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Title: The role of connexins in skin wound healing events
Author: Lorraine, Claire
ISNI:       0000 0004 5989 7289
Awarding Body: Glasgow Caledonian University
Current Institution: Glasgow Caledonian University
Date of Award: 2015
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Chronic non-healing wounds are a major health problem. The protein connexin 43(Cx43), known to be dynamically remodelled during wound healing, is abnormally up-regulated in chronic wounds. Both anti sense and peptidomimetic technologies targeting Cx43 are emerging as prime therapeutic agents for wound healing. It remains unresolved whether inhibiting Cx43 gene expression or blocking Cx43 channel function is the key regulator in these events. Previous in vitro studies using primary dermal fibroblasts determined that the connexin mimetic peptide (CMP) Gap27 inhibited connexin channel function, improved scrape wound closure rates and altered expression of genes associated with the extracellular matrix (ECM). In this study we investigated the role of both Cx43 channel function and Cx43 gene expression in scrape wound closure and in relation to the ECM. Our in vitro data has contributed to the validation of theoretical descriptions produced by the in silico mathematical model in respect of normal wound healing. Image trajectory analysis of time-lapse scrape wound closure in human dermal fibroblasts identified increased velocity in cells at the edge of the scraped area compared to those adjacent to the wound edge. Gap27 (1 OO~M) treatment increased the velocity of cells adjacent to the wound edge to a level comparable with that of the wound edge cells, enhancing scrape wound closure in these cells. In addition, Gap27 (100nM and 1 OO~M) together with two derivatives of Gap27, Compound A (100nM) and Compound B (100nM), attenuated ATP release in human dermal fibroblasts. Scrape wound assays were also performed in mouse dermal fibroblasts isolated from Cx43 wild type (Cx43+ 1+) and Cx43 knockout (Cx43-1-) C57/BL neonatal mice. Real-time PCR and Western blot analysis determined the loss of Cx43 in cells sourced from the Cx43-1 - mice. Gap27 (1 OO~M) enhanced scrape wound closure in Cx43+ 1+ fibroblasts and attenuated ATP release in these cells but had no effect on hemichannel activity in Cx43+ fibroblasts. Scrape wound closure in Cx43-1 fibroblasts was slower than in Cx43+1+ cells, even when cells were plated on different extracellular matrices. In contrast, knockdown of Cx43 protein (-50%) in a keratinocyte cell line (HaCaT cells), determined by both real-time PCR and Western blot analysis, enhanced scrape wound closure. Real-time PCR analysis determined that MMP-9 levels were enhanced in Cx43-1- fibroblasts and myofibroblast transition was also restricted in these cells. The data from this study provides further support that Cx43 plays diverse roles in co-ordinating wound closure events. The results indicate that both channel and nonchannel functions of Cx43 may be involved in this process. The inverse correlation between Cx43 and MMP-9 gene expression observed suggests a role for Cx43 in regulating the ECM. While both CMPs and antisense oligonucleotides hold therapeutic potential, new roles for Cx43 in non-channel functions relating to cell motility are continually emerging. The data supports the development of CMPs in preference to antisense treatments as therapeutic agents for wound healing, due to CMPs having the capability to maintain Cx43 expression whilst inhibiting channel function.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available