Therapeutic applications of endothelial progenitor cells for diabetic ulcer healing
Kealy, John Brian
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Diabetic ulcer healing is a major problem in health care. Impaired circulation has long been associated with diabetes resulting in an increased risk of foot ulceration and making an increase in blood flow an excellent candidate for a therapeutic intervention. Since being identified in 1997, much interest has surrounded endothelial progenitor cells (EPCs) as a means of facilitating neovascularisation. In this study we aimed to explore the use of EPCs to enhance blood vessel formation in the diabetic ulcer and to examine their effects on the healing of these wounds. Firstly, culture and characterisation of the cells had to be optimised. Subsequently a rabbit ear ulcer model was developed and used to explore the potential therapeutic effects of EPC transplantation in vivo. We isolated two types of cell from peripheral blood. The first type, being consistent with previously described outgrowth endothelial cells, displayed endothelial characteristics and showed good clonogenic potential. These cells correspond to endothelial colony forming cells (ECFC) as described in the review by Hirschi et al (Hirschi et al, 2008). However in spite of the fact that this cell type may represent true endothelial progenitor cells, the relative unreliability of culturing these populations from a given sample of peripheral blood made them unsuitable for subsequent therapeutic in vivo work. ECFCs directly incorporate into newly forming blood vessels. The second type also displayed endothelial characteristics, manifest by uptake of acetylated LDL and binding of isolectin, and was consistent with described early endothelial progenitors. It has recently become evident that these cells are monocytic in nature, do not directly differentiate to endothelial cells and support angiogenesis not by direct incorporation but by paracrine means. These cells have been designated circulating angiogenic cells by Hirschi et al. It was this population of cells, CACs, that was used for the subsequent in vivo experiments. Although ECFCs show greater clonogenic potential and a greater transduction efficiency they were a less suitable cell choice for the work reported here compared to CACs as ECFCs are not as reliably isolated from relatively small volumes of blood. This likely represents relatively fewer numbers circulating in in peripheral blood. Since autologous cells were used in this work, only small volumes of blood could be used from which to isolate cells and so CACs were the cell of choice for this work. Gene therapy is also an area of research being applied to a wide range of topics. In this study we also looked at the use of genetically modified EPCs and their applications to wound healing. Specifically, the gene encoding endothelial nitric oxide synthase was used as the transgene as the angiogenic and vasodilatatory properties of nitric oxide have been well established. We initially explored the use of a variety of vector systems for genetic modification of this cell type. After comparing different viral as well as non-viral vectors, adenovirus was selected as the vector of choice. In addition two different cell subpopulations could be identified based on relative transduction efficiencies. A model of diabetic wound healing was initially established for the in vivo experiments. We chose the alloxan induced diabetic ear wound to avoid problems with wound contraction which can occur in other models using the dorsum of the animal. After establishing the model, we studied the effect of duration of diabetes on wound healing. Diabetes was induced 1 week or 4 weeks before creation of the wound and healing was measured after 1 week. A significant restriction of healing was noted in the group where diabetes was induced 4 weeks before wound induction with less restriction being observed where wounds were induced 1 week after the induction of diabetes mellitus. Next, transduced as well as non-transduced cells were delivered to the wound site by way of a collagen scaffold. In terms of the treatment of the wounds with transduced or non-transduced EPCs, no differences were seen in the healing parameters measured when examined at 1 week wound induction after either 2 or 5 weeks hyperglycaemia. Since diabetic ulcer healing has been long identified as an issue of significant clinical concern the aim of this work was to explore strategies for promoting and accelerating healing. Angiogenesis is integral to wound healing and a growing body of work demonstrates the contribution of EPCs in a variety of clinical and pre-clinical settings through promotion of angiogenesis. NO has also been shown to have significant functions regarding angiogenesis and endothelial function. In addition, functional impairment of EPCs has been demonstrated in the diabetic setting and so the aim of this work was to explore whether functionality could be restored to diabetic CACs through gene modification to overexpress eNOS and the effect on diabetic ulcer healing of treatment with these modified cells. A secondary aim was to establish the most efficient vector for transduction of these cells.
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