Optimizing Therapeutic Revascularization by Endothelial Cell Transplantation

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。在这个更新的应用中，我们寻求与组织工程相关的血管网络快速形成的新方法。我们的两个实验室在设计用于蛋白质输送/组织工程的生物材料和使用基因工程来增强血管细胞存活和体内血管形成方面有着丰富的经验：作为这次合作的结果，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要而困难的问题，肝脏的组织工程。在过去的4年里，我们优化了系统，使原代人内皮细胞（ECs）悬浮在蛋白质凝胶中，在体外自组装成血管导管。这些自组装的导管在植入免疫缺陷小鼠宿主体内后为组织工程移植物提供灌注。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而增强。我们还表明，当内皮细胞与人主动脉平滑肌细胞或周细胞（PCs）共植入时，向功能成熟血管的进展会加快。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产生，其次，将这种方法应用于组织工程，通过在EC/PC凝胶构建中共同移植分化的上皮细胞，以产生新的灌注功能组织。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。在目的1中，我们寻求通过识别和参与Bcl-2介导的关键途径来改善EC功能。为了实现这一目标，我们将利用高通量体外系统（包括蛋白凝胶中的细胞球形悬浮液）确定Bcl-2表达增强血管自组装的分子机制，揭示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。我们将测试与优化PC效应相关的两个假设：a)如果EC经过基因改造以诱导过度表达autotaxin（释放溶血磷脂酸所需的酶），将持续释放的autotaxin诱导分子结合到我们的凝胶系统中，PC可以更有效地招募到EC管中；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-以及在目标1和目标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，以实现肝功能再生。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.