PROJECT SUMMARY Age-related macular degeneration (AMD) is an ocular neurodegenerative disorder that accounts for approximately half of all cases of blindness and visual disability in developed nations. It is estimated that around 288 million people will be diagnosed with AMD worldwide by 2040. The neovascular form of AMD, accounting for about 80% of the severe vision loss in patients with AMD, is characterized by neovascularization in the sub- retinal space. Subretinal fibrosis demarcates end-stage neovascular AMD, causing irreparable vision loss, even with anti-VEGF therapy. Thus, it is imperative to unravel the molecular mechanisms underpinning subretinal fibrosis formation and develop drug-based therapies not only for macular neovascularization (MNV) but also to prevent or treat subretinal fibrosis. Accumulating evidence suggests that epithelial-to-mesenchymal transition (EMT) of the retinal epithelial cell (RPE) and endothelial-to-mesenchymal transition (EndoMT) of either the choroidal or retinal endothelial cells all contribute to the mesenchymal cell population in subretinal fibrotic lesions. Additionally, EndoMT leads to an increase in endothelial barrier permeability and could provide an explanation for the reduced efficacy of anti-VEGF treatment over time. Therefore, therapies designed to inhibit or reverse EMT and EndoMT could stabilize endothelial barrier function and prevent or reduce subretinal fibrosis. Various extracellular cytokines and growth factors are involved in activating MNV, EMT, and EndoMT. We have identified the small GTPase ARF6 as a convergence point in signaling pathways activated by many of these cytokines and growth factors. Thus, we hypothesize that ARF6 activation induces MNV, EMT, and EndoMT, thereby contributing to the onset of subretinal fibrosis, and that genetic loss or pharmacological inhibition of ARF6 will reduce both MNV and subretinal fibrosis in neovascular AMD. To test this hypothesis, we will pursue three aims. In Aim 1, we will determine whether ARF6 activation is required for choroidal and retinal EndoMT and subretinal fibrosis in laser-induced CNV and JR5558 mouse models. We will also use the laser-induced CNV model, endothelial cell lineage tracing, and single cell RNAseq to determine the role of ARF6 in EndoMT. In Aim 2, we will determine whether ARF6 activation is required for RPE EMT and subretinal fibrosis in laser-induced CNV and JR5558 mouse models and use cell lineage tracing and single cell RNAseq to determine the function of ARF6 in EMT. In Aim 3, we will investigate whether EMT and EndoMT act additively or synergistically to promote neovascular AMD and whether pharmacologic inhibition of ARF6 can reduce neovascular AMD in mouse models. This study could have a major impact on the future treatment of neovascular AMD by identifying ARF6 as a potential target for drug development. Moreover, this study could also have important implications for the treatment of other ocular vascular diseases,...