Vascular Endothelial Growth Factor, diagnostic and therapeutic aspects

This thesis focuses on the diagnostic and therapeutic potential of Vascular Endothelial Growth Factor, an angiogenic growth factor. Since the recognition of VEGF’s pivotal role in physiological and pathological angiogenesis research has evolved from cell culture and animal experiments to application in humans. The studies described in this thesis aim to contribute to the evaluation of circulating VEGF as a surrogate marker in the quantification of angiogenesis and of the therapeutic role of VEGF especially in therapeutic angiogenesis using gene therapy. In chapter 2 and chapter 4 are general introductions to the clinical studies on therapeutic angiogenesis and basic research on angiogenesis respectively. A review of the literature, presented in chapter 2, describes several potential clinical applications emerging in animal studies on therapeutic angiogenesis. These studies demonstrated that intramuscular (i.m.) gene therapy with the naked plasmid DNA encoding for VEGF165 could potentially address the limitations observed with the administration of the recombinant VEGF protein. VEGF exerts a site specific effect, due to the ischemia-induced upregulation of VEGF receptors, minimizing the risk of systemic side effects which occurs after systemic administration of the recombinant VEGF protein. Early clinical data (phase I and II studies) in peripheral limb and ischemic heart disease provide evidence of the proof of concept of these principles. Definite efficacy can only be proven by the results of placebo controlled phase III studies as described in chapter 9 of this thesis. In chapter 9 the positive results are presented of a double blind placebo controlled study performed in 54 adult diabetic patients with CLI. In chapter 10 we considered to investigate physical methods, namely the the use of electric pulses (electorporation) and the use of ultrasound (sonoporation).These techniques facilitate DNA uptake into cells by the formation of transient pores in the cell membrane in order to improve the transfection efficiency of plasmid based gene delivery to skeletal muscle. Electroporation was more effecient than the use of ultrasound. The combination of plasmid based gene therapy with physical aid such as with electroporation should be considered in future studies on therapeutic angiogenesis. In chapter 8 Sodium fluoreseine leakage studies is described in the evaluation of patients with diabetes related critical ischaemia chapter 8. Another important direction in angiogenesis research is the quantification of angiogenesis by means of circulating VEGF measurements as a biomarker. In chapter 3 we conclude that the largest pool of the total circulating VEGF is contained in peripheral blood cells. This in support of the findings of other authors who also found that circulating VEGF is derived from various blood cells, in particular the platelets. From this line of evidence it was speculated that VEGF in the blood circulation may, at least in part, be stored and/or produced by peripheral blood cells, whereas originally it was thought to be only tumour derived. A relative new insight from our study, however, is that the predominant part of circulating VEGF resides in the neutrophilic granulocytes as confirmed by fluorescence-activated cell sorting (FACS). Future measurements of circulating VEGF aimed at prognostic or intervention targets in cancer patients, should take the various compartments of cell bound VEGF into account. More research into the mechanisms that influence circulating VEGF levels seems relevant, we attributed studies in chapter 5 and 6.