The two common failure modes of therapeutic vascular interventions are early thrombosis and late myointimal hyperptasia, both likely to be reduced by a functioning endothelial cell (EC) monolayer. We have developed a novel method of controlled local delivery of bioactive growth factors to vascular tissues and grafts yielding potent in vivo angiogenesis and surface endothelialization. Our recent studies generated and characterized mutant FGF-1 species with likely more efficacious biological properties. The overall hypotheses are that 1) the R136K mutation of FGF-1 will yield resistance to proteolytic degradation while maintaining or augmenting bioactivity, 2) that additional logically derived structural alterations will increase EC specificity and potency of the mitogenic and angiogenic signals and that 3) local delivery of the derived construct via fibrin hydrogels or as a chimera with a collagen binding domain wilt promote in vivo endothetiaUzation without a concomitant myointimal hyperplasia. These:hypotheses are supported by strong in vitro data that FGF.1 specificity and potency can be altered by mutagenesis, that the RI36K mutation at the proteolytic site promotes molecular and biological :stability and that fusing the peptides to HB-GAM promotes EC specificity. The proposal has 3 Specific Aims.
Specific Aim #1 A): Characterization of existing and next generation R136K based mutant/chimeric proteins based on existing assays of molecular stability and of relevant biological functions using our established method of local delivery, and 1B): elucidation of mechanisms of action responsible for increased R136K mitogenic activity and incorporation of that knowledge into the design of novel mutant chimeras likely to exhibit desirable in vivo activities.
Specific Aim #2 : Determine using in vitro assays whether a chimeric protein: consisting of the R136K form of FGF-1 and a collagen binding domain derived :from C. histolyticum cotlagenase would further promote endothelialization using vascular intramural delivery.
Specific Aim #3 : Evaluate in vivo cell-specific responses to the in vitro-optimized mutant/chimera using application-specific delivery by fibdn glue or by matrix collagen targeting in well characterized animal models of bypass grafting and of endarterectomy. We strongly believe that results will be directly clinically applicable in vascular interventions and should provide an enabling technology for a broad range of tissue engineering strategies.
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