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

Abstract

We tested the hypothesis that the NAD(P)H oxidase-dependent generation of superoxide anion ([Formula: see text]) mediates tumor necrosis factor-α (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 p47phoxand p22phoxwere assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species [Formula: see text] 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) p47phoxtranslocation, 2) an increase in p22phoxprotein, 3) increased localization of p47phoxwith p22phox, 4) [Formula: see text] generation, and 5) increased permeability to albumin. p22phoxantisense oligonucleotide prevented the TNF-induced effect on p22phox, p47phox, [Formula: see text], and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in [Formula: see text] and permeability to albumin was also prevented by the [Formula: see text] scavenger Cu-Zn superoxide dismutase (100 U/ml). The results indicate that the activation of NAD(P)H oxidase, via the generation of [Formula: see text], mediates TNF-induced barrier dysfunction in PMEM.