Rho GTPase Signalling in Cilia and Blood Vessel Lumen Formation

Vascular endothelial cells, the cells that line blood vessels, play roles both in the development of the circulatory system through providing blood vessel integrity, and its homeostasis through responding to changes in the local environment. During development, the acquisition of functionality of new blood vessels, some of which form from pre-existing ones through the process of angiogenesis, requires generation of endothelial lumens to carry the blood, during a process known as lumenogenesis. Throughout new blood vessel formation, endothelial cells go through coordinated and complex cell shape changes and polarity changes driven by Rho GTPase activity. Rho GTPases control intracellular actin dynamics and therefore affect all stages of new vessel growth. Precise control of Rho GTPase signalling is through a range of upstream activator and downstream effector proteins. Here we have investigated the role of two proteins involved in the antagonistic Rho/ Rac pathways during angiogenesis and lumen formation. Results presented in this thesis describe a role for the Rac guanine nucleotide exchange factor (GEF) DOCK4, and Cdc42 GEF DOCK9, in controlling lumen formation in an organotypic in vitro angiogenesis assay, through both common and distinct cellular mechanisms. The RhoA effector ROCK1/2 controls RhoA- mediated cytoskeletal remodelling and have been shown to control angiogenesis in tissue culture assays. Here we use in vivo knockout mouse model to show that ROCK2 controls actin- based filopodia formation and vessel integrity, as well as other cytoskeletal processes such as ciliogenesis. Further investigation showed that loss of ROCK2 results in aberrant lumen formation. We also highlight a new role for ROCK2 signalling in primary cilia growth and abundance in endothelial cells which may also impact on lumen formation. In summary, this work has identified novel molecular pathways that control lumen formation in endothelial cells. These results build on the current understanding of molecular mechanisms involved in lumen formation, and may also lead to better understanding of the function of ROCK, a potential therapeutic target in therapeutic angiogenesis strategies.