A Novel Ocular Fibrosis Signature for AMD Using the Two-Stage Laser-Induced Subretinal Fibrosis Mouse Model

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Purpose: Subretinal fibrosis is a major cause of irreversible vision loss in neovascular age-related macular degeneration (AMD), yet no effective antifibrotic therapies exist due to poorly defined molecular drivers. This study aimed to derive and validate a persistent ocular fibrosis signature using the two-stage laser-induced subretinal fibrosis mouse model and to assess its translational relevance in human AMD. Methods: RNA sequencing (RNA-seq) was performed on retinal pigment epithelium (RPE)/choroid tissues collected at days 3, 6, and 10 following the second laser injury in the two-stage model. A novel core fibrosis signature of 88 persistently upregulated genes was observed across all time points and cross-validated in an independent mouse dataset (GSE189555) and multiple human AMD RNA-seq datasets (GSE115828, GSE146887, GSE135092), including surgically extracted choroidal neovascularization (CNV) membranes and macular RPE/choroid samples. Key signature genes were further validated by immunofluorescence in human subretinal fibrotic and age-matched tissues. Results: The novel ocular fibrosis signature was enriched in epithelial-mesenchymal transition, complement activation, and inflammatory pathways and showed choroid-specific expression with minimal retinal involvement. Cross-validation on independent chronic mouse data and multiple human AMD datasets (peripheral retina, surgically extracted CNV membranes, and macular RPE/choroid) confirmed a progressive enrichment in advanced disease stages. Immunofluorescence in human fibrotic tissue validated key genes (tenascin C, tissue inhibitor of metalloproteinases 1, and apelin receptor) and showed colocalization with myofibroblast-like cells and microglia. Conclusions: We identified a novel, persistent, choroid-specific ocular fibrosis signature with strong cross-species conservation, highlighting fibrogenic-associated drivers and providing a valuable translational tool to understand subretinal fibrosis development and targeted antifibrotic therapies in neovascular AMD.