In this proposal, we hypothesize that the induction of a functional endothelial lining on the surface of a small diameter vascular prosthesis can be achieved after graft implantation by controlled local delivery of proangiogenic growth factors. Specifically, we postulate that recombinant heparan sulfate-mimetic compounds, when formulated as nanoparticles that are incorporated into a fibrin gel, provide a useful strategy for the efficient delivery of Fibroblast Growth Factor-2 (FGF-2) and sphingosine-1-phosphate (S1P), as synergistic angiogenic factors. Moreover, in association with a membrane-mimetic blood-contacting surface, we postulate that the risk of thrombosis can be minimized during the period prior to complete endothelialization. Specifically, we intend to: (1) Define the features of heparan sulfate proteoglycan-mimics that dictate effective sequestration and local cell mediated delivery of pro-angiogenic growth factors. Recombinant proteoglycan mimics and other self-assembling protein-based copolymers will be formulated as nanoparticles that are incorporated into fibrin gels and examined as delivery vehicles for Fibroblast Growth Factor-2 (FGF-2) and sphingosine-1-phosphate (S1P). Protein particle degradation and compound release rates will be modulated by the presence of proteolytically sensitive sites. The interactive effects of combined FGF-2 and S1P delivery on the kinetics and magnitude of the angiogenic response will be investigated using in vitro and in vivo models. (2) Synthesize and characterize a membrane-mimetic glycocalyx for selective endothelial growth in a thromboresistant microenvironment. Membrane-mimetic thin films presenting both protein and carbohydrate ligands that promote endothelial cell growth will be synthesized on fibrin gels. Endothelial cell adhesive, migratory and proliferative behavior will be defined and selective EC-dependant anti-thrombogenic properties will be measured, including the capacity to activate protein C, dephosphorylate ADP, and produce nitric oxide. (3) Characterize the biomimetic material properties, which influences spontaneous endothelialization and resistance to neointimal hyperplasia in vivo. A small diameter vascular prosthesis will be functionalized with membrane- and proteoglycan-mimetic components and surgical implant studies performed to test the hypothesis that combined local delivery of FGF-2 and S1P potentiates the endothelialization of a thromboresistant interface while minimizing neointimal hyperplasia. In addition to morphometric measurements of graft healing, the functional properties of the induced endothelial lining will be characterized using immunohistochemical and other techniques.
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