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Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells
Published on: December 12, 2014
Macrophage-derived SPP1 promotes subretinal fibrosis by YAP1 phase separation in retinal pigment epithelium
Ruoyi Lin1, Zixin Cai2, Tianyu Zheng3
1Department of Ophthalmology, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai, China; Department of Ophthalmology, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai, China.
Background:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
Insights
Acidic metabolites drive subretinal fibrosis (SRF) in neovascular age-related macular degeneration (nAMD) by promoting macrophage Spp1 expression. This activates the YAP1-2α pathway, leading to RPE EMT and fibrosis progression.
Area of Science:
- Ophthalmology
- Cell Biology
- Molecular Biology
Background:
- Subretinal fibrosis (SRF) is a severe complication of neovascular age-related macular degeneration (nAMD).
- The precise molecular mechanisms driving the transition from choroidal neovascularization (CNV) to SRF are not fully understood.
- Current treatment options for SRF are limited, highlighting the need for mechanistic insights.
Purpose of the Study:
- To elucidate the pathological mechanisms underlying the development of subretinal fibrosis (SRF) in neovascular age-related macular degeneration (nAMD).
- To investigate the role of specific metabolites and gene pathways in SRF progression.
- To identify potential therapeutic targets for SRF.
Main Methods:
- Bulk and single-cell RNA sequencing (RNA-seq) of mouse macrophages and CNV models to identify fibrosis-associated genes.
- In vitro and in vivo experiments using laser-induced CNV and SRF models to assess the pro-fibrotic role of Spp1 and its interaction with CD44.
- Analysis of the CD44/RhoA/YAP1 signaling pathway, including YAP1 isoform identification, liquid-liquid phase separation (LLPS) dynamics, and transcription factor interactions (TEAD4).
Main Results:
- Succinate and lactate were found to upregulate Spp1 expression in macrophages, correlating with Ca2+ influx pathways.
- Spp1-expressing macrophages accumulate during CNV progression, and exogenous Spp1 exacerbates CNV-related fibrosis, which is inhibited by siCD44.
- SPP1 binding to CD44 activates the RhoA/YAP1 pathway, shifting to the YAP1-2α isoform, which undergoes LLPS and promotes pro-fibrotic gene transcription via TEAD4, inducing RPE epithelial-mesenchymal transition (EMT)-like changes.
Conclusions:
- Accumulation of acidic metabolites induces SPP1 overexpression in macrophages, promoting RPE EMT through CD44/RhoA-mediated YAP1-2α liquid-liquid phase separation.
- The YAP1-2α LLPS mechanism, involving TEAD4 co-activation, drives pro-fibrotic gene transcription, revealing novel pathomechanisms in SRF.
- These findings offer new insights into SRF pathogenesis and suggest potential therapeutic strategies targeting the SPP1-YAP1 axis.
