Sfrp2 as a Stem Cell Derived Paracrine Factor for Cardioprotection

Sfrp2 作为干细胞衍生的旁分泌因子，具有心脏保护作用

• 批准号: 7642490
• 负责人: Victor J Dzau
• 金额: $ 52.68万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7642490
• 关键词: Acute myocardial infarction; Animals; Antigens; Apoptosis; Apoptotic; Attenuated; Binding; Biological Assay; Cardiac; Cardiac Myocytes; Cell Survival; Cells; Clinical; Collecting Cell; Conditioned Culture Media; Coronary; Cytoprotection; Data; Dependovirus; Echocardiography; Fibrinogen; Functional disorder; Gene Expression Microarray Analysis; Genes; Genetic Enhancer Element; Heart; Hour; Hypoxia; In Vitro; Infarction; Injection of therapeutic agent; Injury; Interruption; Laboratories; Lead; Ligation; Mediating; Mesenchymal Stem Cells; Mus; Muscle Cells; Myocardial; Myocardial Infarction; Myocardial Ischemia; Myocardium; Nature; Pathway interactions; Pattern; Play; Property; Protein Family; Proteins; Rattus; Recombinants; Reporter; Research Personnel; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Stem cells; Stress; Testing; Therapeutic; Therapeutic Effect; Time; Treatment Efficacy; Up-Regulation; Ventricular Function; Viral; Western Blotting; attenuation; base; frizzled related protein-1; frizzled related protein-3; gene therapy; human SFRP4 protein; in vivo; left coronary artery; member; myocardial infarct sizing; novel; overexpression; paracrine; promoter; protective effect; repaired

DESCRIPTION (provided by applicant): We have previously demonstrated the use of genetically modified mesenchymal stem cells overexpressing Akt (Akt-MSC) for therapeutic myocardial protection and repair. More recently, we have shown that conditioned media from these cells can protect cardiomyocytes from hypoxia induced apoptosis in vitro and the myocardium from ischemic damage in vivo. Based on our observation that the protective effect of the conditioned media can occur earlier than 72 hours, we postulated that the actions of Akt-MSC on ischemic myocardium are paracrine in nature and mediated by specific secreted proteins with cytoprotective properties. Further studies have shown that these cells dramatically upregulated (40X) secreted frizzled related protein (Sfrp 2). Sfrp 2 has been shown to bind and antagonize the effects of members of the Wnt family of proteins some of which modulate cell survival or apoptosis. Indeed, our preliminary data showed that Wnts can be upregulated in hypoxic cardiomyocytes in vitro and in the myocardium following infarction in vivo. Based on these results, we hypothesize that (i) Sfrp 2 is involved in paracrine anti-apoptotic effect of Akt-MSCs in vivo (ii) administration of Sfrp 2 protein or gene will confer therapeutic protection against infarct damage in vivo; (iii) the observed protective effect of Sfrp 2 is due to its interruption of the Wnt signaling pathway by interacting and sequestering specific Wnt(s) that have pro-apoptotic properties. To test these hypotheses, we will knockdown the expression of Sfrp 2 in Akt-MSC by siRNA followed by injection of the cells or the conditioned media from the cells into the infarcted mouse hearts. We will then determine in vivo therapeutic efficacy by intramyocardial injections of purified, recombinant Sfrp 2 for protection against ischemic damage in vivo. In addition we will evaluate the effects of overexpression of Sfrp 2 using AAV-mediated expression using regulated and cell specific promoters. Subsequently, we will examine the mechanism of the protection by determining if Sfrp 2 regulates the canonical or the non-canonical Wnt signaling pathways by using in vitro and in vivo reporter assays. Finally we will find Wnts that have proapoptotic properties and determine the nature of interaction of Sfrp 2 with these proapoptotic Wnts. Our studies, if successful, will lead to discovery of novel pathways, mechanisms and may result in new modes of treatment for acute myocardial infarction.

描述(由申请人提供)：我们之前已经证明了使用过表达Akt的转基因间充质干细胞(Akt-MSC)进行治疗性心肌保护和修复。最近，我们发现这些细胞的条件培养液在体外可以保护心肌细胞免受缺氧诱导的细胞凋亡，在体内可以保护心肌免受缺血损伤。根据我们观察到的条件培养液的保护作用可以早于72小时，我们推测Akt-MSC对缺血心肌的作用本质上是旁分泌的，并由具有细胞保护特性的特定分泌蛋白介导。进一步的研究表明，这些细胞显著上调(40倍)分泌卷曲相关蛋白(SFRP2)。SFRP2已被证明可以结合和拮抗Wnt家族蛋白成员的作用，其中一些蛋白调节细胞存活或凋亡。事实上，我们的初步数据显示，WNTS在体外缺氧的心肌细胞和在体心肌梗死后的心肌细胞中都可以上调。根据这些结果，我们推测：(I)SFRP2参与了Akt-MSCs体内的旁分泌抗凋亡作用；(Ii)SFRP2蛋白或基因在体内对脑梗塞损伤具有治疗性保护作用；(Iii)SFRP2的保护作用是通过与具有促凋亡特性的特异性Wnt(S)相互作用和隔离来阻断Wnt信号通路。为了验证这些假设，我们将通过siRNA敲除Akt-MSC中SFRP2的表达，然后将细胞或细胞的条件培养液注射到梗塞的小鼠心脏中。然后，我们将通过心肌内注射纯化的重组SFRP 2来确定体内治疗效果，以保护体内的缺血损伤。此外，我们还将使用调节启动子和细胞特异性启动子来评估AAV介导的SFRP-2过表达的效果。随后，我们将通过体外和体内报告分析来确定SFRP-2是否调节典型的或非典型的Wnt信号通路，以探讨其保护机制。最后，我们将发现具有促凋亡特性的WNTs，并确定SFRP2与这些促凋亡WNTs相互作用的性质。如果我们的研究成功，将导致发现新的途径和机制，并可能导致新的治疗模式的急性心肌梗死。