Optimizing Therapeutic Revascularization by Endothelial Cell Transplantation

通过内皮细胞移植优化治疗性血运重建

• 批准号: 8529594
• 负责人: JORDAN S POBER
• 金额: $ 38.64万
• 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 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8529594
• 关键词: Acids; Adult; Angiogenic Proteins; Angiopoietin-1; Animal Model; BCL-2 Protein; BCL2 gene; Biocompatible Materials; Biological; Biology; Biomedical Engineering; Blood Vessels; Cell Differentiation process; Cell Survival; Cell Transplantation; Cell physiology; Cells; Clinical; Coculture Techniques; Collaborations; Critical Pathways; Development; Disease; Drug Delivery Systems; Encapsulated; Endothelial Cells; Enzymes; Epithelial Cells; Gel; Generations; Genetic Engineering; Goals; Hepatic Stellate Cell; Hepatocyte; Hepatocyte Growth Factor; Human; Immunodeficient Mouse; Implant; In Vitro; Intestines; Laboratories; Liver; Mediating; Methodology; Methods; Modeling; Modification; Molecular; Monocyte Chemoattractant Protein-1; Natural regeneration; Nucleic Acids; Pancreas; Pathway interactions; Perfusion; Pericytes; Placenta; Productivity; Property; Proteins; Recruitment Activity; Research Project Grants; Resistance; Safety; Small Interfering RNA; Smooth Muscle Myocytes; Structure; Supporting Cell; Suspension substance; Suspensions; System; Techniques; Technology; Testing; Therapeutic; Tissue Engineering; Tissues; Translations; Transplantation; Transplanted tissue; Tube; United States National Institutes of Health; Vascular Endothelial Growth Factors; clinical practice; density; design; experience; implantation; improved; in vivo; in vivo Model; liver function; monolayer; novel strategies; paracrine; rapid technique; scaffold; self assembly; tumor

DESCRIPTION (provided by applicant): This is a competing renewal application for a project that has been supported by NIH since 2006. In this renewal application, we seek new methods for rapid formation of vascular networks that are relevant to tissue engineering. Our two laboratories have significant experience with the design of biomaterials for protein delivery/tissue engineering and the use of genetic engineering to enhance vascular cell survival and blood vessel formation in vivo: as a result of this collaboration, we have produced a new method, which we call vessel self assembly, that is now ready to be applied to a significant and difficult problem, tissue engineering of liver. Over the past 4 years we have optimized systems in which primary human endothelial cells (ECs), suspended in protein gels, self assemble into vascular conduits in vitro. These self assembled conduits provide perfusion to tissue engineered grafts in vivo after implantation into immunodeficient mouse hosts. We have shown that vessel self assembly can be enhanced by incorporating sustained delivery of pro-angiogenic proteins that act on ECs. We have also shown that the progression to functionally mature vessels is enhanced when ECs are co-implanted with human aortic smooth muscle cells or pericytes (PCs). In this renewal application, we propose, first, to improve the generation of vascular networks via self-assembly from isolated cells and, second, to apply this methodology to tissue engineering by co-transplantation of differentiated epithelial cells within the EC/PC gel constructs with the goal of producing a new, perfused functional tissue. To accomplish these goals we have identified three specific aims for this five-year project. In Aim 1, we seek to improve EC function by identifying and engaging critical pathways mediated by Bcl-2. To accomplish this, we will identify the molecular mechanisms by which Bcl-2 expression enhances vascular self-assembly using a high-throughput, in vitro system (involving cell spheroid suspension in protein gels) that reveals an effect of Bcl-2 on EC tube formation. In Aim 2, we will improve vascular self-assembly by identifying and activating critical functions mediated by PCs. We will test two hypotheses related to optimizing PC effects: a) that PCs can be recruited to EC tubes more effectively if the ECs are genetically altered to inducibly over-express autotaxin, the enzyme needed for releasing lysophophatidic acid, incorporating sustained release of an autotaxin-inducing molecule into our gel system; b) that PCs exert some or all of their maturing effect on ECs by paracrine release of angiopoietin 1. In Aim 3, we will apply the approaches we have already developed- and new approaches as they are discovered in Aims 1 and 2-to use vascular self-assembly to create tissues by transplanting EC/PC/hepatocytes co-cultures for regeneration of liver function. Here, we will use our sustained release systems for maintaining differentiated functions of hepatocytes. In all of our approaches, we 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）：这是一个自2006年以来一直由NIH支持的项目的竞争性更新申请。在这个更新的应用程序中，我们寻求新的方法，快速形成血管网络，是相关的组织工程。我们的两个实验室在蛋白质递送/组织工程生物材料的设计以及使用基因工程来增强体内血管细胞存活和血管形成方面具有丰富的经验：由于这种合作，我们已经产生了一种新方法，我们称之为血管自组装，现在已经准备好应用于一个重要而困难的问题，肝脏组织工程。在过去的4年里，我们优化了系统，其中原代人内皮细胞（EC），悬浮在蛋白质凝胶，自组装成血管导管在体外。这些自组装导管在植入免疫缺陷小鼠宿主后为体内组织工程移植物提供灌注。我们已经表明，通过持续递送作用于EC的促血管生成蛋白，可以增强血管自组装。我们还表明，当EC与人主动脉平滑肌细胞或周细胞（PC）共同植入时，功能成熟血管的进展得到增强。在这个更新的应用程序中，我们建议，第一，以提高通过自组装从分离的细胞血管网络的生成，第二，应用这种方法的EC/PC凝胶结构内的分化的上皮细胞的共移植的组织工程的目标是产生一个新的，灌注的功能组织。为了实现这些目标，我们为这个五年项目确定了三个具体目标。在目标1中，我们试图通过识别和参与Bcl-2介导的关键途径来改善EC功能。为了实现这一点，我们将确定Bcl-2表达增强血管自组装的分子机制，使用高通量，在体外系统（涉及蛋白凝胶中的细胞球状体悬浮液），揭示了Bcl-2对EC管形成的影响。在目标2中，我们将通过识别和激活PC介导的关键功能来改善血管自组装。我们将测试两个假设相关的优化PC的影响：a）PC可以招募到EC管更有效地，如果EC的遗传改变，诱导过表达autotaxin，释放溶血磷脂酸所需的酶，纳入持续释放的autotaxin诱导分子到我们的凝胶系统; B）PC发挥其部分或全部的成熟效应，通过旁分泌释放血管生成素1的EC。在目标3中，我们将应用我们已经开发的方法-以及在目标1和2中发现的新方法-通过移植EC/PC/肝细胞共培养物来使用血管自组装来创建组织以再生肝功能。在这里，我们将使用我们的缓释系统来维持肝细胞的分化功能。在我们所有的方法中，我们依赖于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。