We have shown previously that the shear stress induced augmentation of endothelial NO synthase expression is dependent on the composition of the extracellular matrix. This observation led us to suggest that the extracellular matrix may decisively modulate the responsiveness of endothelial cells to shear stress. The composition of the matrix can be modulated by an altered cellular synthesis of matrix proteins as well as by the effects of proteases released from endothelial cells or from the extracellular matrix. Indeed, work of several laboratories has demonstrated that endothelial cell exposed to shear stress react by altering the release of proteases, thereby altering the extracellular matrix composition in a way that may lead to cell activation via integrins. As an example, we could demonstrate that exposure to shear stress (16 dyn/cm2) elicited the release of a protease with elastolytic properties from porcine aortic endothelial cells (PAEC). The protease dependent activation of the integrin αvβ3, and a signalling cascade involving p38 and Hsp 27, ultimately resulted in the release of fibroblast growth factor-2 (FGF-2) into the supernatant. Though the released protease is not identical with known elastases of identical molecular weight (including neutrophil elastase and proteinase 3) it was found that addition of a pancreatic elastase induced, similarly as shear stress, the release of FGF-2. We, therefore, studied whether treatment of the extracellular matrix of cultured PAECs with exogenous elastase could activate regulatory compounds of the αvβ3 related signalling pathway as shear stress. To this end, PAECs grown to confluence on a matrix which initially consisted of collagen I were stimulated for 60 minutes with pancreatic elastase (0.5 U/ml) and afterwards stained for laminin, elastin or fibronectin. Interestingly, while the macroscopic distribution of elastin (as studied by immunofluorescence) was not changed during elastase incubation, the extracellular network of laminin and especially that of fibronectin was clearly altered suggesting that these matrix alterations could possibly lead to re-clustering of integrins. Indeed, addition of exogenous elastase led to a transient disappearance of existing focal contacts followed by a distinct topical re-arrangement. This effect correlated with time dependent changes of Tyr phosphorylation of focal adhesion kinase (FAK). Inhibition of αvβ3 integrins by an antibody (abciximabR) did prevent p38 phosphorylation and FGF-2 release both, under conditions of shear stress and exposure to the exogenous elastase. We, therefore, conclude that a significant component of shear stress induced signalling events occurs at the abluminal side of endothelial cells via matrix dependent integrin signalling. This offers a novel pathway regulating the apparent responsiveness of endothelial cells to shear stress which may play a prominent role in growth factor dependent vascular remodelling processes elicited by shear stress.