Nano-sized Cell Guidance System for Ischemic Tissue Repair

描述（由申请人提供）：在美国，心肌和外周组织缺血是心力衰竭和组织坏死的主要原因。缺血性疾病在临床上通过药物施用和手术治疗，其在永久性基础上仍然面临许多治疗挑战。最近，通过血管生成、血管发生或两者来重建缺血组织的血管网络的再血管化治疗正在被广泛研究，以恢复各种组织中的血液灌注。多种干细胞和祖细胞与几种血管生成细胞因子和生长因子一起是有前途的血管重建药物。通常，这些细胞通过冠状动脉内注射移植，但是由于缺乏引导细胞到达受损内皮的信号而导致细胞的显著损失，移植细胞的治疗功效大大降低。这项研究的目的是开发一种纳米级的细胞导向分子，并将其附着在移植细胞上，使移植细胞能够精确定位受损的内皮细胞，从而改善缺血组织的血液灌注。我们假设，与移植细胞结合的表位和与血管细胞粘附分子（VCAM）-1结合的表位连接的超支化聚（甘油）将精确地引导移植细胞到受损的内皮，因为内皮损伤刺激内皮细胞过度表达VACM-1。最终，这种细胞引导的调整将显著改善缺血组织中血液灌注的恢复。我们将使用来自猪脐带血的内皮祖细胞（EPCs）来检验这一假设。含有RGD序列的寡肽（RGD肽）将用作EPC结合表位，含有VHSPNKK序列的寡肽（VHSPNKK肽）将用作VCAM 1结合表位。寡肽结构将变化以提高对细胞和VCAM-1的结合亲和力。这两个寡肽将化学连接到聚（甘油）。寡肽取代聚（甘油）的程度将通过体外分析进一步优化。具体来说，我们将使用荧光共振能量转移（FRET）技术，我们以前开发的定量的聚（甘油）结合到内皮祖细胞的数量。我们将完成这项拟议的研究，首先官能化聚（甘油）与RGD肽[RGD-聚（甘油）]，并分析聚（甘油）与EPC结合（目的1），二次修饰RGD-聚与VHSPNKK肽[RGD-poly（甘油）-VHSPNKK]，并分析其引导EPCs至合成内皮的能力（目的2），最终证明生物活性聚使用具有缺血后肢的免疫缺陷小鼠在体内使用甘油（Aim 3）。本研究将通过组织工程师（Kong，研究者）、化学家（齐默尔曼）和生物学家（Schook）之间的跨学科合作进行。Kong和齐默尔曼的小组负责生物活性聚甘油的合成，并在体外和体内评价其增强移植细胞与靶缺血组织粘附的能力。将由Schook小组评价脐带血细胞分离和表征。我们相信，这项研究的成功完成将显着减少移植细胞的损失，提高EPCs修复缺血组织的治疗效力。我们的体外和体内研究结果将很容易转化为大规模的临床前和临床试验，并有助于将基于细胞的新血管形成疗法推广到临床环境。最后，这种细胞引导系统的设计策略和与细胞和靶组织结合的分子的定量分析将广泛适用于用于治疗许多疾病的干细胞和祖细胞的广泛阵列。 公共卫生相关性：这项拟议研究的成功完成将创建一个精确的细胞引导系统，这将大大提高治疗细胞的再生效率，并加快细胞在缺血性疾病临床治疗中的使用。具体而言，细胞导向系统的体外和体内分析将加快本研究结果转化为临床试验。最终，本研究将有助于挽救一批患有心肌和外周组织缺血性疾病的患者。

DESCRIPTION (provided by applicant): Ischemia in myocardial and peripheral tissues is a leading cause of heart failure and tissue necrosis in the United States. Ischemic diseases are clinically treated with drug administration and surgery, which still meet many challenges for treatment on a permanent basis. Recently, revascularization therapy to rebuild the vascular network of ischemic tissue via angiogenesis, vasculogenesis or both is being extensively studied to restore blood perfusion in various tissues. A variety of stem and progenitor cells are promising revascularization medicines in conjunction with several angiogenic cytokines and growth factors. Commonly, these cells are transplanted via intracoronary injection, but the therapeutic efficacy of transplanted cells is greatly reduced by a significant loss of cells due to the absence of the signals to guide the cells to the injured endothelium. The objectives of this proposed study are to develop a nano-sized cell guidance molecule and attach it to the transplanted cells, so the transplanted cells can pinpoint the injured endothelium and subsequently improve blood perfusion of ischemic tissue. We hypothesize that a hyper-branched poly(glycerol) linked with both epitopes binding with transplanted cells and those binding with vascular cell adhesion molecules (VCAM)-1 will precisely guide transplanted cells to the injured endothelium because the endothelial injury stimulates endothelial cells to over-express VACM-1. Ultimately, this tuning of cell guidance will significantly improve restoration of blood perfusion in the ischemic tissue. We will examine this hypothesis using endothelial progenitor cells (EPCs) derived from a porcine cord blood. The oligopeptide containing RGD sequence (RGD peptide) will be used as the EPC-binding epitope and that containing VHSPNKK sequence (VHSPNKK peptide) will be used as the VCAM1- binding epitope. The oligopeptide structure will be varied to improve the binding affinity to cells and VCAM-1. These two oligopeptides will be chemically linked to the poly(glycerol). The degree of oligopeptides substitution to poly(glycerol) will be further optimized with in vitro analysis. Specifically, we will use a fluorescence resonance energy transfer (FRET) technique we previously developed to quantify the number of poly(glycerol) bound to EPCs. We will complete this proposed study by first functionalizing poly(glycerol) with RGD peptides [RGD- poly(glycerol)] and analyzing the amount of poly(glycerol) bound with EPCs (Aim 1), secondly modifying RGD-poly(glycerol) with VHSPNKK peptides [RGD-poly(glycerol)-VHSPNKK] and analyzing its ability to guide EPCs to the synthetic endothelium (Aim 2) and finally demonstrate the function of bioactive poly(glycerol) in vivo using the immunodeficient mouse with an ischemic hindlimb (Aim 3). This study will be performed through the interdisciplinary collaboration between a tissue engineer (Kong, investigator), chemist (Zimmerman) and biologist (Schook). Kong and Zimmerman's groups are responsible for the synthesis of bioactive poly(glycerol) and evaluation of its ability to enhance the transplanted cell adhesion to the target ischemic tissue in vitro and in vivo. The cell isolation from a cord blood and characterization will be evaluated by the Schook group. We believe that the successful completion of this proposed study will significantly minimize the loss of transplanted cells and improve the therapeutic potency of EPCs for repairing ischemic tissue. Results from our in vitro and in vivo studies will be readily translated into the large scale preclinical and clinical trials, and aid the expedition of cell-based neovascularization therapies to the clinical setting. Finally, this design strategy of a cell guidance system and quantitative analysis of the molecular binding with cells and target tissue will be widely applicable to a broad array of stem and progenitor cells for the treatment of many diseases. PUBLIC HEALTH RELEVANCE: The successful completion of this proposed study will create a precision cell guidance system that will greatly improve the regenerative efficacy of therapeutic cells and expedite the use of cells in clinical treatment of ischemic disease. Specifically, the through in vitro and in vivo analysis of cell guidance system will expedite the translation of the results of this study into the clinical trials. In the end, this study will aid saving a number of patients who suffer from the ischemic disorders of myocardial and peripheral tissues.