- Title
- In vitro and in vivo investigations of the α-integrins regulated by Hypoxia Inducible Factor (HIF)-1 signalling during mucosal wound healing
- Creator
- Goggins, Bridie Jane
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2019
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Inflammatory Bowel Disease (IBD), which includes Crohn’s disease and ulcerative colitis, affects over 5 million people worldwide and the rate of incidence is increasing. IBD is characterised by chronic, immune-mediated inflammation of the gastrointestinal (GI) tract, which contributes to repeated cycles of epithelial wounding and repair in the intestine. In healthy individuals, the intestinal epithelium functions as a selective barrier and is critical for mucosal protection. During intestinal inflammation, damage to the intestinal epithelium leads to a loss of barrier function and epithelial repair is critical to restore GI integrity and homeostasis. Due to vascular damage and increased metabolic demands, epithelial wound healing occurs in an hypoxic environment. Hypoxia inducible factor (HIF)-1 is an oxygen-sensing transcription factor that is key to the co-ordination of mucosal wound healing. HIF-1 is an αβ heterodimeric protein that regulates expression of genes important for cell protection, survival and repair. Previous work has shown that pharmacological stabilisation of HIF-1 by prolyl hydroxylase inhibitors (PHDi) is protective in murine models of colitis. In addition, it has been shown that HIF-1 mediated induction of the cell adhesion protein, integrin-β1 is critical for epithelial and fibroblast function in wound healing in vitro and associated with the progression of wound healing in vivo. Integrin proteins function as heterodimers and mediate epithelial migration through their roles in cell adhesion and migration. Given that integrin-β1 is a HIF-responsive gene, we hypothesised that pharmacological HIF-1 stabilisation accelerates epithelial restitution through integrin-dependent pathways. The aim of this study was to examine the functional role as well as the transcriptional and post-translational activity of epithelial α-integrins dimerising with integrin-β1 to promote HIF-1-mediated wound healing. In this thesis, in vitro cell migration scratch assays, performed in T84 human intestinal epithelial cell lines, showed accelerated cell migration in response to HIF-1 stabilisation. We screened mRNA for α-integrins increased in response to HIF-1 stabilisation and found HIF-increased expression of ITGA5, as well as hypoxia-responsive increases in ITGA2. Integrin-function blocking experiments revealed the functional contribution HIF-1 mediated integrin-α2 and integrin-α5 in accelerated wound closure in vitro. Fluorescent staining of integrins-α2 and α5 revealed an increase in integrin-α2 at the leading edge of migrating cell monolayers and re-localisation of integrin-α5 from perinuclear compartments to the cell cytoplasm following treatment with PHDi. These results suggest that HIF-1 signalling augments epithelial migration through increased integrin-α2 positive adhesions at the leading edge of the wound as a point of anchor, in conjunction with re-localisation of integrin-α5 to the cell periphery to facilitate cell propulsion. In vitro investigations also outlined microtubule-based motor protein; kinesin family of proteins-1C (KIF1C) as a potential HIF-1 mediated transport mechanism for trafficking integrin-α5 during accelerated wound closure. In vivo investigations demonstrated the therapeutic efficacy of HIF-1 stabilisation in mucosal wound healing using two animal models including a mucosal wound-biopsy model and the 2,4,6-Trinitrobenzenesulfonic acid (TNBS) mouse model of colitis. Accelerated healing was associated with increased staining of integrin-α5 and to a lesser extent KIF1C in fibroblast populations in the granulation tissue of mucosal wounds and inflamed tissue, as well as increased KIF1C in the epithelium. These investigations suggest the induction of integrin-α5 and KIF1C, through stabilisation of HIF-1 contributes to accelerated healing of both injured and inflamed mucosa in vivo. Combined, these studies have outlined the potential therapeutic use of HIF-1 stabilisation and its down-stream signalling to integrins and their transport mechanisms to accelerate mucosal wound healing. This therapeutic approach may be used to augment mucosal wound healing processes in the treatment of inflammatory mucosal diseases such as IBD.
- Subject
- inflammatory bowel disease; hypoxia inducible factor; HIF-1; wound healing; cell migration
- Identifier
- http://hdl.handle.net/1959.13/1397968
- Identifier
- uon:34379
- Rights
- Copyright 2019 Bridie Jane Goggins
- Language
- eng
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 11 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 601 KB | Adobe Acrobat PDF | View Details Download |