Growth Factor Mediation of Healing of Vascular Grafts

描述(申请人提供)：治疗性血管介入治疗的两种常见失败模式是早期血栓形成和晚期肌内膜肥大，这两种模式都可能被功能正常的内皮细胞(EC)单层减少。我们开发了一种新的方法，可控制地将生物活性生长因子局部输送到血管组织和移植物上，从而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成纤维细胞生长因子-1物种，可能具有更有效的生物学特性。总体假设是：1)成纤维细胞生长因子-1的R136K突变在保持或增强生物活性的同时将产生对蛋白水解性降解的抵抗力，2)额外的逻辑派生的结构变化将提高EC的特异性和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带有胶原结合结构域的嵌合体局部递送衍生的构建体将促进体内的内皮化而不伴随伴随的肌内膜增生。这些假设得到了强大的体外数据的支持，即FGF.1的特异性和效力可以通过突变来改变，蛋白水解点的RI36K突变促进了分子和生物稳定性，以及将多肽与HB-GAM融合提高了EC的特异性。该提案有3个具体目标。具体目标#1A)：基于现有的分子稳定性和使用我们建立的局部递送方法的相关生物学功能的分析，对现有和下一代基于R136K的突变/嵌合蛋白的表征，以及1B)：阐明导致R136K有丝分裂活性增加的作用机制，并将这一知识纳入可能显示出理想体内活性的新型突变嵌合体的设计中。具体目标#2：通过体外试验确定一种嵌合蛋白：由R136K形式的成纤维细胞生长因子-1和源自溶组链球菌胶原结合结构域组成的嵌合蛋白是否通过血管壁内传递进一步促进内皮化。具体目标#3：在体外优化的突变体/嵌合体的体内，使用纤维胶或基质胶原靶向体外优化的突变体/嵌合体，在具有良好特征的旁路移植和动脉内膜切除术的动物模型中评估细胞特异性反应。我们坚信，这些结果将直接应用于血管介入治疗，并将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.