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Title: The role of Toll-like Receptor 4 in diabetic foot ulceration
Author: Portou, Mark James
ISNI:       0000 0004 8500 4077
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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Introduction Diabetes Mellitus has reached epidemic proportions. Foot ulceration is a multifactorial complication of diabetes associated with marked morbidity and mortality. Innate-immune Toll-like receptor 4 (TLR4) mediated inflammation has been implicated in the systemic pathogenesis of diabetes and may contribute to impairment of wound healing. This thesis investigates the effect of high glucose and hypoxic conditions on TLR4 activation and signalling in vitro and in vivo. Method Human skin biopsies were stained for H&E and TLR4. Fibroblasts cultured at physiological glucose concentration (5.5mM) were exposed to pathological glucose concentrations (25mM), with duplicate samples placed in a hypoxia chamber. The effect of TLR4 and its signalling pathway was assessed through specific inhibitors. Diabetes was induced in wild-type (WT) and TLR4 knock-out C57BL/6 mice by intra-peritoneal injection of low-dose streptozocin. Hindlimb ischaemia was induced by femoral artery ligation four weeks post- streptozocin, and a full thickness skin wound inflicted below the knee. Wound healing was assessed via digital planimetry at days 3, 7 and 14 post surgery. Results Diabetic-ischaemic ulcers demonstrated greater inflammatory cell infiltration (p=0.0001) and TLR4 expression (p=0.038). Hypoxic and high glucose (25mM), conditions led to an increase in TLR4 protein expression, apoptosis and IL-6 release. Inhibition with MyD88 inhibitory-peptide, TLR4 neutralising-antibody and specific TLR4 antagonist ameliorated the effects of high glucose and ischaemia (p < 0.05). In vivo, wound healing was significantly impaired in the diabetic-ischaemic group at day 14 (p < 0.05). Diabetic-ischaemic wounds in TLR4 KO mice exhibited significantly improved healing rates compared to those in WT mice at all time points. Conclusion Diabetic-ischaemic ulceration is associated with increased cellular inflammation and TLR4 expression. Hypoxia stimulates up-regulation of TLR4 protein expression and this effect is exaggerated by hyperglycaemia. Inhibition of TLR4 significantly reduced TLR4 protein expression, apoptosis and IL-6 release, suggesting a protective effect. In TLR4 KO mice, there is a significant improvement in the healing of diabetic-ischaemic wounds compared to WT. We propose a synergistic effect between hypoxia and hyperglycaemia impairing wound healing exists, through TLR4-mediated inflammation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available