Sfrp2 as a Stem Cell Derived Paracrine Factor for Cardioprotection

描述（由申请人提供）：我们先前已经证明了使用过表达Akt的遗传修饰间充质干细胞（Akt-MSC）进行治疗性心肌保护和修复。最近，我们已经证明来自这些细胞的条件培养基可以保护心肌细胞免受体外缺氧诱导的细胞凋亡和体内心肌免受缺血性损伤。基于我们的观察，条件培养基的保护作用可以发生早于72小时，我们推测，Akt-MSC对缺血心肌的作用是旁分泌的性质和介导的特定分泌蛋白的细胞保护特性。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（Sfrp 2）。已经显示Sfrp 2结合并拮抗Wnt蛋白家族成员的作用，其中一些调节细胞存活或凋亡。事实上，我们的初步数据表明，Wnt可以上调缺氧心肌细胞在体外和心肌梗死后在体内。基于这些结果，我们假设：（i）Sfrp 2参与Akt-MSC在体内的旁分泌抗凋亡作用;（ii）Sfrp 2蛋白或基因的施用将赋予针对体内梗塞损伤的治疗性保护;（iii）观察到的Sfrp 2的保护作用是由于其通过相互作用和隔离具有促凋亡特性的特定Wnt而中断Wnt信号传导途径。为了验证这些假设，我们将通过siRNA敲低Akt-MSC中Sfrp 2的表达，然后将细胞或来自细胞的条件培养基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，以保护体内免受缺血性损伤。此外，我们将评估过表达Sfrp 2的影响，使用AAV介导的表达，使用调节和细胞特异性启动子。随后，我们将通过使用体外和体内报告基因测定来确定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，将导致发现新的途径，机制，并可能导致新的模式治疗急性心肌梗死。

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.