Nano-sized Cell Guidance System for Ischemic Tissue Repair

描述（由申请人提供）：心肌和外周组织缺血是美国心力衰竭和组织坏死的主要原因。缺血性疾病在临床上主要通过药物和手术治疗，但在永久性治疗方面仍面临许多挑战。近年来，通过血管生成、血管生成或两者同时进行的重建缺血组织血管网络的血运重建治疗正在被广泛研究，以恢复各种组织的血液灌注。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联合使用是很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，但由于缺乏将细胞引导到受损内皮的信号，移植细胞的显著损失大大降低了治疗效果。本研究的目的是开发纳米级的细胞引导分子，并将其贴附于移植细胞上，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。我们推测，由于内皮损伤刺激内皮细胞过度表达VACM-1，一种与移植细胞结合的表位和与血管细胞粘附分子(VCAM)-1结合的超支聚甘油会精确地引导移植细胞到达受损的内皮。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。我们将使用从猪脐带血中提取的内皮祖细胞（EPCs）来检验这一假设。将含有RGD序列的寡肽（RGD肽）作为epc结合表位，将含有VHSPNKK序列的寡肽（VHSPNKK肽）作为VCAM1结合表位。寡肽结构会发生变化，以提高与细胞和VCAM-1的结合亲和力。这两个寡肽在化学上与聚甘油相连。通过体外分析进一步优化寡肽对聚甘油的取代程度。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聚甘油结合到EPCs的数量。我们将完成这个提议研究首先构建聚(丙三醇)和RGD肽RGD -聚(丙三醇)和聚(丙三醇)绑定的数量分析epc(目标1),其次修改RGD-poly(甘油)VHSPNKK肽(RGD-poly(甘油)-VHSPNKK]和分析的能力引导内皮祖细胞合成内皮(目标2)最后证明生物活性聚(丙三醇)的函数体内使用免疫缺陷小鼠的缺血后肢(目标3)。本研究将通过组织工程师（Kong，研究者），化学家（Zimmerman）和生物学家（Schook）的跨学科合作进行。Kong和Zimmerman的团队负责合成生物活性聚甘油，并在体外和体内评估其增强移植细胞与靶缺血组织粘附的能力。从脐带血中分离的细胞和特性将由Schook小组进行评估。我们相信，本研究的成功完成将显著减少移植细胞的损失，提高EPCs修复缺血组织的治疗效力。我们体外和体内研究的结果将很容易转化为大规模的临床前和临床试验，并帮助以细胞为基础的新生血管治疗进入临床环境。最后，这种细胞引导系统的设计策略和与细胞和靶组织的分子结合的定量分析将广泛适用于广泛的干细胞和祖细胞，用于治疗许多疾病。

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.