117 Extracellular vesicles from endometrial fibroblasts exposed to a chronic inflammation stimulus induce an up-regulation of fibrotic related genes in mare endometrial epithelial cells

Postbreeding inflammation is a physiological event in mares, nevertheless in some animals there is a prolonged and aggravated response leading to endometritis. Repeated acute inflammation might lead to endometrosis, a degenerative disease with a profound impact on the reproductive performance of mares. The unremitting presence of proinflammatory cytokines (IL-1b, IL-6, and TNFa), profibrotic TGFb, as well as profibrotic miRNAs causes differentiation of resident cells into myofibroblasts and over-deposition of extracellular matrix, which leads to fibrosis affecting both the stroma and epithelium of the endometrium. We hypothesise that extracellular vesicles (EV) from myofibroblasts can transmit the fibrotic phenotype to endometrial epithelial cells. Materials and methods: confluent endometrial fibroblasts cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 1% of fetal bovine serum depleted of EV (FBS-d) were exposed to a fibrotic cocktail (IL-1b, IL-6, TNFa, and TGFb, 10 ng/mL each) for 24 h in 5% CO₂ and full humidity. After this period, the cell supernatant was collected, EVs were isolated by ultracentrifugation, quantified by nanoparticle tracking analysis (NTA) and confirmed by western blotting for EV proteins CD9 and CD63. Endometrial epithelial cells previously isolated and characterised in the laboratory were cultured as indicated for fibroblasts. When confluent, the medium was removed, and the cells were washed three times with serum-free phosphate-buffered saline (PBS) and changed to DMEM with 1% FBSd + 1 × 10e10 EVs/mL for 48 h. Controls were DMEM with 1% FBS-d (not exposed to any cells) as negative control, and DMEM with 1% FBSd + fibrotic cocktail as for induced fibroblasts (positive control). At 48 h, the cellular mRNA was isolated and the expression of candidate (fibrotic) genes was analysed by quantitative polymerase chain reaction (DDCT method). Individual genes analysed were aSMA and CGTF, as well as the ratio of expression of MMP9/TIMP1, MMP2/TIMP2 and Col1a1/Col3a1. Results were expressed as fold change (FC); ANOVA and Tukey post hoc with P < 0.05. As expected, epithelial cells in the positive control displayed a significant up-regulation of aSMA, CGTF, MMP9/TIMP1, MMP2/TIMP2, and Col1a1/Col3a1 ratios (P < 0.05). No significative changes were found for the analysed genes in the negative control group, whereas in the epithelial cells exposed to EV from fibroblasts challenged with the fibrotic cocktail, the expression pattern was coincident with the positive control (P = 0.3) and significantly different (P < 0.05) to the negative control. The EVs released from fibroblast exposed to chronic inflammation promote fibrotic phenotype of epithelial cells and probably their trans differentiation into myofibroblasts. This can be of use for the understanding of endometrosis in mares.

Funding for this study was provided by FONDECYT 1210349 and VRID 219.153.027-INV.