In previous experiments we could show that porcine aortic endothelial cells (PAEC) that are exposed to laminar shear stress (16 dyn/cm²) release an enzyme with elastolytic activity that causes the release of fibroblast growth factor 2 (FGF-2). Moreover we found that an activation of the integrin avß3 by an as yet unknown mechanism was essential for the shear induced FGF-2 release. We therefore investigated whether treatment of the extracellular matrix of cultured PAECs with elastase could activate regulatory compounds of the avß3 related focal adhesion complex.

PAECs (passage 3 - 5) were incubated with pancreatic elastase (0,5 U/ml) for 5 to 60 minutes and the distribution and phosphorylation of the focal adhesion kinase (FAK) and the adaptor molecule SHC was studied by immunohistochemistry and by western blot. In all series at least 3 experiments were performed. After 5 minutes of stimulation with elastase focal contacts virtually disappeared. However, after 10 minutes they reappeared in a distinctly different arrangement with further changes during the remaining observation period. A similar time-dependency of FAK phosphorylation could be observed: Tyr397 as well as Tyr576/577 and Tyr925 were dephosphorylated after 5 minutes and afterwards rephosphorylated. Furthermore we found an increased interaction of the SHC adapator molecule isoforms with the integrin avß3 after 10 min of elastase treatment (coimmunoprecipitation, up to 3,3 fold) similar as described previously in bovine aortic endothelial cells under exposure to shear stress. These findings demonstrate that elastase, probably by partial proteolysis of matrix proteins, induces a dynamic change of focal adhesion related signaling in endothelial cells. Whether this is due to exposure of new integrin binding sites (matricryptins) or an altered matrix arrangement remains to be shown. The signaling processes observed here appear to be an essential part of the shear induced release of FGF-2 from endothelial cells.