Regulation of multiple angiogenic pathways by D114 and Notch in human umbilical vein endothelial cells

The Notch ligand, D114, is essential for angiogenesis during embryonic vascular development, and is involved in tumour angiogenesis. Several recent publications demonstrated that blockade of D114 signalling inhibits tumour growth, suggesting that it may constitute a good candidate for anti-cancer therapy. In order to understand the role of D114 at the cellular level, we performed an analysis of D114-regulated genes in HUVECs. The genes identified included several angiogenic signalling pathways, such as VEGF, FGF and HGF. In particular we identified downregulation (VEGFR2, placenta growth factor PIGF) of VEGF pathway components resulting in the overall effect of limiting the response of HUVEC to VEGF. However extensive upregulation of VEGFR1 was observed allowing continued response to its ligand PIGF but the soluble form of the VEGFR1, sVEGFR1 was also upregulated. PIGF enhanced tubulogenesis of HUVEC suggesting that downregulation of PIGF and upregulation of VEGFR1 including sVEGFR1 are important mechanisms by which D114 attenuates PIGF and VEGF signalling. D114-stimulated HUVECs had impaired ERK activation in response to VEGF and HGF indicating that D114 signalling negatively regulates these pathways. D114 expression reduced vessel sprout length in a 3D tubulogenesis assay confirming that D114 signalling inhibits angiogenesis. Altogether, our data suggest that D114 expression acts as a switch from the proliferative phase of angiogenesis to the maturation and stabilisation phase by blocking endothelial cell proliferation and allowing induction of a more mature, differentiated phenotype. The regulation of sVEGFR1 provides a novel mechanism for D114 signalling to regulate cells at distance, not just in adjacent cells. (c) 2007 Elsevier Inc. All rights reserved.