Very long chain fatty acids activate NADPH oxidase in human dermal fibroblasts

Very long chain fatty acids (VLCFAs) are exclusively oxidized in peroxisomes and their levels are significantly increased in tissues of patients with peroxisomal disorders. Although the biochemical indicators of peroxisomal dysfunction, such as elevated VLCFAs, are well known, the mechanisms of pathogenesis of peroxisomal diseases are unclear. In this study we have examined the effect of VLCFAs on NADPH oxidase (NOX), a complex enzyme system responsible for the production of superoxide anions, in order to understand the oxidative stress-mediated mechanisms involved in pathology of peroxisomal disorders. Varying concentrations (2.5 to 10 microg ml(-1)) of VLCFAs, lignoceric acid and cerotic acid, significantly (p < 0.001) increased the enzymic activity of NOX in cultures of human dermal fibroblasts. VLCFAs did not affect the expression of gp91phox or p22phox whereas the mRNA and protein levels of p47phox were significantly (two or three-fold) increased following treatment of fibroblasts with lignoceric acid or cerotic acid. VLCFAs also caused a significant (p < 0.01) increase in lipid peroxidation in dermal fibroblasts which could be markedly reversed by treatment with apocyanin (10 mM) or superoxide dismutase (SOD, 25 U ml(-1)). With these results, we report for the first time that VLCFAs enhance NOX activity and superoxide anion-mediated lipid peroxidation in cultured dermal fibroblasts. This study proposes a mechanism that may be taking place in vivo during peroxisomal dysfunction and that leads to oxidative stress-mediated pathogenesis.     

Reorganization of mouse sperm lipid rafts by capacitation

One of the hallmarks of mammalian sperm capacitation is the loss of cholesterol from the plasma membrane. Cholesterol has been associated with the formation of detergent insoluble membrane microdomains in many cell types, and sperm from several mammalian species have been shown to contain detergent-resistant membranes (DRMs). The change in cholesterol composition of the sperm plasma membrane during capacitation raises the question of whether the contents of DRMs are altered during this process. In this study, we investigated changes in protein composition of DRMs isolated from uncapacitated or capacitated mouse sperm. TX-100 insoluble membranes were fractionated by sucrose flotation gradient centrifugation and analyzed by Western and lectin blotting, and capacitation-related differences in protein composition were identified. Following capacitation, the detergent insoluble fractions moved to lighter positions on the sucrose gradients, reflecting a global change in density or composition. We identified several individual proteins that either became enriched or depleted in DRM fractions following capacitation. These data suggest that the physiological changes in sperm motility, ability to penetrate the zona pellucida (ZP), ZP responsiveness, and other capacitation-dependent changes, may be due in part to a functional reorganization of plasma membrane microdomains.     

NADPH oxidase activation is required for migration by LIGHT in human monocytes

Homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus (HSV) glycoprotein D (gD) for herpes virus entry mediator (HVEM; TR2) (LIGHT), a ligand of herpes virus entry mediator (HVEM), increased reactive oxygen species (ROS) and enhanced the destruction of bacteria in human monocytes. In this study, rhLIGHT was found to increase the expression of the chemokine receptors, chemokine receptor 1 (CCR1) and CCR2, as well as to accelerate the migration activity of human monocytes. Additionally, rhLIGHT was found to increase ROS via NADPH oxidase p47^phox phosphorylation, which was found to be required for LIGHT-induced NF-κB activation, CCR1 and CCR2 expression, migration and IL-8 and TNF-α production. Taken together, these results indicate that NADPH oxidase activation is required for rhLIGHT-induced migration in human monocytes.     

Cytochrome P450 oxidase activity and its role in NADPH dependent lipid peroxidation

A comparison is made between microsomal NADPH-dependent H₂O₂ production and malondialdehyde (MDA) formation in rat liver microsomes, obtained from phenobarbital pretreated rats. An increase in H₂O₂ formation was observed during NADPH-dependent disposition (10 min) of 100 μM diazepam (33%) and 2 mM hexobarbital (69%). In contrast orphenadrine (100 μM) and its mono-N-demethylated metabolite tofenacine (100 μM) decreased the H₂O₂ formation (35% and 55%, respectively). However, all these substrates were found to inhibit NADPH-dependent lipid peroxidation (60 min), estimated by measuring MDA formation, to various extents. These data strongly suggest that the oxidase activity of cytochrome P450 (H₂O₂ production) is not involved in a rate-limiting step in NADPH-dependent lipid peroxidation.     

Kaempferol suppresses collagen-induced platelet activation by inhibiting NADPH oxidase and protecting SHP-2 from oxidative inactivation

Reactive oxygen species (ROS) generated upon collagen stimulation act as second messengers to propagate various platelet-activating events. Among the ROS-generating enzymes, NADPH oxidase (NOX) plays a prominent role in platelet activation. Thus, NOX has been suggested as a novel target for anti-platelet drug development. Although kaempferol has been identified as a NOX inhibitor, the influence of kaempferol on the activation of platelets and the underlying mechanism have never been investigated. Here, we studied the effects of kaempferol on NOX activation, ROS-dependent signaling pathways, and functional responses in collagen-stimulated platelets. Superoxide anion generation stimulated by collagen was significantly inhibited by kaempferol in a concentration-dependent manner. More importantly, kaempferol directly bound p47^phox, a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47^phox and NOX activation. In accordance with the inhibition of NOX, ROS-dependent inactivation of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was potently protected by kaempferol. Subsequently, the specific tyrosine phosphorylation of key components (Syk, Vav1, Btk, and PLCγ2) of collagen receptor signaling pathways was suppressed by kaempferol. Kaempferol also attenuated downstream responses, including cytosolic calcium elevation, P-selectin surface exposure, and integrin-α_IIbβ₃ activation. Ultimately, kaempferol inhibited platelet aggregation and adhesion in response to collagen in vitro and prolonged in vivo thrombotic response in carotid arteries of mice. This study shows that kaempferol impairs collagen-induced platelet activation through inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2. This effect suggests that kaempferol has therapeutic potential for the prevention and treatment of thrombovascular diseases.     

Rapid increase in serum lipid peroxide 4-hydroxynonenal (HNE) through monocyte NADPH oxidase in early endo-toxemia

We have developed a time-resolved fluoroimmunoassay (TR-FIA) for a lipid peroxide 4-hydroxynonenal (HNE), which is 100-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA) and is an easier technique to use for a large number of samples without pre-treatment. By this assay, we found that a low dose of bacterial lipo-polysaccharide (LPS), injected intra-peritoneally (0.5 mg/kg), increased serum HNE level by 28-folds, with a peak at 20 min. LPS also increased HNE in vitro to a much higher level in the monocyte-enriched plasma than in the leukocyte-enriched plasma, with a peak at 10 min. The HNE production after LPS treatment was inhibited by apocynin, a specific NADPH oxidase inhibitor in vivo and in vitro, and to a lesser extent by dimethylsulfoxide a solvent for apocynin and a hydroxyl radical scavenger in vitro. These data suggest that monocyte NADPH oxidase is involved in the lipid peroxidation (HNE formation) in the LPS-challenged rat. This is the first clear demonstration of the link between an inflammatory stimulus and lipid peroxidation in the blood.     

Analysis of dihydroethidium fluorescence for the detection of intracellular and extracellular superoxide produced by NADPH oxidase

All methods used for quantitation of superoxide have limitations when it comes to differentiating between extracellular and intracellular sites of superoxide production. In the present study, we monitored dihydroethidium (DHE)-derived fluorescence at 570 nm, which indicates hydroxyethidium derived from reaction with superoxide produced by human leukemia cells (HL-60) and microvascular endothelial cells (HMEC-1). Phorbol-12-myristate 13-acetate (PMA; 100 ng/ml) caused an increase in fluorescence and lucigenin chemiluminescence in HL-60, which was abolished by superoxide dismutase (SOD; 600 U/ml) indicating that DHE detects extracellular superoxide. Furthermore, both HL-60 cells and HMEC-1 generated a fluorescence signal in the presence of DHE under resting conditions, which was unaffected by SOD, but abolished by polyethylene glycosylated-SOD (PEG-SOD) (100 U/ml) and MnTmPyP (25 μM), indicating that DHE also detects superoxide produced intracellularly. In HMEC-1, silencing of either Nox2 or Nox4 components of NADPH oxidase with small interference RNA (siRNA) resulted in a significant reduction in superoxide detected by both DHE fluorescence (Nox2 siRNA; 71 ± 6% and Nox4 siRNA 83 ± 7% of control) and lucigenin chemiluminescence (Nox2; 54 ± 6% and Nox4 74 ± 4% of control). In conclusion, DHE-derived fluorescence at 570 nm is a convenient method for detection of intracellular and extracellular superoxide produced by phagocytic and vascular NADPH oxidase.     

Analysis of dihydroethidium fluorescence for the detection of intracellular and extracellular superoxide produced by NADPH oxidase

All methods used for quantitation of superoxide have limitations when it comes to differentiating between extracellular and intracellular sites of superoxide production. In the present study, we monitored dihydroethidium (DHE)-derived fluorescence at 570 nm, which indicates hydroxyethidium derived from reaction with superoxide produced by human leukemia cells (HL-60) and microvascular endothelial cells (HMEC-1). Phorbol-12-myristate 13-acetate (PMA; 100 ng/ml) caused an increase in fluorescence and lucigenin chemiluminescence in HL-60, which was abolished by superoxide dismutase (SOD; 600 U/ml) indicating that DHE detects extracellular superoxide. Furthermore, both HL-60 cells and HMEC-1 generated a fluorescence signal in the presence of DHE under resting conditions, which was unaffected by SOD, but abolished by polyethylene glycosylated-SOD (PEG-SOD) (100 U/ml) and MnTmPyP (25 μM), indicating that DHE also detects superoxide produced intracellularly. In HMEC-1, silencing of either Nox2 or Nox4 components of NADPH oxidase with small interference RNA (siRNA) resulted in a significant reduction in superoxide detected by both DHE fluorescence (Nox2 siRNA; 71 ± 6% and Nox4 siRNA 83 ± 7% of control) and lucigenin chemiluminescence (Nox2; 54 ± 6% and Nox4 74 ± 4% of control). In conclusion, DHE-derived fluorescence at 570 nm is a convenient method for detection of intracellular and extracellular superoxide produced by phagocytic and vascular NADPH oxidase.     

The effect of high-density lipoproteins on the formation of lipid/protein conjugates during in vitro oxidation of low-density lipoprotein

High-density lipoprotein (HDL) incubated with low-density lipoprotein (LDL) under oxidising conditions has previously been reported to decrease the accumulation of lipid peroxides on LDL and to diminish the biological effects of LDL, which would have been present had it been oxidatively modified in the absence of HDL. Thus far direct evidence that oxidative modification of LDL is diminished by HDL has, however, been lacking. We used electrospray ionisation mass spectrometry (ESI-MS) to detect 4-hydroxy-2-nonenal (HNE)-modified histidine residues of tryptic fragments of LDL which had been subject to Cu(2+) induced oxidation both in the presence and absence of human or avian HDL. HNE-modified angiotensin II was introduced into the incubation mixture as an internal standard and to check that HDL did not interfere in the detection of HNE-modified peptides non-specifically. Our results confirmed earlier reports that HNE modification of histidine occurs during the oxidation of LDL and for the first time revealed a marked attenuation of the process in the presence of human HDL with no effect on the detection of HNE-modified angiotensin II by ESI-MS. Avian HDL, which lacks the anti-oxidative enzyme paraoxonase, did not affect the formation of apo B adducts. Our findings therefore suggest that covalent linkage of lipid peroxidation products to LDL protein as well as the accumulation of lipid peroxides on LDL is diminished in the presence of HDL containing paraoxonase.     

Restraint of spreading-dependent activation of polymorphonuclear leukocyte NADPH oxidase in an acidified environment

Elucidation of the mechanisms by which environmental pH affects or regulates the functions of polymorphonuclear leukocytes (PMNs) is important because severe acidification of the microenvironment often prevails at sites of inflammation where they act in host defense. In the present study, we investigated the effect of an acidic environment on spreading-dependent activation of O2- -producing NADPH oxidase in PMNs. We found that PMNs underwent spreading spontaneously over type I collagen and plastic surfaces at both neutral and acidic pH, although spreading over fibrinogen surfaces, for which cellular stimulation with H2O2 is required, was inhibited by acidic pH. At acidic pH, however, PMNs were unable to undergo spreading-dependent production of O2-. Pharmacological experiments showed that p38 mitogen-activated protein kinase (MAPK) was involved in the signaling pathways mediating the spreading-dependent activation of NADPH oxidase, and that its spreading-dependent phosphorylation of Thr-180 and Tyr-182, a hallmark of activation, was impaired at acidic pH. Furthermore, the inhibition by acidic pH of O2- production as well as p38 MAPK phosphorylation subsequent to spreading induction was reversible; environmental neutralization and acidification after induction of spreading at acidic and neutral pH, respectively, up- and down-regulated the two phenomena. Acidic pH did not affect the O2- production activity of NADPH oxidase pre-activated by phorbol 12-myristate 13-acetate (PMA). These results suggest that, in PMNs, the p38 MAPK-mediated signaling pathway functions as a pH-sensing regulator of spreading-dependent NADPH oxidase activation.     

Ischemia-activated microglia induces neuronal injury via activation of gp91phox NADPH oxidase

Although glial cells play a major role in the pathogenesis of many neurological diseases by exacerbating neuronal and non-neuronal cell death, the mechanisms involved are unclear. We examined the effects of microglia-(MCM) or astrocyte-(ACM) conditioned media obtained by chemical ischemia on the neuronal injury in SH-SY5Y cells. Chemical ischemia was induced by the treatment with NaN₃ and 2-deoxy-d-glucose for 2 h. MCM-treated SH-SY5Y cells showed reduced the viability, increased caspase-3 activity, decreased Bcl-2/Bax ratio, and increased cytochrome c release, increased inflammatory cytokines, and increased reactive oxygen species (ROS) generation. MCM also increased gp91phox nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which was inhibited by NADPH oxidase inhibitor, apocynin, and gp91phox siRNA. However, ACM did not show any significant changes. The results suggest that microglia activated by ischemic insult may increase reactive oxygen species generation via activation of gp91phox NADPH oxidase, resulting in neuronal injury.     

Inhibition of NADPH oxidase promotes alternative and anti-inflammatory microglial activation during neuroinflammation

Like macrophages, microglia are functionally polarized into different phenotypic activation states, referred as classical and alternative. The balance of the two phenotypes may be critical to ensure proper brain homeostasis, and may be altered in brain pathological states, such as Alzheimer's disease. We investigated the role of NADPH oxidase in microglial activation state using p47(phox) and gp91(phox) -deficient mice as well as apocynin, a NADPH oxidase inhibitor during neuroinflammation induced by an intracerebroventricular injection of LPS or Aβ????. We showed that NADPH oxidase plays a critical role in the modulation of microglial phenotype and subsequent inflammatory response. We demonstrated that inhibition of NADPH oxidase or gene deletion of its functional p47(phox) subunit switched microglial activation from a classical to an alternative state in response to an inflammatory challenge. Moreover, we showed a shift in redox state towards an oxidized milieu and that subpopulations of microglia retain their detrimental phenotype in Alzheimer's disease brains. Microglia can change their activation phenotype depending on NADPH oxidase-dependent redox state of microenvironment. Inhibition of NADPH oxidase represents a promising neuroprotective approach to reduce oxidative stress and modulate microglial phenotype towards an alternative state.     

Inhibition of thrombin-induced microglial activation and NADPH oxidase by minocycline protects dopaminergic neurons in the substantia nigra in vivo

The present study shows that activation of microglial NADPH oxidase and production of reactive oxygen species (ROS) is associated with thrombin-induced degeneration of nigral dopaminergic neurons in vivo. Seven days after thrombin injection in the rat substantia nigra (SN), tyrosine hydroxylase immunocytochemistry showed a significant loss of nigral dopaminergic neurons. This cell death was accompanied by localization of terminal deoxynucleotidyl transferase-mediated fluorecein UTP nick-end labelling (TUNEL) staining within dopaminergic neurons. This neurotoxicity was antagonized by the semisynthetic tetracycline derivative, minocycline, and the observed neuroprotective effects were associated with the ability of minocycline to suppress NADPH oxidase-derived ROS production and pro-inflammatory cytokine expression, including interleukin-1beta and inducible nitric oxide synthase, from activated microglia. These results suggest that microglial NADPH oxidase may be a viable target for neuroprotection against oxidative damage.     

Characterisation of electron currents generated by the human neutrophil NADPH oxidase

Electron transport by the human neutrophil NADPH oxidase is an important microbicidal weapon for phagocytes. The electron current (I_e) generated by the neutrophil NADPH oxidase is poorly characterised due to the lack of appropriate electrophysiological data. In this study, I fully characterise the neutrophil generated I_e when the NADPH oxidase is activated by NADPH and GTPγS. The neutrophil I_e was markedly voltage-dependent in the entire voltage range in comparison to those electron currents measured after chloride was removed from the external bath solution. The difference in I_e measured in chloride free conditions was not due to a change in the activation kinetics of voltage-gated proton channels. The I_e depolarises the neutrophil plasma membrane at a rate of 2.3 V s⁻¹ and this depolarisation was opposed when voltage-gated proton channels are activated. 3 mM ZnCl₂ depolarised the membrane potential to +97.8 ± 2.5 mV (n = 4), and this depolarisation was abolished after NADPH oxidase inhibition.     

Brain-derived neurotrophic factor can act as a pronecrotic factor through transcriptional and translational activation of NADPH oxidase

Several lines of evidence suggest that neurotrophins (NTs) potentiate or cause neuronal injury under various pathological conditions. Since NTs enhance survival and differentiation of cultured neurons in serum or defined media containing antioxidants, we set out experiments to delineate the patterns and underlying mechanisms of brain-derived neurotrophic factor (BDNF)-induced neuronal injury in mixed cortical cell cultures containing glia and neurons in serum-free media without antioxidants, where the three major routes of neuronal cell death, oxidative stress, excitotoxicity, and apoptosis, have been extensively studied. Rat cortical cell cultures, after prolonged exposure to NTs, underwent widespread neuronal necrosis. BDNF-induced neuronal necrosis was accompanied by reactive oxygen species (ROS) production and was dependent on the macromolecular synthesis. cDNA microarray analysis revealed that BDNF increased the expression of cytochrome b558, the plasma membrane-spanning subunit of NADPH oxidase. The expression and activation of NADPH oxidase were increased after exposure to BDNF. The selective inhibitors of NADPH oxidase prevented BDNF-induced ROS production and neuronal death without blocking antiapoptosis action of BDNF. The present study suggests that BDNF-induced expression and activation of NADPH oxidase cause oxidative neuronal necrosis and that the neurotrophic effects of NTs can be maximized under blockade of the pronecrotic action.     

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

Abstract

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H₂O₂ were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H₂O₂ in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H₂O₂ via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.