INCORPORATION OF FIBRONECTIN INTO MATRIX DECREASES TNF-INDUCED INCREASE IN ENDOTHELIAL MONOLAYER PERMEABILITY

Plasma fibronectin, a dimeric adhesive protein in blood, incorporates into the subendothelial and interstitial matrix in the lung especially during vascular injury. Fibronectin in the matrix is believed to infl uence cell-cell interaction and endothelial cell adhesion to the colla gen-rich extracellular matrix. We previously observed that addition of purified soluble human plasma fibronectin (hFn) to cultured pulmonary endothelial monolayers attenuates the increase in protein permeabilit y of such monolayers exposed to tumor necrosis factor-alpha (TNF-alpha ). In the current study, we determined the specificity of this permeab ility response to fibronectin by comparing hFn to two other purified a dhesive proteins in human plasma, i.e., vitronectin (Vn) and fibrinoge n (Fg). We also determined whether matrix incorporation was essential for this hFn-mediated protective response by comparing normal intact h Fn to either hFn alkylated with N-ethylmaleimide (NEM) or to purified 160/180-kDa hFn fragments, since these alternate forms of fibronectin are believed to exhibit limited ability to incorporate into matrix. Ca lf pulmonary artery endothelial (CPAE) monolayers (3-4 days postseedin g) were exposed to human recombinant TNF-alpha for 18 h at a medium co ncentration of 200 U/ml followed by assessment of protein permeability using transendothelial I-125-labeled albumin clearance. Dimeric hFn ( 600 ug/ml) significantly (P < 0.05) reduced the TNF-induced increase i n endothelial monolayer permeability. Vn or Fg, added at equal molar c oncentrations to the hFn, were unable to attenuate endothelial permeab ility. Immunofluorescent analysis utilizing antibodies specific to eit her hFn, human Vn, or human Fg revealed incorporation of the exogenous hFn into the extracellular matrix, but no matrix incorporation of Vn or Fg. Both NEM-treated dimeric hFn as well as purified 160/180-kDa fr agments of hFn, which cannot incorporate into the matrix, were also un able to prevent the TNF-induced increase in protein permeability. Thus the ability for soluble hFn to reduce the TNF-induced increase in lun g endothelial monolayer permeability was specific and dependent on its incorporation into the extracellular matrix.