Macrophage-derived SPP1 promotes subretinal fibrosis by YAP1 phase separation in retinal pigment epithelium.

Subretinal fibrosis (SRF) is a critical end-stage feature of neovascular age-related macular degeneration (nAMD) with limited treatment options. However, the pathological mechanism during the transformation of choroidal neovascularization (CNV) into SRF remains unclear. Bulk RNA-seq of mouse macrophages treated with succinate or lactate in acidic hypoxia identified Spp1 as a key fibrosis-associated gene. Dynamic Spp1 expression was tracked by single-cell RNA-seq in a CNV model, while laser-induced CNV and SRF models assessed the pro-fibrotic role of Spp1 in vivo. In vitro, the binding of SPP1 to RPE CD44 was identified through bioinformatics and Co-IP, and Western blotting examined downstream CD44/RhoA/YAP1 pathway proteins. qPCR quantified nine YAP1 isoforms to identify the predominant one after SPP1 intervention. The specific YAP1 isoform undergoing liquid-liquid phase separation (LLPS) was determined by visualizing intracellular localization and biomolecular condensates via EGFP-tagged plasmid transfection, with LLPS characteristics confirmed by live-cell imaging and fluorescence recovery after photobleaching (FRAP). ATAC-seq identified the transcription factors co-activated with YAP1 driving fibrosis, while EMT phenotypes were evaluated using pro-fibrotic gene expression, migration, and collagen contraction assays. Succinate and lactate upregulated Spp1 and activated correlated Ca2+ influx pathways. An expanding Spp1+ Mφ population was found in early-to-mid CNV. SRF mice showed elevated Spp1in Mφs, and intravitreal Spp1 worsened CNV fibrosis, which blocked by small interfering extracellular matrix receptor III (siCD44). In vitro, SPP1 bound to CD44 activated the RhoA/YAP1 pathway, characterized by a predominant isoform shift from YAP1-1α to YAP1-2α. The nuclear translocation and LLPS of YAP1-2α facilitated pro-fibrotic gene transcription by binding to TEAD4, thereby promoting EMT-like changes in RPE. Under accumulation of acidic metabolites, SPP1-overexpressing Mφs promote EMT in the RPE via CD44/RhoA-mediated YAP1-2α LLPS. This process involves binding to TEAD4 to co-activate pro-fibrotic gene transcription, revealing novel pathomechanisms involved in SRF progression.