Abstract 17143: RASSF5, a New Mediator of Angiogenesis, Mediates Integrin Endocytosis in Endothelial Cells and Vessel Stability in vivo
Integrins are critical mediators of angiogenesis. In our previous work, we identified the GTPase Rap1 as a mediator of integrin activation in endothelial cells (EC) and angiogenesis. RASSF5/RAPL is an effector protein of Rap1 promoting integrin activation and adhesion in lymphocytes. However, the role of RASSF5 for integrin regulation in EC and angiogenesis is unclear. Silencing of RASSF5 with siRNA significantly decreased angiogenic sprouting of HUVEC in a 3-dimensional spheroidal system (83 % inhibition) and blocked VEGF-induced invasion (68 % inhibition), while not inhibiting EC proliferation. Surprisingly, while silencing of Rap1 reduced HUVEC adhesion, silencing of RASSF5 unexpectedly significantly increased integrin-dependent adhesion suggesting distinct roles for RASSF5 and Rap1 in integrin regulation in EC. In line with these results, silencing of RASSF5 significantly increased surface expression of beta1-integrin as assessed by FACS, while not affecting total beta1-integrin protein levels in HUVEC. It is conceivable, that the enhanced surface expression of beta1-integrins upon silencing of RASSF5 is caused by a redistribution of the integrin from an intracellular pool to the cell surface. Therefore, we studied the role of RASSF5 for the endocytosis of beta1-integrins in HUVEC. Strikingly, silencing of RASSF5 blocked beta1-integrin endocytosis by 43 ± 6 %. Interestingly, silencing of RASSF5 preferentially reduced the internalization of activated (matrix-bound) alpha5beta1-integrins. Endogenous alpha5-integrin co-immunoprecipitated with overexpressed RASSF5. In addition, silencing of RASSF5 enhanced actin polymerization and Rac1 activity in EC. Strikingly, in vivo silencing of the RASSF5 homologue in the zebrafish embryo with morpholino led to hemorrhages in the newly formed vessels. In conclusion, our results demonstrate that RASSF5 is binding to the alpha5beta1-integrin and mediating integrin internalization and actin cytoskeleton reorganization in EC, thereby promoting angiogenic sprouting and migration. Our data identify a new angiogenic signaling pathway and shed light on the relevance of alpha5beta1-integrin endocytosis for angiogenic functions such as sprouting, migration and vessel stability.
- © 2011 by American Heart Association, Inc.