Regulation of Blood Vessel Development via Rho Family GTPase Signaling
Barry, David Michael
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The Rho family of small GTPases has been shown to be required in endothelial cells (ECs) during blood vessel formation. However, the underlying cellular events controlled by different GTPases remain unclear. Here, we assess the cellular mechanisms by which Rho family GTPase proteins and their regulators regulate mammalian vascular morphogenesis and maintenance. First we assessed the role of the Rho GTPase Cdc42 during vasculogenesis and angiogenesis. We find that Cdc42 is essential for organization of EC adhesion, as its loss results in disorganized cell-cell junctions and reduced focal adhesions. Endothelial polarity is also rapidly lost upon Cdc42 deletion, as seen by failed localization of apical podocalyxin (PODXL) and basal actin. We link observed failures to a defect in F-actin organization, both in vitro and in vivo, which secondarily impairs EC adhesion and polarity. Subsequently, Rho GTPase regulatory proteins were further investigated to decipher their role during vascular development. Rasip1 was identified as a promising anti-angiogenesis candidate, which is required for the formation of continuous vascular lumens in growing vessels. Molecular bottlenecks were elucidated during vessel formation by dissecting the cellular events that require Rasip1. We show that Rasip1 controls different GTPase signaling pathways that converge upon the actomyosin contractility machinery. We find that different pools of NMII, downstream of Rasip1, control two different processes in endothelial cells: 1. NMII mediates the removal of pre-apical membrane adhesions to form a lumen. 2. NMII then restrains apical membrane expansion, thereby limiting lumen diameter during vessel growth. In the first process, Rasip1 promotes actin contractility via Cdc42 and Pak4 along ribbons of adhesions at the center of EC cords, causing adhesions to clear from the pre-apical membrane. This allows opening of lumens. Subsequently, Rasip1 inhibits NMII and membrane contractility via RhoA suppression to allow regulated lumen expansion. These novel and distinct spatiotemporal molecular and cellular events define the stepwise process of blood vessel morphogenesis and differentiation.