NOX2 Complex Research Articles

Increased Susceptibility of S1PR3-Deficient Mice to Escherichia coli Sepsis Is Associated with an Impaired Bactericidal Activity in Macrophage

Abstract Background Sepsis, a leading cause of death in critically ill patients worldwide, is characterized with imbalanced proinflammatory responses and compromised bacterial clearance in host immunity. Monocytes/macrophages play a crucial role in elimination of bacteria through innate immunity receptors. Methods We explored the effect of Sphingosine-1-phosphate receptor 3 (S1PR3) gene deficiency on mortality, organ injury, and bacterial clearance in cecal ligation and puncture (CLP) or Escherichia coli (E. coli)-induced sepsis. Signaling molecules responsible for S1PR3-mediated bactericidal function were dissected in peritoneal macrophages. Furthermore, S1PR3 specific agonist (GPS-725.017) was evaluated for treatment of sepsis. Results After sepsis onset, S1pr3−/− mice showed increased mortality and organ injury, which attributed to markedly decreased E. coli killing. Mechanically, S1PR3 regulated activity of the NADPH oxidase NOX2 complex and phagolysosomal maturation through recruiting the intracellular class III phosphatidylinositol kinase Vps34 to the phagosome. S1PR3 specific agonist (GPS-725.017) enhanced NOX2 activity in E. coli-containing phagosomes and bactericidal function in wild-type macrophages. Additionally, the survival of mice treated with GPS-725.017 was improved when compared with those treated with vehicle. Conclusion S1PR3 is an important receptor to connect bacterial phagosome to ubiquitous cellular machinery responsible for control of bacterial killing in macrophage. Interventions targeting S1PR3 signaling may serve as promising therapeutic approaches for sepsis.

Nox Complex signal and MAPK cascade pathway are cross-linked and essential for pathogenicity and conidiation of mycoparasite Coniothyrium minitans.

The NADPH oxidase complex of a sclerotial mycoparasite Coniothyrium minitans, an important biocontrol agent against crop diseases caused by Sclerotinia sclerotiorum, was identified and its functions involved in conidiation and mycoparasitism were studied. Gene knock-out and complementary experiments indicated that CmNox1, but not CmNox2, is necessary for conidiation and parasitism, and its expression could be significantly induced by its host fungus. CmNox1 is regulated by CmRac1-CmNoxR and interacts with CmSlt2, a homolog of Saccharomyces cerevisiae Slt2 encoding cell wall integrity-related MAP kinase. In ΔCmNox1, CmSlt2-GFP fusion protein lost the ability to localize to the cell nucleus accurately. The defect of conidiation in ΔCmRac1 could be partially restored by over-expressing CmSlt2, indicating that CmSlt2 was a downstream regulatory factor of CmNox1 and was involved in conidiation and parasitism. The expressions of mycoparasitism-related genes CmPks1, Cmg1 and CH1 were suppressed in the knock-out mutants of the genes in CmNox1-CmSlt2 signal pathway when cultivated either on PDA. Therefore, our study infers that CmRac1-CmNoxR regulates CmNox1-CmSlt2 pathway in regulating conidiation and pathogenicity of C. minitans.

Aminoguanidine treatment increased NOX2 response in diabetic rats: Improved phagocytosis and killing of Candida albicans by neutrophils

In this study, we show that aminoguanidine (AMG), an inhibitor of protein glycation, increases the NOX2 (phagocyte NADPH oxidase) response and microbicidal activity by neutrophils, regardless of diabetic status. The non-enzymatic glycation of proteins, yielding irreversible advanced glycation end products (AGEs), is involved in the development of diabetes complications, including alterations of signaling pathways and the generation of reactive oxygen species by phagocytes. The phagocytes produce ROS (reactive oxygen species) through activation of the NOX2 complex, which generates superoxide. The purpose of this study was to evaluate the effect of hyperglycemia and the glycation of proteins on the NOX2 activity of neutrophils and its implications for cellular physiology, with a focus on the microbicidal activity of these cells. We treated diabetic rats with AMG and evaluated neutrophil ROS generation and Candida albicans killing ability. We observed a large increase in the microbicidal activity of peritoneal neutrophils from AMG-treated rats. The increase was independent of diabetic status and myeloperoxidase activity. Collectively, our results suggest that AMG has an immunomodulator role that triggers an increase in the microbicidal response of neutrophils mainly related to reactive oxygen species production by NOX2.

Carbon Nanotube Degradation in Macrophages: Live Nanoscale Monitoring and Understanding of Biological Pathway.

Despite numerous applications, the cellular-clearance mechanism of multiwalled carbon nanotubes (MWCNTs) has not been clearly established yet. Previous in vitro studies showed the ability of oxidative enzymes to induce nanotube degradation. Interestingly, these enzymes have the common capacity to produce reactive oxygen species (ROS). Here, we combined material and life science approaches for revealing an intracellular way taken by macrophages to degrade carbon nanotubes. We report the in situ monitoring of ROS-mediated MWCNT degradation by liquid-cell transmission electron microscopy. Two degradation mechanisms induced by hydroxyl radicals were extracted from these unseen dynamic nanoscale investigations: a non-site-specific thinning process of the walls and a site-specific transversal drilling process on pre-existing defects of nanotubes. Remarkably, similar ROS-induced structural injuries were observed on MWCNTs after aging into macrophages from 1 to 7 days. Beside unraveling oxidative transformations of MWCNT structure, we elucidated an important, albeit not exclusive, biological pathway for MWCNT degradation in macrophages, involving NOX2 complex activation, superoxide production, and hydroxyl radical attack, which highlights the critical role of oxidative stress in cellular processing of MWCNTs.

NAD(P)H oxidase subunit p47phox is elevated, and p47phox knockout prevents diaphragm contractile dysfunction in heart failure.

Patients with chronic heart failure (CHF) have dyspnea and exercise intolerance, which are caused in part by diaphragm abnormalities. Oxidants impair diaphragm contractile function, and CHF increases diaphragm oxidants. However, the specific source of oxidants and its relevance to diaphragm abnormalities in CHF is unclear. The p47(phox)-dependent Nox2 isoform of NAD(P)H oxidase is a putative source of diaphragm oxidants. Thus, we conducted our study with the goal of determining the effects of CHF on the diaphragm levels of Nox2 complex subunits and test the hypothesis that p47(phox) knockout prevents diaphragm contractile dysfunction elicited by CHF. CHF caused a two- to sixfold increase (P < 0.05) in diaphragm mRNA and protein levels of several Nox2 subunits, with p47(phox) being upregulated and hyperphosphorylated. CHF increased diaphragm extracellular oxidant emission in wild-type but not p47(phox) knockout mice. Diaphragm isometric force, shortening velocity, and peak power were decreased by 20-50% in CHF wild-type mice (P < 0.05), whereas p47(phox) knockout mice were protected from impairments in diaphragm contractile function elicited by CHF. Our experiments show that p47(phox) is upregulated and involved in the increased oxidants and contractile dysfunction in CHF diaphragm. These findings suggest that a p47(phox)-dependent NAD(P)H oxidase mediates the increase in diaphragm oxidants and contractile dysfunction in CHF.

Functional genomics identifies negative regulatory nodes controlling phagocyte oxidative burst.

The phagocyte oxidative burst, mediated by Nox2 NADPH oxidase-derived reactive oxygen species, confers host defense against a broad spectrum of bacterial and fungal pathogens. Loss-of-function mutations that impair function of the Nox2 complex result in a life-threatening immunodeficiency, and genetic variants of Nox2 subunits have been implicated in pathogenesis of inflammatory bowel disease (IBD). Thus, alterations in the oxidative burst can profoundly impact host defense, yet little is known about regulatory mechanisms that fine-tune this response. Here we report the discovery of regulatory nodes controlling oxidative burst by functional screening of genes within loci linked to human inflammatory disease. Implementing a multi-omics approach, we define transcriptional, metabolic and ubiquitin-cycling nodes controlled by Rbpj, Pfkl and Rnf145, respectively. Furthermore, we implicate Rnf145 in proteostasis of the Nox2 complex by endoplasmic reticulum-associated degradation. Consequently, ablation of Rnf145 in murine macrophages enhances bacterial clearance, and rescues the oxidative burst defects associated with Ncf4 haploinsufficiency.

Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins.

LC3-associated phagocytosis (LAP) is a process wherein elements of autophagy conjugate LC3 to phagosomal membranes. We characterize the molecular requirements for LAP, and identify Rubicon as being required for LAP but not autophagy. Rubicon is recruited to LAPosomes and is required for the activity of a Class III PI(3)K complex containing UVRAG but lacking ATG14 and Ambra1. This allows for the sustained localization of PtdIns(3)P, which is critical for recruitment of downstream autophagic proteins and stabilization of the NOX2 complex to produce reactive oxygen species. Both PtdIns(3)P and reactive oxygen species are required for conjugation of LC3 to LAPosomes and subsequent association with LAMP1(+) lysosomes. LAP is induced by engulfment of Aspergillus fumigatus, a fungal pathogen that commonly afflicts immunocompromised hosts, and is required for its optimal clearance in vivo. Therefore, we have identified molecules that distinguish LAP from canonical autophagy, thereby elucidating the importance of LAP in response to A. fumigatus infection.

Intrinsic resistance triggered under acid loading within normal esophageal epithelial cells: NHE1- and ROS-mediated survival.

The transition to a pathological phenotype such as Barrett's esophagus occurs via induction of resistance upon repeated contact with gastric refluxate in esophagus. This study examined the molecular changes within normal esophageal epithelial cells (EECs) under short-term acid loading and the role of these changes in defensive resistance against acidic cytotoxicity. After primary cultured EECs were exposed to pH 4-acidified medium (AM4), cell viability was determined by the MTT assay. Reactive oxygen species (ROS) and NAD(P)H oxidase (NOX) activity were measured. Activation of the mitogen-activated protein kinases (MAPKs) MEK/ERK1/2, p38 and JNK; phosphoinositol-3-kinase (PI3K)/Akt, and nuclear factor-kappa B (NF-κB) were detected by Western blot analysis or immunofluorescence staining. AM4 incubation induced intracellular ROS generation accompanied by increase in NOX activity, which was further increased by Na(+) /H(+) exchange-1 (NHE1)-dependent inhibition but was prevented by inhibition of NOX or mitochondria complex I. AM4 also induced phosphorylation of MEK/ERK1/2, p38 MAPK, PI3K/Akt, and nuclear translocation of NF-κB, and all these effects, except for p38 MAPK phosphorylation, were abolished by inhibition of ROS. ROS-dependent PI3K/Akt activation, which mediates NF-κB nuclear translocation, was inhibited by protein tyrosine kinase (PTK) inhibitors and NHE1-specific inhibitor. All inhibitors of NHE, ROS, PTK, PI3K, or NF-κB further decreased AM4-induced cell viability. Acid loading in the presence of NHE1-dependent protection induced ROS generation by activating NOX and mitochondria complex I, which stimulated PTK/PI3K/Akt/NF-κB-dependent survival in EEC. Our data indicate that normal EEC initially respond to acid loading through intrinsic survival activation.

Conservation of fungal and animal nicotinamide adenine dinucleotide phosphate oxidase complexes.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox) are a group of eukaryotic flavoenzymes that catalyse the reduction of dioxygen to the superoxide anion using electrons provided by NADPH. An integral membrane flavocytochrome b558 heterodimer, composed of the catalytic subunit gp91(phox) and the adaptor protein p22(phox), is essential for catalytic activity of the mammalian Nox2 complex. Two homologues of the mammalian gp91(phox), NoxA and NoxB, have been identified in fungi and shown to be crucial for distinct fungal cell differentiation and developmental processes, but to date, no homologue of the p22(phox) adaptor protein has been identified. Isolation of a mutant from Podospora anserina with a phenotype identical to a previously characterised PaNox1 mutant, combined with phylogenetic analysis, identified a fungal homologue of p22(phox) called PaNoxD. The same adaptor protein was shown to be a component of the Botrytis cinerea NoxA complex as supported by the identical phenotypes of the bcnoxA and bcnoxD mutants and direct physical interaction between BcNoxA and BcNoxD. These results suggest that NoxA/NoxD is the fungal equivalent of the mammalian gp91(phox)/p22(phox) flavocytochrome complex. Tetraspanin (Pls1) mutants of P. anserina and B. cinerea have identical phenotypes to noxB mutants, suggesting that Pls1 is the corresponding integral membrane adaptor for assembly of the NoxB complex.

Molecular control of PtdIns(3,4,5)P3 signaling in neutrophils.

Neutrophils play critical roles in innate immunity and host defense. However, excessive neutrophil accumulation or hyper-responsiveness of neutrophils can be detrimental to the host system. Thus, the response of neutrophils to inflammatory stimuli needs to be tightly controlled. Many cellular processes in neutrophils are mediated by localized formation of an inositol phospholipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), at the plasma membrane. The PtdIns(3,4,5)P3 signaling pathway is negatively regulated by lipid phosphatases and inositol phosphates, which consequently play a critical role in controlling neutrophil function and would be expected to act as ideal therapeutic targets for enhancing or suppressing innate immune responses. Here, we comprehensively review current understanding about the action of lipid phosphatases and inositol phosphates in the control of neutrophil function in infection and inflammation.

BcNoxD, a putative ER protein, is a new component of the NADPH oxidase complex in Botrytis cinerea.

NADPH oxidases (Nox) are major enzymatic producer of reactive oxygen species (ROS). In fungi these multi-enzyme complexes are involved in sexual differentiation and pathogenicity. However, in contrast to mammalian systems, the composition and recruitment of the fungal Nox complexes are unresolved. Here we introduce a new Nox component, the membrane protein NoxD in the grey mold fungus Botrytis cinerea. It has high homology to the ER protein Pro41 from Sordaria macrospora, similar functions to the catalytic Nox subunit BcNoxA in differentiation and pathogenicity, and shows similarities to phagocytic p22phox. BcNoxA and BcNoxD interact with each other. Both proteins are involved in pathogenicity, fusion of conidial anastomosis tubes (CAT) and formation of sclerotia and conidia. These data support our earlier view based on localization studies, for an ER-related function of the Nox complex. We present the first evidence that some functions of the BcNoxA complex are indeed linked to the ER, while others clearly require export from the ER.

Abstract 511: EGFR-initated NADPH oxidase activity regulates Fyn expression in glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) constitute the most aggressive and common form of primary brain tumors, conferring the worst clinical prognosis. Epidermal growth factor receptor (EGFR) amplification, mutation and re-arrangement are commonly observed genetic lesions in GBM. The most frequently occurring EGFR variant in GBM, EGFRvIII, is characterized by a truncated extracellular domain, leading to a receptor which is unable to bind ligand yet rendered constitutively active. In addition to increasing proliferation and survival signaling, EGFRvIII increases the production of reactive oxygen species (ROS); redox signaling in GBM, however, remains an understudied oncogenic feature. Consistent with published findings, our studies indicate that EGFRvIII elevates ROS levels in comparison to wild-type EGFR or vector-transfected control in U87-MG and the observed increase in ROS levels is alleviated by pharmacological and genetic means of EGFR inhibition. ROS increases were determined to occur independent of augmented spare mitochondrial respiratory capacity, but did coincide with elevated basal levels of non-mitochondrial oxygen consumption as measured by Seahorse extracellular flux analysis. Given this finding, our studies focused on the NADPH oxidase (NOX), where chemical inhibition of NOX by apocynin effectively reduced ROS by greater than 50% in EGFRvIII expressing cells. Likewise, EGFRvIII increased the consumption of NADPH greater than 2.0-fold relative to vector control, as measured by cellular NADP/NADPH ratios, and the resulting increases in NADPH consumption were diminished by chemical inhibition of EGFRvIII as well as apocynin inhibition of NOX. These findings indicated that EGFRvIII-induced ROS increases might be related to increased activity of the NOX complex. To this end, siRNA knockdown of p47phox, an essential component of the NOX-2 complex, markedly reduced ROS content as well as cellular proliferation in these cells, thus reinforcing a possible link between aberrant EGFR signaling and ROS production through NOX. Our studies additionally indicate that EGFRvIII-mediated elevations in ROS up-regulate mRNA and protein level expression (&amp;gt;3.0-fold) of the SRC family kinase member Fyn, as Fyn expression is up-regulated in an EGFRvIII-dependent manner and concordantly down-regulated via chemical and genetic means of NOX inhibition. Moreover, siRNA knockdown of Fyn effectively lowers cellular proliferation, survival and increases sensitivity to EGFR kinase inhibition in EGFRvIII, thus indicating that redox-initiated signaling through Fyn, in part, mediates GBM proliferation. Taken together, our results suggest that EGFRvIII increases NOX-2 generated ROS through p47phox, enhancing the proliferative capacity of GBM, in part, via increased Fyn expression, suggesting that targeting redox alterations may prove beneficial in improving clinical outcomes in GBM. Citation Format: Blake Johnson, Joya Chandra. EGFR-initated NADPH oxidase activity regulates Fyn expression in glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 511. doi:10.1158/1538-7445.AM2014-511

Reactive oxygen species deficiency induces autoimmunity with type 1 interferon signature.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the phagocyte reactive oxygen species (ROS)-producing NOX2 enzyme complex and characterized by recurrent infections associated with hyperinflammatory and autoimmune manifestations. A translational, comparative analysis of CGD patients and the corresponding ROS-deficient Ncf1(m1J) mutated mouse model was performed to reveal the molecular pathways operating in NOX2 complex deficient inflammation. A prominent type I interferon (IFN) response signature that was accompanied by elevated autoantibody levels was identified in both mice and humans lacking functional NOX2 complex. To further underline the systemic lupus erythematosus (SLE)-related autoimmune process, we show that naïve Ncf1(m1J) mutated mice, similar to SLE patients, suffer from inflammatory kidney disease with IgG and C3 deposits in the glomeruli. Expression analysis of germ-free Ncf1(m1J) mutated mice reproduced the type I IFN signature, enabling us to conclude that the upregulated signaling pathway is of endogenous origin. Our findings link the previously unexplained connection between ROS deficiency and increased susceptibility to autoimmunity by the discovery that activation of IFN signaling is a major pathway downstream of a deficient NOX2 complex in both mice and humans. We conclude that the lack of phagocyte-derived oxidative burst is associated with spontaneous autoimmunity and linked with type I IFN signature in both mice and humans.

Involvement of fibrinolytic regulators in adhesion of monocytes to vascular endothelial cells induced by glycated LDL and to aorta from diabetic mice

Diabetes mellitus accelerates the development of atherosclerotic cardiovascular diseases. Monocyte adhesion is an early cellular event of atherogenesis. Elevated levels of glyLDL were common in diabetic patients. Our previous studies indicated that HSF1 and p22-phox (a subunit of the NOX complex) were involved in glyLDL-induced up-regulation of PAI-1 in vascular EC. The present study demonstrated that glyLDL significantly increased the adhesion of monocytes to the surface of cultured human umbilical vein or PAEC. Transfection of siRNA for PAI-1, p22-phox, or HSF1 in EC prevented glyLDL-induced monocyte adhesion to EC. uPA siRNA increased monocyte adhesion to EC. Exogenous uPA reduced monocyte adhesion induced by glyLDL or uPA siRNA. Exogenous PAI-1 restored monocyte adhesion to EC inhibited by PAI-1 siRNA or uPA. GlyLDL-induced monocyte adhesion to EC was inhibited by treatment of EC with RAP, an antagonist for LRP, and enhanced by uPAR antibody. The adhesion of monocytes to aorta from leptin db/db diabetic mice was significantly greater than to that from control mice, which was associated with elevated contents of PAI-1, uPA, p22-phox, and HSF1 in hearts of db/db mice. The results suggest that oxidative stress and fibrinolytic regulators (PAI-1, uPA, and uPAR) are implicated in the modulation of glyLDL-induced monocyte adhesion to vascular endothelium, which may play a crucial role in vascular inflammation under diabetes-associated metabolic disorder.

Angiotensin II receptor blockade protects against the slow to fast fiber type shift and type I fiber atrophy in the rat soleus with 7 days of hindlimb unloading (LB804)

Reduced mechanical loading during spaceflight and immobilization results in weakening and atrophy of antigravity skeletal muscles. Data indicates that oxidative stress contributes to inactivity‐induced muscle atrophy. Recently we observed that the Nox2 isoform of NADPH oxidase is upregulated during unloading‐induced soleus muscle atrophy. Clues from other tissue types indicate activation of angiotensin II type 1 receptor (AT1R) as an upstream event leading to Nox2 complex formation and activity. Here, we used an AT1R blocker (Losartan) to test the hypothesis that AT1R activation is an upstream signaling event driving Nox2 complex activity, and thus atrophy in the mechanically unloaded soleus. Rats were divided into 4 groups: ambulatory control (CON), ambulatory + Losartan (40 mg/kd/day) (CON‐L), hindlimb unloaded for 7 days (HU), and HU + Losartan (HU‐L). Losartan treatment did not protect against the reduction in soleus mass during 7 days of unloading. However, the increase in Type II muscle fibers seen with unloading was mitigated by Losartan administration. Losartan also protected against atrophy of Type I muscle fibers. In addition, Losartan reduced the signal intensity and membrane localization of the Nox2 subunit p67phox in the unloaded soleus. Our findings suggest a potential role of AT1R activation as an upstream signaling event leading to Nox2 complex formation, activation, and slow to fast fiber‐type shift during mechanical unloading.Grant Funding Source: Supported by NASA (NNX13AE45G), Predoctoral Space Physiology Grant from ACSM/NASA, and the Sydney and J.L. Huffines Institute

Xanthine oxidase activation mediates intermittent hypoxia increased NADPH oxidase activity (710.3)

We previously showed that NADPH oxidases (Nox) especially Nox2 mediate systemic and cellular responses to intermittent hypoxia (IH). However, the mechanisms by which IH activates Nox2 have not been examined. We recently reported that xanthine oxidase (XO) activation by IH generates ROS which leads to increased intracellular calcium. Given that calcium activated PKC is required for Nox2 activation, we tested the hypothesis that XO activation mediates IH increased Nox activity via ROS augmented calcium. Allopurinol, a XO inhibitor or silencing of XO in PC12 cells prevented IH‐induced Nox2 activation by inhibiting Nox2 complex formation at the membrane. Treatment of cells with Xanthine/XO under normoxia mimicked the effects of IH. Our results further demonstrate that ROS generated by XO, contribute to IH induced Nox activation by increasing intracellular calcium levels. IH augmented PKC activity by phosphorylation of alpha subunit which was inhibited by allopurinol and BAPTA (calcium chleator). More importantly, inhibition of PKC activity by allopurinol abolished Nox2 complex formation. These results demonstrate that ROS generated by XO activates PKC alpha via calcium resulting in IH augmented Nox activity. Supported HL‐ ‐90554.Grant Funding Source: HL‐90554

NADPH Oxidase Complex-Derived Reactive Oxygen Species, the Actin Cytoskeleton, and Rho GTPases in Cell Migration

Rho GTPases are historically known to be central regulators of actin cytoskeleton reorganization. This affects many processes including cell migration. In addition, members of the Rac subfamily are known to be involved in reactive oxygen species (ROS) production through the regulation of NADPH oxidase (Nox) activity. This review focuses on relationships between Nox-regulated ROS, Rho GTPases, and cytoskeletal reorganization, in the context of cell migration. It has become clear that ROS participate in the regulation of certain Rho GTPase family members, thus mediating cytoskeletal reorganization. The role of the actin cytoskeleton in providing a scaffold for components of the Nox complex needs to be examined in the light of these new advances. During cell migration, Rho GTPases, ROS, and cytoskeletal organization appear to function as a complex regulatory network. However, more work is needed to fully elucidate the interactions between these factors and their potential in vivo importance. Ultrastructural analysis, that is, electron microscopy, particularly immunogold labeling, will enable direct visualization of subcellular compartments. This in conjunction with the analysis of tissues lacking specific Rho GTPases, and Nox components will facilitate a detailed examination of the interactions of these structures with the actin cytoskeleton. In combination with the analysis of ROS production, including its subcellular location, these data will contribute significantly to our understanding of this intricate network under physiological conditions. Based on this, in vivo and in vitro studies can then be combined to elucidate the signaling pathways involved and their targets.

NADPH Oxidase 1, a Novel Molecular Source of ROS in Hippocampal Neuronal Death in Vascular Dementia

Chronic cerebral hypoperfusion (CCH) is a common pathological factor that contributes to neurodegenerative diseases such as vascular dementia (VaD). Although oxidative stress has been strongly implicated in the pathogenesis of VaD, the molecular mechanism underlying the selective vulnerability of hippocampal neurons to oxidative damage remains unknown. We assessed whether the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, a specialized superoxide generation system, plays a role in VaD by permanent ligation of bilateral common carotid arteries in rats. Male Wistar rats (10 weeks of age) were subjected to bilateral occlusion of the common carotid arteries (two-vessel occlusion [2VO]). Nox1 expression gradually increased in hippocampal neurons, starting at 1 week after 2VO and for approximately 15 weeks after 2VO. The levels of superoxide, DNA oxidation, and neuronal death in the CA1 subfield of the hippocampus, as well as consequential cognitive impairment, were increased in 2VO rats. Both inhibition of Nox by apocynin, a putative Nox inhibitor, and adeno-associated virus-mediated Nox1 knockdown significantly reduced 2VO-induced reactive oxygen species generation, oxidative DNA damage, hippocampal neuronal degeneration, and cognitive impairment. We provided evidence that neuronal Nox1 is activated in the hippocampus under CCH, causing oxidative stress and consequential hippocampal neuronal death and cognitive impairment. This evidence implies that Nox1-mediated oxidative stress plays an important role in neuronal cell death and cognitive dysfunction in VaD. Nox1 may serve as a potential therapeutic target for VaD.

Roles of NADPH oxidase 2 and 4 in endothelial cell survival and death under serum depletion

Oxidative stress and redox-signal pathways are known to be involved in endothelial apoptosis induced by serum depletion. However, the associated mechanism is not well understood and thus, was investigated in the present study focusing on NADPH oxidases (NOX). Serum removal from the culture medium led to an increase in reactive oxygen species (ROS) production and apoptotic death of human umbilical vein endothelial cells. Serum depletion also increased the gene expression of the NOX2 and NOX4 subunits. The selective suppression of NOX4 expression by small interfering RNA (siRNA) attenuated ROS production and cell death due to serum-depletion whereas siRNA for NOX2 increased cell death. Expression of exogenous NOX2 or NOX4 subunit alone had no significant effects on ROS production or cell death. Coexpression of the subunits of the NOX4 complex (NOX4 and p22phox) or the NOX2 complex (NOX2, p22phox, p47phox and p67phox) increased ROS production and cell death under serum-depleted conditions. This study suggests that endothelial cell survival and death are differentially regulated by expression levels of the subunits of NOX2 and NOX4 complexes.

Nuclear Nox4-Derived Reactive Oxygen Species in Myelodysplastic Syndromes

A role for intracellular ROS production has been recently implicated in the pathogenesis and progression of a wide variety of neoplasias. ROS sources, such as NAD(P)H oxidase (Nox) complexes, are frequently activated in AML (acute myeloid leukemia) blasts and strongly contribute to their proliferation, survival, and drug resistance. Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop AML. The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is the genomic instability. NADPH oxidases are now recognized to have specific subcellular localizations, this targeting to specific compartments for localized ROS production. Local Nox-dependent ROS production in the nucleus may contribute to the regulation of redox-dependent cell growth, differentiation, senescence, DNA damage, and apoptosis. We observed that Nox1, 2, and 4 isoforms and p22phox and Rac1 subunits are expressed in MDS/AML cell lines and MDS samples, also in the nuclear fractions. Interestingly, Nox4 interacts with ERK and Akt1 within nuclear speckle domain, suggesting that Nox4 could be involved in regulating gene expression and splicing factor activity. These data contribute to the elucidation of the molecular mechanisms used by nuclear ROS to drive MDS evolution to AML.