Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.6.3.1 (NADPH oxidase)
 11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Inhibition of thrombin-induced microglial activation and NADPH oxidase by minocycline protects dopaminergic neurons in the substantia nigra in vivo. 1621 27

Platelets play a crucial role in the physiology of primary hemostasis and pathophysiological processes such as arterial thrombosis. Accumulating evidence suggests a key regulatory role of both NO and reactive oxygen species (ROS) in platelets. While the inhibitory role of NO/cGMP signaling in both murine and human platelets is well established, recent data suggest that intracellular ROS generation is involved in platelet activation. Thrombin-induced intracellular ROS production was inhibited by NAD(P)H oxidase inhibitors (DPI and apocynin), cyclooxygenase inhibitor (acetylsalicylic acid), and superoxide scavengers (tiron and MnTMPyP). Furthermore, thrombin (Trap6)-induced platelet aggregation and thrombus formation on collagen under high shear was inhibited by NAD(P)H oxidase inhibitors (DPI and apocynin), whereas secretion and platelet shape change were not affected. Inhibition of alphaIIbbeta3 activation by NAD(P)H oxidase inhibitors and superoxide scavengers was independent of NO/cGMP signaling demonstrating a direct role of platelet NAD(P)H oxidase-generated ROS for integrin alphaIIbbeta3 activation.
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PMID:Platelet regulation by NO/cGMP signaling and NAD(P)H oxidase-generated ROS. 1646 12

The stress-responsive serum- and glucocorticoid-inducible kinase Sgk-1 is involved in osmoregulation and cell survival and may contribute to fibrosis and hypertension. However, the function of Sgk-1 in vascular remodeling and thrombosis, 2 major determinants of pulmonary hypertension (PH), has not been elucidated. We investigated the role of Sgk-1 in thrombin signaling and tissue factor (TF) expression and activity in pulmonary artery smooth muscle cells (PASMC). Thrombin increased Sgk-1 activity and mRNA and protein expression. H2O2 similarly induced Sgk-1 expression. Antioxidants, dominant-negative Rac, and depletion of the NADPH oxidase subunit p22phox diminished thrombin-induced Sgk-1 expression. Inhibition of p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and phosphoinositide-dependent kinase-1 prevented thrombin-induced Sgk-1 expression. Thrombin or Sgk-1 overexpression enhanced TF expression and procoagulant activity, whereas TF upregulation by thrombin was diminished by kinase-deficient Sgk-1 and was not detectable in fibroblasts from mice deficient in sgk-1 (sgk1(-/-)). Similarly, dexamethasone treatment failed to induce TF expression and activity in lung tissue from sgk1(-/-) mice. Transcriptional induction of TF by Sgk-1 was mediated through nuclear factor kappaB. Finally, Sgk-1 and TF proteins were detected in the media of remodeled pulmonary vessels associated with PH. These data show that thrombin potently induces Sgk-1 involving NADPH oxidases, phosphatidylinositol 3-kinase, p38 mitogen-activated protein kinase, and phosphoinositide-dependent kinase-1, and that activation of nuclear factor kappaB by Sgk-1 mediates TF expression and activity by thrombin. Because enhanced procoagulant activity can promote pulmonary vascular remodeling, and Sgk-1 and TF were present in the media of remodeled pulmonary vessels, this pathway may play a critical role in vascular remodeling in PH.
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PMID:The serum- and glucocorticoid-inducible kinase Sgk-1 is involved in pulmonary vascular remodeling: role in redox-sensitive regulation of tissue factor by thrombin. 1648 15

To characterize novel signaling pathways that underlie NAD(P)H oxidase-mediated signaling in atherosclerosis, we first examined differences in thrombin-induced gene expression between wild-type and p47phox(-/-) (NAD[P]H oxidase-deficient) VSMC. Of the 9000 genes analyzed by cDNA microarray method at the G1/S transition point, 76 genes were similarly and significantly modulated in both the cell types, whereas another 22 genes that encompass various functional groups were regulated in NAD(P)H oxidase-dependent manner. Among these 22 genes, thrombin-induced NAD(P)H oxidase-mediated regulation of Klf15, Igbp1, Ak4, Adamts5, Ech1, Serp1, Sec61a2, Aox1, Aoh1, Fxyd5, Rai14, and Serpinh1 was shown for the first time in VSMC. The role of NAD(P)H oxidase in the regulation of a subset of these genes (CD44, BMP4, Id1, and Id3) was confirmed using modulators of reactive oxygen species (ROS) generation, a ROS scavenger and in gain-of-function experiments. We then characterized regulation of these genes in restenosis and atherosclerosis. In both apoE(-/-) mice and in a mouse vascular injury model, these genes are regulated in NAD(P)H oxidase-dependent manner during vascular lesion formation. Based on these findings, we propose that NAD(P)H oxidase-dependent gene expression in general, and the CD44 and BMP4-Id signaling pathway in particular, is important in restenosis and atherosclerosis.
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PMID:Thrombin and NAD(P)H oxidase-mediated regulation of CD44 and BMP4-Id pathway in VSMC, restenosis, and atherosclerosis. 1660 Dec 25

Osteopontin (OPN) is a cytokine upregulated in diabetic vascular disease. To better understand its role in vascular remodeling, we assessed how OPN controls metalloproteinase (MMP) activation in aortic adventitial myofibroblasts (AMFs) and A7r5 vascular smooth muscle cells (VSMCs). By zymography, OPN and tumor necrosis factor (TNF)-alpha preferentially upregulate pro-matrix metalloproteinase 9 (pro-MMP9) activity. TNF-alpha upregulated pro-MMP9 in AMFs isolated from wild-type (OPN(+/+)) mice, but pro-MMP9 induction was abrogated in AMFs from OPN(-/-) mice. OPN treatment of VSMCs enhanced pro-MMP9 activity, and TNF-alpha induction of pro-MMP9 was inhibited by anti-OPN antibody and apocynin. Superoxide and the oxylipid product 8-isoprostaglandin F(2) alpha-isoprostane (8-IsoP) were increased by OPN treatment, and anti-OPN antibody suppressed 8-IsoP production. Like OPN and TNF-alpha, 8-IsoP preferentially activated pro-MMP9. Superoxide, 8-IsoP, and NADPH oxidase 2 (Nox2) subunits were reduced in OPN(-/-) AMFs. Treatment of A7r5 VSMCs with OPN upregulated NADPH oxidase subunit accumulation. OPN structure/function studies mapped these activities to the SVVYGLR heptapeptide motif in the thrombin-liberated human OPN N-terminal domain (SLAYGLR in mouse OPN). Treatment of aortic VSMCs with SVVYGLR upregulated pro-MMP9 activity and restored TNF-alpha activation of pro-MMP9 in OPN(-/-) AMFs. Injection of OPN-deficient OPN(+/-) mice with SVVYGLR peptide upregulated pro-MMP9 activity, 8-IsoP levels, and Nox2 protein levels in aorta and increased panmural superoxide production (dihydroethidium staining). At equivalent hyperglycemia and dyslipidemia, 8-IsoP levels and aortic pro-MMP9 were reduced with complete OPN deficiency in a model of diet-induced diabetes, achieved by comparing OPN(-/-)/LDLR(-/-) versus OPN(+/-)/LDLR(-/-) siblings. Thus, OPN provides a paracrine signal that augments vascular pro-MMP9 activity, mediated in part via superoxide generation and oxylipid formation.
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PMID:An osteopontin-NADPH oxidase signaling cascade promotes pro-matrix metalloproteinase 9 activation in aortic mesenchymal cells. 1679 91

The present study investigated whether thrombin can induce the production of reactive oxygen species (ROS) through activation of neuronal NADPH oxidase and whether this contributes to oxidative damage and consequently to neurodegeneration. Immunocytochemical and biochemical evidence demonstrated that, in neuron-enriched hippocampal cultures, thrombin induces neurodegeneration in a dose-dependent manner. In parallel, ROS production was evident as assessed by analyzing DCF and hydroethidine. Real-time PCR analysis, at various time points after thrombin treatment, also demonstrated that expression of NADPH oxidase subunits (p47(phox) and p67(phox)) occurs. In addition, Western blot analysis and double-label immunocytochemistry showed an up-regulation in the expression of cytosolic components (Rac 1 and p67(phox)), the translocation of cytosolic proteins (p47(phox) and p67(phox)) to the membrane, and the localization of gp91(phox) or p47(phox) expression in hippocampal neurons of cultures and CA1 layer. The thrombin-induced ROS production, protein oxidation, and loss of cultured hippocampal neurons were partially attenuated by an NADPH oxidase inhibitor and/or by several antioxidants. Collectively, the present study is the first to demonstrate that, in cultured hippocampal neurons, thrombin-induced neurotoxicity is, at least in part, caused by neuronal NADPH oxidase-mediated oxidative stress. This strongly suggests that thrombin can act as an endogenous neurotoxin, and inhibitors of thrombin and/or antioxidants can be useful agents for treating oxidative stress-mediated hippocampal neurodegenerative diseases, such as Alzheimer's disease.
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PMID:Thrombin-induced oxidative stress contributes to the death of hippocampal neurons: role of neuronal NADPH oxidase. 1818 16

The statins, hydroxy-3-methylglutaryl-CoA reductase inhibitors that lower serum cholesterol, exhibit myriad clinical benefits, including enhanced vascular integrity. One potential mechanism underlying increased endothelial cell (EC) barrier function is inhibition of geranylgeranylation, a covalent modification enabling translocation of the small GTPases Rho and Rac to the cell membrane. While RhoA inhibition attenuates actin stress fiber formation and promotes EC barrier function, Rac1 inhibition at the cell membrane potentially prevents activation of NADPH oxidase and subsequent generation of superoxides known to induce barrier disruption. We examined the relative regulatory effects of simvastatin on RhoA, Rac1, and NADPH oxidase activities in the context of human pulmonary artery EC barrier protection. Confluent EC treated with simvastatin demonstrated significantly decreased thrombin-induced FITC-dextran permeability, a reflection of vascular integrity, which was linked temporally to simvastatin-mediated actin cytoskeletal rearrangement. Compared with Rho inhibition alone (Y-27632), simvastatin afforded additional protection against thrombin-mediated barrier dysfunction and attenuated LPS-induced EC permeability and superoxide generation. Statin-mediated inhibition of both Rac translocation to the cell membrane and superoxide production were attenuated by geranylgeranyl pyrophosphate (GGPP), indicating that these effects are due to geranylgeranylation inhibition. Finally, thrombin-induced EC permeability was modestly attenuated by reduced Rac1 expression (small interfering RNA), whereas these effects were made more pronounced by simvastatin pretreatment. Together, these data suggest EC barrier protection by simvastatin is due to dual inhibitory effects on RhoA and Rac1 as well as the attenuation of superoxide generation by EC NADPH oxidase and contribute to the molecular mechanistic understanding of the modulation of EC barrier properties by simvastatin.
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PMID:Endothelial cell barrier protection by simvastatin: GTPase regulation and NADPH oxidase inhibition. 1865 77

Reactive oxygen species generated by NADPH oxidase enhance aortic vascular smooth muscle cell proliferation and migration which play an important role in the pathophysiology of atherosclerosis. We investigated the role of NADPH oxidase in the cellular cholesterol metabolism in vascular smooth muscle cells using p47phox-deficient cells. Wild-type and p47phox knockout vascular smooth muscle cells were loaded with cholesterol for 72 h by using 10 mg/L cholesterol:methyl-beta-cyclodextrin complexes and then incubated with or without 0.3 mg/L thrombin for 10 min. Foam cell formation was determined by accumulation of intracellular cholesterol, oil Red O-stained lipid droplets. After cholesterol loading, cellular lipid droplets raised sharply, cellular cholesterol increased from (31.4+/-2.0) to (61.0+/-2.1) mg/g protein (P<0.05) in wild-type cells, and from (29.8+/-2.5) to (51.3+/-3.1) mg/g protein (P<0.05) in p47phox deficient cells, but the difference between the two cell types was not significant. Immunostaining showed decreased levels of smooth muscle alpha-actin and increased levels of macrophage marker Mac-2 in both wild-type and p47phox deficient vascular smooth muscle cells. One of the macrophage-related inflammation genes, monocyte chemoattractant protein-1 (MCP-1) expression did not change in both two cell types detected by immunostaining. Although additional incubating with thrombin, another macrophage-related inflammation gene, vascular cell adhesion molecule-1 (VCAM-1) expression was similar in all groups analyzed by real-time RT-PCR. However, the expression of ATP-binding cassette transporter A1 (ABCA1), acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), the key proteins in cellular cholesterol metabolism, were similarly increased (P<0.05) in both two cell types as determined by quantitative real-time RT-PCR and Western blot, and it was not related to the state of oxidative stress. Interestingly, the expression of adipophilin, the lipid droplet related protein, had the similar results with ABCA1 and ACAT1, but, in wild-type cells, its expression also increased merely incubating with thrombin as determined by quantitative real-time RT-PCR. Together, these results suggest that p47phox-dependent NADPH oxidase is not involved in transdifferentitation of vascular smooth muscle cells into macrophage-like state after cholesterol loading. Deleting p47phox gene does not affect the cellular cholesterol metabolism in vascular smooth muscle cells.
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PMID:[NADPH oxidase activity does not affect cellular cholesterol loading in vascular smooth muscle cells]. 1869 Mar 94

Platelet-activating factor (PAF) is a potent, bioactive phospholipid that acts on multiple cells and tissues through its G protein-coupled receptor (GPCR). PAF is not stored but is rapidly generated via enzymatic acetylation of the precursor 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPAF). The bioactivity of PAF is effectively and tightly regulated by PAF acetylhydrolases, which convert PAF back to lysoPAF. Previous studies report that lysoPAF is an inactive precursor and metabolite of PAF. However, lysoPAF has not been carefully studied in its own context. Here we report that lysoPAF has an opposing effect of PAF in the activation of neutrophils and platelets. Whereas PAF potentiates neutrophil NADPH oxidase activation, lysoPAF dose-dependently inhibits this function. Inhibition by lysoPAF is not affected by the use of a PAF receptor antagonist or genetic deletion of the PAF receptor gene. The mechanism of lysoPAF-mediated inhibition of neutrophils involves an elevation in the intracellular cAMP level, and pharmacological blockade of adenylyl cyclase completely reverses the inhibitory effect of lysoPAF. In addition, lysoPAF increases intracellular cAMP levels in platelets and inhibits thrombin-induced platelet aggregation, which can be reversed by inhibition of protein kinase A. These findings identify lysoPAF as a bioactive lipid with opposing functions of PAF and suggest a novel and intrinsic regulatory mechanism for balance of the potent activity of PAF.
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PMID:Opposing effects of platelet-activating factor and lyso-platelet-activating factor on neutrophil and platelet activation. 1893 Oct 35

Anti-platelet integrin GPIIIa49-66 antibody (Ab) induces complement-independent platelet oxidative fragmentation and death by generation of platelet peroxide following NADPH oxidase activation. A C-terminal 385-amino acid fragment of ADAMTS-18 (a disintegrin metalloproteinase with thrombospondin motifs produced in endothelial cells) induces oxidative platelet fragmentation in an identical kinetic fashion as anti-GPIIIa49-66 Ab. Endothelial cell ADAMTS-18 secretion is enhanced by thrombin and activated by thrombin cleavage to fragment platelets. Platelet aggregates produced ex vivo with ADP or collagen and fibrinogen are destroyed by the C-terminal ADAMTS-18 fragment. Anti-ADAMTS-18 Ab shortens the tail vein bleeding time. The C-terminal fragment protects against FeCI3-induced carotid artery thrombosis as well as cerebral infarction in a postischemic stroke model. Thus, a new mechanism is proposed for platelet thrombus clearance, via platelet oxidative fragmentation induced by thrombin cleavage of ADAMTS-18.
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PMID:C-terminal ADAMTS-18 fragment induces oxidative platelet fragmentation, dissolves platelet aggregates, and protects against carotid artery occlusion and cerebral stroke. 1952 Aug 14


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