title: TNF-alpha disruption of lung endothelial integrity: reduced integrin mediated adhesion to fibronectin.
abstract: Tumor necrosis factor-alpha (TNF-alpha) causes an increase in transendothelial protein permeability of confluent monolayers of calf pulmonary artery endothelial (CPAE) cells, and the addition of plasma fibronectin (pFn) to the culture medium can attenuate this increase in permeability. We determined if reduced integrin function had a role in decreased endothelial cell adhesion to immobilized Fn after exposure of the endothelial monolayers to TNF-alpha. TNF-alpha also causes a reorganization of the subendothelial Fn rich matrix and a significant loss in RGD-dependent adhesion of TNF-alpha treated CPAE cells to pFn coated surfaces. However, flow cytometry revealed no decrease in alpha(5)beta(1) or total beta(1) integrin expression on the surface of the CPAE cells after TNF-alpha. Reduced CPAE adhesion to immobilized Fn was, in part, due to a loss of beta(1)-integrin function since the beta(1)-integrin blocking antibody mAb 13 significantly (P < 0.05) prevented the adhesion of normal control CPAE cells but did not further reduce the adhesion of TNF-alpha-treated cells. In addition, antibodies which activate beta(1) integrins restored (P < 0.05) adhesion of TNF-alpha-treated cells to immobilized pFn but did not alter the adhesion of control cells. Despite reduced ability to adhere to immobilized Fn, TNF-alpha-treated CPAE monolayers demonstrated increased binding and incorporation of fluid-phase pFn into the subendothelial extracellular matrix (ECM) as measured by the analysis of the deoxycholate (DOC) detergent insoluble pool of (125)I-Fn in the cell layer. In contrast to the RGD-mediated adhesion of CPAE cells to matrix Fn, the increased binding of soluble pFn after TNF-alpha was not inhibited by RGD peptides or mAb 13. Thus reduced integrin-dependent adhesion of the CPAE cells to matrix Fn as well as disruption of the Fn matrix may contribute to the increased protein permeability of previously confluent endothelial monolayer after TNF-alpha. In addition, increased ability for the monolayer to incorporate fluid-phase Fn into the ECM after TNF-alpha via a non-beta(1)- integrin dependent mechanism may be a compensatory response to stabilize the Fn matrix and the endothelial barrier.
authors.affiliation: Department of Physiology and Cell Biology, Neil Hellman Medical Research Building, Albany Medical College, Albany, New York 12208, USA.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Rotundo
authors.forename: Theresa M
authors.initials: TM
authors.lastname: Curtis
authors.forename: Melissa D
authors.initials: MD
authors.lastname: Shah
authors.forename: Baochong
authors.initials: B
authors.lastname: Gao
authors.forename: Anthony
authors.initials: A
authors.lastname: Mastrangelo
authors.forename: Susan E
authors.initials: SE
authors.lastname: LaFlamme
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Saba
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000954:Antigens, Surface / Q000032:analysis; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D000939:Epitopes / Q000032:analysis; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D005434:Flow Cytometry; D007457:Iodine Radioisotopes; D008345:Manganese / Q000494:pharmacology; D008857:Microscopy, Interference; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D011651:Pulmonary Artery / Q000166:cytology; D017469:Receptors, Fibronectin / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D000939:Epitopes; D005353:Fibronectins; D007457:Iodine Radioisotopes; D009842:Oligopeptides; D017469:Receptors, Fibronectin; D014409:Tumor Necrosis Factor-alpha; D008345:Manganese; C047981:arginyl-glycyl-aspartic acid