Optimizing Revascularization by EC Transplantation

通过 EC 移植优化血运重建

• 批准号: 7135566
• 负责人: W. Mark Saltzman
• 金额: $ 40.3万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-15 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7135566
• 关键词: BCL2 gene /protein; SCID mouse; angiogenesis; bioengineering /biomedical engineering; biotechnology; cell morphology; cell transplantation; clinical research; cytokine receptors; disease /disorder model; human subject; ischemia; muscle cells; polymers; protein tyrosine kinase; therapy design /development; tissue /cell culture; tissue engineering; tissue support frame; tumor necrosis factor alpha; vascular endothelium; vascular smooth muscle

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.

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，可生物降解的细胞粘附聚合物微粒已被用于将脑细胞组装成新组织；微球控制释放神经生长因子可增强移植后脑细胞的存活和功能。 Pober 实验室已开发出血管细胞的分离、培养和逆转录病毒转导的条件，并且悬浮在天然生物聚合物凝胶中的 Bcl-2 转导的人脐静脉内皮细胞 (EC) 已被证明可形成能够吻合的微血管网络与宿主血管结合并诱导宿主重塑，从而增加局部组织灌注。 Pober 实验室还广泛表征了 EC 对肿瘤坏死因子 (TNF) 的反应，最近表明，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/上皮细胞的途径发挥作用。酪氨酸激酶（Etk）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，以产生能够快速、稳健和可靠地对缺血组织进行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，在聚合物支架的组成、添加控释功能和适当的细胞选择方面优化细胞/聚合物可移植系统将改善缺血模型中血流的治疗恢复和临床结果。为了检验这一假设，我们建议： 1. 比较将野生型和突变型 Bcl-2、TNF-R2 和 Etk 引入人类 EC 对支架内血运重建的影响； 2.通过掺入野生型或修饰的血管平滑肌细胞(VSMC)或通过修饰天然蛋白质聚合物或支架组合物的组成来优化转导的EC的移植条件； 3. 在支架设计中引入药物的控释，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具有灵活性，因此一旦概念验证建立，它们就是测试其他溶血作用的理想构建体。我们的方法依赖于 FDA 在临床环境中已经接受的材料；因此，我们在动物模型中的结果将准备好转化为临床实践。