Growth Factor Mediation of Healing of Vascular Grafts

描述（由申请人提供）：治疗性血管干预的两种常见失败模式是早期血栓形成和晚期肌内膜增生，两者都可能被功能正常的内皮细胞（EC）单层减少。我们已经开发了一种新的方法，可以控制局部递送生物活性生长因子到血管组织和移植物中，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突变的FGF-1物种，可能具有更有效的生物学特性。总体假设是：1)FGF-1的R136K突变在维持或增强生物活性的同时，会产生对蛋白水解降解的抗性；2)额外的逻辑衍生结构改变将增加EC的特异性和有丝分裂和血管生成信号的有效性；3)通过纤维蛋白水凝胶或作为胶原结合域的嵌合体局部递送衍生构建体将促进体内内皮化，而不会伴随肌内膜增生。这些假设得到了大量体外实验数据的支持，即FGF.1的特异性和效力可通过诱变而改变，蛋白水解位点的RI36K突变可促进分子和生物学稳定性，将肽与HB-GAM融合可促进EC特异性。该提案有3个具体目标。具体目标#1A)：利用我们建立的局部传递方法，基于现有的分子稳定性和相关生物学功能测定，对现有和下一代基于R136K的突变/嵌合蛋白进行表征；1B)：阐明导致R136K有丝分裂活性增加的作用机制，并将这些知识纳入可能表现出理想体内活性的新型突变嵌合体的设计中。特定目的#2：通过体外实验确定由FGF-1的R136K形式和胶原结合结构域组成的嵌合蛋白是否会通过血管内输送进一步促进内皮化。特定目标#3：评估体外优化的突变体/嵌合体的体内细胞特异性反应，使用纤维蛋白胶或基质胶原靶向的应用特异性递送，在具有良好特征的动物模型中进行旁路移植术和动脉内膜切除术。我们坚信，这些结果将直接应用于血管干预的临床，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.