Abstract 515: Discovery of a Novel Cysteine-Rich Survival Signaling Protein from Akt Transduced Mesenchymal Stem Cells
Previously we have shown that injection of conditioned medium from mesenchymal stem cells overexpressing the survival gene Akt (Akt-MSCs) into infarcted hearts dramatically reduced the infarct size and prevented ventricular dysfunction as early as 72 hours after treatment. These data strongly suggest that the observed protective effects are due to release of cardio-protective paracrine factors from the Akt-MSCs. To identify potential paracrine factors, we performed microarray analysis of Akt-MSCs which led to the identification of 46 novel upregulated ESTs/transcripts, and among these, 5 novel transcripts potentially encoding secreted proteins were identified. The open reading frames of these novel transcripts were cloned and expressed in E. coli as maltose binding protein (MBP) fusion proteins. Compared with MBP alone, one of the MBP-novel fusion proteins (Protein #12) significantly reduced the H2O2-induced apoptosis in H9C2 myocytes. Protein 12 was re-cloned into pET vector to allow rapid purification as a 6xHis tagged recombinant protein. Since Protein 12 is cysteine rich, purification was performed under denaturing condition and the protein was refolded by dialysis with a redox pair to promote disulfide bond formation. To test the potential protective effects of Protein 12, we examined the effects of addition of this protein on H2O2-induced apoptosis in H9C2 myocytes. Myocytes were treated with 100 μM H2O2 or vehicle and the levels of apoptosis was assessed by FACS analysis following Annexin V/PI staining. As expected, H2O2 induced high levels of early apoptosis, yielding approximately 30% Annexin V positive cells with less than 5% necrotic cells (PI positive). Interestingly, pre-treatment of the cells with 10 nM of Protein 12 for 30 min reduced early apoptosis by nearly 50%. Moreover, this protein significantly reduced H2O2 induced caspase 9 activity in adult rat cardiomyocytes by 38.5%, dramatically inhibited the mitochondrial release of cytochrome C and increased the total survival rate by 28%. Taken together, these results suggest that this cysteine-rich Protein 12 may be responsible, in part, for the cardio-protective effects of Akt-MSCs. Further studies are underway to identify the signaling mechanism of this novel protein.