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.
Highlights
Blood vessels consist of a layer of interconnected endothelial cells delineating a luminal space through which blood flows
Using a combination of loss of function studies in mouse and zebrafish and in vitro cell biology approaches, we show that Rasa3, a GTPase activating protein of the GAP1 family, controls Rap1 activation, endothelial cell adhesion and migration as well as formation of vascular lumens
We found that inactivation of Rasa3 in mouse endothelial cells lead to embryonic bleeding and death at mid-gestation, recapitulating the phenotype observed in full Rasa3 knock-out mice
Summary
Blood vessels consist of a layer of interconnected endothelial cells (ECs) delineating a luminal space through which blood flows. Through loss-of-function experiments, these studies identified several molecular regulators crucial for lumenogenesis, including cell surface and polarity proteins, kinases and phosphatase, actin interactors and regulators, EC-ECM adhesion proteins and small GTPase signaling components [2,3,4,5,6,7,8,9,10,11]. The small GTPase superfamily, which includes the Ras, Rho, Ran, Rab and Arf families, is composed of proteins that act as molecular switches in important signaling pathways. These pathways, which relate to cell proliferation and survival, cell-matrix and cell-cell adhesion, and cytoskeleton dynamics are critical for normal development and physiology and, when deregulated, cause severe life-threatening syndromes and pathologies. While R-Ras has been extensively studied due to its involvement in cancer, Rap has recently attracted a lot of attention due to its central role in development and morphogenesis of higher organisms, especially in the cardiovasculature [14]
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