Infarct Repair with Mesenchymal Stem Cell Subpopulation

间充质干细胞亚群修复梗塞

基本信息

批准号：
8826806
负责人：
BUDDHADEB DAWN
金额：
$ 37.18万
依托单位：
UNIVERSITY OF KANSAS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-08 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8826806
关键词：
Accounting Actins Adherence Adult Anatomy Antigens Apoptosis Attention Autologous BCL2 gene Biology Bone Marrow Bromodeoxyuridine C57BL/6 Mouse CASP8 and FADD-like apoptosis regulating protein CXCR4 gene Calcium Cardiac Cardiac Myosins Cause of Death Cell Adhesion Molecules Cell Culture Techniques Cell Transplantation Cells Cellular biology Confocal Microscopy Country Data Discipline E-Cadherin E-Selectin ENG gene Echocardiography Electron Microscopy Female Fibroblast Growth Factor 2 Fibrosis Flow Cytometry Gelatinase A Growth Factor Health Heart Heart failure Hydrogen Peroxide Hypertrophy Hypoxia In Vitro Infarction Injection of therapeutic agent Integrin alpha4beta1 Intercellular adhesion molecule 1 Left Left Ventricular Function Left ventricular structure Length MCM5 gene Mesenchymal Stem Cells Methods Modeling Molecular Molecular Biology Morbidity - disease rate Morphology Mus Muscle Cells Myocardial Myocardial Infarction Myocardial Ischemia Myocardium Myosin Heavy Chains Natural regeneration Nature Outcome PECAM1 gene PTGS2 gene PTPRC gene Pathway interactions Patients Perfusion Phenotype Phosphotransferases Population Population Control Predisposition Property Protein Chemistry Proteins Proteomics Proto-Oncogene Protein c-kit Public Health Quality of life Reporting Resistance Role STAT3 gene Signal Transduction Small Interfering RNA Staining method Stains Stem cells Stromal Cell-Derived Factor 1 Structural Protein Structure TNF gene Techniques Testing Time Transplantation Treatment Efficacy Troponin T VWF gene Validation Vascular Cell Adhesion Molecule-1 Vascular Endothelial Growth Factor Receptor-2 Vascular Endothelial Growth Factors Ventricular angiogenesis annexin A5 base cardiac repair cell type hemodynamics immunocytochemistry improved in vitro Model in vivo inhibitor/antagonist insight interdisciplinary approach male matrigel morphometry mouse model myogenesis novel paracrine progenitor regenerative repaired stem cell population transcription factor uptake

项目摘要

DESCRIPTION (provided by applicant): The outcomes of cardiac repair with bone marrow mesenchymal stem cell (MSC), a highly heterogeneous cell population, have been variable and generally suboptimal. The efficacy of specific and homogenous MSC subsets defined by antigen expression has not been investigated. Based on preliminary data, our fundamental hypothesis states that transplantation of antigenically-defined CD45-/c-kit-/Sca- 1+/CD90+/CD105- (abbreviated as CD105-) MSC subpopulation will confer greater reparative and regenerative benefits after myocardial infarction (MI) by virtue of enhanced survival, increased retention, improved angiogenesis, greater differentiation into cardiac lineages, and favorable modulation of myocardial matrix. This hypothesis will be tested in a well-established cell culture model in vitro and a mouse model of reperfused MI in vivo, which will yield conclusive results. Aim 1 will determine whether CD105- MSCs will be more resistant to apoptosis and washout. The susceptibility of murine bone marrow MSCs (Unfractionated, CD45-/c-kit-/Sca-1+/CD90+/CD105+ [abbreviated as CD105+, the precise antigenic control population], and CD105-) to apoptosis will be examined, and the underlying molecular basis elucidated. The expression of adhesion molecules on MSCs will be assessed, and cell retention in vivo will be tested following myocardial injection 2 d after a reperfused MI. Aim 2 will investigate whether CD105- MSCs produce greater amounts of cardioprotective and angiogenic molecules, and acquire endothelial phenotype. Endothelial and cardiac commitment will be determined by morphology, and assessment of transcription factors and structural proteins. The molecular signaling underlying the angiogenic effects will be elucidated using specific inhibitors and siRNA, with particular attention to VEGFR2-activated pathways. Aim 3 will establish whether transplantation of CD105- MSCs will induce superior cardiac repair in vivo, and identify the mechanistic basis in a definitive fashion. Unfractionated, and CD105+ and CD105- MSCs will be injected into the infarct borderzone 2 d after a reperfused MI in C57BL/6 mice. Serial echocardiography and a terminal hemodynamic study will be performed to assess left ventricular (LV) function and anatomy. LV structure, infarct size, fibrosis, and myocyte hypertrophy will be assessed by morphometry. Quantitative immunohistochemical methods will be used to precisely determine the contribution of myocyte salvage from apoptosis, angiogenesis, myocyte proliferation, myocyte regeneration, activation of cardiac progenitors, and modulation of calcium handling proteins. Focused proteomic analysis will be performed to identify novel changes in the matrix. For the first time, these studies will critically evaluate th biology of CD105- MSC subset and their efficacy in infarct repair in a comprehensive and thoroughly mechanistic fashion. The impact will be two-fold: (i) biologically, this project will yild novel insights into properties of antigenically-defined MSC populations; and (ii) clinically, the identification and validation of an optimal cell type for cardiac repair will benefit patients with ischemic heart disease and post-MI heart failure.

描述（由申请人提供）：使用骨髓间充质干细胞（MSC）（一种高度异质的细胞群）进行心脏修复的结果是可变的并且通常不是最理想的。尚未研究由抗原表达定义的特异性和同质 MSC 亚群的功效。根据初步数据，我们的基本假设表明，移植抗原定义的 CD45-/c-kit-/Sca-1+/CD90+/CD105-（缩写为 CD105-）MSC 亚群将在心肌梗塞后带来更大的修复和再生益处（MI）凭借增强的存活率、增加的保留、改善的血管生成、更大程度地分化为心脏谱系以及有利的心肌基质调节。这一假设将在成熟的体外细胞培养模型和体内再灌注心肌梗死小鼠模型中进行测试，这将产生结论性的结果。目标 1 将确定 CD105-MSC 是否更能抵抗细胞凋亡和清除。检测小鼠骨髓间充质干细胞（Unfractionated、CD45-/c-kit-/Sca-1+/CD90+/CD105+[缩写为CD105+，精确抗原对照群体]和CD105-）对细胞凋亡的敏感性，阐明了潜在的分子基础。将评估 MSC 上粘附分子的表达，并在再灌注 MI 后 2 天心肌注射后测试体内细胞保留。目标 2 将研究 CD105-MSC 是否产生更多的心脏保护和血管生成分子，并获得内皮表型。内皮和心脏的承诺将通过形态学以及转录因子和结构蛋白的评估来确定。将使用特定抑制剂和 siRNA 阐明血管生成效应背后的分子信号传导，特别关注 VEGFR2 激活途径。目标 3 将确定 CD105-MSC 移植是否会诱导体内良好的心脏修复，并以明确的方式确定其机制基础。 C57BL/6 小鼠再灌注 MI 后 2 天，将未分割的 CD105+ 和 CD105- MSC 注射到梗塞边界区。将进行连续超声心动图和末期血流动力学研究以评估左心室（LV）功能和解剖结构。左心室结构、梗死面积、纤维化和肌细胞肥大将通过形态测定法进行评估。定量免疫组织化学方法将用于精确确定细胞凋亡、血管生成、肌细胞增殖、肌细胞再生、心脏祖细胞激活和钙处理蛋白调节对心肌细胞挽救的贡献。将进行重点蛋白质组分析，以确定基质中的新变化。这些研究将首次以全面、彻底的机制方式严格评估 CD105-MSC 亚群的生物学及其在梗塞修复中的功效。其影响将是双重的：(i) 从生物学角度来看，该项目将为抗原定义的 MSC 群体的特性带来新的见解； (ii) 在临床上，识别和验证用于心脏修复的最佳细胞类型将使患有以下疾病的患者受益：缺血性心脏病和心肌梗死后心力衰竭。