Optimizing Revascularization by EC Transplantation

描述（由申请人提供）：我们的两个实验室在设计用于组织工程的生物材料和使用基因工程来提高血管细胞存活和血管形成方面具有重要经验。例如，在萨尔茨曼实验室，可生物降解的细胞黏附聚合物微粒已被用于将脑细胞组装成新组织；微球控制神经生长因子的释放，增强移植后脑细胞的存活和功能。在Pober实验室，已经开发了血管细胞的分离、培养和逆转录病毒转导的条件，bcl -2转导的人脐静脉内皮细胞（EC）悬浮在天然生物聚合物凝胶中，已被证明可以形成一个微血管网络，能够与宿主血管吻合，并在宿主体内诱导重塑，从而增加局部组织灌注。Pober实验室还广泛表征了ECs对肿瘤坏死因子（TNF）的反应，最近的研究表明，在人体器官培养中，TNF可以通过涉及TNF受体2 （TNF- r2）和下游激酶内皮/上皮酪氨酸激酶（Etk）的途径起作用，以刺激细胞生长和组织修复。在这里，这些技术将被结合和优化，以产生能够快速，稳健和可靠的缺血组织血运重建的工程系统。这些系统将在动物模型中进行测试，以允许解剖导致缺血后肢体血运重建的细胞和分子特征。我们的工作假设是，优化细胞/聚合物可移植系统，包括聚合物支架的组成，增加控释功能，以及适当的细胞选择，将改善缺血模型的血流恢复和临床结果。为了验证这一假设，我们建议：1。比较野生型和突变型Bcl-2、TNF-R2和Etk对支架内血运重建的影响；2. 通过加入野生型或修饰的血管平滑肌细胞（VSMC）或通过修改天然蛋白质聚合物或支架组合物的组成来优化转导EC移植的条件；和3。在支架设计中引入药物的控制释放，例如通过TNF- r2而不是TNF- r1发出信号的TNF突变蛋白。由于这些实验系统在转导基因、蛋白质释放、聚合物表面修饰和细胞来源方面是灵活的，一旦概念证明建立，它们是测试其他假说的理想结构。我们的方法依赖于已经被FDA在临床环境中接受的材料；因此，我们在动物模型上的结果将准备好转化为临床实践。

DESCRIPTION (provided by applicant): Our two laboratories have significant experience with the design of biomaterials for tissue engineering and the use of genetic engineering to enhance vascular cell survival and blood vessel formation in vivo. For example, in the Saltzman laboratory, biodegradable cell-adhesive polymer microparticles have been used for assembly of brain cells into neotissues; controlled release of nerve growth factor by the microspheres enhanced brain cell survival and function after transplantation. In the Pober laboratory, conditions have been developed for isolation, culture and retroviral transduction of vascular cells and Bcl-2-transduced human umbilical vein endothelial cells (EC) suspended in gels of natural biopolymers have been shown to form a microvascular network capable of anastomosis with host vessels and to induce remodeling in the host so as to increase local tissue perfusion. The Pober laboratory has also extensively characterized the responses of ECs to tumor necrosis factor (TNF), most recently showing that in human organ culture, TNF can act through a pathway involving TNF receptor 2 (TNF-R2) and the downstream kinase endothelial/epithelial tyrosine kinase (Etk) to stimulate cell growth and tissue repair. Here, these techniques will be combined and optimized to produce engineered systems that are capable of rapid, robust, and reliable revascularization of ischemic tissue. These systems will be tested in animal models that permit dissection of the cellular and molecular features that lead to revascularization of limbs after ischemia. Our working hypothesis is that optimization of cell/polymer transplantable systems with respect to composition of the polymer scaffold, addition of controlled-release functions, and appropriate selection of cells will lead to improved therapeutic recoveries in blood flow and clinical outcomes in ischemic models. To test this hypothesis, we propose to: 1. compare the effect of introduction into human EC of wild type and mutant forms of Bcl-2, TNF-R2 and Etk on revascularization within scaffolds; 2. optimize the conditions for transplantation of transduced EC by incorporation of wild type or modified vascular smooth muscle cells (VSMC) or by modifications in the composition of the natural protein polymers or of the scaffold composition; and 3. introduce controlled release of agents into the scaffold design such as a TNF mutein that signals via TNF-R2 but not TNF-R1. Since these experimental systems are flexible with respect to transduced genes, protein release, polymer surface modification, and cell source, they are ideal constructs for testing additional lypotheses once proof of concept is established. Our approaches rely on materials that are already acceptable to the FDA in clinical settings; therefore, our results in animal models will be ready for translation into clinical practice.