237 Interplay between horse adipose mesenchymal stem cells and TGF-β1 signalling results in changes in the antifibrotic properties of MSC: implications for the treatment of endometriosis

TGF-β1 is a pleiotropic cytokine central to the resolution of inflammation; its persistent expression leads to myofibroblast differentiation, dysregulation of extracellular matrix, excessive collagen-I deposition, compromise of the functionality of the endometrial glands, and endometrosis. Currently, there is no cure for endometriosis. Mesenchymal stem cells (MSC) have been used to treat it with little success. We hypothesie that, in the scenario of endometrosis, exogenous MSC under continuous exposition to TGF-β1 will change their phenotypes, depending on the time and intensity of exposition, from antifibrotic to pro-fibrotic. Here, we analysed the changes on gene and protein expression in response to TGF-β1 signalling in MSC, their extracellular vesicles (EV), miRNA cargo, and the capacity of revert fibrotic in an in vitro model of endometrial fibrosis. Experiment 1: MSC were exposed to TGF-β1 (10 ng/mL) for 0, 4, and 24 h in DMEM + 1% fetal bovine serum depleted of EV. At the indicated time points, cellular proteins were isolated and assayed by Western blot (aSMA, pSMAD2, pSMAD3, b-Catenin). Total RNA was isolated and the ratio of expression of Col1A1/Col3a1, MMP9/TIMP1 and MMP2/TIMP2 were studied by qPCR. The EV were separated by ultracentrifugation, nanotracked, and validated by Western blotting. The cargo of fibrotic-related miRNAs was evaluated using qPCR for the antifibrotic miRNAs (mir-29, mir-122, mir-486, and mir-19) and the profibrotic miRNAs (mir-192 and mir-199). Experiment 2: Endometrial fibroblasts were challenged for 24 h with IL1, IL6, TNFa and TGF-β1 (10 ng/mL of each) or mock (PBS). After this, endometrial fibroblasts were incubated with 1 × 10⁹ EV/mL collected after 48 h of culture of MSC (Experiment 1) and exposed for 0, 4, and 24 h to TGF-β1. The cellular mRNA was isolated and aSMA, CGTF, and Col1a1/Col3a1 ratio expression tested using DDCT method. Statistics: ANOVA and Tukey post hoc with 0 h as reference (Experiment 1) or wild type (Experiment 2), P < 0.05. Results: In Experiment 1, at 4 h of exposition to TGF-β1, there was an increment of antifibrotic mirNAs 29b, 122, 486, and 19 (P < 0.05). Exposition for 24 h hours led to a rise in the expression of profibrotic mirNAs 192 and 199; of mRNA for COL1A1/COL3A1; and proteins aSMA, pSMAD2, pSMAD3, and b-Catenin as well as a decrease of MMP2/TIMP2, MMP9/TIMP1 mRNA ratio and PGE₂ and IL6 release to the medium. In Experiment 2, EVs from 4 h TGF-β1 produced an inhibitory effect at the mRNA level on aSMA and CGTF expression in the endometrial fibroblasts (P < 0.05) and non-appreciable change in Col1A1/Col3a1 mRNA ratio. Treatment for 24 h incremented aSMA, CGTF, and Col1A1/Col3a1 mRNA ratio (P < 0.05). We confirm that exposition to TGF-β1 modulates the antifibrotic/profibrotic properties of MSC and that EV from treated MSC could finely tune the TGF-β1 signalling pathway. This can be a potential tool for regenerative therapies of endometriosis and others fibrosis-related diseases.

This research was supported by FONDECYT 1210349 and VRID 219.153.027-INV.