Abstract
Purpose :
Subretinal fibrosis occurs in multiple retinal diseases, including wet Age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Myofibroblast is a major cell type that contributes to fibrosis. However, origin of myofibroblast during subretinal fibrosis is still unknown. The purpose of the project is to determine the myofibroblast origins and to define the function of miR-24 in subretinal fibrosis.
Methods :
Perostin (Postn)-Cre;R26-tdTomato and Postn-MCM;R26-DTA mice were used to label myofibroblast and test its role in laser-induced subretinal fibrosis. The contribution of specific cell lineages was confirmed using five cre-expressing mouse lines. MiR-24 function in epithelial-mesenchymal transition (EMT), endothelial-mesenchymal transition (EndMT) and fibrosis were examined by overexpression in ARPE-19 cells and HUVEC. Novel miR-24 target genes were confirmed by Western blot and luciferase assays. Nuclear translocation of MRTF-A was tested after TGF-β2 treatment. Functional study of miR-24 in vivo has been done using a modified PVR model and is ongoing using laser injury model.
Results :
By genetic lineage tracing, Postn-Cre can exclusively label the myofibroblasts and its critical contribution was confirmed by DTA mediated specific cell depleting. RPE cells, ECs, pericytes, and macrophages were found to undergo EMT, EndMT, pericyte-myofibroblast transition and macrophage-mesenchymal transition (MMT), and contribute to myofibroblasts in a laser-induced subretinal fibrosis model. miR-24 overexpression repressed EMT and EndMT as shown by Western blot and immunostaining. LIMK2 was confirmed as target of miR-24 in RPE cells. This is consistent with repressed stress fiber formation, increased G-actin/F-actin ration, and blunted TGF-β-induced nuclear translocation of MRTF, a key gene involved in EMT, EndMT and fibrosis. SMAD3 was identified as a novel miR-24 target gene in human cells. In the in vivo PVR model, overexpression of miR-24 in the injected ARPE-19 cells suppressed PVR formation.
Conclusions :
Myofibroblasts critically contribute to subretinal fibrosis. RPE cells, ECs, pericytes, and macrophages are the major contributors to myofibroblasts in laser-induced subretinal fibrosis. MiR-24 overexpression prevents EMT, EndMT and fibrosis by targeting TGF/SMAD3 and LIMK2/MTRF pathways. MiR-24 could be a potential therapeutic agent for treating fibrotic eye diseases.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.