A novel inhibitory peptide of Dll4-Notch1 signaling and its proangiogenic functions.

BACKGROUND AND PURPOSE Dll4-Notch1 signaling pathway plays an important role in sprouting angiogenesis, vascular remodeling, and arterial or venous specification. It is widely reported that genetic or pharmacologic inhibition of Dll4-Notch1 signaling leads to excessive sprouting angiogenesis. However, transcriptional inhibitors of Dll4-Notch1 signaling remains elusive. EXPERIMENTAL AND APPROACH We designed a new peptide targeting Notch signaling, named TAT-ANK, and the role of this peptide in angiogenesis was studied. In vitro, tube formation and fibrin gel bead assay were performed in human umbilical vein endothelial cells (HUVECs). In vivo, Matrigel plug angiogenesis assay, developmental retinal model and tumor model in mice were used. The mechanisms of TAT-ANK were investigated by immunochemistry, western blotting, immunoprecipitation, qRT-PCR and luciferase reporter assay. KEY RESULTS We demonstrated that amino acids (aa 179-191) in G-protein-coupled receptor-kinase interacting protein-1 (GIT1 ankyrin domain) were crucial for GIT1 binding to the Notch transcription repressor, RBP-J and designed TAT-ANK based on GIT1 aa 179-191. We found that TAT-ANK significantly inhibited Dll4 expression and Notch 1 activation in HUVECs by competing with activated Notch1 to bind to RBP-J. The biological function analysis showed that TAT-ANK promoted angiogenesis in vitro and in vivo by inhibiting Dll4-Notch1 signaling. CONCLUSIONS AND IMPLICATIONS We developed TAT-ANK peptide, a new inhibitor of Notch signaling. This peptide will be of significant interest to the scientists studying Dll4-Notch1 signaling as well as to clinicians who perform clinical trials using Notch signaling inhibitors. Furthermore, our findings will have important conceptual and therapeutic implications on angiogenesis related diseases.