Mesenchymal stromal cells as vectors for revascularization of the heart
Description
The present investigations are designed to create components of an integrated system for delivery of angiogenic protein using tissue culture engineered cells. Mesenchymal stromal cells (MSCs) are genetically engineered to produce angiogenic proteins for delivery into heart. Bicistronic cellular vectors are engineered including the HIV-1 Lentivirus vector NL-VEGF-EGFP as well as the as well as the mammalian expression vector pbetaa-VEGF-IRES2-EGFP to produce the protein vascular endothelial cell growth factor (VEGF) and the fluorescent marker enhanced green fluorescent protein (EGFP). Quantitative levels of VEGF are measured using a novel biological assay to quantify endothelial response to protein. Vascular endothelial cells also provide a means to measure NL-VEGF-EGFP based lentiviral production of material. Experimental production of VEGF by co-culture shows that production of VEGF is limited by cellular proliferation and angiogenic tubule formation during angiogenic sprouting. The final components of an angiogenic assay shows limited biological response to feeder layers of cells producing increasing quantities of angiogenic protein. Angiogenic cellular proliferation provides a means to semi-quantitatively analyze the effects of tissue engineered protein production in vitro. Bicistronic protein production provides a means to precisely quantify EGFP and therefore the level of VEGF mRNA by flow cytometry for EGFP. This analysis shows that VEGF measured by enzyme linked immunosobant assay (ELISA) for the 164 murine isoform produce comparable levels of EGFP Therapeutic delivery of engineered tissue into pathogenic myocardial tissue is performed by cardiomyoplasty in a large animal model. Development of a vascular based infusion system indicates selective occlusion and delivery into pathogenic myocardial tissue. This system has the capacity to monitor real time events and prevents damage to myocardium. Basic fluid flow and pressure analysis of the delivery system is tested for both antegrade and retrograde delivery of microspheres and engineered cells into heart. These experiments show that during time course delivery there is an increase in the pressure required to drive dispersions into distal tissues. As microcapillary beds are progressively occluded there is an increase in the resistance required to drive fluid through tissue beds