Rasa3 controls turnover of endothelial cell adhesion and vascular lumen integrity by a Rap1-dependent mechanism

Abstract

Rasa3 is a GTPase activating protein of the GAP1 family which targets R-Ras and Rap1. Although catalytic inactivation or deletion of Rasa3 in mice leads to severe hemorrhages and embryonic lethality, the biological function and cellular location of Rasa3 underlying these defects remains unknown. Here, using a combination of loss of function studies in mouse and zebrafish as well as in vitro cell biology approaches, we identify a key role for Rasa3 in endothelial cells and vascular lumen integrity. Specific ablation of Rasa3 in the mouse endothelium, but not in megakaryocytes and platelets, lead to embryonic bleeding and death at mid-gestation, recapitulating the phenotype observed in full Rasa3 knock-out mice. Reduced plexus/sprouts formation and vascular lumenization defects were observed when Rasa3 was specifically inactivated in mouse endothelial cells at the postnatal or adult stages. Similar results were obtained in zebrafish after decreasing Rasa3 expression. In vitro, depletion of Rasa3 in cultured endothelial cells increased β1 integrin activation and cell adhesion to extracellular matrix components, decreased cell migration and blocked tubulogenesis. During migration, these Rasa3-depleted cells exhibited larger and more mature adhesions resulting from a perturbed dynamics of adhesion assembly and disassembly which significantly increased their life time. These defects were due to a hyperactivation of the Rap1 GTPase and blockade of FAK/Src signaling. Finally, Rasa3-depleted cells showed reduced turnover of VE-cadherin-based adhesions resulting in more stable endothelial cell-cell adhesion and decreased endothelial permeability. Altogether, our results indicate that Rasa3 is a critical regulator of Rap1 in endothelial cells which controls adhesions properties and vascular lumen integrity; its specific endothelial cell inactivation results in occluded blood vessels, hemorrhages and early embryonic death in mouse, mimicking thus the Rasa3-/- mouse phenotype.