Oxidized Low-density Lipoprotein Impairs Bone Marrow Stem Cell Survival

 描述（由申请人提供）：骨髓干细胞（BMSC）的细胞治疗仍然是组织修复和再生的可行选择。基于细胞的治疗的一个主要挑战是移植后细胞的存活率差。植入的BMSC存活受损的确切机制仍有待确定。MG 53是一种重要的细胞膜修复蛋白，在骨骼肌和心肌中天然产生，并存在于血液中，介导MG 53的组织保护功能。氧化低密度脂蛋白（ox-LDL）是血液中的天然产物。我们观察到，ox-LDL在与冠状动脉疾病患者血清ox-LDL水平相容的浓度下对BMSCs产生显著的膜损伤，这反映在从细胞释放乳酸脱氢酶（LDH）和荧光染料FM 1 -43进入细胞中。重组人MG 53蛋白（rhMG 53）可减少ox-LDL诱导的BMSCs LDH释放和FM 1 -43染料进入。进一步的体内研究表明，静脉内ox-LDL给药导致血清MG 53水平降低，并损害植入正常C57 B6小鼠的BMSC的存活。这项拟议的研究将检验这一假设，即“ox-LDL通过直接损伤细胞膜损害BMSCs的存活，而ox-LDL介导的循环中MG 53水平的降低是BMSCs存活有限的重要因素，细胞膜修复能力受损，细胞治疗的疗效降低”。具体目标是：1）确定ox-LDL在体外和体内对BMSC的膜完整性的影响;和2）确定ox-LDL介导的对MG 53的作用和细胞移植中BMSC的存活的机制。我们将阐明ox-LDL对培养体系中BMSCs的损伤机制，并通过实验评价rhMG 53对BMSCs机械性或缺氧性膜损伤的保护作用。我们将进一步设计表达MG 53的BMSC，以确定持续的MG 53生产是否对ox-LDL处理的小鼠和高脂血症动物体内BMSC的存活具有有益作用。将努力确定ox-LDL如何导致MG 53介导的膜修复受损的机制。我们将确定ox-LDL是否会导致肌细胞MG 53分泌减少或增加其从循环中的清除。我们将使用MG 53基因敲除小鼠和转基因小鼠模型进一步检验这一假设，在转基因小鼠模型中，可以用强力霉素驱动的启动子系统定制循环中可诱导和可逆的MG 53表达。这些研究将揭示MG 53介导的膜修复在细胞治疗中的作用，为植入BMSCs的不良存活的分子机制提供重要信息，并有助于探索新的策略，如靶向ox-LDL和循环MG 53，以优化BMSCs的存活和增强基于细胞的治疗心血管疾病的疗效。1

 DESCRIPTION (provided by applicant): Cell therapy with bone marrow stem cells (BMSCs) remains a viable option for tissue repair and regeneration. One major challenge for cell-based therapy is the poor survival of the cells after transplantation. The exact mechanism(s) for impaired survival of the implanted BMSCs remains to be defined. MG53 is an important cell membrane repair protein that is naturally produced in skeletal and cardiac muscles, and present in blood that mediates the tissue protective function for MG53. Oxidized low-density lipoprotein (ox-LDL) is a natural product in blood. We observed that ox-LDL at the concentrations compatible with the serum ox-LDL levels in patients with coronary artery disease produced significant membrane damage to BMSCs as reflected by the release of lactate dehydrogenase (LDH) from the cell and the entry of fluorescent dye FM1-43 into the cell. Treatment with recombinant human MG53 (rhMG53) protein could reduce LDH release and FM1-43 dye entry in BMSCs exposed to ox-LDL. Further in vivo studies showed that intravenous ox-LDL administration led to decreased serum MG53 level, and impaired survival of BMSCs implemented into normal C57B6 mice. The proposed study will test the hypothesis that "ox-LDL impairs the survival of BMSCs through direct damage to their cell membrane, and ox-LDL-mediated reduction of MG53 level in circulation is an important factor for the limited survival of BMSCs with compromised cell membrane repair capacity and reduced efficacy of cell-based therapy". The specific aims are: 1) to determine the effect of ox-LDL on the membrane integrity of BMSCs both in vitro and in vivo; and 2) to define the mechanism(s) for ox-LDL-mediated action on MG53 and the survival of BMSCs in cell transplantation. We will elucidate the mechanisms that underlie the ox-LDL-induced damage to BMSCs in culture system, and also conduct experiments to evaluate the protective effect of rhMG53 on BMSCs following mechanical or hypoxia-induced membrane injuries. We will further engineer BMSCs that express secretary MG53 to determine if sustained MG53 production has beneficial effects on the survival of BMSCs in vivo in ox-LDL-treated mice and in hyperlipidemic animals. Efforts will be made to determine the mechanism on how ox-LDL causes impairment in MG53-mediated membrane repair. We will determine whether ox-LDL causes decreased MG53 secretion from muscle cells or increased its clearance from circulation. We will further test the hypothesis with the use of MG53 knockout mice and transgenic mouse models where inducible and reversible MG53 expression in circulation can be tailored with a doxycycline-driven promoter system. These studies will reveal the role for MG53-mediated membrane repair in cell-based therapy, provide important information on the molecular mechanisms for the poor survival of implanted BMSCs, and help to explore new strategies like targeting ox- LDL and circulating MG53 to optimizing the survival of BMSCs and enhancing the efficacy of cell-based therapy for cardiovascular diseases. 1