ABSTRACT: Clinical observations and epidemiological surveys indicated that the prevalence of hypertension and heart diseases is increased in cold regions or during winter. Cold exposure increased NADPH oxidase gp91(phox) protein expression in heart, kidneys, and aorta in rats. The aim of this study was to investigate if RNA interference (RNAi) silencing of gp91(phox) would attenuate cold-induced hypertension and cardiovascular and renal damage. The recombinant adeno-associated virus serotype 2 (AAV-2) vector carrying gp91(phox)-shRNA (gp91-shRNA) was constructed for inhibiting gp91(phox) protein expression in cold-exposed rats. Blood pressure (BP) was monitored using a telemetry system. BP was increased in the Control-shRNA and PBS groups within 1 week of exposure to moderate cold (5°C) and reached a plateau after 7 weeks. The cold-induced increase in BP was attenuated significantly by intravenous delivery of gp91-shRNA (1.25×10(10) particles/rat, 0.5?mL). One single dose of gp91-shRNA controlled hypertension for up to 10 weeks. In addition, gp91-shRNA reversed cold-induced vascular dysfunction. gp91-shRNA abolished the cold-induced up-regulation of gp91(phox) protein expression in heart, kidneys, and aorta, confirming effective silencing of gp91(phox). The cold-induced increases in NADPH oxidase activity and superoxide production were eliminated by silencing of gp91(phox), suggesting that the cold-induced up-regulation of NADPH oxidase activity may be attributed to the increased gp91(phox) protein expression. RNAi silencing of gp91(phox) abolished cold-induced cardiac and renal hypertrophy and attenuated aortic, coronary, and renal remodeling. The up-regulation of gp91(phox) may play a critical role in cold-induced cardiovascular dysfunction and organ damage. AAV delivery of gp91-shRNA may be a new and effective therapeutic approach for cold-related cardiovascular disorders.
Project description:Myeloperoxidase (MPO) is an important enzyme involved in the genesis and development of atherosclerosis. Vascular peroxidase 1 (VPO1) is a newly discovered member of the peroxidase family that is mainly expressed in vascular endothelial cells and smooth muscle cells and has structural characteristics and biological activity similar to those of MPO. Our specific aims were to explore the effects of VPO1 on endothelial cell apoptosis induced by oxidized low-density lipoprotein (ox-LDL) and the underlying mechanisms. The results showed that ox-LDL induced endothelial cell apoptosis and the expression of VPO1 in endothelial cells in a concentration- and time-dependent manner concomitant with increased intracellular reactive oxygen species (ROS) and hypochlorous acid (HOCl) generation, and up-regulated protein expression of the NADPH oxidase gp91(phox) subunit and phosphorylation of p38 MAPK. All these effects of ox-LDL were inhibited by VPO1 gene silencing and NADPH oxidase gp91(phox) subunit gene silencing or by pretreatment with the NADPH oxidase inhibitor apocynin or diphenyliodonium. The p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor DEVD-CHO significantly inhibited ox-LDL-induced endothelial cell apoptosis, but had no effect on intracellular ROS and HOCl generation or the expression of NADPH oxidase gp91(phox) subunit or VPO1. Collectively, these findings suggest for the first time that VPO1 plays a critical role in ox-LDL-induced endothelial cell apoptosis and that there is a positive feedback loop between VPO1/HOCl and the now-accepted dogma that the NADPH oxidase/ROS/p38 MAPK/caspase-3 pathway is involved in ox-LDL-induced endothelial cell apoptosis.
Project description:Clinical trials and animal studies have revealed a role for the renin-angiotensin system in the enhanced thrombus development that is associated with hypertension. Because T lymphocytes have been implicated in the vascular dysfunction and blood pressure elevation associated with increased angiotensin II (Ang II) levels, we evaluated the role of the adaptive immune system in mediating the enhanced thrombosis during Ang II-induced hypertension. Light/dye-induced thrombosis was induced in cremaster arterioles of wild-type, immunodeficient Rag-1(-/-), CD8(+), or CD4(+) lymphocyte-deficient and NADPH oxidase (gp91(phox))-deficient mice implanted with an Ang II-loaded pump for 2 weeks. Chronic Ang II infusion enhanced arteriolar thrombosis in wild-type mice but not in Rag-1(-/-), CD4(+) T-cell-deficient, or gp91(phox-/-) mice. CD8(+) T-cell(-/-) mice exhibited partial protection. Adoptive transfer of T cells derived from wild-type or gp91(phox-/-) mice into Rag-1(-/-) restored the prothrombotic phenotype induced by Ang II. T lymphocytes (CD4(+) and, to a lesser extent, CD8(+)) play a major role in mediating the accelerated microvascular thrombosis associated with Ang II-induced hypertension. NADPH oxidase-derived reactive oxygen species, produced by cells other than T lymphocytes, also appear critical for the Ang II-enhanced, T cell-dependent thrombosis response.
Project description:Sepsis syndrome is characterized by a dysregulated inflammatory response to infection. NADPH oxidase-dependent reactive oxygen species (ROS) play significant roles in the pathophysiology of sepsis. We previously showed that disruption of Nrf2, a master regulator of antioxidant defenses, caused a dysregulation of innate immune response that resulted in greater mortality in a polymicrobial sepsis and LPS shock model; however, the underlying mechanisms are unclear. In the current study, compared with wild-type (Nrf2(+/+)) macrophages, we observed greater protein kinase C-induced NADPH oxidase-dependent ROS generation in Nrf2-disrupted (Nrf2(-/-)) macrophages that was modulated by glutathione levels. To address the NADPH oxidase-mediated hyperinflammatory response and sepsis-induced lung injury and mortality in Nrf2(-/-) mice, we used double knockout mice lacking Nrf2 and NADPH oxidase subunit, gp91(phox) (Nrf2(-/-)//gp91(phox-/-)). Compared with Nrf2(+/+) macrophages, LPS induced greater activation of TLR4 as evident by TLR4 surface trafficking and downstream recruitment of MyD88 and Toll/IL-1R domain-containing adaptor in Nrf2(-/-) macrophages that was diminished by ablation of gp91(phox). Similarly, phosphorylation of IkappaB and IFN regulatory factor 3 as well as cytokine expression was markedly higher in Nrf2(-/-) macrophages; whereas, it was similar in Nrf2(+/+) and Nrf2(-/-)//gp91(phox-/-). In vivo studies showed greater LPS-induced pulmonary inflammation in Nrf2(-/-) mice that was significantly reduced by ablation of gp91(phox). Furthermore, LPS shock and polymicrobial sepsis induced early and greater mortality in Nrf2(-/-) mice; whereas, Nrf2(-/-)//gp91(phox-/-) showed prolonged survival. Together, these results demonstrate that Nrf2 is essential for the regulation of NADPH oxidase-dependent ROS-mediated TLR4 activation and lethal innate immune response in sepsis.
Project description:BACKGROUND/AIMS:Activated stellate cells are considered the principal mediators of chronic alcoholic pancreatitis/fibrosis. However the mechanisms of alcohol action on pancreatic stellate cells (PaSCs) are poorly understood. The aims of this study were to determine the presence and role of the NADPH oxidase system in mediating alcohol effects on PaSCs with specific emphasis on proliferation. METHODS:PaSC NADPH oxidase components mRNA and protein were determined by RT-PCR and Western blot. The NADPH oxidase activity was measured by detecting the production of reactive oxygen species using lucigenin-derived chemiluminescence assay. PaSC DNA synthesis, a measure of proliferation, was performed by determining the [3H] thymidine incorporation into DNA. RESULTS:mRNA for NADPH oxidase components Nox1, gp91(phox), Nox4, p22(phox), p47(phox) and p67(phox) and protein for NADPH oxidase subunits gp91(phox), p22(phox), p47(phox) and p67(phox) are present in PaSCs. Treatment with platelet-derived growth factor (PDGF) significantly increased the NADPH oxidase activity and DNA synthesis in cultured PaSCs. Alcohol treatment markedly augmented both the NADPH oxidase activity and the DNA synthesis caused by PDGF, which was prevented by antioxidant N-acetyl-L-cysteine, ROS scavenger tiron, and the NADPH oxidase inhibitor diphenylene iodium. The effects of PDGF on NADPH oxidase activity and DNA synthesis were prevented in PaSCs isolated from the pancreas of mice with a genetic deficiency of p47(phox). CONCLUSIONS:Ethanol causes proliferation of stellate cells by augmenting the activation of the cell's NADPH oxidase system stimulated by PDGF. These results provide new insights into the mechanisms of alcohol-induced fibrosing disorders.
Project description:Carbon monoxide (CO), a byproduct of heme catabolism by heme oxygenase (HO), confers potent antiinflammatory effects. Here we demonstrate that CO derived from HO-1 inhibited Toll-like receptor (TLR) 2, 4, 5, and 9 signaling, but not TLR3-dependent signaling, in macrophages. Ligand-mediated receptor trafficking to lipid rafts represents an early event in signal initiation of immune cells. Trafficking of TLR4 to lipid rafts in response to LPS was reactive oxygen species (ROS) dependent because it was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase, and in gp91(phox)-deficient macrophages. CO selectively inhibited ligand-induced recruitment of TLR4 to lipid rafts, which was also associated with the inhibition of ligand-induced ROS production in macrophages. TLR3 did not translocate to lipid rafts by polyinosine-polycytidylic acid (poly(I:C)). CO had no effect on poly(I:C)-induced ROS production and TLR3 signaling. The inhibitory effect of CO on TLR-induced cytokine production was abolished in gp91(phox)-deficient macrophages, also indicating a role for NADPH oxidase. CO attenuated LPS-induced NADPH oxidase activity in vitro, potentially by binding to gp91(phox). Thus, CO negatively controlled TLR signaling pathways by inhibiting translocation of TLR to lipid rafts through suppression of NADPH oxidase-dependent ROS generation.
Project description:Flavocytochrome b(558), the catalytic core of the phagocyte NADPH oxidase (NOX2), mediates electron transfer from NADPH to molecular oxygen to generate superoxide, the precursor of highly ROS for host defense. Flavocytochrome b(558) is an integral membrane heterodimer consisting of a large glycosylated subunit, gp91(phox), and a smaller subunit, p22(phox). We recently showed in murine macrophages that flavocytochrome b(558) localizes to the PM and Rab11-positive recycling endosomes, whereas in primary hMDMs, gp91(phox) and p22(phox) reside in the PM and the ER. The antimicrobial activity of macrophages, including ROS production, is greatly enhanced by IFN-?, but how this is achieved is incompletely understood. To further define the mechanisms by which IFN-? enhances macrophage NADPH oxidase activity, we evaluated changes in flavocytochrome b(558) expression and localization, along with NADPH oxidase activity, in IFN-? stimulated RAW 264.7 cells and primary murine BMDMs and hMDMs. We found that enhanced capacity for ROS production is, in part, a result of increased protein expression of gp91(phox) and p22(phox) but also demonstrate that IFN-? induced a shift in the predominant localization of gp91(phox) and p22(phox) from intracellular membrane compartments to the PM. Our results are the first to show that a cytokine can change the distribution of macrophage flavocytochrome b(558) and provide a potential, new mechanism by which IFN-? modulates macrophage antimicrobial activity. Altogether, our data suggest that the mechanisms by which IFN-? regulates antimicrobial activity of macrophages are more complex than previously appreciated.
Project description:Our previous studies have shown that NOD-like receptor protein (NALP3) inflammasome activation is importantly involved in podocyte dysfunction and glomerular sclerosis induced by hyperhomocysteinemia (hHcys). The present study was designed to test whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated redox signaling contributes to homocysteine (Hcys)-induced activation of NALP3 inflammasomes, an intracellular inflammatory machinery in podocytes in vitro and in vivo.In vitro confocal microscopy and size-exclusion chromatography revealed that upon NADPH oxidase inhibition by gp91(phox) siRNA, gp91ds-tat peptide, diphenyleneiodonium, or apocynin, aggregation of inflammasome proteins NALP3, apoptosis-associated speck-like protein (ASC), and caspase-1 was significantly attenuated in mouse podocytes. This NADPH oxidase inhibition also resulted in diminished Hcys-induced inflammasome activation, evidenced by reduced caspase-1 activity and interleukin-1? production. Similar findings were observed in vivo where gp91(phox-/-) mice and mice receiving a gp91ds-tat treatment exhibited markedly reduced inflammasome formation and activation. Further, in vivo NADPH oxidase inhibition protected the glomeruli and podocytes from hHcys-induced injury as shown by attenuated proteinuria, albuminuria, and glomerular sclerotic changes. This might be attributed to the fact that gp91(phox-/-) and gp91ds-tat-treated mice had abolished infiltration of macrophages and T-cells into the glomeruli during hHcys.Our study for the first time links NADPH oxidase to the formation and activation of NALP3 inflammasomes in podocytes.Hcys-induced NADPH oxidase activation is importantly involved in the switching on of NALP3 inflammasomes within podocytes, which leads to the downstream recruitment of immune cells, ultimately resulting in glomerular injury and sclerosis.
Project description:The role of NADPH oxidase activation in pneumonia is complex because reactive oxygen species contribute to both microbial killing and regulation of the acute pulmonary infiltrate. The relative importance of each role remains poorly defined in community-acquired pneumonia.We evaluated the contribution of NADPH oxidase-derived reactive oxygen species to the pathogenesis of pneumococcal pneumonia, addressing both the contribution to microbial killing and regulation of the inflammatory response.Mice deficient in the gp91(phox) component of the phagocyte NADPH oxidase were studied after pneumococcal challenge.gp91(phox)(-/-) mice demonstrated no defect in microbial clearance as compared with wild-type C57BL/6 mice. A significant increase in bacterial clearance from the lungs of gp91(phox)(-/-) mice was associated with increased numbers of neutrophils in the lung, lower rates of neutrophil apoptosis, and enhanced activation. Marked alterations in pulmonary cytokine/chemokine expression were also noted in the lungs of gp91(phox)(-/-) mice, characterized by elevated levels of tumor necrosis factor-alpha, KC, macrophage inflammatory protein-2, monocyte chemotactic protein-1, and IL-6. The greater numbers of neutrophils in gp91(phox)(-/-) mice were not associated with increased lung injury. Levels of neutrophil elastase in bronchoalveolar lavage were not decreased in gp91(phox)(-/-) mice.During pneumococcal pneumonia, NADPH oxidase-derived reactive oxygen species are redundant for host defense but limit neutrophil recruitment and survival. Decreased NADPH oxidase-dependent reactive oxygen species production is well tolerated and improves disease outcome during pneumococcal pneumonia by removing neutrophils from the tight constraints of reactive oxygen species-mediated regulation.
Project description:The generation of superoxide by the NADPH oxidase of neutrophils is accompanied by the efflux of H+ ions through a H+ channel. gp91-phox, a protein component of the oxidase, has been shown previously to function as a H+ channel [Henderson, Banting and Chappell (1995) J. Biol. Chem. 270, 5909-5916]. We have constructed a CHO cell line (CHO-N) that expresses an N-terminal fragment of gp91-phox containing the predicted multiple transmembrane domains of the protein. These cells exhibit H+ fluxes in response to an imposed proton motive force and in the presence of arachidonate (to open the channel). The H+ fluxes were indistinguishable from those observed in cells expressing full-length gp91-phox. Therefore the N-terminal 230 amino acids of gp91-phox contain all that is required to function as the NADPH oxidase-associated H+ channel.
Project description:NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NADPH oxidase 2 (NOX2)-type complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F-actin content, retrograde F-actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91(phox) localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40(phox) . p40(phox) itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91(phox) and p40(phox) with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in colocalization of p40(phox) with NOX2/gp91(phox) at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth. We have previously shown that reactive oxygen species (ROS) are critical for actin organization and dynamics in neuronal growth cones as well as neurite outgrowth. Here, we report that the cytosolic subunit p40(phox) of the NOX2-type NADPH oxidase complex is partially associated with F-actin in neuronal growth cones, while ROS produced by this complex regulates F-actin dynamics and neurite growth. These findings provide evidence for a bidirectional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones.