Abstract 346: The Notch ligand, Delta-like 1 (Dll1), is Required for Postnatal Arteriogenesis in Response to Ischemia
Introduction: Neovascularization with competent arteries is essential for regeneration of ischemic organs. Notch signaling regulates arterial development downstream of VEGF and upstream of EphrinB2 (Efnb2) ligand, which is essential for arterial remodelling. However, the role of Notch signaling in adult arteriogenesis is unknown. Delta-like 1 (Dll1) is one of four mammalian Notch ligands and is essential for embryonic development. Its function in the vascular system is so far unknown.
Methods and Results: LacZ staining of adult Dll1lacZ/+ heterozygous mice, in which one endogenous allele has been replaced by the lacZ reporter gene, demonstrated restricted Dll1 expression in the endothelium of arteries, while capillaries or veins lacked Dll1. After induction of hindlimb ischemia (HLI), Dll1lacZ/+ mice demonstrated impaired recovery of perfusion by laser doppler flowmetry compared to wildtype (wt) mice (p<0.05), which lead to foot amputation exclusively in Dll1lacZ/+ mice (61% vs. wt 0%, n>9). Histologically, collateral growth in wt animals resulted in a significant increase in collateral lumen and wall area, which was absent in Dll1lacZ/+ collaterals (p<0.01). In growing collaterals Dll1, and VEGF, were strongly upregulated, which induced activation of endothelial Notch1 (N1) and upregulation of Efnb2. In contrast, in Dll1lacZ/+ collaterals Dll1 was only weakly induced, and induction of N1 and Efnb2 was absent, while VEGF was induced to similar levels. In vitro, VEGF and bFGF synergistically induced Dll1, N1 activation and Efnb2 in human aortic endothelial cells, and enhanced the angiogenic response in tube formation assays. A specific inhibitor of Notch activation, DAPT, blocked VEGF/bFGF-induced N1 activation, Efnb2 induction and tube formation, but not Dll1 upregulation. This demonstrates that Dll1 regulates angiogenesis upstream of endothelial N1 and Efnb2. Furthermore, Dll1lacZ/+ mouse aortic endothelial cells displayed severly impaired tube formation and migration (p<0.01, n=3 each).
Conclusion: Dll1 is expressed in arterial endothelium. Dll1 is upregulated by angiogenic growth factors and regulates postnatal arteriogenesis through activation of endothelial Notch signaling and upregulation of Efnb2.