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Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation

Nature volume 454, pages 656–660 (2008)Cite this article

Abstract

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is a key process in several pathological conditions, including tumour growth and age-related macular degeneration1. Vascular endothelial growth factors (VEGFs) stimulate angiogenesis and lymphangiogenesis by activating VEGF receptor (VEGFR) tyrosine kinases in endothelial cells2. VEGFR-3 (also known as FLT-4) is present in all endothelia during development, and in the adult it becomes restricted to the lymphatic endothelium3. However, VEGFR-3 is upregulated in the microvasculature of tumours and wounds4,5. Here we demonstrate that VEGFR-3 is highly expressed in angiogenic sprouts, and genetic targeting of VEGFR-3 or blocking of VEGFR-3 signalling with monoclonal antibodies results in decreased sprouting, vascular density, vessel branching and endothelial cell proliferation in mouse angiogenesis models. Stimulation of VEGFR-3 augmented VEGF-induced angiogenesis and sustained angiogenesis even in the presence of VEGFR-2 (also known as KDR or FLK-1) inhibitors, whereas antibodies against VEGFR-3 and VEGFR-2 in combination resulted in additive inhibition of angiogenesis and tumour growth. Furthermore, genetic or pharmacological disruption of the Notch signalling pathway led to widespread endothelial VEGFR-3 expression and excessive sprouting, which was inhibited by blocking VEGFR-3 signals. Our results implicate VEGFR-3 as a regulator of vascular network formation. Targeting VEGFR-3 may provide additional efficacy for anti-angiogenic therapies, especially towards vessels that are resistant to VEGF or VEGFR-2 inhibitors.

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Figure 1: VEGFR-3 is expressed in the tumour vasculature and localizes to endothelial tip cells.
Figure 2: VEGFR-3-function-blocking antibodies inhibit angiogenic sprouting.
Figure 3: Regulation and activation of VEGFR-3 by VEGF family ligands.
Figure 4: Notch signalling downregulates VEGFR-3 in endothelial cells.

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Acknowledgements

We would like to thank P. Haiko, W. Holnthoner, T. Holopainen and D. Tvorogov for help with the experiments, as well as A. Ristimäki for the MKN45 cells, and T. Takahashi for NCI-H460-LNM35 cells. We also thank S. Fanta, C. Heckman, K. Helenius and T. Petrova for critical comments on the manuscript. The Biomedicum Molecular Imaging Unit is acknowledged for microscopy services, and M. Helanterä, P. Hyvärinen, A. Kotronen, T. Laakkonen, S. Lampi, K. Makkonen, A. Malinen, T. Tainola and S. Wallin for technical assistance, as well as A. Lehtonen and T. Taina for animal husbandry. Electron microscopy was carried out in collaboration with the Electron Microscopy Unit, Institute of Biotechnology at the University of Helsinki. This work was supported by grants from the NIH (5 R01 HL075183-02), The European Union (Lymphangiogenomics, LSHG-CT-2004-503573) and the Louis Jeantet Foundation (K.A.), as well as the Association for International Cancer Research (UK) and IngaBritt and Anne Lundberg Foundation (C.B.). T.T. was supported by personal grants from the Finnish Cancer Organizations, the Finnish Cultural Foundation, Nylands Nation, The Paulo Foundation and the Helsinki Biomedical Graduate School.

Author Contributions T.T. designed, directed and performed embryo, retina, mouse ear and xenograft experiments, immunohistochemistry and data analysis, interpreted results and wrote the paper; G.Z. performed xenograft experiments, immunohistochemistry, electron microscopy and ELISA, analysed data and interpreted results; E.W. designed and performed qRT–PCR and data analysis, and interpreted results; A.M. performed embryo and retina experiments, immunohistochemistry, and data analysis; S.S. performed intraocular injections and immunohistochemistry, analysed data and interpreted results; M. Wirzenius designed and performed mouse ear experiments and immunohistochemistry, analysed data and interpreted results; M. Waltari performed xenograft experiments, immunohistochemistry and data analysis; M. H. directed experiments and interpreted results; T.S. performed Rip1Tag2 tumour experiments, analysed data and interpreted results; R.P. performed immunohistochemistry and analysed data; C.F. performed intraocular injections; A.D. provided the Dll4+/- mice; H.I. performed surgery and provided clinical tumour samples; P.L. directed experiments and interpreted results; G.C. directed experiments and interpreted results; S.Y.-H. developed and provided adenovirus vectors; M.S. generated and provided K14–VEGF and K14–VEGF-E transgenic mice; B.P. generated and provided monoclonal VEGFR function-blocking antibodies; A.E. directed experiments and interpreted results; C.B. designed experiments, interpreted results and helped write the paper; K.A. designed experiments, interpreted results and wrote the paper.

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  1. Elisabet Wallgard and Aino Murtomäki: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Biomedicum Helsinki and the Haartman Institute University of Helsinki, PO Box 63 (Haartmaninkatu 8), 00014 Helsinki, Finland ,

    Tuomas Tammela, Georgia Zarkada, Aino Murtomäki, Maria Wirzenius, Marika Waltari, Pirjo Laakkonen & Kari Alitalo

  2. Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden

    Elisabet Wallgard, Mats Hellström & Christer Betsholtz

  3. Institut National de la Santé et de la Recherche Médicale U833, Collège de France, 11 Place Marcelin Berthelot, 75005 Paris, France ,

    Steven Suchting, Catarina Freitas & Anne Eichmann

  4. Department of Clinical-Biological Sciences, Center of Biomedicine, University of Basel, Mattenstrasse 28, CH-4058 Basel, Switzerland,

    Tibor Schomber & Gerhard Christofori

  5. Department of Transplantation and Hepatic Surgery, Helsinki University Central Hospital, PO Box 263, 00029 Helsinki, Finland,

    Reetta Peltonen & Helena Isoniemi

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

    Antonio Duarte

  7. A. I. Virtanen Institute and Department of Medicine, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland,

    Seppo Ylä-Herttuala

  8. Department of Molecular Oncology, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan,

    Masabumi Shibuya

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

    Bronislaw Pytowski

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  1. Tuomas Tammela
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Correspondence to Kari Alitalo.

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Competing interests

K.A. is the chairman of the scientific advisory board of Vegenics Limited, an Australian biotech company partly owned by LICR and Licentia Ltd.

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Tammela, T., Zarkada, G., Wallgard, E. et al. Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation. Nature 454, 656–660 (2008). https://doi.org/10.1038/nature07083

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