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

基本信息

批准号：
7145291
负责人：
Victor J Dzau
金额：
$ 50.83万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-15 至 2010-06-30
项目状态：
已结题

项目摘要

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过表达 Akt 的转基因间充质干细胞（Akt-MSC）用于治疗性心肌保护和修复的用途。最近，我们发现这些细胞的条件培养基可以在体外保护心肌细胞免受缺氧诱导的细胞凋亡，并在体内保护心肌免受缺血性损伤。根据我们观察到条件培养基的保护作用可以在 72 小时之前发生，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，这些细胞分泌的卷曲相关蛋白 (Sfrp 2) 显着上调 (40X)。 Sfrp 2 已被证明可以结合并拮抗 Wnt 蛋白家族成员的作用，其中一些蛋白调节细胞存活或凋亡。事实上，我们的初步数据表明，Wnts 可以在体外缺氧心肌细胞和体内梗塞后的心肌中上调。基于这些结果，我们假设 (i) Sfrp 2 参与 Akt-MSC 体内的旁分泌抗凋亡作用 (ii) 施用 Sfrp 2 蛋白或基因将赋予体内针对梗塞损伤的治疗保护作用； (iii) 观察到的 Sfrp 2 保护作用是由于它通过相互作用和隔离具有促凋亡特性的特定 Wnt 来中断 Wnt 信号传导途径。为了测试这些假设，我们将通过 siRNA 敲低 Akt-MSC 中 Sfrp 2 的表达，然后将细胞或细胞的条件培养基注射到梗塞小鼠心脏中。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，以防止体内缺血性损伤。此外，我们将评估使用 AAV 介导的表达（使用受调节的细胞特异性启动子）过度表达 Sfrp 2 的效果。随后，我们将通过使用体外和体内报告基因检测确定 Sfrp 2 是否调节经典或非经典 Wnt 信号通路来检查保护机制。最后，我们将找到具有促凋亡特性的 Wnt，并确定 Sfrp 2 与这些促凋亡 Wnt 相互作用的性质。我们的研究如果成功，将发现新的途径、机制，并可能产生治疗急性心肌梗塞的新模式。