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Title: Effects of diabetes and Hoxa3 upon macrophage function
Author: Burgess, Matthew
ISNI:       0000 0004 6059 914X
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2016
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Chronic non-healing wounds commonly present in patients with diabetes. These wounds are characterised by elevated numbers of immature leukocytes and M1 macrophages and reduced numbers of endothelial cells and M2 macrophages, impairing wound healing resolution. Topical treatment of murine diabetic wounds with a Hoxa3 gene expression vector redresses the balance of inflammatory and pro-healing cells within the lesion, reducing excessive inflammation and rescuing the wound healing phenotype. In this thesis I present experiments to further understanding of how diabetes alters the macrophage phenotype and how this may cause the decreased endothelial cell and M2 macrophage numbers in the diabetic wound. In vitro culture was used to characterize the intrinsic changes of diabetic macrophages isolated from the environmental effects of the diabetic wound milieu. These same systems were used to develop a cell culture system for the promotion of monocytic to endothelial transdifferentiation. Finally the in vitro macrophage culture system was used to assess the effects of Hoxa3 treatment upon diabetic macrophages and how Hoxa3 transcriptional activity in macrophages may contribute to the restoration of wound healing. In vitro cultured diabetic macrophages were observed to raise an increased response to classical and alternative activation signals that may contribute to the excessive inflammatory state of diabetic cutaneous wounds. Treatment of these macrophages for four days with a Hoxa3 conditioned medium protein transduction system upregulated the expression of the plasminogen activator urokinase receptor gene Plaur and enhanced the expression of macrophage maturation markers. These macrophages also exhibit an enhanced response to classical activation stimuli, a reduced alternative activation response. In an in vitro neovascularisation assay Hoxa3 treated macrophages inhibit vessel growth. These effects of Hoxa3 treatment of diabetic macrophages are unexpected based on the rescue of the inflammatory phenotype with Hoxa3 treatment of diabetic wounds. Non-diabetic macrophages were also treated for four days with a Hoxa3 conditioned medium and exhibited upregulation of macrophage maturation markers. These macrophages showed no difference in activation state polarisation compared to macrophages grown in a control conditioned medium but did upregulate activation markers in unstimulated cells. This may be indicative of a priming for response to low levels of activation stimuli. The Hoxa3 treated non-diabetic cells also promoted the formation of vessel networks in a neovascularisation co-culture assay, possibly through the promotion of angiogenesis. These results suggest that diabetes directly effects the maturation and inflammatory phenotype of macrophages and that Hoxa3 treatment rescues the impaired maturation phenotype and may stimulate macrophage populations to a pro-angiogenic state.
Supervisor: Mace, Kimberly Sponsor: Medical Research Council ; Zochonis Charitable Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Diabetes ; Inflammation ; Macrophage ; Wound Healing ; Hoxa3 ; Maturation ; Activation