Tuesday 3 July 2007
The ligand for the Notch receptors, Delta-like ligand 4 (DLL4), is normally induced by VEGF and is a negative-feedback regulator that restrains vascular sprouting and branching. The Dll4-Notch signalling pathway has been shown to regulate angiogenesis during development.
The Dll4 gene is located on chromosome 15q14 and encodes a 685 amino acid single-pass Type I transmembrane protein. Dll4 has been well conserved during evolution with the human and mouse protein sharing 87% sequence identity. The extracellular domain of human Dll4 contains eight epidermal growth factor (EGF)-like repeats, four potential glycosylation sites and a 45 amino acid Delta, Serrate, Lag2 (DSL) domain, which is necessary for the interaction and activation of Notch-1 and Notch-4. As described previously for Dll1, the Dll4 intracellular domain might also be cleaved and translocated into the nucleus to regulate transcription, thus implying bidirectional signalling.
Several factors, including vascular endothelial growth factor (VEGF), fibroblast growth factor (bFGF), interleukin (IL)-6, Hypoxia-inducible factor 1α (HIF1α), Foxc proteins and NICD itself regulate Dll4 expression positively.
VEGF is a potent inducer of angiogenesis. Dll4 expression is up-regulated by VEGF-A in most ECs tested [e.g. in HUVECs (human umbilical vein ECs) and HIAECs (human iliac artery ECs)] but not in microvascular ECs) through the activation of VEGF-R2.
However, blocking VEGF signalling with SU5416 (a selective VEGF-R2 inhibitor) did not affect the level of Dll4 produced in zebrafish, thus suggesting that VEGF induction of Dll4 might have arisen later during evolution.
Similarly, bFGF, another proangiogenic molecule, induces Dll4 expression in HUVECs. IL-6 induces Dll4 expression efficiently in non-endothelial stromal cell lines through signalling by the signal transducer and activator of transcription 3 (STAT3)–Janus kinase pathway (JAK signaling pathway).
Dll4 is restricted largely to the endothelium of developing vessels and to a small number of additional tissues, such as the thymus, the retina, the brain, the neural tube and hematopoietic cells.
Endothelial Dll4 is confined mainly to arteries and capillaries and it is absent from veins. Most interestingly, Dll4 expression is low in the human adult vasculature but is up-regulated in vessels in a variety of solid tumours.
Finally, Dll4 expression in tumours is not restricted to the endothelium but is also present in other tumour cells (our unpublished observations).
Dll4 signalling acts downstream of the VEGF signalling pathway (MIM.192240) to prevent excessive angiogenesis and to trigger terminal differentiation of newly forming vessels.
VEGF-A is a prime regulator of vessel elongation, acting through the activation of EC proliferation and EC survival.
Activation of VEGF-R2 by VEGF-A occurs early during sprouting angiogenesis. Concomitantly, VEGF-A up-regulates Notch signalling locally through the activation of Dll4 expression.
Dll4 in turn signals through Notch1 and Notch4 to block excessive branching by preventing the tip cell phenotype.
Finally, Dll4 regulates vessel maturation through inhibition of EC proliferation, recruitment of mural cells and potentially through arterial–venous differentiation.
Dll4-dependent activation of the Notch signalling pathway also acts through a negative feedback mechanism to block VEGF signalling through induction of FLT1 (VEGF-R1 MIM.165070) and repression of KDR (VEGF-R2 MIM.191306).
Notch signalling pathway
Notch signalling is crucial for vascular development and this role is further emphasized with the cloning of the Notch ligand, Dll4.
The deletion or inhibition of DLL4 results in excessive, non-productive angiogenesis. This unrestrained angiogenesis unexpectedly and paradoxically decreases tumour growth, even in tumours resistant to anti-VEGF therapies.
DLL4 is up-regulated in vessels in a variety of solid tumours. Dll4 expression in tumours is not restricted to the endothelium but is also present in other tumour cells (17822956).
Based on this expression pattern, different molecules have been generated to block Dll4 signalling. These blocking agents inhibited tumour growth in vivo by triggering excessive but nonfunctional angiogenesis. These molecules constitute a new category of pro-angiogenic yet anticancer agents and offer an exciting alternative to previously described vascular targeting molecules.