Optimizing Revascularization by EC Transplantation

描述(由申请人提供)：我们的两个实验室在组织工程生物材料的设计和利用基因工程增强体内血管细胞存活和血管形成方面拥有丰富的经验。例如，在Saltzman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肿瘤组织；通过微球控制神经生长因子的释放，提高了脑细胞移植后的存活和功能。在Pober实验室，已经开发出分离、培养和逆转录病毒转导血管细胞的条件，并已证明悬浮在天然生物聚合物凝胶中的经Bcl2转导的人脐静脉内皮细胞(EC)能够形成能够与宿主血管吻合的微血管网络，并诱导宿主体内重塑，从而增加局部组织灌注量。Pober实验室还广泛地描述了内皮细胞对肿瘤坏死因子(TNF)的反应，最新的研究表明，在人类器官培养中，肿瘤坏死因子可通过涉及肿瘤坏死因子受体2(TNF-R2)及其下游的内皮/上皮酪氨酸激酶(ETK)的途径发挥作用，以刺激细胞生长和组织修复。在这里，这些技术将被组合和优化，以生产能够快速、稳健和可靠地对缺血组织进行再血管化的工程化系统。这些系统将在动物模型中进行测试，这些模型允许解剖导致肢体缺血后血管重建的细胞和分子特征。我们的工作假设是，针对聚合物支架的组成、添加控释功能和适当选择细胞，优化细胞/聚合物可移植系统将改善缺血模型的血流恢复和临床结果。为了验证这一假说，我们建议：1.比较野生型和突变型Bcl-2、TNF-R2和ETK导入人内皮细胞对支架内血管重建的影响；2.通过掺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聚合物的组成或支架组成来优化转导EC的移植条件；以及3.在支架设计中引入受控释放的药物，如通过TNF-R2而不是TNF-R1信号的肿瘤坏死因子突变体。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，一旦概念得到证实，它们是测试额外脂肪假体的理想结构。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.