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

Nature volume 484, pages 110–114 (2012)Cite this article

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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 processes1. 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 sprouts2,3. DLL4 activates Notch in adjacent cells, which suppresses the expression of VEGF receptors and thereby restrains endothelial sprouting and proliferation2,4,5,6. 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.

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Figure 1: Notch inhibition promotes angiogenesis independently of VEGFR2.
Figure 2: VEGFR2 strongly regulates VEGFR3 protein levels but not DLL4 at the angiogenic front.
Figure 3: Notch regulates VEGFR3 activity independently of VEGFR2.
Figure 4: Inhibition of VEGFR3 kinase activity suppresses Notch-regulated sprouting.

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Acknowledgements

We thank M. Schiller, M. Ehling, M. Pitulescu and M. Nakayama for the help with experiments and discussions, and G. Breier and T. Honjo for floxed Vegfr2 and Rbpj mutant mice, respectively. Funding was provided by the Max Planck Society, the University of Münster and the German Research Foundation (programmes SFB 629 and SPP 1190).

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Authors and Affiliations

  1. Department of Tissue Morphogenesis, Max Planck Institute for Molecular Biomedicine, D-48149 Münster, Germany,

    Rui Benedito, Susana F. Rocha, Marina Woeste, Martin Zamykal & Ralf H. Adams

  2. University of Münster, Faculty of Medicine, D-48149 Münster, Germany,

    Rui Benedito, Susana F. Rocha, Marina Woeste, Martin Zamykal & Ralf H. Adams

  3. Ecole Polytechnique Fédérale de Lausanne (EPFL), Swiss Institute for Experimental Cancer Research (ISREC), CH-1015 Lausanne, Switzerland,

    Freddy Radtke

  4. Translational Research Laboratory, Catalan Institute of Oncology, IDIBELL, 08907 L’Hospitalet de Llobregat, Spain,

    Oriol Casanovas

  5. The Interdisciplinary Centre of Research in Animal Health (CIISA), Faculty of Veterinary Medicine, Technical University of Lisbon, 1300-474 Lisbon, Portugal,

    Antonio Duarte

  6. ImClone Systems, 180 Varick Street, New York, 10014, New York, USA

    Bronislaw Pytowski

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  1. Rui Benedito
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Contributions

R.B. and R.H.A. designed the experiments, interpreted the results and wrote the manuscript. R.B. generated and characterized the mutant mouse lines. R.B. directed M.W. and M.Z. and carried out the immunohistochemistry, immunoblots, qRT–PCR, confocal imaging and quantifications. S.F.R. developed the immunoprecipitation and immunoblotting assays and carried out the confocal imaging and quantifications. A.D. and F.R. provided the Dll4floxed and Notch1floxed mice, respectively. O.C. and B.P. generated and provided the monoclonal VEGFR2- and VEGFR3-blocking antibodies (ImClone Systems).

Corresponding authors

Correspondence to Rui Benedito or Ralf H. Adams.

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Competing interests

B.P. is an employee and shareholder of ImClone Systems.

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Benedito, R., Rocha, S., Woeste, M. et al. Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF–VEGFR2 signalling. Nature 484, 110–114 (2012). https://doi.org/10.1038/nature10908

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