Cell Cycle Control of Arterial-Venous Specification

PROJECT SUMMARY 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 mechanisms that coordinately regulate endothelial cell growth suppression and phenotypic specialization during vascular network formation, which has created significant roadblocks for clinical therapies, tissue engineering and regenerative medicine. We aim to circumvent these roadblocks by elucidating fundamental mechanisms of vessel network formation and repair. Our previous studies, in the postnatal murine retinal vascularization model of sprouting angiogenesis, revealed 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 state, which enables TGF-b1-induced arterial gene expression (Fang 2017; Chavkin 2022). Conversely, preliminary data show that flow magnitudes typical of veins/venules induce early G1, which we found enables BMP4-induced upregulation of venous genes (Chavkin 2022). Our preliminary human stem cell studies are consistent with these findings and show that vessel-specific shear stresses (venous vs. arterial) promote differential growth responses (early vs. late G1 states), as well as venous vs. arterial gene expression. However, blood flow is not required for the initiation of arterial-venous specification during embryonic vascular development (Chong 2011), and whether endothelial cell cycle control is required for this process is unknown. Defining the role of endothelial cell cycle regulation in embryonic vascular development could reveal a universal mechanism required to establish arterial-venous networks. Furthermore, identifying novel key regulators of endothelial cell cycle state and fate can provide new targets for the treatment of diseases associated with endothelial cell hyperproliferation, including vascular malformations and tumor angiogenesis. Thus, our hypothesis is that endothelial cell cycle control is required for embryonic arterial and venous specification, its dysregulation promotes arterial-venous malformations, and manipulation of endothelial cell cycle state can prevent or regress vascular malformations. We further hypothesize that mechanistic insights gained from these in vivo studies can be used to optimize the specification of human stem cell-derived arterial and venous endothelial cells. To ensure scientific rigor, we will test these hypotheses in vivo in models of arterial-venous network formation and repair, and in vitro in human stem cell-derived endothelial cell culture systems that allow genetic and flow manipulation to define mechanisms that regulate endothelial cell cycle state and arterial-venous fate. Evaluation of these hypotheses will yield novel fundamental insights into blood vessel formation that could improve vascular pathology treatments and human microvasculature engineering to sustain a variety of tissues.