Substrate stiffness modifies gene expression and transcriptional response of equine endometrial fibroblasts to TGF-β1

Equine endometrial fibrosis is a leading cause of subfertility in aging mares. Fibrosis is a reparative response involving excess deposition of extracellular matrix (ECM) and increasing tissue stiffness. Augmented rigidity itself can drive fibrosis, by stimulating transition from fibroblasts to myofibroblasts. Myofibroblasts release latent transforming growth factor beta 1 (TGF-β1) from the ECM, thereby activating this profibrotic cytokine. Tissue culture polystyrene (TCP) is commonly used for in vitro experiments. The endometrium, however, is considerably softer than TCP. This study critically evaluated the use of hydrogels versus TCP. Differences in transcript abundance between equine endometrial fibroblasts cultured on TCP and hydrogels of decreasing stiffness (25 kPa to 2 kPa) and their transcriptional response to TGF-β1, were examined. Cells cultured on substrates of varying stiffness exhibited visual variations concerning adherence, morphology, and cell density, besides differences in basal gene expression and transcriptional response to TGF-β1. On stiffer substrates, the smooth muscle genes TAGLN and ACTA2, alongside the transcripts encoding the signaling proteins PDGFB, CCN2, and SERPINE1 were expressed at higher levels. This pattern was also evident for integrin receptor subunit ITGAV, while ITGB5 was expressed at lower levels on stiffer substrates. While ITGB3 demonstrated a response to TGF-β1 exposure independent of stiffness, an increase in transcript abundance of PDGFB, ITGAV, and ITGB5 towards TGF-β1 was only observed on softer hydrogels. The results highlight the importance of stiffness in cellular regulatory patterns, particularly relevant to fibrosis research. We recommend critically reconsidering the use of TCP when designing experiments in vitro.