P27 Modelling and mechanisms of keloid fibroblast plasticity

Abstract Introduction and aims Incorrect wound repair can lead to keloid scars, which are unsuccessfully managed clinically because of limited understanding of aetiology, a lack of modelling of key scar features, and reliance on insufficient biomarkers. Recently, we discovered that keloid lesions have cartilage-like extracellular matrix, suggesting that keloid fibroblasts misdifferentiate to an inappropriate phenotype. Therefore, we hypothesize that keloid fibroblasts are more plastic than normal fibroblasts and aim to test their differentiation potential and underpinning epigenetic and transcriptional mechanisms. Methods Primary keloid dermal fibroblasts (KDF) and adjacent normal skin (NDF) were cultured in standard two-dimensional (2D) conditions, then analysed with bulk Assay for Transposase-Accessible Chromatin (ATAC) and RNA sequencing. To investigate whether KDFs have distinct plasticity, cells were exposed to differentiating culture conditions. Results The chromatin was globally more accessible in the KDF vs. NDF, a feature associated with stemness and plasticity. Indeed, keloid differentially accessible regions (DARs) were found to extensively overlap with the epigenetic signature of induced pluripotent stem (iPS) cells. Gene ontology (GO) enrichment analysis of keloid DARs further confirmed that ‘cell differentiation’ and ‘embryonic morphogenesis’ are distinguishing features. Transcription factor motifs enriched in keloid revealed master regulators of chondrogenic and adipogenic differentiation (e.g. RUNX1/2, TEAD). To compare the amenability of NDF vs. KDF to adopt different phenotypes, cells were subjected to prochondrogenic and proadipogenic conditions. In chondrogenic media in 2D monolayers and three-dimensional (3D) micromass culture conditions, alcian blue-positive staining was uniquely observed in KDF grown in both 2D and 3D, akin to keloid histology. Interestingly, KDF also showed more lipid accumulation compared with NDF in proadipogenic conditions. Conclusions The findings demonstrate the unique plasticity of keloid cells and provides evidence that this feature is in part regulated epigenetically. Pharmacological manipulation of the epigenetic and transcriptional regulation of the cartilage-like phenotype observed in disease is hypothesized to be of therapeutic value.