"Myeloid PFKFB3 in subretinal fibrosis"

REVISED PROJECT SUMMARY Subretinal fibrosis is an end-stage fibrous plaque/disciform scar that progresses from choroidal neovascularization (CNV) during neovascular age-related macular degeneration (nAMD). Subretinal fibrosis compromises highly organized anatomical layers and tightly coordinated cellular interactions, inevitably leading to irreversible visual impairment. Current treatment for subretinal fibrosis is limited and thus, therapeutic strategies for the inhibition of subretinal fibrosis are imperative. Multiple cell types, including endothelial cells (ECs), retinal pigment epithelium (RPE) cells, macrophages, and glial cells, contribute to subretinal fibrosis by either differentiating into mesenchymal-like cells and further differentiating into α-smooth muscle actin-positive myofibroblasts and/or producing profibrotic and proinflammatory factors. However, the underlying metabolic mechanisms for these cellular and molecular activities remain poorly defined. Glycolysis is a metabolic pathway utilized by many proliferative cells. Our preliminary data show that cells in subretinal fibrotic areas are hyper-glycolytic, as evidenced by high levels 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. We have demonstrated that high levels of glycolytic enzymes including Pfkfb3 are present in the RPE/choroid complex isolated from laser-induced and spontaneous subretinal fibrosis in C57BL/6j mice and very low–density lipoprotein receptor deficient (Vldlr-/-) mice and that the area of subretinal fibrosis is markedly decreased in Pfkfb3-/+ mice. Our in vitro studies have also shown that PFKFB3/Pfkfb3 deletion in myeloid cells inhibits their transition to myofibroblast cells as well as reducing their production of proinflammatory and profibrotic factors. We hypothesize that Pfkfb3-mediated glycolysis in myeloid cells induces their transition to myofibroblasts and induces their production of profibrotic and proinflammatory factors by activating HIFs pathways, eventually leading to the development of subretinal fibrosis. To test our hypothesis, we have generated a variety of genetic mice and established mouse subretinal fibrosis models with laser-induced CNV and spontaneous CNV in Vldlr-/- mice. We will investigate the effect on subretinal fibrosis of Pfkfb3 deficiency or inhibition in myeloid cells using specific genetic and pharmacological tools with an integrated approach of in vivo and in vitro models. Our study will define the role of PFKFB3-mediated metabolism in myeloid cells in the development of subretinal fibrosis and validate inhibition of myeloid PFKFB3 as a novel strategy for the treatment of subretinal fibrosis.