MSC-EVs attenuate subretinal fibrosis in choroidal neovascularization through miR-21-5p-mediated inhibition of EMT and MMT and suppression of inflammation.

Subretinal fibrosis causes irreversible vision loss in neovascular age-related macular degeneration (nAMD). Sustained macular inflammation drives the initiation and progression of fibrosis by activating profibrotic cells and perpetuating tissue damage. This study investigated the therapeutic potential of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) in mitigating nAMD-associated subretinal fibrosis. MSC-EVs were prepared from human bone marrow-derived MSCs and characterized using nanoparticle tracking analysis, transmission electron microscopy, and Western Blotting. Subretinal fibrosis was induced in C57BL/6J mice using the two-stage laser-induced model. MSC-EVs were injected either intravitreally (1 × 108 particles/eye, single injection) or retro-orbitally (1 × 108 particles, two injections four days apart) immediately after the second laser. Eyes were collected 10 days post-second laser for immunostaining of collagen-1 and CD31 or iso-lectin B4. In vitro, primary human RPE and ARPE-19 cells were treated with TGF-β2 (10 ng/mL) to induce epithelial-mesenchymal transition (EMT); peritoneal macrophages were treated with TGF-β1 (10 ng/mL) to induce macrophage-to-myofibroblast transition (MMT). After 48 h, cells were treated with MSC-EVs (cell-to-MSC-EV ratio = 1:2000) for 3 days. Myofibroblast markers (αSMA, fibronectin, and collagen-1) were examined by immunocytochemistry and quantitative PCR (qPCR). Human iPCS-derived macrophages (iMACs), bone-marrow-derived macrophages, peritoneal macrophages, and BV2 microglia were treated with LPS (100 ng/mL) and IFN-γ (20 ng/mL) for 24 h with or without MSC-EVs (1:2000). Small RNA sequencing was used to identify specific functional molecules within MSC-EVs. Immune-related gene expressions were evaluated by qPCR. Intravitreal and retroorbital administration of MSC-EVs reduced collagen-1+ fibrotic lesions by 46% and 30%, respectively, and significantly inhibited infiltrating Iba-1+ cells. In vitro, MSC-EVs attenuated TGF-β2-induced upregulation of αSMA, fibronectin, and collagen-1 at both protein and mRNA levels in RPE cells. Similarly, the expression of Acta2, Fn1, and Col1a1 in TGF-β1-treated macrophages was also significantly reduced following MSC-EV treatment. In LPS + IFN-γ-stimulated immune cells, MSC-EVs significantly suppressed the expression of Il6 and Il1b in all cell types, and reduced the expression of Inos, Tnfa, and Cd86 in iMACs, peritoneal macrophages, and BV2 cells. Enriched hsa-miR-21-5p was identified in MSC-EVs and involved in the TGF-β-related signaling pathway. Overexpression of miR-21-5p mimic abrogated the TGF-β1-driven upregulation of pro-fibrotic markers in RPE and macrophages. Local administration of MSC-EVs effectively mitigated subretinal fibrosis and reduced inflammation in the mouse model of nAMD, potentially via miR-21-5p-mediated attenuation of EMT and MMT, and suppression of inflammation. MSC-EVs represent a novel cell-free therapeutic strategy for macular fibrosis in nAMD.