The VEGF receptor-2 (KDR) pathway in vascular endothelial cells (EC) promotes cell survival and migration, essential steps for vascular repair and remodeling. Culture of peripheral blood also results in an EC-like population of cells termed blood outgrowth endothelial cells (BOEC), which contribute to vascular remodeling through a poorly-defined mechanism of paracrine regulation of vascular EC. In our PRELIMINARY STUDIES, we have 1) identified the multifunctional cytokine, nitric oxide (NO) as a key paracrine factor released by BOEC, 2) elucidated an S-nitrosylation dependent mechanism by which NO activates dynamin GTPase, an important regulator of EC signaling and vesicle trafficking, and 3) identified a novel mechanism by which dynamin GTPase activity positively regulates the KDR pathway of EC migration and survival through both regulatory protein interaction and compartmentalization. These key observations will allow us to rigorously and mechanistically test our CENTRAL HYPOTHESIS that BOEC-derived NO stimulates KDR dependent migration and proliferation of native vascular EC through dynamin nitrosylation and GTPase activity regulation, thereby promoting vascular remodeling. Thus, in our SPECIFIC AIMS, we will: 1) examine the mechanism by which NO nitrosylates dynamin by mapping nitrosylation of individual cysteine residues in the dynamin GTPase domain and then establish the causative role of nitrosylation in the process of NO activation of dynamin function, 2) delineate the regulatory role of dynamin and its nitrosylation on KDR subcellular compartmentalization, activation, and function in vitro and in vivo, by studying dynamin-KDR protein interactions and dynamin dependent KDR internalization, 3) perform studies in BOEC and EC in coculture models and in an in vivo model of vascular remodeling, to directly test how BOEC-derived NO activates KDR- dependent EC migration, survival, endothelial reconstitution, and function. To address these aims, we will utilize a variety of feasible and state-of-the-art approaches including mechanistic recombinant protein studies, retroviral and siRNA based gain and loss of function in BOEC and vascular EC, and validated cell delivery approaches in in vivo models. Thus, these multidisciplinary studies will delineate novel paracrine signaling pathways between vascular EC and circulation-derived cells in the process of vascular remodeling.
Vascular remodeling is an important physiologic process with therapeutic relevance in the setting of a broad number of vascular diseases. Thus, this work has physiologic, pathobiologic, and therapeutic importance.
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