Vascular remodelling can be often observed as consequence of chronic changes in blood flow. Whereas formation and modification of collaterals also known as arteriogenesis is induced by elevated flow, a decrease in flow may result in plaque formation or atherosclerosis. Both processes are tightly controlled not only by specific growth factors and proteases but also by their individual inhibitors. We have previously shown that porcine aortic endothelial cells (PAEC) when exposed to shear stress (16 dyn/cm²) actively release FGF-2, a growth factor, which is known for its proangiogenic activity. The release mechanism was dependent on the activation of the integrin a_vb₃, because its inhibition reduced shear stress induced FGF-2 release significantly. The aim of this study was to investigate the process of shear stress induced integrin activation and to identify key steps of the corresponding signal cascades.

PAEC exposed to shear stress showed a significant higher elastolytic activity in their supernatants compared to corresponding static control cells (static 0.0619 U/ml ± 0.0206, shear 0.2036 U/ml ± 0.0577). Inhibition of the integrin a_vb₃ (Abciximab, 1 mg/ml) had no influence on the shear stress induced elastase release, indicating that although activation of a_vb₃ is a necessary step for shear stress mediated FGF-2 release it is not relevant for the release of elastase. Moreover, in parallel experiments we were able to show that this shear stress induced elastase activity led to an activation of integrin a_vb₃. Likewise, as proof of concept, the exogenous application of elastase activated a_vb₃ and induced the release of FGF-2 similar as shear stress did. To further analyze elastase mediated a_vb₃ activation, we performed coimmunoprecipitation experiments with integrin a_vb₃ and its adaptor protein Shc, indicating an active integrin clustering. After 10 minutes of elastase stimulation a significant higher amount of all three Shc isoforms was complexed with the integrin (44 kDa: 2.7 fold more, 52 kDa: 1.7 fold more, 66 kDa: 3.3 fold more). By the use of specific elastase inhibitors (MeOSuc-Ala-Ala-Pro-Val-chloromethylketone, 50 mM) it could be verified that also shear stress induced activation of integrin a_vb₃ was elastase mediated.

Our results demonstrate that shear stress induced FGF-2 release is proteolytically controlled by an elastase dependent activation of integrin a_vb₃.