Abstract 329: Discovery of a Novel Paracrine Angiogenic Factor from Akt-Transduced Mesenchymal Stem Cells for Cell/Protein-based Therapy in angiogenesis
Previously we have shown that injection of mesenchymal stem cells overexpressing the survival gene Akt (Akt-MSCs) into infarcted rodent hearts dramatically reduced the infarct size and improved ventricular function as early as 72 hours after treatment. Furthermore, injection of conditioned medium isolated from these cells reproduced the similar effects. These data strongly suggest that the observed beneficial effects are due to paracrine factors released from the Akt-MSCs. Since angiogenesis has been shown previously to reduce ischemia injury, we reasoned that part of the protective effects include an angiogenic component. To help identify potential paracrine factors, we performed microarray analysis of Akt-MSCs and identified 46 novel differentially expressed 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. These 5 novel MBP fusion proteins (10nM) were tested in an endothelial tube formation assay using Human Umbilical Vein Endothelial Cells (HUVECs). Compared to MBP control or fresh serum free media, one of the MBP-novel fusion proteins has a pronounced tube forming ability (n = 8, p < 0.001). Bioinformatic analysis reveals that this novel angiogenic factor contains two fibronectin type III domains and a short hydrophobic ~20 amino acid as the signal peptide at amino-terminus. Full-length human cDNA (1704 base pairs) of this novel angiogenic factor was cloned into pcDNA-DEST40 plasmid without the stop codon, in the translational frame fused with V5-epitope at the carboxyl terminus. Upon transient transfection into HEK293 cells with this expression construct, this novel angiogenic factor secreted into culture medium as ~63 KDa protein evidenced by western blotting using anti-V5 antibody. Further studies will dissect the signaling mechanisms of this novel factor and our ongoing in vivo studies will further explore the possibilities of using protein and/or cell-based approach for novel therapeutic angiogenesis for the injured heart.