NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-α

We tested the hypothesis that the NAD(P) H oxidase-dependent generation of superoxide anion (O-2(is approximately equal to)) 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 p47(phox) and p22(phox) were assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species O-2(is approximately equal to) was measured by the fluorescence of 6-carboxy- 2', 7'-dichlorodihydrofluorescein diacetate-di( 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) p47(phox) translocation, 2) an increase in p22phox protein, 3) increased localization of p47phox with p22(phox), 4) O-2 (is approximately equal to) generation, and 5) increased permeability to albumin. p22(phox) antisense oligonucleotide prevented the TNF-induced effect on p22(phox), p47(phox), O-2 (is approximately equal to) , and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in O-2 (is approximately equal to) and permeability to albumin was also prevented by the O-2 (is approximately equal to) scavenger Cu-Zn superoxide dismutase ( 100 U/ml). The results indicate that the activation of NAD( P) H oxidase, via the generation of O-2 (is approximately equal to), mediates TNF-induced barrier dysfunction in PMEM.