In wound healing and tissue repair, the presence of α-smooth muscle actin (α-SMA) containing myofibroblasts leads to wound closure and collagen-rich scar formation. This thesis investigated mechanisms of transforming growth factor-β1 (TGF-β1)-mediated differentiation and the dysfunctions involved in age-associated loss of differentiation. The loss of epidermal growth factor receptor (EGFR) and hyaluronan (HA) production, and diminished interaction of HA with its receptor CD44 (compromising its function) were the principal contributors to aged fibroblast resistance to differentiation. In response to TGF-β1, CD44 relocated to EGFR held in cholesterol-rich membrane-bound lipid rafts. This was HA-dependent, as hyaluronidase or 4-methylumbelliferone treatments restricted CD44 motility and prevented CD44-EGFR co-localisation. Additionally the intracellular signalling cascade was found to be a sequential phosphorylation of extracellular signal regulated kinase 1 & 2 followed by Ca2+/calmodulin kinase II. The activation of both proteins was required for differentiation. Elevated microRNA-7 (miR-7) expression was found in aged fibroblasts. Overexpressing miR-7 in young fibroblasts attenuated the expression of EGFR and inhibited differentiation. When miR-7 was inhibited, EGFR and hyaluronan synthase 2 expression, CD44 membrane motility, and TGF-β1-mediated differentiation in aged fibroblasts were restored. Activation of EGFR drove miR-7 promoter activity and expression in a JAK/STAT1 dependent manner. Treatments of aged fibroblasts with 17β-estradiol (E2) resulted in decreased miR-7 expression and, when TGF-β1 was added, restored the α-SMA-positive phenotype. E2 treatments had no impact on STAT1 phosphorylation; leading to the hypothesis that E2 regulation of inflammatory mediators may be involved. The data demonstrated different points of intervention for the promotion or prevention of TGF-β1-regulated myofibroblast differentiation. The interactions between HA-CD44 and EGFR were crucial elements in the differentiation process and the importance of miR-7 was apparent. The mechanisms shown here may have direct implications for modifying the wound healing response, particularly for developing therapeutic strategies to improve healing in the elderly.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:600617 |
| Date | January 2014 |
| Creators | Midgley, Adam Christopher |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/59241/ |
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