dysfunction and fibrosis, effects that were variably enhanced in LinA3/Nox4 mice. We also observed that the fibrosis in vessels from Nox4 mice was not associated with inflammation. These results suggest that Nox4 may be cardiovascular protective, which when downregulated leads to blood pressure elevation and vascular injury, processes that may be amplified by Ang II-dependent hypertension.

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.

NADPH oxidase 2 plays a role in experimental corneal neovascularization.

Corneal neovascularization, the growth of new blood vessels in the cornea, is a leading cause of vision impairment after corneal injury. Neovascularization typically occurs in response to corneal injury such as that caused by infection, physical trauma, chemical burns or in the setting of corneal transplant rejection. The NADPH oxidase enzyme complex is involved in cell signalling for wound-healing angiogenesis, but its role in corneal neovascularization has not been studied. We have now analysed the role of the Nox2 isoform of NADPH oxidase in corneal neovascularization in mice following chemical injury. C57BL/6 mice aged 8-14 weeks were cauterized with an applicator coated with 75% silver nitrate and 25% potassium nitrate for 8 s. Neovascularization extending radially from limbal vessels was observed in corneal whole-mounts from cauterized wild type mice and CD31+ vessels were identified in cauterized corneal sections at day 7. In contrast, in Nox2 knockout (Nox2 KO) mice vascular endothelial growth factor-A (Vegf-A), Flt1 mRNA expression, and the extent of corneal neovascularization were all markedly reduced compared with their wild type controls. The accumulation of Iba-1+ microglia and macrophages in the cornea was significantly less in Nox2 KO than in wild type mice. In conclusion, we have demonstrated that Nox2 is implicated in the inflammatory and neovascular response to corneal chemical injury in mice and clearly VEGF is a mediator of this effect. This work raises the possibility that therapies targeting Nox2 may have potential for suppressing corneal neovascularization and inflammation in humans.