Deletion Of Nox4 Research Articles

Endothelium-specific deletion of Nox4 delays retinal vascular development and mitigates pathological angiogenesis.

NADPH oxidase 4 (Nox4) is a major isoform of NADPH oxidases playing an important role in many biological processes. Previously we have shown that Nox4 is highly expressed in retinal blood vessels and is upregulated in oxygen-induced retinopathy (OIR). However, the exact role of endothelial Nox4 in retinal angiogenesis remains elusive. Herein, using endothelial cell (EC)-specific Nox4 knockout (Nox4EC-KO) mice, we investigated the impact of endothelial Nox4 deletion on retinal vascular development and pathological angiogenesis during OIR. Our results show that deletion of Nox4 in ECs led to retarded retinal vasculature development with fewer, blunted-end tip cells and sparser, dysmorphic filopodia at vascular front, and reduced density of vascular network in superficial, deep, and intermediate layers in postnatal day 7 (P7), P12, and P17 retinas, respectively. In OIR, loss of endothelial Nox4 had no effect on hyperoxia-induced retinal vaso-obliteration at P9 but significantly reduced aberrant retinal neovascularization at P17 and decreased the deep layer capillary density at P25. Ex vivo study confirmed that lack of Nox4 in ECs impaired vascular sprouting. Mechanistically, loss of Nox4 significantly reduced expression of VEGF, p-VEGFR2, integrin αV, angiopoietin-2, and p-ERK1/2, attenuating EC migration and proliferation. Taken together, our results indicate that endothelial Nox4 is important for retinal vascular development and contributes to pathological angiogenesis, likely through regulation of VEGF/VEGFR2 and angiopoietin-2/integrin αV/ERK pathways. In addition, our study suggests that endothelial Nox4 appears to be essential for intraretinal revascularization after hypoxia. These findings call for caution on targeting endothelial Nox4 in ischemic/hypoxic retinal diseases.

NADPH oxidase 4 mediates the protective effects of physical activity against obesity-induced vascular dysfunction.

Physical activity is one of the most potent strategies to prevent endothelial dysfunction. Recent evidence suggests vaso-protective properties of hydrogen peroxide (H2O2) produced by main endothelial NADPH oxidase isoform 4 (Nox4) in the vasculature. Therefore, we hypothesized that Nox4 connects physical activity with vaso-protective effects. Analysis of the endothelial function using Mulvany Myograph showed endothelial dysfunction in wild-type (WT) as well as in C57BL/6J/ Nox4-/- (Nox4-/-) mice after 20 weeks on high-fat diet (HFD). Access to running wheels during the HFD prevented endothelial dysfunction in WT but not in Nox4-/- mice. Mechanistically, exercise led to an increased H2O2 release in the aorta of WT mice with increased phosphorylation of eNOS pathway member AKT serine/threonine kinase 1 (AKT1). Both H2O2 release and phosphorylation of AKT1 were diminished in aortas of Nox4-/- mice. Deletion of Nox4 also resulted in lower intracellular calcium release proven by reduced phenylephrine-mediated contraction, whilst potassium-induced contraction was not affected. H2O2 scavenger catalase reduced phenylephrine-induced contraction in WT mice. Supplementing H2O2 increased phenylephrine-induced contraction in Nox4-/- mice. Exercise-induced peroxisome proliferative-activated receptor gamma, coactivator 1 alpha (Ppargc1a), as key regulator of mitochondria biogenesis in WT but not Nox4-/- mice. Furthermore, exercise-induced citrate synthase activity and mitochondria mass were reduced in the absence of Nox4. Thus, Nox4-/- mice became less active and ran less compared with WT mice. Nox4 derived H2O2 plays a key role in exercise-induced adaptations of eNOS and Ppargc1a pathway and intracellular calcium release. Hence, loss of Nox4 diminished physical activity performance and vascular protective effects of exercise.

P6285Protective effects of exercise on vascular function are mediated by NADPH oxidase 4

Abstract Background/Introduction Physical activity is one of the most potent strategies to prevent endothelial dysfunction. Recent evidence indicates vaso-protective properties of H2O2 produced by main endothelial NADPH oxidase isoform 4 (Nox4) in the vasculature. Purpose Therefore, we hypothesized that Nox4 connects physical activity with vaso-protective effects. Methods and results Analysis of endothelial function by Mulvany myograph showed endothelial dysfunction in wild-type as well as in Nox4−/− mice after 20 weeks on high-fat diet. Access to voluntary running wheels during high-fat diet prevented endothelial dysfunction in wild-type but not in Nox4−/− mice. Mechanistically, exercise led to increased H2O2 release in the aorta of wild-type mice with increased phosphorylation of eNOS pathway member AKT serine/threonine kinase 1 (Akt1), subsequently. Both effects were diminished in aortas of Nox4−/− mice. Deletion of Nox4 also led to decreased capacity for intracellular calcium release and reduced phenylephrine-mediated contraction, whereas potassium-induced contraction was unaffected. H2O2 scavenger catalase reduced phenylephrine-contraction in wild-type mice. Supplementation of H2O2 increased phenylephrine-induced contraction in Nox4−/− mice. Exercise induced key regulator of mitochondria biogenesis peroxisome proliferative activated receptor gamma, coactivator 1 alpha (Ppargc1a) in wild-type but not Nox4−/− mice. Furthermore, exercise induced citrate synthase activity and reduced mitochondria mass in the absence of Nox4. Thus, Nox4−/− mice became less active and ran less compared with wild-type mice. Conclusions Nox4 derived H2O2 plays a key role in exercise-induced adaptations of eNOS and Ppargc1a pathway and intracellular calcium release. Hence, loss of Nox4 diminished physical activity performance and vascular protective effects of exercise. Acknowledgement/Funding This work was supported by a research grant of the German Cardiac Society (DGK) (to H.L.) and DFG (Grant MO 1695/4-1 to H.M.).

Hepatocyte-specific deletion of Nox4 induces whole-body insulin resistance

Hepatocyte-specific deletion of Nox4 induces whole-body insulin resistance

Abrogation of transforming growth factor-β-induced tissue fibrosis in mice with a global genetic deletion of Nox4

Excessive connective tissue deposition in skin and various internal organs is characteristic of systemic sclerosis (SSc). The profibrotic growth factor TGF-β plays a crucial role in SSc pathogenesis. The expression of NADPH oxidase 4 (NOX4), a critical mediator of oxidative stress, is potently stimulated by TGF-β. Here, we evaluated the effect of NOX4 on the development of TGF-β-induced tissue fibrosis. C57BL6/J control mice and Nox4 knockout mice were implanted subcutaneously with osmotic pumps containing either saline or 2.5 µg TGF-β1. After 28 days, skin and lung samples were isolated for histopathologic analysis, measurement of hydroxyproline content and gene expression analysis. Histopathology of skin and lungs from normal C57BL6/J mice treated with TGF-β1 showed profound dermal fibrosis and peribronchial and diffuse interstitial lung fibrosis. In contrast, TGF-β-treated Nox4 knockout mice showed normal skin and lung histology. Hydroxyproline levels in TGF-β-treated C57BL6/J mice skin and lungs demonstrated significant increases, however, hydroxyproline content of TGF-β-treated Nox4 knockout mice tissues was not changed. Expression of various profibrotic and fibrosis-associated genes was upregulated in skin and lungs of TGF-β1-treated C57BL6/J mice but was not significantly changed in TGF-β1-treated Nox4 knockout mice. The induction of skin and lung tissue fibrosis by TGF-β1 parenteral administration in mice was abrogated by the genetic deletion of Nox4 confirming that NOX4 is an essential mediator of the profibrotic effects of TGF-β. These results suggest Nox4 inhibition as a potential therapeutic target for SSc and other fibroproliferative disorders.

Nox4 Promotes Neural Stem/Precursor Cell Proliferation and Neurogenesis in the Hippocampus and Restores Memory Function Following Trimethyltin-Induced Injury

Reactive oxygen species (ROS) modulate the growth of neural stem/precursor cells (NS/PCs) and participate in hippocampus-associated learning and memory. However, the origin of these regulatory ROS in NS/PCs is not fully understood. In the present study, we found that Nox4, a ROS-producing NADPH oxidase family protein, is expressed in primary cultured NS/PCs and in those of the adult mouse brain. Nox inhibitors VAS 2870 and GKT137831 or Nox4 deletion attenuated bFGF-induced proliferation of cultured NS/PCs, while lentivirus-mediated Nox4 overexpression increased the production of H2O2, the phosphorylation of Akt, and the proliferation of cultured NS/PCs. Nox4 did not significantly affect the potential of cultured NS/PCs to differentiate into neurons or astrocytes. The histological and functional development of the hippocampus appeared normal in Nox4−/− mice. Although pathological and functional damages in the hippocampus induced by the neurotoxin trimethyltin were not significantly different between wild-type and Nox4−/− mice, the post-injury reactive proliferation of NS/PCs and neurogenesis in the subgranular zone (SGZ) of the dentate gyrus were significantly impaired in Nox4−/− animals. Restoration from the trimethyltin-induced impairment in recognition and spatial working memory was also significantly attenuated in Nox4−/− mice. Collectively, our findings suggest that Nox4 participates in NS/PC proliferation and neurogenesis in the hippocampus following injury, thereby helping to restore memory function.

NADPH oxidases in traumatic brain injury – Promising therapeutic targets?

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Despite intense investigation, no neuroprotective agents for TBI have yet translated to the clinic. Recent efforts have focused on identifying potential therapeutic targets that underlie the secondary TBI pathology that evolves minutes to years following the initial injury. Oxidative stress is a key player in this complex cascade of secondary injury mechanisms and prominently contributes to neurodegeneration and neuroinflammation. NADPH oxidase (NOX) is a family of enzymes whose unique function is to produce reactive oxygen species (ROS). Human post-mortem and animal studies have identified elevated NOX2 and NOX4 levels in the injured brain, suggesting that these two NOXs are involved in the pathogenesis of TBI. In support of this, NOX2 and NOX4 deletion studies have collectively revealed that targeting NOX enzymes can reduce oxidative stress, attenuate neuroinflammation, promote neuronal survival, and improve functional outcomes following TBI. In addition, NOX inhibitor studies have confirmed these findings and demonstrated an extended critical window of efficacious TBI treatment. Finally, the translational potential, caveats, and future directions of the field are highlighted and discussed throughout the review.

Nox4 genetic inhibition in experimental hypertension and metabolic syndrome

Metabolic syndrome is a combination of symptoms including obesity, dyslipidaemia, glucose intolerance and hypertension. Oxidative stress appears to be a pathophysiological factor that links these signs and encourages progression towards heart failure and diabetes. Nox4 is a hydrogen peroxide nicotinamide adenine dinucleotide phosphate(NADPH) oxidase isoform -found in various cardiovascular cells and tissues, but also in tissues such as the liver- which is involved in glucose and lipid homeostasis. To test whether inhibition of the Nox4 enzyme could improve blood pressure and metabolic parameters in mice receiving either angiotensinII or a high-fat diet. Systolic and diastolic arterial pressures, pulse rate and heart rate were obtained in 24male mice (12wild-type [WT] and 12Nox4-/-) before and during 14days of angiotensinII infusion. After angiotensinII infusion, cardiac histological remodeling was assessed. Weight and biochemical parameters were measured in 18male and 18female mice (nine WT and nine Nox4-/- per gender) after 10weeks on a standard chow diet, then 15weeks on a high-fat diet. Glucose tolerance and insulin sensitivity were tested at age 25weeks. Knock-out animals did not demonstrate a baseline blood pressure phenotype, but blocking Nox4 protected against angiotensinII-mediated arterial and pulse pressure increases. No protection against angiotensinII-induced cardiac fibrosis was observed. From a metabolic point of view, Nox4 inhibition reduced plasma triglycerides in male and female mice under a chow diet. However, Nox4 deletion did not affect the metabolic profile under a high-fat diet in males or females, but increased glucose intolerance in females. Our data identify Nox4 as a key source of radical oxygen species involved in hypertension and some metabolic problems.

P 049 - Hydrogen peroxide formation by Nox4 limits malignant transformation

Through the constitutive production of H2O2 the NAPDH Oxidase Nox4 promotes differentiation of cells. Previous observations suggested that the absence of Nox4 promotes inflammation and de-differentiation. Chronic inflammation and lack of differentiation are pre-carcinogenic states. We therefore hypothesized that lack of Nox4 promotes tumor development. Tumor formation was studied in two murine tumor models. In both models tumor burden was massively enhanced in Nox4-deficient mice. Genetic deletion of Nox4 resulted in increased DNA damage. In contrast, the DNA damage marker γH2AX was significantly reduced in Nox4-/- mice. As underlying mechanism we identified that Nox4 oxidizes Akt, which subsequently retains the phosphatase PP2A in the cytosol. In the absence of Nox4 PP2A accumulates in the nucleus. As a consequence, H2AX is dephosphorylated before DNA repair can take place resulting in genomic instability. In line with this, phosphorylated Akt accumulates in the cytosol promoting proliferation and survival of tumor cells as observed. Overexpression of all three Akt isoforms could rescue the described phenotype whereas overexpression of Akt mutants lacking the redox-sensitive cysteines, had no effect on PP2A translocation, percentage of DNA damage and γH2AX phosphorylation. Taken together these results suggest a protective role of endogenous Nox4 for malignant transformation.

277 - Hydrogen Peroxide Formation by Nox4 Limits Malignant Transformation

Through the constitutive production of H2O2 the NAPDH Oxidase Nox4 promotes differentiation of cells and contributes to cellular quiescence. Our previous observations in mesenchymal tissue suggested that the absence of Nox4 promotes inflammation and de-differentiation. Chronic inflammation and lack of differentiation are pre-carcinogenic states. We therefore hypothesized that lack of Nox4 promotes tumor development. The process of inflammation-dependent tumor formation was studied in two carcinogen-induced murine tumor models, leading to the formation of fibro sarcomas and colon carcinomas. In both models tumor burden was massively enhanced in Nox4-deficient mice. Genetic deletion of Nox4 resulted in increased DNA damage as detected by Nicoletti-staining and Comet assay. In contrast, the DNA damage response marker γH2AX was significantly reduced in Nox4-/- mice. As underlying mechanism we identified that Nox4 oxidizes Akt, which subsequently retains the phosphatase PP2A in the cytosol. In the absence of Nox4 PP2A accumulates in the nucleus. As a consequence, H2AX is dephosphorylated before DNA repair can take place resulting in genomic instability. In line with this, phosphorylated Akt accumulates in the cytosol promoting proliferation and survival of tumor cells as observed. Overexpression of all three Akt isoforms could rescue the described phenotype whereas overexpression of Akt mutants lacking the redox-sensitive cysteines, had no effect on PP2A translocation, percentage of DNA damage and γH2AX phosphorylation. Taken together these results suggest a protective role of endogenous Nox4 for malignant transformation. By the oxidation of Akt the enzyme prevents nuclear translocation of PP2A and thereby enables adequate DNA damage repair.

Abstract P225: Nox1 or Nox4 Deletion Prevents Type-1 Diabetes-induced Endothelial Dysfunction

Objective: The prognosis of type-1 diabetes is in part related to the increased risk of vascular complications such as atherosclerosis. Overproduction of reactive oxygen species by NADPH oxidase (NOX) is believed to play an important role in diabetes-related vascular injury. NOX1 may play a role in the macrovascular disease, whereas NOX4 may have protective actions. Nevertheless, their role in diabetic vascular injury is less well understood. We hypothesized that deletion of Nox1 would prevent diabetes-induced endothelial dysfunction and vascular remodeling of small arteries whereas Nox4 would exaggerate vascular injury in atherosclerosis-prone apolipoprotein knockout ( Apoe -/- ) mice. Methods: Diabetes was induced by streptozotocin IP injections (STZ, 55 mg/kg/day) for 5 days in 6-week-old male Apoe -/- mice, Apoe -/- mice deficient in Nox1 ( Apoe -/- / Nox1 y/- ) and Nox4 ( Apoe -/- / Nox4 -/- ). Mice were studied 14 weeks later. Endothelial function and vascular remodeling were assessed in mesenteric arteries (MA) using pressurized myography. Results: Apoe -/- mice presented a maximal endothelium-dependent vasodilatory response (E max ) to acetylcholine of 48±8%, which was further decreased by diabetes to 20±6%. In contrast, endothelium-dependent relaxations to acetylcholine were 1.5-fold higher in diabetic Apoe -/- / Nox1 y/- and Apoe -/- / Nox4 -/- mice compared to non-diabetic Apoe -/- mice (E max : 72±9 and 70±7 vs 48±8%). Diabetes decreased MA stiffness in Apoe -/- mice, as indicated by a rightward displacement of the stress-strain curves (strain at 140 mm Hg: 1.02±0.04 vs 0.75±0.04), which was blunted by Nox1 or Nox4 knockout (strain at 140 mm Hg: 0.77±0.02 and 0.81±0.02). MA media/lumen was unaltered by diabetes. Knockout of Nox4 but not Nox1 increased MA media/lumen 1.4-fold in diabetic Apoe -/- mice (4.1±0.4 and 3.5±0.2 vs 2.9±0.2%). Conclusions: These results suggest that NOX1 and NOX4 play a pathophysiological role in diabetes-induced endothelial dysfunction and contribute to potentially maladaptive changes in vascular stiffness. NOX4 seems to have dual actions on the vasculature, as it is also protective against vascular remodeling of small arteries in type 1 diabetes.

Tyrosine kinase FYN negatively regulates NOX4 in cardiac remodeling.

NADPH oxidases (Noxes) produce ROS that regulate cell growth and death. NOX4 expression in cardiomyocytes (CMs) plays an important role in cardiac remodeling and injury, but the posttranslational mechanisms that modulate this enzyme are poorly understood. Here, we determined that FYN, a Src family tyrosine kinase, interacts with the C-terminal domain of NOX4. FYN and NOX4 colocalized in perinuclear mitochondria, ER, and nuclear fractions in CMs, and FYN expression negatively regulated NOX4-induced O2- production and apoptosis in CMs. Mechanistically, we found that direct phosphorylation of tyrosine 566 on NOX4 was critical for this FYN-mediated negative regulation. Transverse aortic constriction activated FYN in the left ventricle (LV), and FYN-deficient mice displayed exacerbated cardiac hypertrophy and dysfunction and increased ROS production and apoptosis. Deletion of Nox4 rescued the exaggerated LV remodeling in FYN-deficient mice. Furthermore, FYN expression was markedly decreased in failing human hearts, corroborating its role as a regulator of cardiac cell death and ROS production. In conclusion, FYN is activated by oxidative stress and serves as a negative feedback regulator of NOX4 in CMs during cardiac remodeling.

NOX4-derived reactive oxygen species limit fibrosis and inhibit proliferation of vascular smooth muscle cells in diabetic atherosclerosis

Smooth muscle cell (SMC) proliferation and fibrosis contribute to the development of advanced atherosclerotic lesions. Oxidative stress caused by increased production or unphysiological location of reactive oxygen species (ROS) is a known major pathomechanism. However, in atherosclerosis, in particular under hyperglycaemic/diabetic conditions, the hydrogen peroxide-producing NADPH oxidase type 4 (NOX4) is protective. Here we aim to elucidate the mechanisms underlying this paradoxical atheroprotection of vascular smooth muscle NOX4 under conditions of normo- and hyperglycaemia both in vivo and ex vivo. Following 20-weeks of streptozotocin-induced diabetes, Apoe−/− mice showed a reduction in SM-alpha-actin and calponin gene expression with concomitant increases in platelet-derived growth factor (PDGF), osteopontin (OPN) and the extracellular matrix (ECM) protein fibronectin when compared to non-diabetic controls. Genetic deletion of Nox4 (Nox4−/−Apoe−/−) exacerbated diabetes-induced expression of PDGF, OPN, collagen I, and proliferation marker Ki67. Aortic SMCs isolated from NOX4-deficient mice exhibited a dedifferentiated phenotype including loss of contractile gene expression, increased proliferation and ECM production as well as elevated levels of NOX1-associated ROS. Mechanistic studies revealed that elevated PDGF signalling in NOX4-deficient SMCs mediated the loss of calponin and increase in fibronectin, while the upregulation of NOX1 was associated with the increased expression of OPN and markers of proliferation. These findings demonstrate that NOX4 actively regulates SMC pathophysiological responses in diabetic Apoe−/− mice and in primary mouse SMCs through the activities of PDGF and NOX1.

NADPH oxidase 4 deficiency leads to impaired wound repair and reduced dityrosine-crosslinking, but does not affect myofibroblast formation

NADPH oxidases (NOX) mediate redox signaling by generating superoxide and/or hydrogen peroxide, which are involved in biosynthetic pathways, e.g. thyroid hormone generation, dityrosine crosslinking, as well as bacterial killing. Data investigating the role of NOX enzymes in cutaneous wound repair is limited and specifically their function in skin myofibroblast expression is unknown. The isoform NOX4 was recently shown to be a pre-requisite for the differentiation of cardiac and pulmonary myofibroblasts.In this study we investigate the role of NOX4 in wound repair using a wound model in NOX4 knockout mice (n=16) and wildtype mice (n=16). Wounds were photographed daily until complete wound closure. Mice were sacrificed at day 3, 7, 14; wound tissue was harvested.NOX4-deficient mice healed significantly slower (22 days, SD=1.9) than wild-type mice (17 days, SD=1.4, p<0.005). However, there was no difference in myofibroblast expression. Strong dityrosine formation was observed, but was significantly weaker in NOX4-/- mice (p<0.05). NOX2, HIF1α and CD31 expression was significantly weaker in NOX4-/- mice (p<0.05).In this study we show for the first time that NOX4 plays a role in cutaneous wound repair. Our data suggests that NOX4 mediates HIF1α expression and neoangiogenesis during wound repair. NOX4 deletion led to a decreased expression of NOX2, implying a role of NOX4 in phagocytic cell recruitment. NOX4 was required for effective wound contraction but not myofibroblast expression. We suggest that myofibroblast contraction in NOX4-deficient mice is less effective in contracting the wound because of insufficient dityrosine-crosslinking of the ECM, providing the first indication for a physiological function of dityrosine crosslinking in higher animals.

Role of Nox4 and p67phox in ROS production in response to increased tubular flow in the mTAL of Dahl salt‐sensitive (SS) rats

Reactive Oxygen Species (ROS) in the renal outer medulla play an important role in the development of salt‐sensitive hypertension. NADPH oxidases (Nox) are the major source of ROS production in the kidney. We have examined the contribution of Nox4 and p67phox, related to the most abundant isoforms of NADPH‐oxidase Nox4 and Nox2 in the kidney, to the ROS production in response to sodium delivery and tubular flow in medullary thick ascending limb (mTAL) isolated from Dahl salt‐sensitive (SS) rats. We have shown in our previous studies that SSNox4−/− and SSp67phox−/− mutant rats exhibited a significant reduction of salt‐induced hypertension compared to SS rats after 21 day of 4.0% NaCl and a large reduction of albuminuria, tubular casts, and glomerular injury. In this study we have shown that increase in mTAL luminal flow stimulates cellular hydrogen peroxide (H2O2) and superoxide (O2·−), as well as mitochondrial H2O2 production in SS rats indicated by changes in fluorescence of sensitive dyes for detection of ROS (PF6‐AM, DHE, Mito‐PY1). In the present study we have determined that deletion of Nox4 (SSNox4−/−) resulted in significant reduction of intracellular H2O2 compared to SS in response to increased luminal flow in mTAL isolated from rats fed a 4% NaCl diet for 3 days. Deletion of the p67phox subunit in SS rat (SSp67phox−/−) also decreased H2O2 production compared to SS but to a lesser extent than that observed in mTAL of Nox4 rats (SSNox4−/−). There were no differences between cellular O2·− production in mTAL of SSNox4−/− rats compared to SS. However there was a significant decrease in cellular O2·− production in mTAL of mutant SSp67phox−/− rats. We conclude from this study that Nox4 is a major player in the production of cellular H2O2 in response to increased mTAL tubular flow of the SS rat whereas the p67phox subunit of Nox2 is involved in cellular O2·− production. Neither Nox4 nor the p67phox subunit of Nox2 seems to contribute to the mitochondrial H2O2 production in response to increased mTAL luminal flow.Support or Funding InformationHL 122662HL 082798

NADPH oxidase 4 attenuates cerebral artery changes during the progression of Marfan syndrome.

Marfan syndrome (MFS) is a connective tissue disorder that is often associated with the fibrillin-1 (Fbn1) gene mutation and characterized by cardiovascular alterations, predominantly ascending aortic aneurysms. Although neurovascular complications are uncommon in MFS, the improvement in Marfan patients' life expectancy is revealing other secondary alterations, potentially including neurovascular disorders. However, little is known about small-vessel pathophysiology in MFS. MFS is associated with hyperactivated transforming growth factor (TGF)-β signaling, which among numerous other downstream effectors, induces the NADPH oxidase 4 (Nox4) isoform of NADPH oxidase, a strong enzymatic source of H2O2 We hypothesized that MFS induces middle cerebral artery (MCA) alterations and that Nox4 contributes to them. MCA properties from 3-, 6-, or 9-mo-old Marfan (Fbn1(C1039G/+)) mice were compared with those from age/sex-matched wild-type littermates. At 6 mo, Marfan compared with wild-type mice developed higher MCA wall/lumen (wild-type: 0.081 ± 0.004; Marfan: 0.093 ± 0.002; 60 mmHg; P < 0.05), coupled with increased reactive oxygen species production, TGF-β, and Nox4 expression. However, wall stiffness and myogenic autoregulation did not change. To investigate the influence of Nox4 on cerebrovascular properties, we generated Marfan mice with Nox4 deficiency (Nox4(-/-)). Strikingly, Nox4 deletion in Marfan mice aggravated MCA wall thickening (cross-sectional area; Marfan: 6,660 ± 363 μm(2); Marfan Nox4(-/-): 8,795 ± 824 μm(2); 60 mmHg; P < 0.05), accompanied by decreased TGF-β expression and increased collagen deposition and Nox1 expression. These findings provide the first evidence that Nox4 mitigates cerebral artery structural changes in a murine model of MFS.

Reactive Oxygen Species Can Provide Atheroprotection via NOX4-Dependent Inhibition of Inflammation and Vascular Remodeling.

Oxidative stress is considered a hallmark of atherosclerosis. In particular, the superoxide-generating type 1 NADPH oxidase (NOX1) has been shown to be induced and play a pivotal role in early phases of mouse models of atherosclerosis and in the context of diabetes mellitus. Here, we investigated the role of the most abundant type 4 isoform (NOX4) in human and mouse advanced atherosclerosis. Plaques of patients with cardiovascular events or established diabetes mellitus showed a surprising reduction in expression of the most abundant but hydrogen peroxide (H2O2)-generating type 4 isoform (Nox4), whereas Nox1 mRNA was elevated, when compared with respective controls. As these data suggested that NOX4-derived reactive oxygen species may convey a surprisingly protective effect during plaque progression, we examined a mouse model of accelerated and advanced diabetic atherosclerosis, the streptozotocin-treated ApoE(-/-) mouse, with (NOX4(-/-)) and without genetic deletion of Nox4. Similar to the human data, advanced versus early plaques of wild-type mice showed reduced Nox4 mRNA expression. Consistent with a rather protective role of NOX4-derived reactive oxygen species, NOX4(-/-) mice showed increased atherosclerosis when compared with wild-type mice. Deleting NOX4 was associated with reduced H2O2 forming activity and attenuation of the proinflammatory markers, monocyte chemotratic protein-1, interleukin-1β, and tumor necrosis factor-α, as well as vascular macrophage accumulation. Furthermore, there was a greater accumulation of fibrillar collagen fibres within the vascular wall and plaque in diabetic Nox4(-/-)ApoE(-/-) mice, indicative of plaque remodeling. These data could be replicated in human aortic endothelial cells in vitro, where Nox4 overexpression increased H2O2 and reduced the expression of pro-oxidants and profibrotic markers. Interestingly, Nox4 levels inversely correlated with Nox2 gene and protein levels. Although NOX2 is not constitutively active unlike NOX4 and forms rather superoxide, this opens up the possibility that at least some effects of NOX4 deletion are mediated by NOX2 activation. Thus, the appearance of reactive oxygen species in atherosclerosis is apparently not always a nondesirable oxidative stress, but can also have protective effects. Both in humans and in mouse, the H2O2-forming NOX4, unlike the superoxide-forming NOX1, can act as a negative modulator of inflammation and remodeling and convey atheroprotection. These results have implications on how to judge reactive oxygen species formation in cardiovascular disease and need to be considered in the development of NOX inhibitory drugs.

Evidence of the Importance of Nox4 in Production of Hypertension in Dahl Salt-Sensitive Rats.

This study reports the consequences of knocking out NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4 (Nox4) on the development of hypertension and kidney injury in the Dahl salt-sensitive (SS) rat. Zinc finger nuclease injection of single-cell SS embryos was used to create an 8 base-pair frame-shift deletion of Nox4, resulting in a loss of the ≈68 kDa band in Western blot analysis of renal cortical tissue of the knock out of Nox4 in the SS rat (SS(Nox4-/-)) rats. SS(Nox4-/-) rats exhibited a significant reduction of salt-induced hypertension compared with SS rats after 21 days of 4.0% NaCl diet (134±5 versus 151±3 mm Hg in SS) and a significant reduction of albuminuria, tubular casts, and glomerular injury. Optical fluorescence 3-dimensional cryoimaging revealed significantly higher redox ratios (NADH/FAD [reduced nicotinamide adenine dinucleotide/flavin adenine dinucleotide]) in the kidneys of SS(Nox4-/-) rats even when fed the 0.4% NaCl diet, indicating greater levels of mitochondrial electron transport chain metabolic activity and reduced oxidative stress compared with SS rats. Before the development of hypertension, RNA expression levels of Nox subunits Nox2, p67(phox), and p22(phox) were found to be significantly lower (P<0.05) in SS(Nox4-/-) compared with SS rats in the renal cortex. Thus, the mutation of Nox4 seems to modify transcription of several genes in ways that contribute to the protective effects observed in the SS(Nox4-/-) rats. We conclude that the reduced renal injury and attenuated blood pressure response to high salt in the SS(Nox4-/-) rat could be the result of multiple pathways, including gene transcription, mitochondrial energetics, oxidative stress, and protein matrix production impacted by the knock out of Nox4.

NADPH oxidase 4 protects against development of endothelial dysfunction and atherosclerosis in LDL receptor deficient mice.

Endothelial dysfunction is an early step in the development of atherosclerosis. Increased formation of superoxide anions by NADPH oxidase Nox1, 2, and 5 reduces nitric oxide availability and can promote endothelial dysfunction. In contrast, recent evidence supports a vasoprotective role of H2O2 produced by main endothelial isoform Nox4. Therefore, we analysed the impact of genetic deletion of Nox4 on endothelial dysfunction and atherosclerosis in the low-density lipoprotein receptor (Ldlr) knockout model. Ex vivo analysis of endothelial function by Mulvany myograph showed impaired endothelial function in thoracic aorta of Nox4(-/-)/Ldlr(-/-) mice. Further progression of endothelial dysfunction due to high-fat diet increased atherosclerotic plaque burden and galectin-3 staining in Nox4(-/-)/Ldlr(-/-) mice compared with Ldlr(-/-) mice. Under physiological conditions, loss of Nox4 does not influence aortic vascular function. In this setting, loss of Nox4-derived H2O2 production could be partially compensated for by nNOS upregulation. Using an innovative optical coherence tomography approach, we were able to analyse endothelial function by flow-mediated vasodilation in the murine saphenous artery in vivo. This new approach revealed an altered flow-mediated dilation in Nox4(-/-) mice, indicating a role for Nox4 under physiological conditions in peripheral arteries in vivo. Nox4 plays an important role in maintaining endothelial function under physiological and pathological conditions. Loss of Nox4-derived H2O2 could be partially compensated for by nNOS upregulation, but severe endothelial dysfunction is not reversible. This leads to increased atherosclerosis under atherosclerotic prone conditions.

Abstract 15112: NADPH Oxidase 4 Protects Against Development of Endothelial Dysfunction and Atherosclerosis in LDL Receptor Deficient Mice

Introduction: Endothelial dysfunction is an early step in the development of atherosclerosis. Increased formation of superoxide anions by NADPH oxidase Nox1, 2 and 5 reduce NO availability and can promote endothelial dysfunction. In contrast, recent evidence supports a vasoprotective role of H2O2 produced by main endothelial isoform Nox4. Therefore, we assessed the hypothesis genetic deletion of Nox4 influences endothelial dysfunction and atherosclerosis in the low-density lipoprotein receptor (Ldlr) knockout model. Methods and Results: Ex vivo analysis of endothelial function by Mulvany myograph showed impaired endothelial function in thoracic aorta of Nox4-/-/Ldlr-/- mice. Further progression of endothelial dysfunction by high-fat diet increased atherosclerotic plaque burden and galectin-3 staining in Nox4-/-/Ldlr-/- mice compared to Ldlr-/- mice. In addition, we obtained evidence that under physiological conditions the loss of Nox4-derived H2O2 production could partially be compensated by nNOS upregulation. Using a self-developed optical coherence tomography device we were able to analyse endothelial function by flow-mediated vasodilation in the murine saphenous artery in vivo. This new approach observed an altered flow-mediated dilation in Nox4-/- mice, indicating a role for Nox4 under physiological conditions in vivo likewise. Conclusion: Nox4 plays an important role under physiological and pathological conditions to maintain endothelial function. Loss of Nox4-derived H2O2 could partially be compensated by nNOS upregulation, but severe endothelial dysfunction is not reversible. This leads to increased atherosclerosis under atherosclerotic prone conditions.