Abstract 17426: Kindlin-2 Mediates VEGF-Induced Integrin Activation in Endothelial Cells and Angiogenesis
Integrins are mediating adhesion to extracellular matrix (ECM) proteins and are essential for angiogenesis. In our previous work, we identified Rap1 as a mediator of integrin affinity in endothelial cells (EC) and angiogenesis. However, the molecular regulation of integrin activity in EC downstream of Rap1 and their contribution to angiogenesis are unclear. The cytoskeletal proteins talin-1 and kindlin-2 bind to the cytoplasmic tail of beta1-integrin subunits, thereby synergistically mediating the activation of adhesion in several cellular systems. Silencing of either talin-1 or kindlin-2 with siRNA significantly blocked the VEGF-stimulated angiogenic sprouting of HUVEC in a 3-dimensional spheroidal EC culture assay by 67±4 % and 33±7 % and blocked tube formation in the matrigel assay by 57±16 % and 49±5 %, respectively, in comparison to scrambled siRNA-transfected EC. In addition, silencing of either kindlin-2 or talin-1 with siRNA significantly blocked the VEGF-induced migration of EC by 56±11 % and 96±1.4 %, respectively. Integrin-dependent adhesion to ECM proteins is a prerequisite for migration. Silencing of either kindlin-2 or talin-1 significantly blocked, while overexpression of kindlin-2-GFP increased HUVEC adhesion to the beta1-integrin ligand, fibronectin. In order to get insight in the regulation of integrin activity by kindlin-2 in EC, we performed immunofluorescent stainings with an antibody recognizing activated beta1-integrins. Short term stimulation of EC with VEGF increased beta1-integrin activity and induced the co-localization of kindlin-2-GFP with activated beta1-integrins. Interestingly, silencing of kindlin-2 reduced the VEGF-induced activation of beta1-integrin-affinity, suggesting that kindlin-2 regulates integrin activation downstream of VEGF signaling. We additionally explored the role of kindlin-2 as potential downstream mediator of Rap1, which is rapidly activated by VEGF stimulation. Indeed, silencing of kindlin-2 blocked the Rap1-induced activation of beta1-integrins. Taken together, our data reveal a critical role of the protein kindlin-2 as downstream effector of the VEGF/Rap1 signaling pathway in the regulation of integrin-dependent adhesion in EC and angiogenesis.
- © 2010 by American Heart Association, Inc.