Establishment of a 3D cell culture perfused system to investigate mechanisms of blood vessel lumen morphogenesis: the role of cilia and cytoskeletal regulators in flow sensing

Cardiovascular function relies on stable and continuous formation of the blood vessel network by endothelial cells (ECs) to sustain blood flow and tissue viability. Although much is known regarding the molecules that control the initial stages of blood vessel growth, current understanding of the mechanisms by which the blood vessel lumen develops and expands to allow and sustain blood flow remain poorly elucidated. Endothelial primary cilia, microtubule-based organelles that protrude from the plasma membrane into the vessel lumen, mediate mechanotransduction of blood flow. Therefore, this thesis assesses the effects of genetic ablation of cilia, and cytoskeletal regulators DOCK4 and ROCK involved in ciliogenesis, on flow sensing and lumen formation under conditions of fluid flow. A promising approach for understanding the mechanism behind development of a functional circulatory system is the use of perfused endothelial tubes in three- dimensional (3D) organotypic co-cultures of endothelial cells and fibroblasts. The system which was established in this study allowed manipulation of molecules associated with flow sensing under conditions of fluid flow. Molecular mechanisms of vascular lumen formation and lumen expansion were investigated using this system. This study concludes that primary cilia are necessary for vascular tube formation in the co-culture model as their ablation inhibited lumen formation and expansion, and ROCK inhibition leading to disruption of proper cilium assembly inhibits vascular lumen formation. Furthermore, knockdown of GEF DOCK4 under conditions of FGF stimulation, increases angiogenesis in co-culture through higher EC proliferation accompanied by decreased cilia incidence. Finally, during this study a serotonin receptor HTR6-CFP2 fusion protein was cloned into a lentiviral vector and the protein was successfully expressed in ECs for assessment of ciliary dynamics in live cells.