Abstract
Purpose :
The suppression of aberrant neovascular growth in wet AMD (wAMD) by anti-VEGF therapy can lead to subretinal fibrosis and permanent blindness. Damaged RPE undergoes an epithelial mesenchymal transition (EMT) to initiate subretinal fibrosis. However, therapeutic strategies for fibrosis remain to be developed. Previously, in various wAMD models, we identified VLDLR as an endogenous inhibitor of retinal fibrosis; here we investigated whether metabolism reprograming in the RPE could be a trigger of subretinal fibrosis in wAMD
Methods :
Differential expression (DE) of key metabolism genes in the VLDLR KO (VKO) RPE-choroid tissue was analyzed by single-cell RNA sequencing and qRT-PCR. A seahorse analyzer was used to track real-time RPE bioenergetics. SiRNA-mediated knock-down was used to identify fibrosis-associated genes. Co-immunoprecipitation (co-IP) was performed to detect protein-protein interaction. The level and localization of profibrotic markers in VKO mice and human AMD eyes were analyzed by Western blotting and immunofluorescence microscopy. Carnitine palmitoyl transferase 1A (CPT1A), a rate limiting enzyme of the fatty acid oxidation (FAO), was overexpressed to upregulate mitochondria FAO in vitro and in vivo. Electroretinogram was used to evaluate retinal function
Results :
DE and network analysis detected profibrosis and mitochondrial energy metabolism as the top dysregulated pathways in the VKO RPE and identified key downregulated enzymes in the pathways. Functionally, VKO RPE exhibited reduced mitochondrial oxidation, increased glycolysis, and upregulation of profibrotic and EMT markers. Both VKO mice and human AMD retina showed decreased CPT1A and increased collagen 1 expression. Molecularly, transforming growth factor beta receptor (TGFβR) activation in RPE cells upregulated profibrotic factors and EMT markers, while downregulated CPT1A in an ERK-dependent manner. Co-IP identified a direct association of VLDLR and TGFβR, which may contribute to attenuating the fibrotic pathway. Overexpression of CPT1A in both human primary RPE cells and VKO eyes alleviated EMT and fibrotic marker expression and retinal function decline
Conclusions :
Our study suggests that mitochondrial oxidation to glycolysis switch correlates with RPE profibrosis and EMT, and that attenuation of dysregulated mitochondria energetics is a novel therapeutic strategy for subretinal fibrosis in nAMD
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.