Controlled Spatiotemporal Delivery of miRNA Anatgomir for Promoting Vascular Self

描述(由申请人提供)： 以往的工作已经证明，分化良好的人内皮细胞在体内和体外都可以通过蛋白质凝胶自组装成血管管道，然后移植到免疫缺陷小鼠体内。在只含EC的移植物中，血管成熟需要重新招募宿主壁细胞，如血管平滑肌细胞或周细胞(PC)。当内皮细胞与人PC共植入时，血管的成熟被加速和增强。血管自组装也可以通过持续输送作用于内皮细胞或PC的促血管生成蛋白来增强，特别是当EC导向的血管内皮生长因子(VEGF)与PC导向的单核细胞趋化蛋白-1(MCP-1)相结合时。然而，血管的自组装和成熟似乎仍然太慢，无法优化实质细胞的存活，至少需要10天。在涉及microRNAs(MiRNAs)的靶细胞内，促血管生成蛋白的作用可能分别被正反馈环和负反馈环增强或限制。MiRNAs是一种短的、非编码的RNA，通过减少特定的mRNA半衰期或翻译来调节各种发育过程。单个miRNA可以降低多个基因的表达，通常是在相同的途径上。MiRNAs的作用可以被被称为反交配子的互补短RNA序列所抑制。当miRNA以细胞特有的方式表达时，反配子以细胞特有的方式发挥作用。该项目测试了一种假说，即受控释放的对抗剂可以增强血管生成蛋白(如血管内皮生长因子)在血管自组装中的治疗效果。这一假设将通过两个具体目标进行检验。在目标1中，聚合物纳米颗粒(NP)将被用来寻找在3D培养中提供对miRNA和Anagomir递送到内皮细胞细胞质的空间和时间控制的最佳方法。在目标2中，这些NP递送系统将测试它们的能力，以控制将对miR-17/20-已知增强血管内皮生长因子的影响-到3D细胞培养的对抗剂的空间和时间递送的能力，通过将ECs和PC悬浮在胶原和纤维连接蛋白凝胶中产生的。(摘要结束)

DESCRIPTION (provided by applicant): Previous work has demonstrated that well differentiated human endothelial cells (ECs) will self assemble into vascular conduits in protein gels both in vitro and in vivo after implantation into immunodeficient mouse hosts. Vessel maturation in grafts containing only EC requires recruitment of host mural cells, such as vascular smooth muscle cells or pericytes (PCs). The maturation of vessels is accelerated and enhanced when ECs are co-implanted with human PCs. Vessel self assembly can also be enhanced by sustained delivery of pro- angiogenic proteins that act on ECs or PCs, especially when an EC-directed agent, vascular endothelial growth factor (VEGF), is combined with a PC-directed agent, monocyte chemotactic protein -1 (MCP-1). However, vessel self-assembly and maturation still appears too slow to optimize parenchymal cell survival, requiring at least 10 days. The actions of pro-angiogenic proteins may be augmented or limited by positive and negative feedback loops, respectively, within the target cells that involve microRNAs (miRNAs). miRNAs are short, non-coding RNAs that regulate a variety of development processes by reducing specific mRNA half lives or translation. A single miRNA can reduce the expression of multiple genes often in the same pathway. The effects of miRNAs can be inhibited by complementary short RNA sequences referred to as antagomirs. Antagomirs act in a cell-specific manner when the miRNA is expressed in a cell specific manner. This project tests the hypothesis that controlled delivery of an antagomir can enhance the therapeutic benefits of angiogenic proteins such as VEGF in vascular self-assembly. This hypothesis will be tested through two specific aims. In Aim 1, polymer nanoparticles (NP) will be used to find the optimal approaches for providing spatial and temporal control over miRNA and antagomir delivery to the cytoplasm of ECs in 3D culture. In Aim 2, these NP delivery systems will be tested for their ability to control the spatial and temporal delivery of antagomirs to miR-17/20-which is known to augment the effects of VEGF-to 3D cell cultures produced by suspending ECs and PCs in gels of collagen and fibronectin. (End of Abstract)