Anti-VEGF therapies, standard care in neovascular age-related macular degeneration (nvAMD), improve outcomes in less than 50% of patients and do not prevent vision-loss progression due to fibrosis or macular atrophy. We used human physiologically relevant models to gain understanding into the coordination of signaling pathways and cross-talk involved in the activation of choroidal endothelial cells (CECs) to migrate and form macular neovascularization in nvAMD. The scaffolding protein, IQGAP1, sustains activation of the GTPase Rac1, which is necessary for CEC migration. Rac1 is activated by AMD-related stresses involving inflammatory, oxidative and angiogenic factors, as well as by the oxysterol, 7-ketocholesterol (7KC), which accumulates in blood and Bruch’s membrane with increased age and in AMD. 7KC causes CECs to change expression of cell markers from endothelial to mesenchymal ones, suggesting endothelial-mesenchymal transition (EndMT). IQGAP1 appears involved. 7KC also causes fibrosis in models of laser induced injury. Our data support the hypothetical framework that will be tested in the next funding period: that (1) IQGAP1 is critical to EndMT induced by the oxysterol 7KC; and that (2) 7KC triggers transcriptional events that render CECs unable to maintain expression of endothelial markers but to develop into a new phenotype of migratory, mesenchymal cells that develop into fibrosis. We will also test two potential therapies to reduce fibrosis: (a) to inhibit TGFβ signaling in combination with anti-VEGF and (b) to target phosphorylation of IQGAP1 in a novel mutant IQGAP1 mouse that we created by CRISPR-Cas9-induced gene mutation. Specific Aim 1 is to test the prediction that IQGAP1 mediates EndMT-induced migration in CECs exposed to 7KC. Specific Aim 2 is to test the prediction that 7KC, mediated through IQGAP1, decreases the proportion of labeled endothelial positive to mesenchymal positive cells after laser in endothelial specific yellow-fluorescent protein reporter mice. Specific Aim 3 is to test predictions that increased age, TGFβ-signaling, or IQGAP1 serine phosphorylation will increase αSMA-labeled lesions after laser in 7KC-treated eyes and to test strategies as possible future treatments. We will also evaluate the involvement of Müller cells, pericyes, and RPE. Tools include isolated human CECs; high throughput RNA sequencing; flow cytometry; spectral domain optical coherence tomography (sdOCT); 7KC-induced models of EndMT and fibrosis; yellow-fluorescent protein endothelial reporter mice; conditional inducible endothelial Iqgap1 knockout mice; a mutant IQGAP1 mouse through CRISPR-Cas9-technology; intravitreal injections of pharmacologic agents; Micron IV laser induced injury to test 7KC-induced EndMT and lesion formation. These studies will test the role of IQGAP1 in 7KC-induced EndMT as a potential cause of fibrosis, which is poorly responsive to anti-VEGF in nvAMD, and will test two novel treatments to reduce nvAMD and EndMT.