Role of alpha(v)beta(3)-integrin in TNF-alpha-induced endothelial cell migration.

Tumor necrosis factor-alpha (TNF-alpha), one of the major inflammatory cytokines, is known to influence endothelial cell migration. In this study, we demonstrate that exposure of calf pulmonary artery endothelial cells to TNF-alpha caused an increase in the formation of membrane protrusions and cell migration. Fluorescence microscopy revealed an increase in alpha(v)beta(3) focal contacts but a decrease in alpha(5)beta(1) focal contacts in TNF-alpha-treated cells. In addition, both cell-surface and total cellular expression of alpha(v)beta(3)-integrins increased significantly, whereas the expression of alpha(5)beta(1)-integrins was unaltered. Only focal contacts containing alpha(v)beta(3)- but not alpha(5)beta(1)-integrins were present in membrane protrusions of cells at the migration front. In contrast, robust focal contacts containing alpha(5)beta(1)-integrins were present in cells behind the migration front. A blocking antibody to alpha(v)beta(3), but not a blocking antibody to alpha(5)-integrins, significantly inhibited TNF-alpha-induced cell migration. These results indicate that in response to TNF-alpha, endothelial cells may increase the activation and ligation of alpha(v)beta(3) while decreasing the activation and ligation of alpha(5)beta(1)-integrins to facilitate cell migration, a process essential for vascular wound healing and angiogenesis.

TNF-alpha disruption of lung endothelial integrity: reduced integrin mediated adhesion to fibronectin.

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.