RNA-seq analysis reveals the pro-fibrotic role of aged RPE/choroid in a mouse model of subretinal fibrosis

Abstract

Purpose : Old age promotes subretinal fibrosis. This study aimed to understand the age-related RPE/choroid molecular alterations that are responsible for increased subretinal fibrosis secondary to choroidal neovascularization in old age.

Methods : Young (2-2.5 months, n=3) and aged (15-16 months, n=3) mice were subjected to two-stage laser-induced subretinal fibrosis. Twenty days later, RPE/choroidal tissue from mice with subretinal fibrosis and age-matched controls were collected and processed for bulk RNA sequencing (RNA-seq) analysis. DESeq2 was used to determine the differentially expressed genes (DEGs). Gene Ontology (GO) and KEGG were used to analyze the enriched functions and pathways. Murine microenvironment cell populations counter (mMCP-counter) analysis was conducted to quantify infiltrating immune and stromal cells in RPE/choroid. The expression of the selected DEGs were verified by qRT-PCR.

Results : A total of 547 DEGs (log₂(fold change) ≥ 0.5, FDR < 0.05) were uncovered between normal young and aged RPE/choroid. mMCP-counter analysis showed lower vascular and lymphatic cell scores and higher fibroblast scores in aged RPE/choroid compared to that in young RPE/choroid. The top 25 terms in the GO enrichment analysis were related to immune regulation, protein metabolism and ribosome function. The 30 highest degree DEGs calculated by the Cytohubba plugin of Cytoscape belonged to ribosomal protein subunit (e.g. Rpl15, Rpl22, Rpl36, Rps6, Rps21).
After induction of subretinal fibrosis, 139 DEGs were detected in young RPE/choroid lesion and 2266 DEGs in aged lesion. 60 DEGs co-exist between young ang aged RPE/choroid and 2206 DEGs were specific to aged RPE/choroid. GSEA enrichment analysis of 2206 specific DEGs demonstrated that the immune response, angiogenesis, cellular activation and focal adhesion, chemokine and TGFβ signaling pathways were significantly enriched, and core genes of these pathways were upregulated in aged subretinal fibrotic RPE/choroid. The genes related to ribosome biogenesis and mitochondrial respiratory chain complex synthesis were down-regulated in aged subretinal fibrosis RPE/ choroid. mMCP-counter analysis revealed significant higher scores of monocytes/macrophages and fibroblasts in aged subretinal fibrotic RPE/choroid.

Conclusions : Old age promotes subretinal fibrosis through the pro-inflammatory and pro-fibrotic microenvironment of RPE/choroid.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.