Abstract
Purpose :
Subretinal fibrosis occurs in multiple retinal diseases. Myofibroblast is a major cell type that contributes to fibrosis. However, the origin of myofibroblast is still controversial. EMT is critical in transforming RPE cells into myofibroblasts. The induction of EMT in RPE cells by TGF-β2 involves multiple signaling pathways. This project aims to elucidate myofibroblasts' origins and define the roles of the TGF-β/SMAD3 and LIMK2/MRTF pathways to find a potential therapeutic target.
Methods :
Postn-Cre;R26-tdTomato and Postn-MCM;R26-DTA mice have been used to label myofibroblasts and test its role in the laser-induced subretinal fibrosis. The contribution of specific cell lineages was confirmed using Cre-mice driven by different cell lineage promoters. TGF-β2 was used to induce EMT and fibrosis in ARPE-19 cells. The expression of EMT and fibrosis markers was analyzed by immunostaining. Active collagen accumulation was indicated by F-CHP staining. The contractility of myofibroblasts was assessed through gel contraction assay. The effects of SIS3 (Smad3 inhibitor) and CCG-203971 (MRTFA inhibitor) were investigated using similar experimental setups. We are in the middle of establishing a new ex vivo model of RPE EMT and fibrosis and testing the effect of inhibitors in this model. The function of miR-24 was also tested in a newly developed ex vivo model.
Results :
Postn-Cre can exclusively label the myofibroblasts that contribute to subretinal fibrosis, and its critical contribution was confirmed by DTA-mediated specific cell depletion. RPE cells, ECs, macrophages, and pericytes were found to undergo EMT, EndMT, MMT, and PMT, respectively, and contribute to myofibroblasts in a laser model. SIS3, or CCG-203971 can partially inhibit TGF-β2-induced EMT and fibrosis in RPE cells. The combination of the two can strongly repress this process. Overexpression of miR-24 can repress EMT and fibrosis in vitro and ex vivo.
Conclusions :
Myofibroblast critically contributes to subretinal fibrosis. RPE cells, ECs, macrophages, and pericytes contribute to myofibroblast in laser-induced subretinal fibrosis. The combination of SIS3 and CCG-203971 can repress EMT and fibrosis in RPE cells by regulating TGF/SMAD3 and the LIMK2/MTRF pathways. Overexpression of miR-24 represses EMT and fibrosis in vitro and ex vivo, suggesting its potential as a therapeutic agent for treating subretinal fibrosis in diseases.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.