Therapeutic angiogenesis : attempts to influence the survival of rat epigastric island flaps

• Main Author: Raine, Cameron
• Published: University of Edinburgh 2004
• Subjects: 615.19
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.660882

Recent work has suggested that therapeutic angiogenesis may be achieved using gene therapy techniques based on the cytokine vascular endothelial growth factor (VEGF). This project will investigate the hypothesis that such techniques may be used to induce angiogenesis in an experimental model of flap ischaemia and improve flap survival. All members of the VEGF family of cytokines stimulate angiogenesis. This work was based on VEGF beta (VEGFB). Two delivery strategies were investigated: intra-arterial administration of ‘raw’ plasmid DNA encoding the gene for VEGFB and subcutaneous injection of cultured cells genetically modified to produce the VEGFB cytokine. The rat epigastric island flap is designed to undergo partial ischaemic necrosis to a predictable extent. In this series mean flap survival was 85% (95% C.I. 82,89) at one week. Flap vascularity was further characterised histologically and angiographically at two time points: immediately following elevation and at one week when demarcation of the non-viable tissue had occurred. A pattern of physiological angiogenesis occurring within the ischaemic flaps was apparent from the analysis of these results. Optical conditions for gene transfer and expression were first investigated <i>in vitro</i> using the green fluorescent protein (GFP) reporter system and cultured endothelial cells. use of the transfection agent DOTAP significantly improved the number of cells expressing GFP at all observations. The substitution of a mammalian expression plasmid containing the VEGFB gene (pEF-BOS-VEGFB) for the reporter plasmid resulted in positive expression of VEGFB by the endothelial cells as detected by immunostaining. Successful transfer of the VEGFB gene to the endothelial cells with exogenous protein production had therefore been achieved. Replication of these conditions <i>in vivo</i> using the rat epigastric island flap model however, failed to produce any evidence of either gene transfer to the arterial wall within the flap pedicle or stimulated angiogenesis within the flap itself. It is likely that these systems failed as a result of their inability to deliver sufficient active cytokine to the tissues of the flap. In addition, evidence is emerging that therapeutic angiogenesis may best be performed using a combination of agents where additional cytokines are employed in conjunction with primary angiogenic agent to promote both the stability and function of immature endothelial cells. The optimisation of delivery strategies for the transfer of genetic material to experimental flaps coupled with a greater understanding of the most effective agent, or combination of agents, needed to produce new vessels which are both functional and permanent are essential requirements for the ultimate success of this technology.