Our two laboratories have significant experience with the design of biomaterials for tissue engineering and the use of genetic engineering to enhance vascular cell survival and blood vessel formation in vivo. For example, in the Saltzman laboratory, biodegradable cell-adhesive polymer microparticles have been used for assembly of brain cells into neotissues; controlled release of nerve growth factor by the microspheres enhanced brain cell survival and function after transplantation. In the Pober laboratory, conditions have been developed for isolation, culture and retroviral transduction of vascular cells and Bcl-2-transduced human umbilical vein endothelial cells (EC) suspended in gels of natural biopolymers have been shown to form a microvascular network capable of anastomosis with host vessels and to induce remodeling in the host so as to increase local tissue perfusion. The Pober laboratory has also extensively characterized the responses of ECs to tumor necrosis factor (TNF), most recently showing that in human organ culture, TNF can act through a pathway involving TNF receptor 2 (TNF-R2) and the downstream kinase endothelial/epithelial tyrosine kinase (Etk) to stimulate cell growth and tissue repair. Here, these techniques will be combined and optimized to produce engineered systems that are capable of rapid, robust, and reliable revascularization of ischemic tissue. These systems will be tested in animal models that permit dissection of the cellular and molecular features that lead to revascularization of limbs after ischemia. Our working hypothesis is that optimization of cell/polymer transplantable systems with respect to composition of the polymer scaffold, addition of controlled-release functions, and appropriate selection of cells will lead to improved therapeutic recoveries in blood flow and clinical outcomes in ischemic models. To test this hypothesis, we propose to: 1. compare the effect of introduction into human EC of wild type and mutant forms of Bcl-2, TNF-R2 and Etk on revascularization within scaffolds; 2. optimize the conditions for transplantation of transduced EC by incorporation of wild type or modified vascular smooth muscle cells (VSMC) or by modifications in the composition of the natural protein polymers or of the scaffold composition; and 3. introduce controlled release of agents into the scaffold design such as a TNF mutein that signals via TNF-R2 but not TNF-R1. Since these experimental systems are flexible with respect to transduced genes, protein release, polymer surface modification, and cell source, they are ideal constructs for testing additional lypotheses once proof of concept is established. Our approaches rely on materials that are already acceptable to the FDA in clinical settings; therefore, our results in animal models will be ready for translation into clinical practice. ? ? ?
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