Abstract
Purpose :
Subretinal fibrosis is one of major causes for patients with neovascular age-related macular degeneration (nAMD) who are unresponsive to anti-VEGF treatments. Epithelial-to-Mesenchymal Transition (EMT) is an important process in the development of subretinal fibrosis. However, it is unknown how metabolism affects transition of retinal pigment epithelial (RPE) cells to mesenchymal-like cells under conditions of subretinal fibrosis. In this study, we tested whether 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase isoform 3 (PFKFB3)-mediated glycolysis in RPE cells induces EMT by activating hypoxia-inducible factors (HIFs) pathways, eventually leading to the development of subretinal fibrosis.
Methods :
Retinal samples of human nAMD were stained with antibodies against PFKFB3 and ACTA2. Laser-induced choroidal neovascularization (CNV) and spontaneous CNV in very low–density lipoprotein receptor-deficient (Vldlr-/-) mice were used as models of mouse subretinal fibrosis. Global and RPE-specific Pfkfb3 deficient mice in C57 or Vldlr-/- background were used for laser injury-induced or spontaneous subretinal fibrosis, respectively. We collected the RPE/choroidal complex of mice for immunostaining with antibodies against isolectin B4, collagen I and ACTA2. In in vitro studies, we initiated EMT by treating cultured human primary RPEs with transforming growth factor beta-2 (TGFβ-2). With loss- and gain-of HIFs function approaches, we examined whether and how HIF participates into PFKFB3-associated EMT with quantitative PCR and Western blotting.
Results :
We found that high levels of glycolytic enzymes including PFKFB3 were present in the RPE/choroid complex isolated from laser-induced subretinal fibrosis in C57BL/6j mice, spontaneous lesions in Vldlr-/- mice and RPE layer of nAMD patients. We also observed that the area of subretinal fibrosis was markedly decreased in Pfkfb3 global and RPE-specific deficient mice. In in vitro studies, PFKFB3 knockdown in RPE cells inhibited their transition to mesenchymal and reduced their production of proinflammatory and profibrotic factors by regulating the HIFs signaling pathway.
Conclusions :
Our study reveals an important role of PFKFB3-mediated metabolism in RPE cells in the development of subretinal fibrosis and indicates that PFKFB3 inhibition is a novel strategy for the treatment of subretinal fibrosis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.