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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 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.

References

  1. Lohela, M., Bry, M., Tammela, T. & Alitalo, K. VEGFs and receptors involved in angiogenesis versus lymphangiogenesis. Curr. Opin. Cell Biol. 21, 154–165 (2009)

    CAS  Article  Google Scholar 

  2. Suchting, S. et al. The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching. Proc. Natl Acad. Sci. USA 104, 3225–3230 (2007)

    ADS  CAS  Article  Google Scholar 

  3. Lobov, I. B. et al. Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting. Proc. Natl Acad. Sci. USA 104, 3219–3224 (2007)

    ADS  CAS  Article  Google Scholar 

  4. Phng, L. K. & Gerhardt, H. Angiogenesis: a team effort coordinated by Notch. Dev. Cell 16, 196–208 (2009)

    CAS  Article  Google Scholar 

  5. Tammela, T. et al. Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation. Nature 454, 656–660 (2008)

    ADS  CAS  Article  Google Scholar 

  6. Siekmann, A. F. & Lawson, N. D. Notch signalling limits angiogenic cell behaviour in developing zebrafish arteries. Nature 445, 781–784 (2007)

    ADS  CAS  Article  Google Scholar 

  7. Benedito, R. et al. The Notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis. Cell 137, 1124–1135 (2009)

    CAS  Article  Google Scholar 

  8. Hellström, M. et al. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature 445, 776–780 (2007)

    ADS  Article  Google Scholar 

  9. Noguera-Troise, I. et al. Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis. Nature 444, 1032–1037 (2006)

    ADS  CAS  Article  Google Scholar 

  10. Siekmann, A. F., Covassin, L. & Lawson, N. D. Modulation of VEGF signalling output by the Notch pathway. Bioessays 30, 303–313 (2008)

    CAS  Article  Google Scholar 

  11. Thurston, G. & Kitajewski, J. VEGF and Delta–Notch: interacting signalling pathways in tumour angiogenesis. Br. J. Cancer 99, 1204–1209 (2008)

    CAS  Article  Google Scholar 

  12. Haigh, J. J. et al. Cortical and retinal defects caused by dosage-dependent reductions in VEGF-A paracrine signaling. Dev. Biol. 262, 225–241 (2003)

    CAS  Article  Google Scholar 

  13. Claxton, S. et al. Efficient, inducible Cre-recombinase activation in vascular endothelium. Genesis 46, 74–80 (2008)

    CAS  Article  Google Scholar 

  14. Wang, Y. et al. Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Nature 465, 483–486 (2010)

    ADS  CAS  Article  Google Scholar 

  15. Koch, U. et al. Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment. J. Exp. Med. 205, 2515–2523 (2008)

    CAS  Article  Google Scholar 

  16. Han, H. et al. Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision. Int. Immunol. 14, 637–645 (2002)

    CAS  Article  Google Scholar 

  17. Harrington, L. S. et al. Regulation of multiple angiogenic pathways by Dll4 and Notch in human umbilical vein endothelial cells. Microvasc. Res. 75, 144–154 (2008)

    CAS  Article  Google Scholar 

  18. Taylor, K. L., Henderson, A. M. & Hughes, C. C. Notch activation during endothelial cell network formation in vitro targets the basic HLH transcription factor HESR-1 and downregulates VEGFR-2/KDR expression. Microvasc. Res. 64, 372–383 (2002)

    CAS  Article  Google Scholar 

  19. Sainson, R. C. et al. Cell-autonomous notch signaling regulates endothelial cell branching and proliferation during vascular tubulogenesis. FASEB J. 19, 1027–1029 (2005)

    CAS  Article  Google Scholar 

  20. Hogan, B. M. et al. Vegfc/Flt4 signalling is suppressed by Dll4 in developing zebrafish intersegmental arteries. Development 136, 4001–4009 (2009)

    CAS  Article  Google Scholar 

  21. Zhang, L. et al. VEGFR-3 ligand-binding and kinase activity are required for lymphangiogenesis but not for angiogenesis. Cell Res. 20, 1313–1331 (2010)

    ADS  Google Scholar 

  22. Pytowski, B. et al. Complete and specific inhibition of adult lymphatic regeneration by a novel VEGFR-3 neutralizing antibody. J. Natl Cancer Inst. 97, 14–21 (2005)

    CAS  Article  Google Scholar 

  23. Kirkin, V. et al. Characterization of indolinones which preferentially inhibit VEGF-C- and VEGF-D-induced activation of VEGFR-3 rather than VEGFR-2. Eur. J. Biochem. 268, 5530–5540 (2001)

    CAS  Article  Google Scholar 

  24. Tammela, T. et al. VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling. Nature Cell Biol. 13, 1202–1213 (2011)

    CAS  Article  Google Scholar 

  25. Galvagni, F. et al. Endothelial cell adhesion to the extracellular matrix induces c-Src-dependent VEGFR-3 phosphorylation without the activation of the receptor intrinsic kinase activity. Circ. Res. 106, 1839–1848 (2010)

    CAS  Article  Google Scholar 

  26. Stenzel, D. et al. Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo. EMBO Rep. 12, 1135–1143 (2011)

    CAS  Article  Google Scholar 

  27. Estrach, S. et al. Laminin-binding integrins induce Dll4 expression and Notch signaling in endothelial cells. Circ. Res. 109, 172–182 (2011)

    CAS  Article  Google Scholar 

  28. Lemmon, M. A. & Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010)

    CAS  Article  Google Scholar 

  29. Lux, A., Llacer, H., Heussen, F. M. & Joussen, A. M. Non-responders to bevacizumab (Avastin) therapy of choroidal neovascular lesions. Br. J. Ophthalmol. 91, 1318–1322 (2007)

    Article  Google Scholar 

  30. Jubb, A. M. & Harris, A. L. Biomarkers to predict the clinical efficacy of bevacizumab in cancer. Lancet Oncol. 11, 1172–1183 (2010)

    CAS  Article  Google Scholar 

  31. Duarte, A. et al. Dosage-sensitive requirement for mouse Dll4 in artery development. Genes Dev. 18, 2474–2478 (2004)

    CAS  Article  Google Scholar 

  32. Radtke, F. et al. Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 10, 547–558 (1999)

    CAS  Article  Google Scholar 

  33. Manley, P. W. et al. Advances in the structural biology, design and clinical development of VEGF-R kinase inhibitors for the treatment of angiogenesis. Biochim. Biophys. Acta 1697, 17–27 (2004)

    CAS  Article  Google Scholar 

  34. Manley, P. W. et al. Anthranilic acid amides: a novel class of antiangiogenic VEGF receptor kinase inhibitors. J. Med. Chem. 45, 5687–5693 (2002)

    CAS  Article  Google Scholar 

  35. Prewett, M. et al. Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors. Cancer Res. 59, 5209–5218 (1999)

    CAS  PubMed  Google Scholar 

  36. Liang, W. C. et al. Cross-species vascular endothelial growth factor (VEGF)-blocking antibodies completely inhibit the growth of human tumor xenografts and measure the contribution of stromal VEGF. J. Biol. Chem. 281, 951–961 (2006)

    CAS  Article  Google Scholar 

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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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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).

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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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