Sfrp2 as a Stem Cell Derived Paracrine Factor for Cardioprotection

描述（由申请人提供）：我们之前已经证明了将过表达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。最近，我们已经证明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介质的保护作用可早于72小时发生，我们假设Akt-MSC对缺血心肌的作用本质上是旁分泌的，由具有细胞保护特性的特异性分泌蛋白介导。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（Sfrp 2）。Sfrp 2已被证明可以结合和拮抗Wnt蛋白家族成员的作用，其中一些Wnt蛋白家族成员调节细胞存活或凋亡。事实上，我们的初步数据显示，wnt可以在体外缺氧心肌细胞和体内梗死后的心肌中上调。基于这些结果，我们假设(i) Sfrp 2参与了体内Akt-MSCs的旁分泌抗凋亡作用（ii）给药Sfrp 2蛋白或基因将在体内对梗死损伤提供治疗性保护；（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异性Wnt(s)来中断Wnt信号通路。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死小鼠心脏中。然后，我们将通过在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，我们将利用aav介导的调控和细胞特异性启动子来评估Sfrp 2过表达的影响。随后，我们将通过体外和体内报告基因试验来确定Sfrp 2是否调节典型或非典型Wnt信号通路，从而研究这种保护的机制。最后，我们将发现具有促凋亡特性的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，将导致新的途径和机制的发现，并可能导致治疗急性心肌梗死的新模式。

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.