PROJECT SUMMARY Neovascular age-related macular degeneration (nvAMD) is a leading cause of central vision loss in individuals over the age of 50 years. Vision loss occurs when choroidal endothelial cells (CECs) transmigrate the retinal pigment epithelium (RPE) into the neurosensory retina and form choroidal neovascularization (CNV). Current standard of care includes agents that inhibit the bioactivity of vascular endothelial growth factor (VEGF). Although anti-VEGF agents have reduced CNV and improved visual acuity in patients, there are concerns about possible adverse effects on neuronal and glial health in the retina from long-term VEGF inhibition. Thus, better understanding of downstream VEGF signaling in CECs is necessary to identify more targeted therapeutic approaches. One of the downstream effectors is Rac1, a Rho GTPase that in the GTP-bound state regulates CEC transmigration. VEGF-mediated Rac1GTP is sustained by binding IQ motif containing GTPase activating protein 1 (IQGAP1), a multidomain GTPase-binding protein, at a specific domain. Therefore, interfering with Rac1GTP/IQGAP1 binding may be an approach to inhibit CEC migration and prevent vision- threatening CNV. Cultured CECs that express constitutively active Rap1a, a Ras GTPase that also binds to IQGAP1, had reduced VEGF-mediated Rac1GTP and Rac1GTP/IQGAP1 interactions. Together, these results suggest that increased Rap1aGTP/IQGAP1 binding in CECs reduces VEGF-induced Rac1GTP, CEC migration and CNV by interfering with Rac1GTP/IQGAP1 binding. This proposal will determine the mechanistic role of Rap1GTP binding the IQ domain of IQGAP1 on Rac1GTP/IQGAP1 interactions in Specific Aim 1 and in vitro angiogenesis in Specific Aim 2. In Specific Aim 3, this proposal will determine the mechanistic role of Rap1aGTP/IQGAP1 interactions in endothelial cells during CNV development. Methods include: isolation of CECs from adult human donor eyes; transfection of CECs with mutant IQGAP1 constructs; pharmacologic activation of Rac1 and Rap1; co-immunoprecipitation; western blot; Matrigel migration assay; tube formation assay; proliferation assay; Rap1b null mice and tamoxifen-inducible endothelial-IQGAP1 knockout mice; murine laser-induced CNV model; intravitreal injections of agents that activate Rap1; flow cytometry; immunohistochemistry of RPE/choroid flat mounts and cryo-sections; Micron IV imaging; and spectral domain optical coherence tomography. These studies will elucidate a potential mechanism of regulating Rac1GTP/IQGAP1 interactions, which has an important role in VEGF-induced CEC migration and CNV pathogenesis, and potentially identify targeted therapeutic approaches that can reduce risks from current standard of care VEGF inhibitors. Furthermore, this fellowship application outlines a detailed research and clinical training plan to help the candidate become a successful physician-scientist in ophthalmology.