Growth Factor Mediation of Healing of Vascular Grafts

描述（由申请人提供）：治疗性血管介入的两种常见失败模式是早期血栓形成和晚期肌内膜增生，两者都可能通过功能性内皮细胞（EC）单层减少。我们已经开发了一种新的方法，控制局部递送生物活性生长因子的血管组织和移植物产生有效的体内血管生成和表面内皮化。我们最近的研究产生并表征了可能具有更有效生物学特性的突变FGF-1物种。总体假设是：1）FGF-1的R136 K突变将产生对蛋白水解降解的抗性，同时维持或增加生物活性，2）额外的逻辑衍生的结构改变将增加EC特异性和促有丝分裂和血管生成信号的效力，以及3）通过纤维蛋白水凝胶或作为具有胶原结合结构域的嵌合体局部递送衍生构建体将促进体内内皮化而没有伴随的肌内膜增生。这些假设得到了强有力的体外数据的支持，即FGF. 1特异性和效力可以通过诱变改变，蛋白水解位点处的RI 36 K突变促进分子和生物学稳定性，并且将肽融合到HB-GAM促进EC特异性。该提案有三个具体目标。具体目标#1A）：使用我们建立的局部递送方法，基于分子稳定性和相关生物学功能的现有测定，表征现有和下一代基于R136 K的突变体/嵌合蛋白，和1B）：阐明负责增加R136 K促有丝分裂活性的作用机制，并将该知识结合到可能表现出期望的体内活性的新突变体嵌合体的设计中。具体目标#2：使用体外试验确定是否存在由FGF-1的R136 K形式和衍生自C.溶组织菌cotlagenase将进一步促进血管壁内递送内皮化。具体目标#3：在旁路移植术和动脉内膜切除术的充分表征的动物模型中，使用通过生物胶或通过基质胶原靶向的应用特异性递送，评价对体外优化的突变体/嵌合体的体内细胞特异性反应。我们坚信，这些结果将直接应用于血管介入的临床，并为广泛的组织工程策略提供一种使能技术。

DESCRIPTION (provided by applicant): 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 #1A): 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.