NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-alpha.
We tested the hypothesis that the NAD(P)H oxidase-dependent generation of superoxide anion (O2-*) mediates tumor necrosis factor-alpha (TNF)-induced alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The NAD(P)H oxidase subcomponents p47phox and p22phox were assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species O2-* was measured by the fluorescence of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetatedi(acetoxymethyl ester), 5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester, and dihydroethidium. TNF treatment (50 ng/ml for 4.0 h) induced 1) p47phox translocation, 2) an increase in p22phox protein, 3) increased localization of p47phox with p22phox, 4) O2-* generation, and 5) increased permeability to albumin. p22phox antisense oligonucleotide prevented the TNF-induced effect on p22phox, p47phox, O2-*, and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in O2-* and permeability to albumin was also prevented by the O2-* scavenger Cu-Zn superoxide dismutase (100 U/ml). The results indicate that the activation of NAD(P)H oxidase, via the generation of O2-*, mediates TNF-induced barrier dysfunction in PMEM.
Gertzberg
Nancy
N
Center for Cardiovascular Science, Albany Medical College, NY 12208, USA.
Neumann
Paul
P
Rizzo
Victor
V
Johnson
Arnold
A
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D002452:Cell Count; D002470:Cell Survival; D002478:Cells, Cultured; D004396:Coloring Agents; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D026901:Membrane Transport Proteins; D008833:Microcirculation / Q000502:physiology; D009252:NADPH Dehydrogenase / Q000235:genetics / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D016376:Oligonucleotides, Antisense; D018384:Oxidative Stress / Q000502:physiology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011652:Pulmonary Circulation / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism; D014343:Trypan Blue; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000970:Antineoplastic Agents; D004396:Coloring Agents; D026901:Membrane Transport Proteins; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D014343:Trypan Blue