Regulation of <em>Notch1</em> and <em>Dll4</em> by Vascular Endothelial Growth Factor in Arterial Endothelial Cells: Implications for Modulating Arteriogenesis and Angiogenesis
Notch and its ligands play critical roles in cell fate determination. Expression of Notch and ligand in vascular endothelium and defects in vascular phenotypes of targeted mutants in the Notch pathway have suggested a critical role for Notch signaling in vasculogenesis and angiogenesis. However, the angiogenic signaling that controls Notch and ligand gene expression is unknown. We show here that vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor can induce gene expression of Notch1 and its ligand, Delta-like 4 (Dll4), in human arterial endothelial cells. The VEGF-induced specific signaling is mediated through VEGF receptors 1 and 2 and is transmitted via the phosphatidylinositol 3-kinase/Akt pathway but is independent of mitogen-activated protein kinase and Src tyrosine kinase. Constitutive activation of Notch signaling stabilizes network formation of endothelial cells on Matrigel and enhances formation of vessel-like structures in a three-dimensional angiogenesis model, whereas blocking Notch signaling can partially inhibit network formation. This study provides the first evidence for regulation of Notch/Delta gene expression by an angiogenic growth factor and insight into the critical role of Notch signaling in arteriogenesis and angiogenesis.
Notch signaling is highly conserved through evolution and plays a fundamental role in the determination of cell fate (1, 48). It also affects cell cycle progression and apoptosis. In humans, there are four Notch receptors, Notch 1 to 4, and five ligands, including Jagged1 and -2 and Dll1, -3, and -4. Activation of Notch upon ligand binding is accompanied by proteolytic processing that releases an intracellular domain of Notch (NICD) from the membrane. The NICD then translocates into the nucleus and associates with the CSL [CBF-1 (RBP-Jκ)/Su(H)/Lag-1] family of DNA-binding proteins to form a transcriptional activator, which turns on transcription of a set of target genes, including the E(spl) (Enhancer of Split) group and others (28). Most of the Notch target genes encode transcription regulators, which in turn modulate cell fate by affecting the function of tissue-specific basic helix-loop-helix transcription factors or through other molecular targets, such as NF-κB (2).
Vasculogenesis and angiogenesis are processes of the formation of new vascular networks, which involve sprouting, branching, splitting, and differential growth of vessels from the primary plexus or existing vessel into a functioning circulation system (4, 10). Vessels develop into specific types, including arteries, veins, capillaries, and lymphatics. In adults, physiological angiogenesis occurs during the female reproductive cycle and in wound healing, while abnormal angiogenesis can be observed in solid tumor and rheumatoid arthritis. A number of cellular signaling pathways, such as vascular endothelial growth factor (VEGF) and its receptor (VEGFR), basic fibroblast growth factor (bFGF), transforming growth factor beta, and platelet-derived growth factor with their receptors, angiopoietin/Tie and ephrin/Eph, have been implicated in regulating vasculogenesis and angiogenesis (50). Among angiogenic regulators, VEGF family members VEGF-A (VEGF), -B, -C, -D, and -E and placenta growth factor and VEGFRs [VEGFR1 (Flt-1), VEGFR2 (KDR/Flk-1), and VEGFR3 (Flt-4)] are key mediators. VEGF stimulates vascular endothelial cells through VEGFR1 and VEGFR2, whereas VEGF-C and -D bind to VEGFR2 and VEGFR3 and primarily affect lymphangiogenesis (44).
Growing evidence suggests involvement of Notch signaling in the regulation of vascular formation. For instance, the human degenerative vascular disease cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) has been associated with mutations in Notch3 (16). In vertebrates, Notch1, Notch4, Jagged1, Jagged2, Dll1, and Dll4 are expressed in vascular endothelium (22, 25, 35, 42, 41, 46). Targeted Notch-1, Notch1/Notch 4, Jagged-1, and Dll-1 mutations all result in vascular defects (13, 15, 19, 49). Notch signaling must also be appropriately regulated in order to maintain normal vascular development, since expression of activated Notch4 in mouse embryonic endothelium results in vascular patterning defects (43). In zebrafish, development of the aorta requires the gridlock gene, a homologue of mammalian HES (Hairy/Enhancer of Split), which is regulated by Notch activation (51). Moreover, the essential roles of Jagged-1 and HES related 1 (HESR1) in modulating vessel formation in vitro have been well demonstrated (12, 53).
The precise role of Notch signaling in governing endothelial cell behavior remains unclear. In Notch1 and Notch1/Notch4 mouse embryos, the primary vascular plexus appeared to form normally, but embryos failed to remodel the plexus to form large and small blood vessels, indicating that Notch signaling is essential for angiogenic vascular morphogenesis and remodeling (15, 20). Notch signaling also plays a role in defining arterial endothelial cells and determining the formation of arteries through repression of venous fate. In zebrafish, DeltaC, a ligand for the Notch, is expressed in endothelial cells that contribute to the dorsal aorta but not the posterior cardinal vein at least 6 h before the onset of blood flow (36). The phenotype of the zebrafish gridlock gene mutant revealed a defect in the formation of the dorsal aorta but not in the vein (51). More direct evidence supporting the crucial role of gridlock in the control of the artery-vein decision in zebrafish embryos has been provided very recently (52). In mammals, Dll4, a newly identified ligand responsible for the activation of Notch1 and Notch4, is preferentially expressed in arterial endothelium (35), suggesting a potential role for Dll4 in modulating arterial development (arteriogenesis).
The angiogenic signaling pathways controlling Notch/Delta gene expression are unknown. The relationship between Notch signaling and other angiogenic regulators, such as VEGF, bFGF, transforming growth factor beta, platelet-derived growth factor, angiopoietin/Tie, and ephrin/Eph, has not been well investigated. Here we asked whether soluble angiogenic factors can regulate Notch/Delta gene expression and which specific signaling pathway delivers the initiation signal in human endothelial cells. Furthermore, the biological significance of expression of Notch1/Dll4 on endothelial cells has been addressed by inducing the activation of Notch signaling in arterial endothelial cells. Our findings provide the first example of regulation of Notch/Delta gene expression by a soluble growth factor and thus establish a functional linkage between two important angiogenic signaling pathways and also give insight into a critical role for Notch signaling in regulating arteriogenesis/angiogenesis.
We thank P. Carmeliet, D. W. Ball, and W. Ogawa for providing different recombinant adenoviruses; M. Detmar, M. Skobe, M. Shibuya, T. Kodach, and T. Honjo for various plasmids; and T. Sudo for anti-HES-1 antibody.
This work was supported by the McCabe Fund and grants from the National Institutes of Health (CA47159, CA25874, and CA10815).
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