Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF–VEGFR2 signalling

Abstract

Developing tissues and growing tumours produce vascular endothelial growth factors (VEGFs), leading to the activation of the corresponding receptors in endothelial cells. The resultant angiogenic expansion of the local vasculature can promote physiological and pathological growth processes. Previous work has uncovered that the VEGF and Notch pathways are tightly linked. Signalling triggered by VEGF-A (also known as VEGF) has been shown to induce expression of the Notch ligand DLL4 in angiogenic vessels and, most prominently, in the tip of endothelial sprouts. DLL4 activates Notch in adjacent cells, which suppresses the expression of VEGF receptors and thereby restrains endothelial sprouting and proliferation. Here we show, by using inducible loss-of-function genetics in combination with inhibitors in vivo, that DLL4 protein expression in retinal tip cells is only weakly modulated by VEGFR2 signalling. Surprisingly, Notch inhibition also had no significant impact on VEGFR2 expression and induced deregulated endothelial sprouting and proliferation even in the absence of VEGFR2, which is the most important VEGF-A receptor and is considered to be indispensable for these processes. By contrast, VEGFR3, the main receptor for VEGF-C, was strongly modulated by Notch. VEGFR3 kinase-activity inhibitors but not ligand-blocking antibodies suppressed the sprouting of endothelial cells that had low Notch signalling activity. Our results establish that VEGFR2 and VEGFR3 are regulated in a highly differential manner by Notch. We propose that successful anti-angiogenic targeting of these receptors and their ligands will strongly depend on the status of endothelial Notch signalling.

Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells

“…Blocking of Notch signaling in mouse retina also led to significant increase in vascular density [33,39] with deregulated endothelial growth but without any increase in VEGFR2 levels, the main marker for sprouting [33]. Our previous investigation using VEGF inhibitors in mouse tumor model and kidney regeneration after Thy1 nephritis indicated that IA occurs in a VEGF independent manner and that the tumor could escape anti-VEGF therapy by switch from sprouting to intussusceptive angiogenesis [40,41].…”

“…Similarly, DLL4 inhibition causes formation of a disorganized capillary network in ischemic muscles [32]. Notch inhibition induces deregulated endothelial sprouting and proliferation, independently of VEGFR2, in mouse retina as well [33]. Notch signaling has been mostly related to sprouting neo-angiogenesis.…”

Integrin β1 controls VE-cadherin localization and blood vessel stability

“…All immunostainings were carried out with littermate tissues processed simultaneously under the same conditions, as described previously 59,60 . In brief, to analyse (and quantitate) the morphology of the retinal vasculature and preserve intact endothelial filopodia and sprouts at the angiogenic front, whole animal eyes were fixed in 4% paraformaldehyde (PFA) at 4°C overnight.…”

Vascular Endothelial Growth Factor Receptor 3 Signaling Contributes to Angioobliterative Pulmonary Hypertension

“…8 To investigate these mechanisms of lung vessel injury and repair, rat models of severe angioobliterative PAH and right heart failure, 9,10 which are based on chronic hyp-oxic exposure 11 or immunoinsufficiency 5 together-and very paradoxically-with the administration of an antiangiogenic vascular endothelial growth factor (VEGF) inhibitor, are now being examined. 8,12 This apparent paradox of antiangiogenic drug-induced PAH can now be explained by taking into account an improved understanding of the role played by VEGF in vascular biology [13][14][15] and of the mechanisms of resistance of tumor cells to antiangiogenic drugs. 16,17 Collectively, a large number of new studies illustrate the multitude of biological effects that are attributable to VEGF-A, its various splice variants, and signals transmitted via several membrane and nuclear VEGF receptors.…”