Milk fat globule EGF and factor V/VIII domain containing (MFGE8) as a novel player in equine endometrial fibrosis

Abstract With increasing age, mares are frequently affected by endometrial fibrosis. Transforming growth factor beta 1 (TGF-β1) promotes fibrosis, while milk fat globule EGF and factor V/VIII domain containing (MFGE8) primarily exerts antifibrotic effects. We used in situ hybridization analysis (ISH) to investigate MFGE8 , TGFB1 and its downstream effectors cellular communication network factor 2 ( CCN2 ) and transgelin ( TAGLN ) in equine endometrial fibrosis. Additionally, transcriptional effects of TGF-β1 and MFGE8 on equine endometrial fibroblasts in vitro were investigated by single cell sequencing. Fibrotic areas overexpressing TGFB1 displayed increased CCN2 and TAGLN expression. The vast majority of glandular areas overexpressing MFGE8 were affected by fibrosis, confirming its relevance in equine endometrial fibrosis. Single cell sequencing following TGF-β1 exposure of fibroblasts confirmed its profibrotic effects. MFGE8 treatment effects were subtler, including decreased profibrotic leukemia inhibitory factor expression and an upregulation of interferon alpha and beta signaling in a subset of cells. In a fibroblast subpopulation, upregulation of phosphatase and tensin homolog signaling, indicated an antifibrotic effect of MFGE8. In conclusion, our study reaffirms the profibrotic effects of TGF-β1 and highlights MFGE8 as a novel player in equine endometrial fibrosis with our results suggesting an antifibrotic effect of MFGE8.