An important goal in treating ischemic heart disease is to improve the thromhoresistance of the cardiac circulation. As one approach to this problem, we have established germ line and somatic cell delivery systems which allow for the expression of heterologous genes within the mice and micro-and macrovascular beds of the heart. For example, we have shown in transgenic mice that discrete regions of the human von Willebrand factor (vWF) gene contain information for regional expression within the vascular tree. Specifically, we have identified sequences within the 5' region and/or first intron that contain information for expression within microvessels of the heart. In other studies, we have successfully isolated murine endothelial cell precursors and subsequently incorporated them into the developing cardiovascular system. Taken together, these preliminary results strongly support the feasibility of overexpressing natural anticoagulant enzymes within the coronary circulation. The long range goals of this proposal are to enhance the thromboresistance of the coronary vessels through the delivery of genes encoding limiting components involved in the generation of heparin sulfate, activated protein C and plasmin as well as the destruction of ADP. Using the microvascular bed-specific vWF promoter as a starting point, we will attempt to augment its expression, render its action inducible and extend its specificity to the epicardial vessels of the heart. We hope that these modifications of the promoter will allow for both spatial and temporal control of gene expression. The cardiac endothelial cell-specific promoters will then be used to overexpress the natural anticoagulants in transgenic mouse hearts. The biological effect of the enzymes and their therapeutic potential will he analyzed using a number of genetic and environmentally-induced thrombotic models. We will then isolate murine endothelial cells at various stages of differentiation and test their potential to become incorporated into the cane circulation of the chick embryo and adult mouse. The seeding capacity of these cells will be optimized through the expression of specific growth factors and/or cell surface receptors. The precursor endothelial cells will then be genetically engineered ex to express natural anticoagulants and used in somatic cell delivery systems to augment thromboresistance of the coronary nation. Finally, we will use viral vectors to deliver the vWF-2 promoter-driven genes and the genetically altered endothelial cells to the blood vessels of the heart Together, the above studies represent a diversified yet complementary approach to achieving successful gene transfer within the coronary circulation.
