A novel monoclonal antibody reveals the enrichment of NADPH oxidase 5 in human splenic endothelial cells.

Members of the NOX/DUOX family of NADPH oxidases are responsible for regulated ROS production in diverse cells and tissues. Detection of NOX/DUOX proteins at the protein level remains an important challenge in the field. Here we report the development and characterization of a novel anti-NOX5 monoclonal antibody, which recognizes the human NOX5 protein in both Western blot, immunocytochemistry, and histochemistry applications. With the help of the antibody we could successfully detect both heterologously and endogenously expressed NOX5 in mammalian cells. Furthermore, we could also detect NOX5 protein in the human spleen, testis, and ovary. Immunohistochemical studies on human testis revealed that NOX5 localized to spermatogenic cells. This expression pattern was also supported by the result of in silico analysis of single-cell RNA sequencing data that indicated that NOX5 protein is present in developing spermatids and spermatocytes. Mature spermatozoa, however, did not contain detectable NOX5. In the human ovary, both immunostaining and single-cell RNA sequencing suggest that NOX5 is expressed in interstitial fibroblasts and theca cells. We also analyzed vascular cells for the presence of NOX5 and we found that NOX5 expression is a fairly specific feature of splenic endothelial cells.

Endothelial NOX5 overexpression induces changes in the cardiac gene profile: potential impact in myocardial infarction?

Cardiovascular diseases and the ischemic heart disease specifically constitute the main cause of death worldwide. The ischemic heart disease may lead to myocardial infarction, which in turn triggers numerous mechanisms and pathways involved in cardiac repair and remodeling. Our goal in the present study was to characterize the effect of the NADPH oxidase 5 (NOX5) endothelial expression in healthy and infarcted knock-in mice on diverse signaling pathways. The mechanisms studied in the heart of mice were the redox pathway, metalloproteinases and collagen pathway, signaling factors such as NFκB, AKT or Bcl-2, and adhesion molecules among others. Recent studies support that NOX5 expression in animal models can modify the environment and predisposes organ response to harmful stimuli prior to pathological processes. We found many alterations in the mRNA expression of components involved in cardiac fibrosis as collagen type I or TGF-ß and in key players of cardiac apoptosis such as AKT, Bcl-2, or p53. In the heart of NOX5-expressing mice after chronic myocardial infarction, gene alterations were predominant in the redox pathway (NOX2, NOX4, p22phox, or SOD1), but we also found alterations in VCAM-1 and ß-MHC expression. Our results suggest that NOX5 endothelial expression in mice preconditions the heart, and we propose that NOX5 has a cardioprotective role. The correlation studies performed between echocardiographic parameters and cardiac mRNA expression supported NOX5 protective action.

Structure, regulation, and physiological functions of NADPH oxidase 5 (NOX5).

NOX5 is the last member of the NADPH oxidase (NOXs) family to be identified and presents some specific characteristics differing from the rest of the NOXs. It contains four Ca2+ binding domains at the N-terminus and its activity is regulated by the intracellular concentration of Ca2+. NOX5 generates superoxide (O2•-) using NADPH as a substrate, and it modulates functions related to processes in which reactive oxygen species (ROS) are involved. Those functions appear to be detrimental or beneficial depending on the level of ROS produced. For example, the increase in NOX5 activity is related to the development of various oxidative stress-related pathologies such as cancer, cardiovascular, and renal diseases. In this context, pancreatic expression of NOX5 can negatively alter insulin action in high-fat diet-fed transgenic mice. This is consistent with the idea that the expression of NOX5 tends to increase in response to a stimulus or a stressful situation, generally causing a worsening of the pathology. On the other hand, it has also been suggested that it might have a positive role in preparing the body for metabolic stress, for example, by inducing a protective adipose tissue adaptation to the excess of nutrients supplied by a high-fat diet. In this line, its endothelial overexpression can delay lipid accumulation and insulin resistance development in obese transgenic mice by inducing the secretion of IL-6 followed by the expression of thermogenic and lipolytic genes. However, as NOX5 gene is not present in rodents and human NOX5 protein has not been crystallized, its function is still poorly characterized and further extensive research is required.

Regorafenib induces NOX5-mediated endoplasmic reticulum stress and potentiates the anti-tumor activity of cisplatin in non-small cell lung cancer cells.

Lung cancer is one of the most commonly diagnosed cancers worldwide. Although cisplatin-based chemotherapy regimens serve a pivotal role in non-small cell lung cancer (NSCLC) treatment, drug resistance and serious side effects limited its further clinical application. Regorafenib, a small-molecule multi-kinase inhibitor, was demonstrated to have promising anti-tumor activity in various solid tumors. In the present study, we found that regorafenib markedly enhanced cisplatin-induced cytotoxicity in lung cancer cells by activating reactive oxygen species (ROS)-mediated endoplasmic reticulum stress (ER Stress), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. Regorafenib increased ROS generation by promoting NADPH oxidase 5 (NOX5) expression, and knocking down NOX5 attenuated ROS-mediated cytotoxicity of regorafenib in lung cancer cells. Additionally, mice xenograft model validated that synergistic anti-tumor effects of combined treatment with regorafenib and cisplatin. Our results suggested that combination therapy with regorafenib and cisplatin may serve as a potential therapeutic strategy for some NSCLC patients.

Ca2+ -binding-region-dependent cell surface localization of NADPH oxidase Nox5.

Six gene splice variants of superoxide-generating NADPH oxidase 5 (Nox5) have been identified in humans, and they differ in the sequence of their N-terminal cytoplasmic domains, which comprise four EF-hand motifs. Here, we demonstrated that the Ca2+ -dependent association and dissociation between the N- and C-terminal cytoplasmic domains of the Nox5ß variant are affected by the alanine substitution of the conserved Ile-113 or Leu-115 at the connecting loop between the third and fourth EF-hand motifs. These substitutions impair the cell surface localization of Nox5ß. In addition, the Nox5ε/S variant, lacking all EF-hand motifs, does not localize to the plasma membrane. Thus, the Ca2+ -sensitive intramolecular interaction determines the Nox5 subcellular localization, that is, whether Nox5 variants generate superoxide in the extracellular or intracellular space.

NOX2 and NOX5 are increased in cardiac microvascular endothelium of deceased COVID-19 patients.

BACKGROUND: Cardiac injury and inflammation are common findings in COVID-19 patients. Autopsy studies have revealed cardiac microvascular endothelial damage and thrombosis in COVID-19 patients, indicative of microvascular dysfunction in which reactive oxygen species (ROS) may play a role. We explored whether the ROS producing proteins NOX2, NOX4 and NOX5 are involved in COVID-19-induced cardio-microvascular endothelial dysfunction. METHODS: Heart tissue were taken from the left (LV) and right (RV) ventricle of COVID-19 patients (n = 15) and the LV of controls (n = 14) at autopsy. The NOX2-, NOX4-, NOX5- and Nitrotyrosine (NT)-positive intramyocardial blood vessels fractions were quantitatively analyzed using immunohistochemistry. RESULTS: The LV NOX2+, NOX5+ and NT+ blood vessels fractions in COVID-19 patients were significantly higher than in controls. The fraction of NOX4+ blood vessels in COVID-19 patients was comparable with controls. In COVID-19 patients, the fractions of NOX2+, NOX5+ and NT+ vessels did not differ significantly between the LV and RV, and correlated positively between LV and RV in case of NOX5 (r = 0.710; p = 0.006). A negative correlation between NOX5 and NOX2 (r = -0.591; p = 0.029) and between NOX5 and disease time (r = -0.576; p = 0.034) was noted in the LV of COVID-19 patients. CONCLUSION: We show the induction of NOX2 and NOX5 in the cardiac microvascular endothelium in COVID-19 patients, which may contribute to the previously observed cardio-microvascular dysfunction in COVID-19 patients. The exact roles of these NOXes in pathogenesis of COVID-19 however remain to be elucidated.

Endothelial reactive oxygen-forming NADPH oxidase 5 is a possible player in diabetic aortic aneurysm but not atherosclerosis.

Atherosclerosis and its complications are major causes of cardiovascular morbidity and death. Apart from risk factors such as hypercholesterolemia and inflammation, the causal molecular mechanisms are unknown. One proposed causal mechanism involves elevated levels of reactive oxygen species (ROS). Indeed, early expression of the ROS forming NADPH oxidase type 5 (Nox5) in vascular endothelial cells correlates with atherosclerosis and aortic aneurysm. Here we test the pro-atherogenic Nox5 hypothesis using mouse models. Because Nox5 is missing from the mouse genome, a knock-in mouse model expressing human Nox5 in its physiological location of endothelial cells (eNOX5ki/ki) was tested as a possible new humanised mouse atherosclerosis model. However, whether just on a high cholesterol diet or by crossing in aortic atherosclerosis-prone ApoE-/- mice with and without induction of diabetes, Nox5 neither induced on its own nor aggravated aortic atherosclerosis. Surprisingly, however, diabetic ApoE-/- x eNOX5ki/ki mice developed aortic aneurysms more than twice as often correlating with lower vascular collagens, as assessed by trichrome staining, without changes in inflammatory gene expression, suggesting that endothelial Nox5 directly affects extracellular matrix remodelling associated with aneurysm formation in diabetes. Thus Nox5-derived reactive oxygen species are not a new independent mechanism of atherosclerosis but may enhance the frequency of abdominal aortic aneurysms in the context of diabetes. Together with similar clinical findings, our preclinical target validation opens up a first-in-class mechanism-based approach to treat or even prevent abdominal aortic aneurysms.

Impact of calcium and reactive oxygen species on human sperm function: Role of NOX5.

NOX5 is introduced as a new therapeutic target for infertility treatment. This study aimed to compare the basal and stimulated reactive oxygen species (ROS) production and sperm function in human teratozoospermic (n = 15) and normozoospermic (n = 17) semen samples following calcium overload and NOX5 activation. Washed spermatozoa incubated for 1 h under five various conditions: control group, adding a calcium ionophore A23187, phorbol myristate acetate (PMA), A23187 + PMA, and diphenylene iodonium (DPI) + A23187 + PMA. ROS generation was measured immediately after treatment for 30 min. Motility, viability, acrosome reaction, and apoptosis were evaluated after 1-h incubation. ROS production significantly increased when A23187 or PMA was added to the sperm medium. DPI had suppressive effects on ROS generation. Progressive and total motility significantly decreased following calcium elevation and NOX5 activation, which was somewhat returned by DPI. Necrotic and live cells in teratozoospermia was, respectively, higher and lower than normozoospermia samples. Incubation with A23187 significantly increased the percentage of early and late apoptosis. Teratozoosperm are more vulnerable than normal spermatozoa, and produce more basal and stimulated ROS. It seems that calcium overload induces apoptosis in spermatozoa and loss of viability through MPT pore opening and increased intracellular ROS.

Independent of Renox, NOX5 Promotes Renal Inflammation and Fibrosis in Diabetes by Activating ROS-Sensitive Pathways.

Excessive production of renal reactive oxygen species (ROS) plays a major role in diabetic kidney disease (DKD). Here, we provide key findings demonstrating the predominant pathological role of the pro-oxidant enzyme NADPH oxidase 5 (NOX5) in DKD, independent of the previously characterized NOX4 pathway. In patients with diabetes, we found increased expression of renal NOX5 in association with enhanced ROS formation and upregulation of ROS-sensitive factors early growth response 1 (EGR-1), protein kinase C-α (PKC-α), and a key metabolic gene involved in redox balance, thioredoxin-interacting protein (TXNIP). In preclinical models of DKD, overexpression of NOX5 in Nox4-deficient mice enhances kidney damage by increasing albuminuria and augmenting renal fibrosis and inflammation via enhanced ROS formation and the modulation of EGR1, TXNIP, ERK1/2, PKC-α, and PKC-ε. In addition, the only first-in-class NOX inhibitor, GKT137831, appears to be ineffective in the presence of NOX5 expression in diabetes. In vitro, silencing of NOX5 in human mesangial cells attenuated upregulation of EGR1, PKC-α, and TXNIP induced by high glucose levels, as well as markers of inflammation (TLR4 and MCP-1) and fibrosis (CTGF and collagens I and III) via reduction in ROS formation. Collectively, these findings identify NOX5 as a superior target in human DKD compared with other NOX isoforms such as NOX4, which may have been overinterpreted in previous rodent studies.

Beneficial effects of PCSK9 inhibition with alirocumab in familial hypercholesterolemia involve modulation of new immune players.

Familial hypercholesterolemia (FH) is associated with low-grade systemic inflammation, a key driver of premature atherosclerosis. We investigated the effects of inhibiting proprotein convertase subtilisin/kexin type 9 (PCSK9) function on inflammatory state, endothelial dysfunction and cardiovascular outcomes in patients with FH. Fourteen patients with FH were evaluated before and 8 weeks after administration of a PCSK9 blocking monoclonal antibody (alirocumab, 150 mg/subcutaneous/14 days). In vivo and ex vivo analysis revealed that alirocumab blunted the attachment of leukocytes to TNFα-stimulated human umbilical arterial endothelial cells (HUAEC) and suppressed the activation of platelets and most leukocyte subsets, which was accompanied by the diminished expression of CX3CR1, CXCR6 and CCR2 on several leukocyte subpopulations. By contrast, T-regulatory cell activation was enhanced by alirocumab treatment, which also elevated anti-inflammatory IL-10 plasma levels and lowered circulating pro-inflammatory cytokines. Plasma levels of IFNγ positively correlated with levels of total and LDL-cholesterol, whereas circulating IL-10 levels negatively correlated with these key lipid parameters. In vitro analysis revealed that TNFα stimulation of HUAEC increased the expression of PCSK9, whereas endothelial PCSK9 silencing reduced TNFα-induced mononuclear cell adhesion mediated by Nox5 up-regulation and p38-MAPK/NFκB activation, concomitant with reduced SREBP2 expression. PCSK9 silencing also decreased endothelial CX3CL1 and CXCL16 expression and chemokine generation. In conclusion, PCSK9 inhibition impairs systemic inflammation and endothelial dysfunction by constraining leukocyte-endothelium interactions. PCSK9 blockade may constitute a new therapeutic approach to control the inflammatory state associated with FH, preventing further cardiovascular events in this cardiometabolic disorder.

Nicotine promotes vascular calcification via intracellular Ca2+-mediated, Nox5-induced oxidative stress, and extracellular vesicle release in vascular smooth muscle cells.

AIMS: Smokers are at increased risk of cardiovascular events. However, the exact mechanisms through which smoking influences cardiovascular disease resulting in accelerated atherosclerosis and vascular calcification are unknown. The aim of this study was to investigate effects of nicotine on initiation of vascular smooth muscle cell (VSMC) calcification and to elucidate underlying mechanisms. METHODS AND RESULTS: We assessed vascular calcification of 62 carotid lesions of both smoking and non-smoking patients using ex vivo micro-computed tomography (µCT) scanning. Calcification was present more often in carotid plaques of smokers (n = 22 of 30, 73.3%) compared to non-smokers (n = 11 of 32, 34.3%; P < 0.001), confirming higher atherosclerotic burden. The difference was particularly profound for microcalcifications, which was 17-fold higher in smokers compared to non-smokers. In vitro, nicotine-induced human primary VSMC calcification, and increased osteogenic gene expression (Runx2, Osx, BSP, and OPN) and extracellular vesicle (EV) secretion. The pro-calcifying effects of nicotine were mediated by Ca2+-dependent Nox5. SiRNA knock-down of Nox5 inhibited nicotine-induced EV release and calcification. Moreover, pre-treatment of hVSMCs with vitamin K2 ameliorated nicotine-induced intracellular oxidative stress, EV secretion, and calcification. Using nicotinic acetylcholine receptor (nAChR) blockers α-bungarotoxin and hexamethonium bromide, we found that the effects of nicotine on intracellular Ca2+ and oxidative stress were mediated by α7 and α3 nAChR. Finally, we showed that Nox5 expression was higher in carotid arteries of smokers and correlated with calcification levels in these vessels. CONCLUSION: In this study, we provide evidence that nicotine induces Nox5-mediated pro-calcific processes as novel mechanism of increased atherosclerotic calcification. We identified that activation of α7 and α3 nAChR by nicotine increases intracellular Ca2+ and initiates calcification of hVSMCs through increased Nox5 activity, leading to oxidative stress-mediated EV release. Identifying the role of Nox5-induced oxidative stress opens novel avenues for diagnosis and treatment of smoking-induced cardiovascular disease.

Central role of c-Src in NOX5- mediated redox signalling in vascular smooth muscle cells in human hypertension.

AIMS: NOX-derived reactive oxygen species (ROS) are mediators of signalling pathways implicated in vascular smooth muscle cell (VSMC) dysfunction in hypertension. Among the numerous redox-sensitive kinases important in VSMC regulation is c-Src. However, mechanisms linking NOX/ROS to c-Src are unclear, especially in the context of oxidative stress in hypertension. Here, we investigated the role of NOX-induced oxidative stress in VSMCs in human hypertension focusing on NOX5, and explored c-Src, as a putative intermediate connecting NOX5-ROS to downstream effector targets underlying VSMC dysfunction. METHODS AND RESULTS: VSMC from arteries from normotensive (NT) and hypertensive (HT) subjects were studied. NOX1,2,4,5 expression, ROS generation, oxidation/phosphorylation of signalling molecules, and actin polymerization and migration were assessed in the absence and presence of NOX5 (melittin) and Src (PP2) inhibitors. NOX5 and p22phox-dependent NOXs (NOX1-4) were down-regulated using NOX5 siRNA and p22phox-siRNA approaches. As proof of concept in intact vessels, vascular function was assessed by myography in transgenic mice expressing human NOX5 in a VSMC-specific manner. In HT VSMCs, NOX5 was up-regulated, with associated oxidative stress, hyperoxidation (c-Src, peroxiredoxin, DJ-1), and hyperphosphorylation (c-Src, PKC, ERK1/2, MLC20) of signalling molecules. NOX5 siRNA reduced ROS generation in NT and HT subjects. NOX5 siRNA, but not p22phox-siRNA, blunted c-Src phosphorylation in HT VSMCs. NOX5 siRNA reduced phosphorylation of MLC20 and FAK in NT and HT. In p22phox- silenced HT VSMCs, Ang II-induced phosphorylation of MLC20 was increased, effects blocked by melittin and PP2. NOX5 and c-Src inhibition attenuated actin polymerization and migration in HT VSMCs. In NOX5 transgenic mice, vascular hypercontractilty was decreased by melittin and PP2. CONCLUSION: We define NOX5/ROS/c-Src as a novel feedforward signalling network in human VSMCs. Amplification of this system in hypertension contributes to VSMC dysfunction. Dampening the NOX5/ROS/c-Src pathway may ameliorate hypertension-associated vascular injury.

The evolutionary origins of peroxynitrite signalling.

Peroxynitrite is a reactive intermediate formed in vivo through uncatalysed reaction of superoxide and nitric oxide radicals. Despite significant interest in detecting peroxynitrite in vivo and understanding its production, little attention has been given to the evolutionary origins of peroxynitrite signalling. Herein we focus on two enzymes that are key to the biosynthesis of superoxide and nitric oxide, NADPH oxidase 5 (NOX5) and endothelial nitric oxide synthase (eNOS), respectively. Multiple sequence alignments of both enzymes including homologues from all domains of life, coupled with a phylogenetic analysis of NOX5, suggest eNOS and NOX5 are present in animals as the result of horizontal gene transfer from ancestral cyanobacteria to ancestral eukaryotes. Therefore, biochemical studies from other laboratories on a NOX5 homologue in Cylindrospermum stagnale and an eNOS homologue in Synechococcus sp. PCC 7335 are likely to be of relevance to human NOX5 and eNOS and to the production of superoxide, nitric oxide and peroxynitrite in humans.

NOX5 mediates the crosstalk between tumor cells and cancer-associated fibroblasts via regulating cytokine network.

Activation of cancer-associated fibroblasts (CAFs) is a crucial feature for tumor malignancy. The reciprocal interplay between tumor cells and CAFs not only facilitates tumor progression and metastasis but also sustains the tumor-promoting function of CAFs. Nevertheless, how tumor cells readily adapt to these functional CAFs is still unclear. NADPH oxidase 5 (NOX5) is a strong reactive oxygen species producer overexpressed in esophageal squamous cell carcinoma (ESCC) cells. In this study, we showed that NOX5-positive ESCC cells induced normal fibroblasts (NFs) or adipose-derived mesenchymal stem cells (MSCs) to express the marker of CAFs-α smooth muscle actin. Moreover, these tumor cells reprogrammed the cytokine profile of the activated CAFs, which further stimulated NFs or MSCs to CAFs and induced lymphangiogenesis to facilitate ESCC malignancy. NOX5 activated intratumoral Src/nuclear factor-κB signaling to stimulate secretion of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and lactate from tumor cells. Subsequently, TNF-α, IL-1ß, and lactate activated CAFs, and facilitated the secretion of IL-6, IL-7, IL-8, CCL5, and transforming growth factor-ß1 from CAFs. These CAFs-derived cytokines reciprocally induced the progression of NOX5-positive ESCC cells. Our findings together indicate that NOX5 serves as the driving oncoprotein to provide a niche that is beneficial for tumor malignant progression.

NADPH oxidase 5 has a crucial role in cellular motility of colon cancer cells.

NADPH oxidases (NOXs) are a family of transmembrane proteins that generate reactive oxygen species. It was previously reported that patients with colon cancer who had high NOX5 expression had poor prognosis. However, no studies have investigated the cellular functions of NOX5 in colon cancer. The present study aimed to clarify the relationship between NOX5 and cancer development using an in vitro model. Reverse transcription­quantitative PCR was performed to determine the NOX5 expression levels of colon cancer cell lines. NOX5­knockdown experiments were conducted, and the effect on cell proliferation, migration, and invasion were analyzed. In addition, mRNA microarray was conducted to assess changes in gene profile. NOX5 mRNA expression was high in HCT116 cells and moderate in SW48 cells. NOX5 knockdown significantly inhibited cell migration and invasion in both HCT116 and SW48 cells; however, NOX5 knockdown reduced cell proliferation in only HCT116 cells. mRNA microarrays revealed a strong relationship between NOX5 expression levels and integrin­linked kinase signaling pathways. The NOX5 expression in colon cancer cells affected cancer progression, especially cell motility. NOX5 may be a novel therapeutic target for the future development of treatments for colon cancer.

Lysophosphatidylcholine induces oxidative stress in human endothelial cells via NOX5 activation - implications in atherosclerosis.

OBJECTIVE: The mechanisms involved in NOX5 activation in atherosclerotic processes are not completely understood. The present study tested the hypothesis that lysophosphatidylcholine (LPC), a proatherogenic component of oxLDL, induces endothelial calcium influx, which drives NOX5-dependent reactive oxygen species (ROS) production, oxidative stress, and endothelial cell dysfunction. APPROACH: Human aortic endothelial cells (HAEC) were stimulated with LPC (10-5 M, for different time points). Pharmacological inhibition of NOX5 (Melittin, 10-7 M) and NOX5 gene silencing (siRNA) was used to determine the role of NOX5-dependent ROS production in endothelial oxidative stress induced by LPC. ROS production was determined by lucigenin assay and electron paramagnetic spectroscopy (EPR), calcium transients by Fluo4 fluorimetry, and NOX5 activity and protein expression by pharmacological assays and immunoblotting, respectively. RESULTS: LPC increased ROS generation in endothelial cells at short (15 min) and long (4 h) stimulation times. LPC-induced ROS was abolished by a selective NOX5 inhibitor and by NOX5 siRNA. NOX1/4 dual inhibition and selective NOX1 inhibition only decreased ROS generation at 4 h. LPC increased HAEC intracellular calcium, important for NOX5 activation, and this was blocked by nifedipine and thapsigargin. Bapta-AM, selective Ca2+ chelator, prevented LPC-induced ROS production. NOX5 knockdown decreased LPC-induced ICAM-1 mRNA expression and monocyte adhesion to endothelial cells. CONCLUSION: These results suggest that NOX5, by mechanisms linked to increased intracellular calcium, is key to early LPC-induced endothelial oxidative stress and pro-inflammatory processes. Since these are essential events in the formation and progression of atherosclerotic lesions, the present study highlights an important role for NOX5 in atherosclerosis.

Nitric oxide and heme-NO stimulate superoxide production by NADPH oxidase 5.

Nitric oxide (NO) is a ubiquitous cell signaling molecule which mediates widespread and diverse processes in the cell. These NO dependent effects often involve activation (e.g. NO binding to the heme group of soluble guanylyl cyclase for cGMP production) or inactivation (e.g. S-nitrosation) of protein targets. We studied the effect of NO and heme-NO on the transmembrane signaling enzyme NADPH oxidase 5 (NOX5), a heme protein which produces superoxide in response to increases in intracellular calcium. We found that treatment with NO donors increases NOX5 activity through heme-dependent effects, and that this effect could be recapitulated by the addition of heme-NO. This work adds to our understanding of NOX5 regulation in the cell but also provides a framework for understanding how NO could cause widespread changes in hemeprotein activity based on different affinities for heme v. heme-NO, and helps explain the opposing roles NO plays in activation and inactivation of hemeprotein targets.

Endothelial Nox5 Expression Modulates Glucose Uptake and Lipid Accumulation in Mice Fed a High-Fat Diet and 3T3-L1 Adipocytes Treated with Glucose and Palmitic Acid.

Obesity is a global health issue associated with insulin resistance and altered lipid homeostasis. It has been described that reactive oxygen species (ROS) derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity are involved in the development of these pathologies. The present study describes the role of endothelial NOX5 expression over adipose tissue by using two experimental systems: NOX5 conditional knock-in mice fed with a high-fat diet and 3T3-L1 adipocytes cultured with conditioned media of NOX5-expressing endothelial cells previously treated with glucose and palmitic acid. Animals expressing NOX5 presented lower body weight gain and less mesenteric and epididymal adipose mass compared to control mice fed with the same diet. NOX5-expressing mice also showed significantly lower glycaemia and improved insulin-induced glucose uptake. In addition, Glut4 and Caveolin 1 (Cav1) expression were significantly increased in the adipose tissue of these animals. Likewise, 3T3-L1 adipocytes treated with conditioned media from NOX5-expressing endothelial cells, incubated with high glucose and palmitic acid, presented a reduction in lipid accumulation and an increase in glucose uptake. Moreover, a significant increase in the expression of Glut4 and Cav1 was also detected in these cells. Taken together, all these data support that, in response to a highly caloric diet, NOX5 endothelial activity may regulate glucose sensitivity and lipid homeostasis in the adipose tissue.

NOX5 is expressed aberrantly but not a critical pathogenetic gene in Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of intramural ganglion cells in the distal gastrointestinal tract (GI), which results in tonic contraction of the aganglionic gut segment and functional intestinal obstruction. Recent studies have suggested NADPH oxidase 5 (NOX5) as a candidate risk gene for HSCR. In this study, we examined the function of NOX5 to verify its role in the development of the enteric nervous system (ENS). METHODS: HSCR tissue specimens (n = 10) were collected at the time of pull-through surgery and control specimens (n = 10) were obtained at the time of colostomy closure in patients. The NOX5 expression in aganglionic and ganglionic segments of HSCR colon and normal colon were analyzed by immunohistochemistry (IHC), western blot and real-time quantitative PCR (qPCR). The gene expression levels and spatiotemporal expression spectrum of NOX5 in different development stages of zebrafish embryo were determined using qPCR and in-situ hybridization (ISH). The enteric nervous system in NOX5 Morpholino (MO) knockdown and wild type (WT) zebrafish embryo was analyzed by whole-mount immunofluorescence (IF). Intestinal transit assay was performed to analyze the gastrointestinal motility in NOX5 knockdown and control larvae. RESULTS: NOX5 is strongly expressed in the ganglion cells in the proximal segment of HSCR colons and all segments of normal colons. Moreover, the expression of NOX5 is markedly decreased in the aganglionic segment of HSCR colon compared to the ganglionic segment. In zebrafish, NOX5 mRNA level is the highest in the one cell stage embryos and it is decreased overtime with the development of the embryos. Interestingly, the expression of NOX5 appears to be enriched in the nervous system. However, the number of neurons in the GI tract and the GI motility were not affected upon NOX5 knockdown. CONCLUSIONS: Our study shows that NOX5 markedly decreased in the aganglionic segment of HSCR but didn't involve in the ENS development of zebrafish. It implies that absence of intestinal ganglion cells may lead to down-regulation of NOX5.