PROJECT SUMMARY Subretinal fibrosis, an end-stage fibrous plaque/disciform scar that progresses from choroidal neovascularization (CNV) of neovascular age-related macular degeneration (nAMD), compromises highly organized anatomical layers and tightly coordinated cellular interactions, inevitably leading to irreversible visual impairment. Current treatment for subretinal fibrosis is limited; thus, therapeutic strategies for the inhibition of subretinal fibrosis are essential. Many types of cells, including retinal pigment epithelium (RPE) cells, contribute to subretinal fibrosis by differentiating into mesenchymal-like cells, α-smooth muscle actin-positive myofibroblasts and/or producing profibrotic and proinflammatory factors. However, the underlying metabolic mechanisms for these cellular and molecular activities are not well defined. Glycolysis is a metabolic pathway utilized by many proliferative cells. my preliminary data show that cells in subretinal fibrotic areas are hyper-glycolytic, as evidenced by increased expression of glycolytic enzymes and glycolytic regulators/activators including 6-phosphofructo-2- kinase/fructose-2, 6-bisphosphatase isoform 3 (PFKFB3), a critical enzyme for activation of glycolysis in various highly proliferative cells. PFKFB3 catalyzes the synthesis of fructose-2,6-bisphosphate (F2, 6P2), which is the most potent allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme for glycolysis. I have found that high levels of glycolytic enzymes including PFKFB3 are present in the RPE/choroid complex isolated from laser-induced subretinal fibrosis in C57BL/6j mice, spontaneous lesion in very low–density lipoprotein receptor deficient (Vldlr-/-) mice and RPE layer of nAMD patients. Furthermore, I have also observed that the area of subretinal fibrosis is markedly decreased in Pfkfb3+/- mice. My in vitro studies have shown that PFKFB3 knockdown in RPE cells inhibits their transition to mesenchymal and reduces their production of proinflammatory and profibrotic factors. I hypothesize that PFKFB3-mediated glycolysis in RPE cells induces their transition to mesenchymal cells and their production of profibrotic and proinflammatory factors by activating HIFs pathways, eventually leading to the development of subretinal fibrosis. To test my hypothesis, I have generated RPE- specific Pfkfb3-deficient mice and established mouse subretinal fibrosis models with laser-induced CNV and spontaneous CNV in Vldlr-/- mice. I will investigate the effect of Pfkfb3 deficiency in RPE cells on subretinal fibrosis using specific genetic tools with an integrated approach of in vivo and in vitro models. My study will define the role of PFKFB3-mediated metabolism in RPE cells in the development of subretinal fibrosis and demonstrate PFKFB3 inhibition as a novel strategy for the treatment of subretinal fibrosis.