The NADPH oxidase NOX4 promotes the directed migration of endothelial cells by stabilizing vascular endothelial growth factor receptor 2 protein.
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ABSTRACT:
SUBMITTER: Miyano K
PROVIDER: S-EPMC7450136 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The free radical theory of aging proposes that ROS (reactive oxygen species) are major driving forces of aging, and are also critically involved in cellular senescence. Besides the mitochondrial respiratory chain, alternative sources of ROS have been described that might contribute to cellular senescence. Noxs (NADPH oxidases) are well-known sources of superoxide, which contribute to the antimicrobial capabilities of macrophages, a process involving the prototypical member of the family referred to as Nox2. However, in recent years non-phagocytic homologues of Nox2 have been identified that are involved in processes other than the host defence. Superoxide anions produced by these enzymes are believed to play a major role in signalling by MAPKs (mitogen-activated protein kinases) and stress-activated kinases, but could also contribute to cellular senescence, which is known to involve oxygen radicals. In HUVECs (human umbilical vein endothelial cells), Nox4 is predominantly expressed, but its role in replicative senescence of HUVECs remains to be elucidated. Using shRNA (small-hairpin RNA)-mediated knockdown of Nox4, implicating lentiviral vectors, we addressed the question of whether lifelong depletion of Nox4 in HUVECs would influence the senescent phenotype. We found a significant extension of the replicative lifespan of HUVECs upon knockdown of Nox4. Surprisingly, mean telomere length was significantly reduced in Nox4-depleted cells. Nox4 depletion had no discernable influence on the activity of MAPKs and stress-activated kinases, but reduced the degree of oxidative DNA damage. These results suggest that Nox4 activity increases oxidative damage in HUVECs, leading to loss of replicative potential, which is at least partly independent of telomere attrition.
| S-EPMC2762686 | biostudies-literature
Project description:RNA-seq analysis was performed in samples from WT and NOX4 Knock-out mice after two-thirds Partial Hepatectomy (PH). Transcriptomic analysis through RNA-seq revealed significant changes mainly 24 hours after PH in NOX4-/- mice and support a relevant role for Myc in a node of regulation of proliferation-related genes.
2021-08-05 | GSE181476 | GEO
Project description:Objective: AIP1 expression is downregulated in human atherosclerotic plaques and global deletion of AIP1 in mice exacerbates atherosclerosis in ApoE-KO mouse models. However, the direct role of AIP1 in endothelium, vascular remodeling and associated vascular diseases has not been determined. Approach and Results: We used endothelial cell (EC)-specific AIP1-deficient (AIP1-ECKO) mice to define the role of AIP1 in vascular remodeling and intima-media thickening in a mouse carotid artery ligation model characterized by both neointimal hyperplasia and inward vessel remodeling. Compared to WT littermates, AIP1-ECKO mice had 2.2-fold larger intima area and 4.4-fold thicker intima as measured by intima/media ratio in arteries with more proliferating vascular smooth muscle cells (VSMCs) at week 2-4 post-injury. Increased reactive oxygen species (ROS) in endothelium at early time points induced inflammation and vessel dysfunction in AIP1-ECKO prior to VSMC accumulations. Moreover, knockdown of AIP1 in human EC enhanced ROS generation which was attenuated by co-silencing of NOX2. Mechanistically, AIP1 via its proline-rich region binds to the SH3 domain of cytosolic subunit p47phox to disrupt formation of an active NOX2 complex, attenuating ROS production. Conclusion: Our study supports that AIP1 regulates vascular remodeling with intima-media thickening by suppressing endothelial NOX2-dependent oxidative stress. Highlights: •In a carotid ligation model, endothelial cell (EC)-specific AIP1-deficient (AIP1-ECKO) mice had much larger media area, thicker vessel wall and augmented neointima formation.•Increased production of reactive oxygen species in vascular EC at early time points concomitant with vessel dysfunction in AIP1-ECKO.•AIP1 via its proline-rich region binds to the SH3 domain of cytosolic subunit p47phox to disrupt formation of an active NOX2 complex, attenuating ROS production.
| S-EPMC5921534 | biostudies-literature
Project description:BACKGROUND- Reactive oxygen species serve signaling functions in the vasculature, and hypoxia has been associated with increased reactive oxygen species production. NADPH oxidase 4 (Nox4) is a reactive oxygen species-producing enzyme that is highly expressed in the endothelium, yet its specific role is unknown. We sought to determine the role of Nox4 in the endothelial response to hypoxia.Hypoxia induced Nox4 expression both in vitro and in vivo and overexpression of Nox4 was sufficient to promote endothelial proliferation, migration, and tube formation. To determine the in vivo relevance of our observations, we generated transgenic mice with endothelial-specific Nox4 overexpression using the vascular endothelial cadherin promoter (VECad-Nox4 mice). In vivo, the VECad-Nox4 mice had accelerated recovery from hindlimb ischemia and enhanced aortic capillary sprouting. Because endothelial nitric oxide synthase (eNOS) is involved in endothelial angiogenic responses and eNOS is activated by reactive oxygen species, we probed the effect of Nox4 on eNOS. In cultured endothelial cells overexpressing Nox4, we observed a significant increase in eNOS protein expression and activity. To causally address the link between eNOS and Nox4, we crossed our transgenic Nox4 mice with eNOS(-/-) mice. Aortas from these mice did not demonstrate enhanced aortic sprouting, and VECad-Nox4 mice on the eNOS(-/-) background did not demonstrate enhanced recovery from hindlimb ischemia.Collectively, we demonstrate that augmented endothelial Nox4 expression promotes angiogenesis and recovery from hypoxia in an eNOS-dependent manner.
| S-EPMC3589548 | biostudies-literature
Project description:Aggregates of the microtubule-associated protein tau are a common marker of neurodegenerative diseases collectively termed as tauopathies, such as Alzheimer's disease (AD) and frontotemporal dementia. Therapeutic strategies based on tau have failed in late stage clinical trials, suggesting that tauopathy may be the consequence of upstream causal mechanisms. As increasing levels of reactive oxygen species (ROS) may trigger protein aggregation or modulate protein degradation and, we had previously shown that the ROS producing enzyme NADPH oxidase 4 (NOX4) is a major contributor to cellular autotoxicity, this study was designed to evaluate if NOX4 is implicated in tauopathy. Our results show that NOX4 is upregulated in patients with frontotemporal lobar degeneration and AD patients and, in a humanized mouse model of tauopathy induced by AVV-TauP301L brain delivery. Both, global knockout and neuronal knockdown of the Nox4 gene in mice, diminished the accumulation of pathological tau and positively modified established tauopathy by a mechanism that implicates modulation of the autophagy-lysosomal pathway (ALP) and, consequently, improving the macroautophagy flux. Moreover, neuronal-targeted NOX4 knockdown was sufficient to reduce neurotoxicity and prevent cognitive decline, even after induction of tauopathy, suggesting a direct and causal role for neuronal NOX4 in tauopathy. Thus, NOX4 is a previously unrecognized causative, mechanism-based target in tauopathies and blood-brain barrier permeable specific NOX4 inhibitors could have therapeutic potential even in established disease.
| S-EPMC8686076 | biostudies-literature
Project description:Liver is a unique organ in displaying a reparative and regenerative response after acute/chronic damage or partial hepatectomy, when all the cell types must proliferate to re-establish the liver mass. The NADPH oxidase NOX4 mediates Transforming Growth Factor-beta (TGF-?) actions, including apoptosis in hepatocytes and activation of stellate cells to myofibroblasts. Aim of this work was to analyze the impact of NOX4 in liver regeneration by using two mouse models where Nox4 was deleted: 1) general deletion of Nox4 (NOX4-/-) and 2) hepatocyte-specific deletion of Nox4 (NOX4hepKO). Liver regeneration was analyzed after 2/3 partial hepatectomy (PH). Results indicated an earlier recovery of the liver-to-body weight ratio in both NOX4-/- and NOX4hepKO mice and an increased survival, when compared to corresponding WT mice. The regenerative hepatocellular fat accumulation and the parenchyma organization recovered faster in NOX4 deleted livers. Hepatocyte proliferation, analyzed by Ki67 and phospho-Histone3 immunohistochemistry, was accelerated and increased in NOX4 deleted mice, coincident with an earlier and increased Myc expression. Primary hepatocytes isolated from NOX4 deleted mice showed higher proliferative capacity and increased expression of Myc and different cyclins in response to serum. Transcriptomic analysis through RNA-seq revealed significant changes after PH in NOX4-/- mice and support a relevant role for Myc in a node of regulation of proliferation-related genes. Interestingly, RNA-seq also revealed changes in the expression of genes related to activation of the TGF-? pathway. In fact, levels of active TGF-?1, phosphorylation of Smads and levels of its target p21 were lower at 24?h in NOX4 deleted mice. Nox4 did not appear to be essential for the termination of liver regeneration in vivo, neither for the in vitro hepatocyte response to TGF-?1 in terms of growth inhibition, which suggest its potential as therapeutic target to improve liver regeneration, without adverse effects.
| S-EPMC7823210 | biostudies-literature
Project description:Aging is characterized by increased aortic stiffness, an early, independent predictor and cause of cardiovascular disease. Oxidative stress from excess reactive oxygen species (ROS) production increases with age. Mitochondria and NADPH oxidases (NOXs) are two major sources of ROS in cardiovascular system. We showed previously that increased mitochondrial ROS levels over a lifetime induce aortic stiffening in a mouse oxidative stress model. Also, NADPH oxidase 4 (NOX4) expression and ROS levels increase with age in aortas, aortic vascular smooth muscle cells (VSMCs) and mitochondria, and are correlated with age-associated aortic stiffness in hypercholesterolemic mice. The present study investigated whether young mice (4 months-old) with increased mitochondrial NOX4 levels recapitulate vascular aging and age-associated aortic stiffness. We generated transgenic mice with low (Nox4TG605; 2.1-fold higher) and high (Nox4TG618; 4.9-fold higher) mitochondrial NOX4 expression. Young Nox4TG618 mice showed significant increase in aortic stiffness and decrease in phenylephrine-induced aortic contraction, but not Nox4TG605 mice. Increased mitochondrial oxidative stress increased intrinsic VSMC stiffness, induced aortic extracellular matrix remodeling and fibrosis, a leftward shift in stress-strain curves, decreased volume compliance and focal adhesion turnover in Nox4TG618 mice. Nox4TG618 VSMCs phenocopied other features of vascular aging such as increased DNA damage, increased premature and replicative senescence and apoptosis, increased proinflammatory protein expression and decreased respiration. Aortic stiffening in young Nox4TG618 mice was significantly blunted with mitochondrial-targeted catalase overexpression. This demonstration of the role of mitochondrial oxidative stress in aortic stiffness will galvanize search for new mitochondrial-targeted therapeutics for treatment of age-associated vascular dysfunction.
| S-EPMC6831838 | biostudies-literature
Project description:Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [NOX] enzymes serve several hemostatic and host defense functions in various lung diseases, but the role of NOX4 signaling in tuberculous pleurisy is not well understood. The role of NOX4 signaling in tuberculous pleural fibrosis was studied using invitro pleural mesothelial cell (PMC) experiments and a murine model of Mycobacterium bovis bacillus Calmette⁻Guérin (BCG) pleural infection. The production of NOX4 reactive oxygen species (NOX4⁻ROS) and the epithelial mesenchymal transition (EMT) in PMCs were both induced by heat-killed mycobacterium tuberculosis (HKMT). In cultured PMCs, HKMT-induced collagen-1 synthesis and EMT were blocked by pretreatment with small interfering RNA (siRNA) NOX4. Moreover, NOX4⁻ROS production and subsequent fibrosis were reduced by treatment with losartan and the toll-like receptor 4 (TLR4) inhibitor TAK-242. The HKMT-induced EMT and intracellular ROS production were mediated by NOX4 via the activation of extracellular signal-regulated kinase (ERK) signaling. Finally, in a BCG-induced pleurisy model, recruitment of inflammatory pleural cells, release of inflammatory cytokines, and thickened mesothelial fibrosis were attenuated by SiNOX4 compared to SiCon. Our study identified that HKMT-induced pleural fibrosis is mediated by NOX4⁻ERK⁻ROS via TLR4 and Angiotensin II receptor type1 (AT1R). There results suggest that NOX4 may be a novel therapeutic target for intervention in tuberculous pleural fibrosis.
| S-EPMC6351931 | biostudies-literature
Project description:NADPH oxidases (Nox enzymes) are critical mediators of both physiologic and pathophysiologic processes. Nox enzymes catalyze NADPH-dependent generation of reactive oxygen species (ROS), including superoxide and hydrogen peroxide. Until recently, Nox4 was proposed to be involved exclusively in normal physiologic functions. Compelling evidence, however, suggests that Nox4 plays a critical role in fibrosis, as well as a host of pathologies and diseases. These considerations led to a search for novel, small molecule inhibitors of this important enzyme. Ultimately, a series of novel tertiary sulfonylureas (23-25) was designed using pharmacophore modeling, synthesized, and evaluated for inhibition of Nox4-dependent signaling.
| S-EPMC5895456 | biostudies-literature
Project description:Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin(-/-)mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.
| S-EPMC4807287 | biostudies-literature