Abstract
Keloids are pathological scars that extend outside the initial wound area and deposit excessive and irregular extracellular matrix (ECM). The reasons behind the formation of these scars are not fully understood and an animal model does not exist. Proteomics studies from the lab have shown that keloid lesions have a cartilage-like ECM composition. Furthermore, keloid tissue is composed of a fibrillar ECM network that is highly aligned, with extensive parallel bundling of collagen and fibronectin. The research presented in this thesis aims to improve understanding of these disease-associated features and discover regulatory mechanisms that might be driving the pathological composition and organisation of the ECM, by using in vitro models.Primary dermal fibroblasts derived from a keloid lesion (KDF) and adjacent normal skin (NDF) were cultured in 2D monolayers in the absence or presence of ascorbic acid to promote the production of ECM. Alignment of the ECM and fibroblasts was analysed using a Matlab script, with KDF shown to be significantly more aligned than NDF. This phenotypic trend was also confirmed with alignment analysis of fibroblasts isolated from other fibrotic diseases, including recessive dystrophic epidermolysis bullosa and normal scarring, in vitro. The transcription profile of NDF was compared to KDF with bulk RNA sequencing. Gene Ontology (GO) enrichment analysis showed that the differentially expressed genes (DEGs) increased in KDF compared to NDF were enriched for ECM and ECM related categories; many of the DEGs were structural constituents of the ECM and enzymes. These ECM components may mediate the aligned fibrotic architecture we describe. Furthermore, KDF also showed an increase in chondrogenic gene expression relative to NDF. To study this phenotype, NDF and KDF were grown with chondrogenic media in 3D micromass culture conditions. Alcian blue-positive staining, which demonstrates cartilage-like ECM, was uniquely observed in KDF grown in 3D chondrogenic differentiation protocols. ATAC-sequencing of fibroblasts grown in traditional (2D) culture conditions was conducted to question whether the KDF are epigenetically distinct from patient-matched and site-matched NDF, and whether this was maintained during fibroblast culturing, pursuing a hypothesis that this may reflect distinct cell plasticity in KDF. This approach revealed significant epigenetic differences between these primary cell lines; interestingly, there was enrichment for genes related to the pathological ECM within or near differentially accessible chromatin regions.
Furthermore, a previously observed cytokine of interest, IL-6, was shown to be elevated in KDF in the RNA-seq. Inhibition of IL-6 decreased the alignment of KDF and their ECM and addition of IL-6 increased the alignment in NDF and their ECM. Components of IL-6 downstream signalling pathways known to regulate ECM production, PI3K/AKT and MAPK/ERK, were also observed both in the RNA-seq and ATAC-seq. However transcriptional changes caused by IL-6 receptor inhibitor did not influence gene expression, indicating that IL-6 does not mediate cell alignment via transcription. This project has revealed important insights into the keloid phenotype, its regulation via IL-6 signalling, and the ability to pharmacologically revert those characteristics in vitro.
| Date of Award | 1 Mar 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Tanya Shaw (Supervisor) & Malcolm Logan (Supervisor) |