Characterising the in vitro and in vivo function of the RhoG effector DOCK4 during angiogenesis and ischemia.

The RAC1 specific GEF, DOCK4, has been identified as an essential component in the Rho GTPase signalling pathway, imperative for correct vascular patterning and lumenisation during sprouting angiogenesis in vitro. As RAC1 has been previously implicated in the signalling events involved in vascular regrowth within a hypoxic environment, it was hypothesized that DOCK4 may be an important effector in the response to vascular injury and oxygen deprivation. To test this hypothesis, a DOCK4 depleted endothelial co-culture assay was carried out in both hypoxic and normoxic conditions. DOCK4 driven activation of RAC1 has been demonstrated under VEGF signalling, however FGF2 signalling pathways have also been strongly implicated in vascular response to blood vessel injury and hypoxia. Therefore, co-culture assays were carried out to assess sprouting angiogenesis with DOCK4 knockdown in response to FGF2 supplementation. Further, a heterozygous DOCK4 depleted murine model in ischemia studies using a model of HLI was employed together with LDI monitoring of vascular response and regrowth, comparing the response of heterozygous Dock4 KO mice and their WT littermate controls. DOCK4 interacts with the CDC42 GEF DOCK9 but the molecular basis of the interaction is unknown, as is the role of GEF heterodimerization in cell signalling. This study aims to further understand the function of DOCK4 within a pathological sprouting angiogenesis while also investigating the mechanism of interaction between DOCK4 and DOCK9. The two pro-angiogenic growth factors VEGFA and FGF2 drive different phenotypical growth responses during sprouting angiogenesis in vitro. DOCK4 was demonstrated as being an important component of FGF2 stimulated angiogenesis under hypoxia, indicating DOCK4 as important for mechanisms involved in the angiogenic response to ischemia. The specific site of DOCK9 which interacts with the SH3 domain of DOCK4 was not elucidated during this study, however it was determined that DOCK9 proline rich regions identified as PRR 2, 3, 4, and 9 were unlikely to be involved in the interaction. The small molecule inhibitor QL-47 was demonstrated to be a potent anti-angiogenic compound with VEGFA stimulated ECs being particularly sensitive to QL-47. However, it is highly unlikely that the anti-angiogenic effects are due to disruption of the DOCK4-DOCK9 interaction, as the p.C628 cysteine residue was found to not be involved in DOCK4 SH3 domain interaction. Understanding how Rho GTPases are regulated and mechanisms underpinning their activity will progress the understanding of events that drive blood vessel growth while gaining insight into dysregulation during angiogenic pathologies.