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Evaluation of the stimulatory effects of EBC-46 on dermal fibroblast and keratinocyte wound healing responses in vitro and correlation to preferential healing in vivo

Moses, Rachael Louise 2016. Evaluation of the stimulatory effects of EBC-46 on dermal fibroblast and keratinocyte wound healing responses in vitro and correlation to preferential healing in vivo. PhD Thesis, Cardiff University.
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Abstract

The novel epoxy-tiglianes, EBC-46 and ‘lesser activity’ EBC-211, are sourced from seeds of the Fountain’s Blushwood Tree (Fontainea picrosperma), indigenous to Queensland Tropical Rainforest. Australian biotechnology company, QBiotics Ltd.,has demonstrated that EBC-46 stimulates exceptional dermal wound healing responses in vivo, following cancer treatment and tumour destruction in domesticated animals. Consequently, QBiotics is developing EBC-46 as a veterinary anti-cancer pharmaceutical and performing human clinical trials. However, little is known on how EBC-46 induces its exceptional healing effects, manifested as accelerated wound re-epithelialisation, closure and reduced scarring. This study aimed to elucidate how EBC-46 and EBC-211 mediates these exceptional wound healing effects in vitro, through analysis of HaCaT keratinocyte and dermal fibroblast/myofibroblast genotypic and phenotypic responses, following epoxytigliane treatment (0.001-100μg/ml). A number of key wound healing responses were assessed, including proliferation, cell cycle progression, scratch wound repopulation; and transforming growth factor-β1 (TGF-β1)-driven, fibroblastmyofibroblast differentiation. Studies demonstrated that both EBC-46 and EBC-211 induced fibroblast and HaCaT cytotoxicity at 100μg/ml. EBC-46 and EBC-211 (0.001-10μg/ml) significantly retarded fibroblast proliferation and delayed S/G2 cell cycle transition, but exerted no significant effects on fibroblast migratory responses. Although EBC-46 had no effects on α-smooth muscle actin (α-SMA) expression, stress fibre organization and myofibroblast formation (0.001-0.01μg/ml and 1-10μg/ml), EBC-46 significantly inhibited α-SMA expression and stress fibre formation at 0.1μg/ml, with cells retaining normal fibroblast morphologies. EBC-211 induced similar effects at 10μg/ml Both EBC-46 and EBC-211 (0.001-10μg/ml) stimulated significant HaCaT proliferation, G1/S and S/G2 cell cycle transitions; and accelerated scratch wound repopulation, even with mitomycin C. Microarray analysis and protein level validation, identified numerous differentially expressed genes in epoxy-tiglianetreated, HaCaTs. Up-regulated genes included certain keratins and others associated with promoting cell cycle progression, proliferation and migration. Down-regulated genes included other keratins and genes associated with inhibiting cell cycle progression and proliferation, including certain cytokines and chemokines. This study has provided evidence to explain the enhanced re-epithelialisation and reduced scarring responses observed in epoxy-tigliane-treated skin. Furthermore, it highlights the potential of epoxy-tiglianes as novel therapeutics for impaired dermal wound healing and excessive scarring situations.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Dentistry
Subjects: R Medicine > RK Dentistry
Uncontrolled Keywords: epoxy-tiglianes; wound healing; skin; fibroblasts; keratinocytes; chronic wounds; scarring.
Funders: QBiotics Ltd, CITER
Date of First Compliant Deposit: 22 December 2016
Last Modified: 26 Jan 2018 02:31
URI: http://orca.cf.ac.uk/id/eprint/97027

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