Establishing a functional vascular network is a rate-limiting step in embryonic development, the repair of injured tissues, and the engineering of tissue replacements. Although we have made progress in identifying factors that promote endothelial cell proliferation and sprouting, we lack understanding of how to properly control endothelial cell growth and phenotypic specialization during vascular remodeling, which has created a significant roadblock for clinical therapies, tissue engineering and regenerative medicine. Although multiple signaling pathways have been implicated in the regulation of arterial-venous network formation, including flow-induced mechanotransduction and Notch signaling, the mechanisms by which these signals coordinately regulate endothelial cell growth suppression and identity were unclear. Our recent studies revealed that remodeling vascular plexi are subject to systemic blood circulation, and that shear stress of different magnitudes promotes differential growth responses and gene expression. That is, arterial/arteriolar shear stress levels promote Notch signaling, and downstream p27-induced late G1 phase arrest that enables arterial gene expression (Fang 2017). Conversely, flow magnitudes typical of veins/venules induce early G1 arrest, and enables upregulation of venous genes. Interestingly, distinct endothelial cell cycle states appear to be maintained in arteries vs. veins postnatally. We know very little about the role of cell cycle control in endothelial cell fate decisions, or the differential signaling pathways induced by vessel-specific flow magnitudes, and how they may coordinately induce and maintain endothelial cell cycle state and identity. The scientific premise of our research is that endothelial cell cycle control is required for proper arterial and venous specification, such that when endothelial cells are in different cell cycle states, they exhibit different propensity for arterial vs. venous gene expression. Support for this idea comes from studies in embryonic stem cells that show cells in early vs. late G1 phase have a propensity for mesoderm/endoderm vs. ectoderm fate, respectively (Paulkin 2014). Thus, our hypothesis is that differential flow forces in arteries and veins induce different intracellular signaling pathways that promote distinct endothelial cell cycle states, creating distinct windows of opportunity for the regulation of arterial vs. venous gene expression. To ensure scientific rigor, we will test this hypothesis in vivo in models of arterial- venous network formation and repair, and in vitro in human endothelial cell culture systems that allow flow manipulation. We will define mechanisms by which vessel-specific flow magnitudes modulate endothelial cell cycle state, determine how distinct endothelial cell cycle states enable differential phenotypic specialization (artery vs. vein), and determine whether manipulation of endothelial cell cycle state can prevent or correct arterial-venou...