Ascorbate inhibits NADPH oxidase subunit p47phox expression in microvascular endothelial cells.
The production of reactive oxygen species (ROS) is central to the etiology of endothelial dysfunction in sepsis. Endothelial cells respond to infection by activating NADPH oxidases that are sources of intracellular ROS and potential targets for therapeutic administration of antioxidants. Ascorbate is an antioxidant that accumulates in these cells and improves capillary blood flow, vascular reactivity, arterial blood pressure, and survival in experimental sepsis. Therefore, the present study tested the hypothesis that ascorbate regulates NADPH oxidases in microvascular endothelial cells exposed to septic insult. We observed that incubation with Escherichia coli lipopolysaccharide (LPS) and interferon-gamma (IFNgamma) increased NADPH oxidase activity and expression of the enzyme subunit p47phox in mouse microvascular endothelial cells of skeletal muscle origin. Pretreatment of the cells with ascorbate prevented these increases. Polyethylene glycol-conjugated catalase and selective inhibitors of Jak2 also abrogated induction of p47phox. Exogenous hydrogen peroxide induced p47phox expression that was prevented by pretreatment of the cells with ascorbate. LPS+IFNgamma or hydrogen peroxide activated the Jak2/Stat1/IRF1 pathway and this effect was also inhibited by ascorbate. In conclusion, ascorbate blocks the stimulation by septic insult of redox-sensitive Jak2/Stat1/IRF1 signaling, p47phox expression, and NADPH oxidase activity in microvascular endothelial cells. Because endothelial NADPH oxidases produce ROS that can cause endothelial dysfunction, their inhibition by ascorbate may represent a new strategy for sepsis therapy.
Wu
Feng
F
Department of Exercise and Nutrition Sciences, University at Buffalo, 3435 Main Street, Buffalo, NY 14214-8028, USA. fengwu@buffalo.edu
Schuster
David P
DP
Tyml
Karel
K
Wilson
John X
JX
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D004926:Escherichia coli / Q000378:metabolism; D005121:Extremities; D006861:Hydrogen Peroxide / Q000494:pharmacology; D050856:Interferon Regulatory Factor-1 / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology; D053614:Janus Kinase 2 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008833:Microcirculation; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D050794:STAT1 Transcription Factor / Q000378:metabolism
D000975:Antioxidants; D050856:Interferon Regulatory Factor-1; C494258:Irf1 protein, mouse; D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; D050794:STAT1 Transcription Factor; C494080:Stat1 protein, mouse; D007371:Interferon-gamma; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D053614:Janus Kinase 2; D001205:Ascorbic Acid