Fibronectin attenuates increased endothelial monolayer permeability after RGD peptide, anti-alpha 5 beta 1, or TNF-alpha exposure

Abstract

Endothelial permeability can be altered by tumor necrosis factor-alpha (TNF-alpha), a cytokine released in association with inflammation-induced tissue injury. In the subendothelial matrix, fibronectin (Fn) influences endothelial cell adhesion by the interaction of integrins with RGD and non-RGD attachment sites in Fn. We compared the effect of TNF-alpha, RGD-containing peptides (GRGDSP), or antibody to alpha 5 beta 1-integrins on the protein permeability of bovine lung endothelial monolayers as assessed by transendothelial 125I-labeled albumin clearance. We also examined the influence of purified human plasma fibronectin (hFn) on this permeability response. TNF-alpha, RGD peptides, and antibodies to alpha 5 beta 1-integrins elicited a dose- and time-dependent increase in protein permeability as well as a reorganization and/or disruption of the endogenous Fn matrix. A control RGE peptide (GRGESP) as well as immunoglobulin G purified from nonimmune rabbit serum did not increase endothelial protein permeability or disrupt the endogenous fibrillar Fn pattern in the matrix. Likewise, a LDV peptide derived from the alternatively spliced type III connecting segment (IIICS) within bovine Fn (bFn) was unable to increase permeability of the bovine endothelial monolayer. Co-incubation of purified soluble hFn (300 or 600 micrograms/ml) with either TNF-alpha, the RGD peptide, or the antibody to alpha 5 beta 1-integrins prevented the increase in endothelial permeability. This protective effect was also observed when the purified hFn (600 micrograms/ml) was added after the TNF-alpha-induced increase in endothelial permeability had taken place. Immunofluorescent analysis confirmed the incorporation of the hFn into the subendothelial matrix and its co-localization with the endogenous bFn. The similar alteration of the subendothelial matrix after exposure to RGD peptides, anti-alpha 5 beta 1-antibodies, or TNF-alpha, coupled with the ability for hFn to attenuate the permeability increase typically elicited by all three agents, suggests that disruption of cell-matrix interactions may be the mechanism by which TNF-alpha alters endothelial permeability.

β3-Integrin Regulates Vascular Endothelial Growth Factor-A–Dependent Permeability

“…Evidence that integrins are involved in maintaining this barrier comes from studies showing that treating endothelial monolayers or isolated coronary venules with RGD peptides (small molecules that disrupt interactions between integrins and the ECM) increase their permeability. [43][44][45] The fact that the increased permeability in these in vitro studies is prevented by the addition of soluble fibronectin or vitronectin suggests that interactions between integrins and the ECM influence vessel permeability. From the data we present here, however, we are able to conclude that ␤3-integrin is not essential for maintaining an intact and functional endothelial barrier.…”

Fibrinogen induces endothelial cell permeability

“…Treatment of EC with anti-α 5 β 1 integrin antibody has been reported to increase endothelial monolayer permeability [39,41]. However, the antibodies used in these studies were either human placenta α 5 β 1 goat antiserum [39], or polyclonal antibody with no indication of an ability to specifically block α 5 β 1 integrin function [41].…”

“…However, the antibodies used in these studies were either human placenta α 5 β 1 goat antiserum [39], or polyclonal antibody with no indication of an ability to specifically block α 5 β 1 integrin function [41]. Therefore, it is possible that these antibodies, upon binding, activate α 5 β 1 integrin, or other integrins that may not even be Fg receptors.…”

Attachment of hyaluronan to metallic surfaces

“…Cell integrins are transmembrane proteins on the cell surface that adhere to the proteins of the ECM such as collagen, fibronectin, or vitronectin. These and other adhesive proteins contain short peptide sequences such as RGD, REDV, YIGSR, PHSRN, KNEED, and PRRARV, [37][38][39][40][41][42][43][44][45][46][47][48][49] which are bound by the integrins to promote cell adhesion, spreading, and proliferation. Although significant work has been done to attach proteins and peptides to metal oxide particles 21 and silicon oxide on Si wafers, 26 relatively little research has been reported on attachment of polypeptides to macroscopic metallic surfaces.…”