Nox4 Protein Research Articles

Dihydromyricetin ameliorates atherosclerosis in LDL receptor deficient mice

Background and aimsDihydromyricetin, the most abundant flavonoid in Ampelopsis grossedentata, exerts numerous pharmacological activities, including anti-inflammatory, antioxidant, hepatoprotective, and lipid regulatory activities; however, its protective effect against atherosclerosis remains poorly understood. The aim of the present study was to evaluate the effects of dihydromyricetin on high fat diet (HFD)-induced atherosclerosis using LDL receptor deficient (LDLr−/−) mice. MethodsBlood samples were collected for determination of serum lipid profiles, oxidized LDL (ox-LDL) and pro-inflammatory cytokines. Histology, hepatic lipid content, quantification of atherosclerosis, assessment of oxidative stress and inflammation were performed on liver and aorta samples by molecular biology methods. The effects of dihydromyricetin on ox-LDL-induced human umbilical vein endothelial cells (HUVECs) dysfunction and foam cell formation were further studied. Results(1) Dihydromyricetin ameliorated hyperlipidemia, reduced serum ox-LDL, IL-6 and TNF-α levels in HFD-fed LDLr−/− mice. Moreover, (2) dihydromyricetin suppressed hepatic lipid accumulation and increased protein expressions of PPARα, LXRα and ABCA1. (3) It inhibited atherosclerotic lesion formation and favoured features of plaque stability. (4) Dihydromyricetin prevented hepatic and aortic inflammation as evidenced by the reduced IL-6 and TNF-α mRNA expression; (5) it prevented hepatic and aortic oxidative stress by normalizing activities of antioxidant enzymes in the liver and suppressing reactive oxygen species generation and NOX2 protein expression in both liver and aorta; (6) it inhibited oxLDL-induced injury, monocytes adhesion and oxidative stress in HUVECs and (7) inhibited macrophage foam cell formation and enhanced cholesterol efflux. ConclusionsThese findings suggest that dihydromyricetin could reduce atherosclerosis via its pleiotropic effects, including improvement of endothelial dysfunction, inhibition of macrophage foam cell formation, amelioration of lipid profiles, anti-inflammatory action and anti-oxidative effect.

Decreased neural precursor cell pool in NADPH oxidase 2-deficiency: From mouse brain to neural differentiation of patient derived iPSC

There is emerging evidence for the involvement of reactive oxygen species (ROS) in the regulation of stem cells and cellular differentiation. Absence of the ROS-generating NADPH oxidase NOX2 in chronic granulomatous disease (CGD) patients, predominantly manifests as immune deficiency, but has also been associated with decreased cognition. Here, we investigate the role of NOX enzymes in neuronal homeostasis in adult mouse brain and in neural cells derived from human induced pluripotent stem cells (iPSC). High levels of NOX2 were found in mouse adult neurogenic regions. In NOX2-deficient mice, neurogenic regions showed diminished redox modifications, as well as decrease in neuroprecursor numbers and in expression of genes involved in neural differentiation including NES, BDNF and OTX2. iPSC from healthy subjects and patients with CGD were used to study the role of NOX2 in human in vitro neuronal development. Expression of NOX2 was low in undifferentiated iPSC, upregulated upon neural induction, and disappeared during neuronal differentiation. In human neurospheres, NOX2 protein and ROS generation were polarized within the inner cell layer of rosette structures. NOX2 deficiency in CGD-iPSCs resulted in an abnormal neural induction in vitro, as revealed by a reduced expression of neuroprogenitor markers (NES, BDNF, OTX2, NRSF/REST), and a decreased generation of mature neurons. Vector-mediated NOX2 expression in NOX2-deficient iPSCs rescued neurogenesis. Taken together, our study provides novel evidence for a regulatory role of NOX2 during early stages of neurogenesis in mouse and human.

APX-115, a first-in-class pan-NADPH oxidase (Nox) inhibitor, protects db/db mice from renal injury

Recent studies have suggested that renal Nox is important in the progression of diabetic nephropathy. Therefore, we investigated the effect of a novel pan-NOX-inhibitor, APX-115, on diabetic nephropathy in type 2 diabetic mice. Eight- week-old db/m and db/db mice were treated with APX-115 for 12 weeks. APX-115 was administered by oral gavage at a dose of 60 mg/kg per day. To compare the effects of APX-115 with a dual Nox1/Nox4 inhibitor, db/db mice were treated with GKT137831 according to the same protocol. APX-115 significantly improved insulin resistance in diabetic mice, similar to GKT137831. Oxidative stress as measured by plasma 8-isoprostane level was decreased in the APX-115 group compared with diabetic controls. All lipid profiles, both in plasma and tissues improved with Nox inhibition. APX-115 treatment decreased Nox1, Nox2, and Nox4 protein expression in the kidney. APX-115 decreased urinary albumin excretion and preserved creatinine level. In diabetic kidneys, APX-115 significantly improved mesangial expansion, but GKT137831 did not. In addition, F4/80 infiltration in the adipose tissue and kidney decreased with APX-115 treatment. We also found that TGF-β stimulated ROS generation in primary mouse mesangial cells (pMMCs) from wild-type, Nox1 KO, and Duox1 KO mice, but did not induce Nox activity in pMMCs from Nox2 knockout (KO), Nox4 KO, or Duox2 KO mice. These results indicate that activating Nox2, Nox4, or Duox2 in pMMCs is essential for TGF-β-mediated ROS generation. Our findings suggest that APX-115 may be as effective or may provide better protection than the dual Nox1/Nox4 inhibitor, and pan-Nox inhibition with APX-115 might be a promising therapy for diabetic nephropathy.

Zinc regulates Nox1 expression through a NF-κB and mitochondrial ROS dependent mechanism to induce senescence of vascular smooth muscle cells

AimsThe role of oxidative stress and inflammation in the development and progression of cardiovascular diseases (CVD) is well established. Increases in oxidative stress can further exacerbate the inflammatory response and lead to cellular senescence. We previously reported that angiotensin II (Ang II) and zinc increase reactive oxygen species (ROS) and cause senescence of vascular smooth muscle cells (VSMCs) and that senescence induced by Ang II is a zinc-dependent process. Zinc stimulated NADPH oxidase (Nox) activity; however, the role of Nox isoforms in zinc effects was not determined. ResultsHere, we show that downregulation of Nox1, but not Nox4, by siRNA prevented both Ang II- and zinc-induced senescence in VSMCs. On the other hand, overexpression of Nox1 induced senescence, which was associated with reduced proliferation, reduced expression of telomerase and increased DNA damage. Zinc increased Nox1 protein expression, which was inhibited by chelation of zinc with TPEN and by overexpression of the zinc exporters ZnT3 and ZnT10. These transporters work to reduce cytosolic zinc, suggesting that increased cytosolic zinc mediates Nox1 upregulation. Other metals including copper, iron, cobalt and manganese failed to upregulate Nox1, suggesting that this pathway is zinc specific. Nox1 upregulation was inhibited by actinomycin D (ACD), an inhibitor of transcription, by inhibition of NF-κB, a known Nox1 transcriptional regulator and by N-acetyl cysteine (NAC) and MitoTEMPO, suggesting that NF-κB and mitochondrial ROS mediate zinc effects. Supporting this idea, we found that zinc increased NF-κB activation in the cytosol, stimulated the translocation of the p65 subunit to the nucleus, and that zinc accumulated in mitochondria increasing mitochondrial ROS, measured using MitoSox. Further, zinc-induced senescence was reduced by inhibition of NF-κB or reduction of mitochondrial ROS with MitoTEMPO. NF-κB activity was also reduced by MitoTEMPO, suggesting that mitochondrial ROS is upstream of NF-κB. Innovation and conclusionOur data demonstrate that altered zinc distribution leading to accumulation of zinc in the mitochondria increases mitochondrial ROS production causing NF-κB activation which in turn upregulates Nox1 expression inducing senescence of VSMCs.

Role of NADPH Oxidase-4 in Human Endothelial Progenitor Cells.

Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. However, the role of NOX4 in EPC function is not completely understood.Methods: EPCs were isolated from human saphenous vein and mammary artery discarded during bypass surgery. NOX4 gene and protein expression in EPCs were measured by real time-PCR and Western blot analysis respectively. NOX4 gene expression was inhibited using an adenoviral vector expressing human NOX4 shRNA (Ad-NOX4i). H2O2 production was measured by Amplex red assay. EPC migration was evaluated using a transwell migration assay. EPC proliferation and viability were measured using trypan blue counts.Results: Inhibition of NOX4 using Ad-NOX4i reduced Nox4 gene and protein expression as well as H2O2 formation in EPCs. Inhibition of NOX4-derived H2O2 decreased both proliferation and migration of EPCs. Interestingly, pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) decreased NOX4 expression and reduced survival of EPCs. However, the survival of EPCs was further diminished by TNF-α in NOX4-knockdown cells, suggesting that NOX4 has a protective role in EPCs.Conclusion: These findings suggest that NOX4-type NADPH oxidase is important for proliferation and migration functions of EPCs and protects against pro-inflammatory cytokine induced EPC death. These properties of NOX4 may facilitate the efficient function of EPCs which is vital for successful neovascularization.

TMEM16A Contributes to Endothelial Dysfunction by Facilitating Nox2 NADPH Oxidase-Derived Reactive Oxygen Species Generation in Hypertension.

Ca2+-activated Cl- channels play a crucial role in various physiological processes. However, the role of TMEM16A in vascular endothelial dysfunction during hypertension is unclear. In this study, we investigated the specific involvement of TMEM16A in regulating endothelial function and blood pressure and the underlying mechanism. Reverse transcription-polymerase chain reaction, Western blotting, coimmunoprecipitation, confocal imaging, patch-clamp recordings, and TMEM16A endothelial-specific transgenic and knockout mice were used. We found that TMEM16A was expressed abundantly and functioned as a Ca2+-activated Cl- channel in endothelial cells. Angiotensin II induced endothelial dysfunction with an increase in TMEM16A expression. The knockout of endothelial-specific TMEM16A significantly lowered the blood pressure and ameliorated endothelial dysfunction in angiotensin II-induced hypertension, whereas the overexpression of endothelial-specific TMEM16A resulted in the opposite effects. These results were related to the increased reactive oxygen species production, Nox2-containing NADPH oxidase activation, and Nox2 and p22phox protein expression that were facilitated by TMEM16A on angiotensin II-induced hypertensive challenge. Moreover, TMEM16A directly bound with Nox2 and reduced the degradation of Nox2 through the proteasome-dependent degradation pathway. Therefore, TMEM16A is a positive regulator of endothelial reactive oxygen species generation via Nox2-containing NADPH oxidase, which induces endothelial dysfunction and hypertension. Modification of TMEM16A may be a novel therapeutic strategy for endothelial dysfunction-associated diseases.

Dysregulation of Alveolar Macrophage PPARγ, NADPH Oxidases, and TGFβ1 in Otherwise Healthy HIV-Infected Individuals.

Despite antiretroviral therapy (ART), respiratory infections increase mortality in individuals living with chronic human immunodeficiency virus (HIV) infection. In experimental and clinical studies of chronic HIV infection, alveolar macrophages (AMs) exhibit impaired phagocytosis and bacterial clearance. Peroxisome proliferator-activated receptor (PPAR)γ, NADPH oxidase (Nox) isoforms Nox1, Nox2, Nox4, and transforming growth factor-beta 1 (TGFβ1) are critical mediators of AM oxidative stress and phagocytic dysfunction. Therefore, we hypothesized that HIV alters AM expression of these targets, resulting in chronic lung oxidative stress and subsequent immune dysfunction. A cross-sectional study of HIV-infected (n = 22) and HIV-uninfected (n = 6) subjects was conducted. Bronchoalveolar lavage (BAL) was performed, and AMs were isolated. Lung H2O2 generation was determined by measuring H2O2 in the BAL fluid. In AMs, PPARγ, Nox1, Nox2, Nox4, and TGFβ1 mRNA (quantitative real-time polymerase chain reaction) and protein (fluorescent immunomicroscopy) levels were assessed. Compared with HIV-uninfected (control) subjects, HIV-infected subjects were relatively older and the majority were African American; ∼86% were on ART, and their median CD4 count was 445, with a median viral load of 0 log copies/ml. HIV infection was associated with increased H2O2 in the BAL, decreased AM mRNA and protein levels of PPARγ, and increased AM mRNA and protein levels of Nox1, Nox2, Nox4, and TGFβ1. PPARγ attenuation and increases in Nox1, Nox2, Nox4, and TGFβ1 contribute to AM oxidative stress and immune dysfunction in the AMs of otherwise healthy HIV-infected subjects. These findings provide novel insights into the molecular mechanisms by which HIV increases susceptibility to pulmonary infections.

Mechanism of angiotensin-converting enzyme 2 overexpression improving collagen synthesis in lung

Objective: To explore the mechanism of angiotensin-converting enzyme 2 (ACE2) overexpression improving collagen synthesis in lung. Methods: Lung fibroblasts of mice over-expressing ACE2 and the wild type (WT) were cultured in vitro and divided into 5 groups: WT-control, WT-AngiotensinⅡ (AngⅡ), ACE2(+ /+) -control, ACE2(+ /+) -AngⅡ and ACE2(+ /+) -AngⅡ+ A779. The protein relative expression levels of ACE2, collagen Ⅰ, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), autophagy-related protein (Beclin1), ubiquitin-binding protein p62 (P62), microtubule-associated proteins light chain 3-Ⅱ (LC3-Ⅱ) were measured by Western blot and triphosadenine (ATP) level was measured by ATP Assay Kit. Fibroblasts over-expressing ACE2 were pretreated with or without the autophagy inhibitor and were separated into 4 groups: ACE2(+ /+) -control, ACE2(+ /+) -AngⅡ, ACE2(+ /+) -AngⅡ+ 3-MA and ACE2(+ /+) -3-MA. In vivo, random allocation was used to averagely divide mice into four groups: WT-control, WT-Bleomycin (BLM), ACE2(+ /+) - control, ACE2(+ /+) -BLM. Wild type and ACE2 over-expressing mice were instilled with bleomycin endotracheally (3.5 mg/kg) or the same volume saline. All mice were sacrificed after 28 days and the lung tissue were used for HE and Masson staining as well as immunohistochemical staining for NOX4, P62 and LC3. Results: The vimentin in lung fibroblasts isolated from mice was proved to be positive by both immunohistochemical and immunofluorescence. The ACE2 protein level of lung fibroblasts over-expressing ACE2 was higher than the wild type (0.202±0.062 and 0.067±0.040, P<0.05). The protein levels of collagenⅠ, NOX4 and NLRP3 in WT-AngⅡ group were obviously higher than the WT-control group (0.861±0.129 and 0.417±0.076, 0.432±0.036 and 0.318±0.058, 0.367±0.125 and 0.045±0.012, all P<0.05). The difference of collagenⅠand NLRP3 between ACE2(+ /+) -AngⅡ group and ACE2(+ /+) -control group had no statistical significance (all P>0.05). CollagenⅠand NOX4 protein level in ACE2(+ /+) -AngⅡ+ A779 group were observably higher than ACE2(+ /+) - AngⅡ group (0.707±0.155 and 0.458±0.108, 0.299±0.038 and 0.149±0.090, all P<0.05). The autophagy related protein levels of Beclin1, P62 and LC3-Ⅱ in ACE2(+ /+) -control group were distinctly higher than WT-control group (0.834±0.051 and 0.274±0.018, 0.467±0.078 and 0.093±0.025, 0.494±0.065 and 0.150±0.054, all P<0.05). However, these protein levels in ACE2(+ /+) -AngⅡ+ A779 group were lower than ACE2(+ /+) -AngⅡ group (1.331±0.203 and 1.565±0.069, 0.298±0.096 and 0.438±0.077, 0.464±0.093 and 0.768±0.071, all P<0.05). ACE2(+ /+) -AngⅡ+ 3-MA group had higher collagenⅠ (0.383±0.125 and 0.032±0.013, P<0.05) and lower LC3-Ⅱ protein level (1.177±0.140 and 1.387±0.183, P<0.05) than AngⅡ group. In bleomycin induced lung fibrosis in mice, ACE2(+ /+) -BLM mice exhibited milder lung fibrosis and lower NOX4 protein level but higher LC3-Ⅱprotein level compared with WT-BLM mice. Conclusion: ACE2 over-expression ameliorated collagen synthesis through enhancing autophagy in lung.

Roux-en-Y gastric bypass improves glucose homeostasis, reduces oxidative stress and inflammation in livers of obese rats and in Kupffer cells via an AMPK-dependent pathway

Oxidative stress and inflammation are implicated in the pathogenesis of steatohepatitis. We hypothesize that Roux-en-Y gastric bypass reduces oxidative stress and inflammation in the liver of obese rats via activation of AMPK-α. Obese Sprague-Dawley male rats underwent either sham operation or Roux-en-Y gastric bypass. Hepatic TNF-α, NF-κB, IRS-2, PI3 kinase, PKC-ζ, NOX2, and AMPK-α were measured. Mechanistic studies were done in a rat Kupffer cell line (RKC1) that was treated with free fatty acids to mimic lipotoxicity and then transfected with AMPK-α siRNA. Reactive oxygen species, TNF-α, NF-κB, AMPK-α, p-AMPK-α, PPAR-γ, and NOX2 were measured. A t test was used. Roux-en-Y gastric bypass lowered nonfasting serum glucose, improved the glucose tolerance test, and induced IRS2/PI3 kinase interaction. Additionally, Roux-en-Y gastric bypass decreased hepatic NOX2, PKC-ζ, TNF-α expression and activation of NF-κB. Free fatty acids increased reactive oxygen species, TNF-α protein, NOX2 protein, and activated NF-κB. Rosiglitazone attenuated the free fatty acids-induced increase in reactive oxygen species, TNF-α, NOX2, and NF-κB; blocking AMPK-α by siRNA abolished the effects of rosiglitazone. Roux-en-Y gastric bypass exhibits antidiabetic properties and is associated with downregulation of proinflammation genes and oxidative stress in the liver and within Kupffer cells via activation of AMPK-α.

Involvement of Nox2 and Nox4 NADPH oxidases in early brain injury after subarachnoid hemorrhage

Oxidative stress is responsible for a poor prognosis of subarachnoid hemorrhage (SAH) patients. Nox2 has been shown to participate in SAH-induced early brain injury (EBI). Nox4 is another major subtype of Nox family widely expressed in central nervous system (CNS). Here, we investigated the role of Nox4 and whether there was a synergistic effect of Nox2 and Nox4 in SAH-induced EBI. Clinical brain biopsies of four patients with traumatic brain injury (TBI) and perihematomal brain tissue from six subjects with SAH were examined. Gp91ds-tat (a specific inhibitor of Nox2), GKT137831 (a specific inhibitor of Nox4), and apocynin (a non-specific Nox inhibitor) were used to test the role of Nox2 and Nox4. The protein levels of Nox2 and Nox4 were elevated in rat neurons and astrocytes at 12 h after SAH, and in cultured brain microvascular endothelial cells at 24 h after exposure to OxyHb. Similarly, there were higher Nox2 and Nox4 protein levels in perihematomal neurons and astrocytes in SAH patients than that in brain tissue from subjects with TBI. In SAH rat model, gp91ds-tat and GKT137831 could reduce SAH-induced neuronal death and degeneration, whereas apocynin did not induce a more intense neuroprotection. Consistently, in in vitro SAH model, siRNA-mediated silencing of Nox2 and Nox4 suppressed the OxyHb-induced neuronal apoptosis, whereas Nox2 and Nox4 co-knockdown also did not show a remarkable overlay effect. In conclusion, Nox4 should contribute to the pathological processes in SAH-induced EBI, and there was not an overlay effect of Nox2 inhibition and Nox4 inhibition on preventing SAH-induced EBI.

Mycoplasma bovis NADH oxidase functions as both a NADH oxidizing and O2 reducing enzyme and an adhesin

Mycoplasma bovis causes considerable economic losses in the cattle industry worldwide. In mycoplasmal infections, adhesion to the host cell is of the utmost importance. In this study, the amino acid sequence of NOX was predicted to have enzymatic domains. The nox gene was then cloned and expressed in Escherichia coli. The enzymatic activity of recombinant NOX (rNOX) was confirmed based on its capacity to oxidize NADH to NAD+ and reduce O2 to H2O2. The adherence of rNOX to embryonic bovine lung (EBL) cells was confirmed with confocal laser scanning microscopy, enzyme-linked immunosorbent assay, and flow cytometry. Both preblocking EBL cells with purified rNOX and preneutralizing M. bovis with polyclonal antiserum to rNOX significantly reduced the adherence of M. bovis to EBL cells. Mycoplasma bovisNOX–expressed a truncated NOX protein at a level 10-fold less than that of the wild type. The capacities of M. bovisNOX– for cell adhesion and H2O2 production were also significantly reduced. The rNOX was further used to pan phage displaying lung cDNA library and fibronectin was determined to be potential ligand. In conclusion, M. bovis NOX functions as both an active NADH oxidase and adhesin, and is therefore a potential virulence factor.

Sex-specific differences in mitochondria biogenesis, morphology, respiratory function, and ROS homeostasis in young mouse heart and brain.

Sex‐specific differences in mitochondrial function and free radical homeostasis are reported in the context of aging but not well‐established in pathogeneses occurring early in life. Here, we examine if sex disparity in mitochondria function, morphology, and redox status starts early and hence can be implicated in sexual dimorphism in cardiac as well as neurological disorders prevalent at young age. Although mitochondrial activity in the heart did not significantly vary between sexes, female brain exhibited enhanced respiration and higher reserve capacity. This was associated with lower H2O2 production in female cardiac and brain tissues. Using transmission electron microscopy, we found that the number of female cardiac mitochondria is moderately greater (117 ± 3%, P = 0.049, N = 4) than male's, which increased significantly for cortical mitochondria (134 ± 4%, P = 0.001, N = 4). However, male's cardiac mitochondria exhibited fragmented, circular, and smaller mitochondria relative to female's mitochondria, while no morphologic sex‐dependent differences were observed in cortical mitochondria. No sex differences were detected in Nox2 and Nox4 proteins or O2‐consuming/H2O2‐producing activities in brain homogenate or synaptosomes. However, a strong trend of increased EPR‐detected NOX superoxide in male synaptosomes hinted at higher superoxide dismutase activity in female brains, which was confirmed by two independent protocols. We also provide direct evidence that respiring mitochondria generally produce an order‐of‐magnitude lower reactive oxygen species (ROS) proportions than currently estimated. Our results indicate that sex differences in mitochondrial biogenesis, bioenergetics, and morphology may start at young age and that sex‐dependent SOD capacity may be responsible for differences in ROS homeostasis in heart and brain.

Spinal Cord Injury Leads to Hyperoxidation and Nitrosylation of Skeletal Muscle Ryanodine Receptor-1 Associated with Upregulation of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4.

Spinal cord injury (SCI) results in marked atrophy and dysfunction of skeletal muscle. There are currently no effective treatments for SCI-induced muscle atrophy or the dysfunction of the remaining muscle tissue. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-4 (Nox4) produces reactive oxygen species (ROS) in sarcoplasmic reticulum (SR) and has been identified as an important O2 sensor in skeletal muscle. Ryanodine receptors (RyRs) are calcium (Ca2+) channels that are responsible for Ca2+ release from SR. In skeletal muscle, type1 RyR (RyR1) is predominantly functional. RyR1 is regulated by multiple proteins, including calstabin1, which assures that they close appropriately once contraction has ceased. RyR1 function is also regulated by oxidation and redox-dependent cysteine nitrosylation. Excessive oxidation/nitrosylation of RyR1 is associated with dissociation of calstabin1 and reduced muscle force generation. However, whether Nox4 levels in skeletal muscle are elevated or whether RyR1 is oxidized or nitrosylated after SCI has not been determined. In this study, we examined Nox4 expression, oxidation/nitrolysation status, and association of calstabin1 with RyR1 in skeletal muscle derived from rats that were subjected to T4 complete transection (SCI), and observed elevated expression of Nox4 messenger RNA and protein in muscle after SCI associated with enhanced binding of Nox4 to RyR1, increased oxidation and nitrosylation of RyR1, and dissociation of calstabin1 from RyR1 in SCI rat muscle. Our data suggest that RyR1 dysfunction resulting from excessive oxidation/nitrosylation may contribute to reduced specific force after SCI and suggest that Nox4 may be the source of ROS responsible for increased oxidation and nitrosylation of RyR1.

NOX1 and NOX4 are required for the differentiation of mouse F9 cells into extraembryonic endoderm.

Mouse F9 cells differentiate to primitive endoderm (PrE) when treated with retinoic acid (RA). Differentiation is accompanied by increased reactive oxygen species (ROS) levels, and while treating F9 cells with antioxidants attenuates differentiation, H2O2 treatment alone is sufficient to induce PrE. We identified the NADPH oxidase (NOX) complexes as candidates for the source of this endogenous ROS, and within this gene family, and over the course of differentiation, Nox1 and Nox 4 show the greatest upregulation induced by RA. Gata6, encoding a master regulator of extraembryonic endoderm is also up-regulated by RA and we provide evidence that NOX1 and NOX4 protein levels increase in F9 cells overexpressing Gata6. Pan-NOX and NOX1-specific inhibitors significantly reduced the ability of RA to induce PrE, and this was recapitulated using a genetic approach to knockdown Nox1 and/or Nox4 transcripts. Interestingly, overexpressing either gene in untreated F9 cells did not induce differentiation, even though each elevated ROS levels. Thus, the data suggests that ROS produced during PrE differentiation is dependent in part on increased NOX1 and NOX4 levels, which is under the control of GATA6. Furthermore, these results suggest that the combined activity of multiple NOX proteins is necessary for the differentiation of F9 cells to primitive endoderm.

Quercetin protects liver injury induced by bile duct ligation via attenuation of Rac1 and NADPH oxidase1 expression in rats

Bile duct ligation (BDL) and subsequent cholestasis are correlated with oxidative stress, hepatocellular injury and fibrosis. Quercetin is a flavonoid with antifibrotic, and hepatoprotective properties. However, the molecular mechanism underlying quercetin-mediated hepatoprotection is not fully understood. The current study was to evaluate mechanisms of hepatoprotective effect of quercetin in BDL rat model. We divided male Wistar rats into 4 groups (n=8 for each): sham, sham+quercetin (30 mg/kg per day), BDL, and BDL+quercetin (30 mg/kg per day). Four weeks later, the rats were sacrificed, the blood was collected for liver enzyme measurements and liver for the measurement of Rac1, Rac1-GTP and NOX1 mRNA and protein levels by quantitative PCR and Western blotting, respectively. Quercetin significantly alleviated liver injury in BDL rats as evidenced by histology and reduced liver enzymes. Furthermore, the mRNA and protein expression of Rac1, Rac1-GTP and NOX1 were significantly increased in BDL rats compared with those in the sham group (P<0.05); quercetin treatment reversed these variables back toward normal (P<0.05). Another interesting finding was that the antioxidant markers e.g. superoxide dismutase and catalase were elevated in quercetin-treated BDL rats compared to BDL rats (P<0.05). Quercetin demonstrated hepatoprotective activity against BDL-induced liver injury through increasing antioxidant capacity of the liver tissue, while preventing the production of Rac1, Rac1-GTP and NOX1 proteins.

Chronic NaHS treatment decreases oxidative stress and improves endothelial function in diabetic mice.

Hydrogen sulphide (H2S) is endogenously produced in vascular tissue and has anti-oxidant and vasoprotective properties. This study investigates whether chronic treatment using the fast H2S donor NaHS could elicit a vasoprotective effect in diabetes. Diabetes was induced in male C57BL6/J mice with streptozotocin (60 mg/kg daily, ip for 2 weeks) and confirmed by elevated blood glucose and glycated haemoglobin levels. Diabetic mice were then treated with NaHS (100 µmol/kg/day) for 4 weeks, and aortae collected for functional and biochemical analyses. In the diabetic group, both endothelium-dependent vasorelaxation and basal nitric oxide (NO•) bioactivity were significantly reduced ( p < 0.05), and maximal vasorelaxation to the NO• donor sodium nitroprusside was impaired ( p < 0.05) in aorta compared to control mice. Vascular superoxide generation via nicotine adenine dinucleotide phosphate (NADPH) oxidase ( p < 0.05) was elevated in aorta from diabetic mice which was associated with increased expression of NOX2 ( p < 0.05). NaHS treatment of diabetic mice restored endothelial function and exogenous NO• efficacy back to control levels. NaHS treatment also reduced the diabetes-induced increase in NADPH oxidase activity, but did not affect NOX2 protein expression. These data show that chronic NaHS treatment reverses diabetes-induced vascular dysfunction by restoring NO• efficacy and reducing superoxide production in the mouse aorta.

CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells.

Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting. NOX4 knockout cell lines showed reduced cell proliferation with an increase of sub-G1 cell population and the decrease of S/G2/M population. Moreover, NOX4 deficiency resulted in a dramatic decrease in invadopodium formation and the invasive activity. In addition, NOX4 deficiency also caused a decrease in focal adhesions and cell migration in HeLa cells. These results suggest that NOX4 is required for both efficient proliferation and invasion of HeLa cells.

Acute Exposure to Cigarette Smoking Followed by Myocardial Infarction Aggravates Renal Damage in an In Vivo Mouse Model.

Cigarette smoking (S) is a risk factor for progressive chronic kidney disease, renal dysfunction, and renal failure. In this study, the effect of smoking on kidney function was investigated in a mouse model of myocardial infarction (MI) using 4 groups: control (C), smoking (S), MI, and S+MI. Histological analysis of S+MI group showed alterations in kidney structure including swelling of the proximal convoluted tubules (PCTs), thinning of the epithelial lining, focal loss of the brush border of PCTs, and patchy glomerular retraction. Molecular analysis revealed that nephrin expression was significantly reduced in the S+MI group, whereas sodium-hydrogen exchanger-1 (NHE-1) was significantly increased, suggesting altered glomerular filtration and kidney functions. Moreover, S+MI group, but not S alone, showed a significant increase in the expression of connective tissue growth factor (CTGF) and fibrotic proteins fibronectin (FN) and α-smooth muscle actin (SMA), in comparison to controls, in addition to a significant increase in mRNA levels of IL-6 and TNF-α inflammatory markers. Finally, reactive oxygen species (ROS) production was significantly accentuated in S+MI group concomitant with a significant increase in NOX-4 protein levels. In conclusion, smoking aggravates murine acute renal damage caused by MI at the structural and molecular levels by exacerbating renal dysfunction.

NADPH oxidase 4 regulates vascular inflammation in aging and atherosclerosis

We recently reported that increased NADPH oxidase 4 (NOX4) expression and activity during aging results in enhanced cellular and mitochondrial oxidative stress, vascular inflammation, dysfunction, and atherosclerosis. The goal of the present study was to elucidate the molecular mechanism(s) for these effects and determine the importance of NOX4 modulation of proinflammatory gene expression in mouse vascular smooth muscle cells (VSMCs). A novel peptide-mediated siRNA transfection approach was used to inhibit Nox4 expression with minimal cellular toxicity. Using melittin-derived peptide p5RHH, we achieved significantly higher transfection efficiency (92% vs. 85% with Lipofectamine) and decreased toxicity (p<0.001 vs. Lipofectamine in MTT and p<0.0001 vs. Lipofectamine in LDH assays) in VSMCs. TGFβ1 significantly upregulates Nox4 mRNA (p<0.01) and protein (p<0.01) expression in VSMCs. p5RHH-mediated Nox4 siRNA transfection greatly attenuated TGFβ1-induced upregulation of Nox4 mRNA (p<0.01) and protein (p<0.0001) levels and decreased hydrogen peroxide production (p<0.0001). Expression of pro-inflammatory genes Ccl2, Ccl5, Il6, and Vcam1 was significantly upregulated in VSMCs in several settings cells isolated from aged vs. young wild-type mice, in atherosclerotic arteries of Apoe−/− mice, and atherosclerotic human carotid arteries and correlated with NOX4 expression. p5RHH-mediated Nox4 siRNA transfection significantly attenuated the expression of these pro-inflammatory genes in TGFβ1-treated mouse VSMCs, with the highest degree of inhibition in the expression of Il6. p5RHH peptide-mediated knockdown of TGFβ-activated kinase 1 (TAK1, also known as Map3k7), Jun, and Rela, but not Nfkb2, downregulated TGFβ1-induced Nox4 expression in VSMCs. Together, these data demonstrate that increased expression and activation of NOX4, which might result from increased TGFβ1 levels seen during aging, induces a proinflammatory phenotype in VSMCs, enhancing atherosclerosis.

Mulberrofuran G Protects Ischemic Injury-induced Cell Death via Inhibition of NOX4-mediated ROS Generation and ER Stress.

The aim of this study was to investigate the neuroprotective effect of mulberrofuran G (MG) in in vitro and in vivo models of cerebral ischemia. MG was isolated from the root bark of Morus bombycis. MG inhibited nicotinamide adenine dinucleotide phosphate oxidase (NOX) enzyme activity and oxygen-glucose deprivation/reoxygenation (OGD/R)-induced NOX4 protein expression in SH-SY5Y cells. MG inhibited the expression of activated caspase-3 and caspase-9 and cleaved poly adenine dinucleotide phosphate-ribose polymerase in OGD/R-induced SH-SY5Y cells. In addition, MG protected OGD/R-induced neuronal cell death and inhibited OGD/R-induced reactive oxygen species generation in SH-SY5Y cells. In in vivo model, MG-treated groups (0.2, 1, and 5 mg/kg) reduced the infarct volume in middle cerebral artery occlusion/reperfusion-induced ischemic rats. The MG-treated groups also reduced NOX4 protein expression in middle cerebral artery occlusion/reperfusion-induced ischemic rats. Furthermore, protein expression of 78-kDa glucose-regulated protein/binding immunoglobulin protein, phosphorylated IRE1α, X-box-binding protein 1, and cytosine enhancer binding protein homologous protein, mediators of endoplasmic reticulum stress, were inhibited in MG-treated groups. Taken together, MG showed protective effect in in vitro and in vivo models of cerebral ischemia through inhibition of NOX4-mediated reactive oxygen species generation and endoplasmic reticulum stress. This finding will give an insight that inhibition of NOX enzyme activity and NOX4 protein expression could be a new potential therapeutic strategy for cerebral ischemia. Copyright © 2016 John Wiley & Sons, Ltd.