Foreign gene regulation and adenoviral mediated gene transfer in models of myocardial ischaemia
Myocardial ischaemia is characterised by a reduction in blood flow sufficient to cause a pathological change in myocardial function. One of the myocardial adaptive responses to ischaemic stress involves a change in gene expression which is regulated by the interaction of DNA binding proteins with specific DNA sequences. Identifying intracellular signalling pathways that link the changes in the extracellular environment during ischaemia to alterations in gene expression may provide important information on how the myocardium adapts to ischaemic stress. Specific regulatory sequences that interact with DNA binding proteins in response to ischaemic stress may be used as part of a gene therapy protocol to regulate foreign gene expression in the ischaemic myocardium. One of the aims of this study was to identify control regions that may play a role in the myocardial response to ischaemic stress. A hybrid promoter, containing myosin heavy chain basal regulatory sequences plus four copies of the erythropoietin HIF-1 binding site, conferred inducible expression of a luciferase reporter gene in response to 15 minutes of ischaemia followed by reperfusion. This induction was rapid and reversible upon reperfusion of the ischaemic myocardium. The human skeletal α-actin (SkAct) promoter was also investigated but showed no significant activation in response to 15 minutes of ischaemia followed by reperfusion in rat and rabbit myocardium at the time points examined. The SkAct promoter was further characterised by analysing the relative expression of mutant and wild-type SkAct promoters in the rat heart after direct DNA injection. This study indicated possible regulatory sequences that may be involved in the cardiac specific regulation of the SkAct gene and indicated for some promoter constructs a possible discordance between in vivo and in vitro data. The extent and severity of the pathological loss of function caused by myocardial ischaemia is critically dependent on the collateral circulation perfusing the ischaemic region. Stimulating angiogenesis in the ischaemic heart by adenovirus mediated overexpression of angiogenic factors may offer the possibility of increasing collateral perfusion to ischaemic areas, improving myocardial function. Adenoviral vectors are capable of delivering foreign genes to the myocardium with high efficiency and low toxicity. With a view to limiting the extent of myocardial ischaemia and preventing further expansion of the ischaemic areas, the present study also aimed to develop a potentially therapeutic adenoviral vector expressing an angiogenic factor. A replication deficient recombinant adenovirus expressing the highly specific and potent angiogenic factor, vascular endothelial growth factor (VEGF), was constructed. Using this viral vector for infection of cell lines in culture, evidence was obtained of foreign expression of the recombinant gene product.