TNF-alpha induces endothelial cell F-actin depolymerization, new actin synthesis, and barrier dysfunction.
Tumor necrosis factor-alpha (TNF-alpha) influences pulmonary vascular endothelial barrier function in vitro. We studied whether recombinant TNF-alpha (rTNF-alpha) regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed to human rTNF-alpha (1,000 U/ml) and evaluated for 1) transendothelial [14C]albumin flux, 2) F-actin organization with fluorescence microscopy, 3) F-actin quantitation by spectrofluorometry, and 4) monomeric G-actin levels by the deoxyribonuclease I inhibition assay. rTNF-alpha induced increments in [14C]albumin flux (P < 0.04) and intercellular gap formation at > or = 2-6 h. During this same time, the endothelial F-actin pool decreased (P = 0.0064), with reciprocal increases in the G-actin pool (P < 0.0001). Prior F-actin stabilization with phallicidin protected against the rTNF-alpha-induced increments in G-actin (P < 0.002) as well as changes in barrier function (P < 0.01). Prior protein synthesis inhibition enhanced the rTNF-alpha-induced decrement in F-actin (P < 0.0001), blunted the G-actin increment (P < 0.002), and increased rTNF-alpha-induced changes in endothelial barrier function (P < 0.003). Therefore, rTNF-alpha induces pulmonary vascular endothelial F-actin depolymerization, intercellular gap formation, and barrier dysfunction. rTNF-alpha also increased total actin (P < 0.02) and new actin synthesis (P < 0.002), which may be a compensatory endothelial cell response to rTNF-alpha-induced F-actin depolymerization.
Goldblum
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Department of Medicine, Department of Veterans Affairs Medical Center, Baltimore, Maryland.
Ding
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Campbell-Washington
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D000199:Actins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015151:Immunoblotting; D007700:Kinetics; D008856:Microscopy, Fluorescence; D011651:Pulmonary Artery; D011994:Recombinant Proteins / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000199:Actins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D003571:Cytochalasin B; D003513:Cycloheximide