Angiogenesis is the formation of new blood vessels from pre-existing ones and it is necessary for physiological processes such as normal development, and pathological
conditions like cancer growth and metastatic cell dissemination. In order to form new blood vessels, endothelial cells, the cells that line the blood vessels, must go through a number of distinct cellular processes. In order to sprout, cells have to initially detach from the vessel wall, migrate and divide. Endothelial cells must then establish new cell junctions and form functional lumens. During these processes the endothelial cell actin cytoskeleton and cell morphology change dramatically. The Rho family of small GTPases are key regulators of such morphological changes. Whilst the prototypical family members Rho, Rac and Cdc42 have been implicated in different aspects of endothelial cell behavior such as migration and cell assembly, other Rho GTPase family members are less characterized. The aim of this study has been to understand the role of the Rho GTPase RhoG in vessel
formation and to identify its regulators and downstream effectors. For this purpose organotypic co-culture system was employed of endothelial cells and fibroblasts that gives
rise to three-dimensional capillary-like tubes as an in vitro tool to model vessel development. Using RNAi-mediated knockdown in the endothelial cells in the in vitro coculture
system, the role of putative regulators (guanine nucleotide exchange factors) and downstream effectors in angiogenesis was analysed and players that control lateral filopdia
formation were identified. Pulldown assays that determine the activation status of Rho GTPases and gene overexpression and protein co-immunoprecipitation assays were then used to confirm phenotypic results seen in the co-culture assay. Using this system, a novel non-canonical SGEF-RhoG-DOCK4-Rac1-DOCK9-Cdc42 signaling module was identified that controls filopodia formation during endothelial cell assembly and sprouting. Knockdown of components of this signaling module block lateral filopodia formation, while tip filopodia persist suggesting that that different sub-populations of endothelial cell filopodia exist that are regulated independently during tubule morphogenesis. Mechanistic studies carried out in the course of this study showed that DOCK4 and DOCK9 interact
with each other and with the RhoG effector ELMO. Complex formation of DOCK9 with ELMO is through DOCK4 whereas DOCK4 interacts directly with DOCK9. Domain deletion studies showed that DOCK4 interacts with DOCK9 via its SH3 domain.
In summary, this work has identified a new signaling cascade that drives vessel formation via lateral filopodia formation and sprouting and demonstrated for the first time an interaction between a Rac1 exchange factor (DOCK4) and a Cdc42 exchange factor (DOCK9). In addition to providing new information about the process of angiogenic sprouting and functional interaction between different GEFs, the signaling components identified in this study may constitute useful therapeutic targets in pathological
angiogenesis in cancer and other diseases
