This competitive renewal application addresses an important question in vascular biology, which concerns the molecular control of how endothelial cell (EC)-lined tubes form in three-dimensional extracellular matrices. We have developed excellent models of this process in vitro and have been identifying key mechanisms and molecules, which are required for these events. Our work has identified the extracellular matrix-integrin-cytoskeletal (MIC) signaling axis as a critical regulator of EC morphogenesis and Rho GTPases as major signaling molecules that control this process. In particular, we have shown that Cdc42 is required for intracellular vacuole and lumen formation while RhoA is required for EC invasion, branching and tube stability. Furthermore, we have shown that GFP-Cdc42 chimeras target to intracellular vacuoles allowing real-time imaging of the lumen formation process in vitro. In addition, transgenic Zebrafish embryos have been created carrying the GFP-Cdc42 chimera. Using time-lapse imaging of sprouting ECs in vivo, intracellular vacuoles are observed and appear to contribute directly to EC lumen development. The lumen formation processes observed in vitro versus in vivo appear remarkably similar. In this proposal, we focus on a molecular characterization of the roles of Cdc42 and RhoA in EC tubular morphogenesis in vitro and in vivo. Also, we characterize the role of known effectors and regulators of these GTPases during these events. Proteomic approaches are proposed to identify and characterize the role of Cdc42-binding proteins in ECs undergoing tubular morphogenesis. We further propose to image developing vessels in vitro in our models of EC invasion and morphogenesis in three-dimensional collagen matrices and compare them to in vivo images obtained using transgenic Zebrafish to better the define the mechanisms controlling EC lumen development. ? ? The specific aims of this application are:
Aim #1. To investigate the role of RhoA, Rael and Cdc42 GTPases during different stages of capillary tube formation in three-dimensional extracellular matrices and during angiogenesis in vivo.
Aim #2. To identify and determine the role of Cdc42 GTPase regulatory molecules which control EC intracellular vacuole and lumen formation in three-dimensional extracellular matrices.
Aim #3. To investigate the role of RhoA GTPase regulatory molecules during EC invasion, branching, tube stabilization and survival in three-dimensional extracellular matrices. ? ? ? ?
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