Abstract P013: Canopy 2 Encodes for a Novel Secreted Proangiogenic Factor that Revascularizes and Regenerates Murine Hearts Post Myocardial Infarction

Through genomic screening of 256 novel genes by RT-PCR in human smooth muscle cells, we discovered that Canopy 2 (CNPY2), previously named as MSAP, TMEM4 or ZSIG9, actually encodes for a secreted pro-angiogenic factor, which was differentially regulated by in vitro hypoxia. Tissue distribution profile reveals that CNPY2 has the highest expression levels in the heart, liver and lung. Surprisingly, CNPY2 was dramatically downregulated (∼2–5 fold) in the murine heart at day 1–7 post myocardial infarction. Recombinant CNPY2 proteins were purified both from E.coli to generate rabbit (blocking) antibodies, and from mammalian cells with proper folding and posttranslational modification to study signaling cascades. We demonstrate that recombinant CNPY2 protein binds to the extracellular domain of VEGFR2 and phosphorylates the intracellular Tyr 1175 and Tyr 1214 of VEGFR2, activates FAK and ERK which resulting in pERK nuclear translocation in human endothelial cells. On the other hand, CNPY2 binds to adhesion molecule desmoglein 1, activates Cdc42, PAK1 and FAK in human smooth muscle cells. CNPY2 reorganizes the dynamic vimentin structure in both cell types, resulting in an accelerated cell migration. In vivo delivery of recombination CNPY2 protein enhanced the blood capillary formation in a retinal ischemia/reperfusion mouse model, and dramatically revascularized blood vessel network, reduced infarct size and improved cardiac function in a rat myocardial infarction model. In line with the paracrine mechanism in (stem) cell-based therapy, our data suggests that CNPY2 might be employed as a promising therapeutic protein for ischemic tissue revascularization and regeneration.