Optimizing Therapeutic Revascularization by Endothelial Cell Transplantation

描述(由申请人提供)：这是一项竞争性的续签申请，该项目自2006年以来一直得到美国国立卫生研究院的支持。在这一更新应用中，我们寻求与组织工程相关的快速形成血管网络的新方法。我们的两个实验室在蛋白质输送/组织工程的生物材料设计以及利用基因工程来增强体内血管细胞存活和血管形成方面拥有丰富的经验：由于这种合作，我们已经产生了一种新的方法，我们称之为血管自组装，现在已经准备好应用于一个重大而困难的问题-肝脏组织工程。在过去的4年里，我们优化了悬浮在蛋白质凝胶中的原代人内皮细胞(ECs)在体外自组装成血管管道的系统。这些自组装管道在植入免疫缺陷小鼠宿主后，为体内的组织工程移植物提供灌流。我们已经证明，血管自组装可以通过结合作用于内皮细胞的促血管生成蛋白的持续递送来增强。我们还表明，当内皮细胞与人主动脉平滑肌细胞或周细胞(PC)共植入时，促进了向功能成熟血管的进展。在这一更新应用中，我们建议，首先，通过分离细胞的自组装来改善血管网络的生成，其次，通过将分化的上皮细胞共同移植到EC/PC凝胶结构中，将这种方法应用于组织工程，目标是产生新的、灌流的功能组织。为了实现这些目标，我们为这个五年计划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由Bcl-2介导的关键通路来改善EC的功能。为了实现这一点，我们将使用高通量的体外系统(包括蛋白质凝胶中的细胞球体悬浮)来确定Bcl-2表达促进血管自组装的分子机制，该系统揭示了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由PC介导的关键功能来改善血管自组装。我们将测试两个与优化PC效应相关的假设：a)如果对EC进行基因改造以诱导其过度表达自体趋化因子，PC可以更有效地被招募到EC管内，自体趋化因子是释放溶血磷脂酸所需的酶，结合自体趋化诱导分子的持续释放到我们的凝胶系统中；b)PC通过旁分泌释放血管生成素1对ECs发挥部分或全部成熟作用。在目标3中，我们将应用我们已经开发的方法-以及在AIMS 1和2中发现的新方法-通过移植EC/PC/肝细胞共培养来创建组织，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。在我们的所有方法中，我们都依赖于已经被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): 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.