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. ? ? ?
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