Abstract 689: Notch Signaling Stimulates Expression Of Wnts And Contributes To Cardiac Markers Gene Expression In Human Progenitor Cell
It has been demonstrated that adult human circulating endothelial progenitor cells (EPC) can differentiate to a cardiomyogenic phenotype. Notch signaling promotes epithelial-to-mesenchymal transformation and plays a prominent role in heart and vessel development. Here, we investigated the role of Notch activation for cardiac differentiation of EPC in a co-culture system with neonatal rat cardiomyocyte (CM). EPC expressed the receptors Notch-1 and Notch-2, whereas CM expressed high level of Notch ligand Jagged-1. Therefore, we hypothesized that CM may activate Notch signaling within EPC. Indeed, after co-culture, Notch activation was detected in EPC by immunohistochemical detection of the intracellular cleavage fragment of Notch-1 (NICD), whereas NICD was only rarely detected in EPC before co-culture. Western blot analysis confirmed Notch cleavage after co-culture. RT-PCR directed against the human specific sequences of the Notch target genes Hey2 and Hes1 demonstrated a transient activation of the transcriptional activity of Notch in human EPC after co-culture with CM. Inhibition of γ-secretase significantly blocked Notch cleavages and NICD translocations. Furthermore, the expression of the cardiac marker protein γ-sarcomeric actinin and troponin T was significantly suppressed by γ-secretase inhibition (55.8 ± 8.1% and 54.0 ± 10.5% of control, respectively) or addition of soluble recombinant Jagged-1, indicating that Notch activation facilitates cardiac marker gene expression. Because non-canonical Wnts have previously been shown to promote cardiac differentiation, we additionally determined the influence of Notch activation on the expression of Wnt5a and. Wnt5a and Wnt11 expressions in the human cells was induced by the co-culture and was blocked by γ-secretase inhibitor. Likewise, stimulation of Notch signaling by immobilized Jagged-1 promoted NICD cleavage and Wnt5a expression in EPC. These data suggested that Notch is transiently activated upon co-culture of EPC with neonatal rat CM. γ-Secretase-dependent Notch activation is required for cardiac gene expression in human cells and induces the expression of non-canonical Wnt proteins, which may act in an paracrine manner to further amplify cardiac differentiation.