Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation

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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.


  1. 1

    Carmeliet, P. Angiogenesis in life, disease and medicine. Nature 438, 932–936 (2005)

  2. 2

    Ferrara, N., Gerber, H. P. & LeCouter, J. The biology of VEGF and its receptors. Nature Med. 9, 669–676 (2003)

  3. 3

    Kaipainen, A. et al. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc. Natl Acad. Sci. USA 92, 3566–3570 (1995)

  4. 4

    Valtola, R. et al. VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer. Am. J. Pathol. 154, 1381–1390 (1999)

  5. 5

    Paavonen, K., Puolakkainen, P., Jussila, L., Jahkola, T. & Alitalo, K. Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing. Am. J. Pathol. 156, 1499–1504 (2000)

  6. 6

    Ferrara, N. et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380, 439–442 (1996)

  7. 7

    Carmeliet, P. et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435–439 (1996)

  8. 8

    Shalaby, F. et al. Failure of blood island formation and vasculogenesis in Flk-1-deficient mice. Nature 376, 62–66 (1995)

  9. 9

    Gille, H. et al. Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants. J. Biol. Chem. 276, 3222–3230 (2001)

  10. 10

    Gerhardt, H. et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J. Cell Biol. 161, 1163–1177 (2003)

  11. 11

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

  12. 12

    Ridgway, J. et al. Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature 444, 1083–1087 (2006)

  13. 13

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

  14. 14

    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)

  15. 15

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

  16. 16

    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)

  17. 17

    Roca, C. & Adams, R. H. Regulation of vascular morphogenesis by Notch signaling. Genes Dev. 21, 2511–2524 (2007)

  18. 18

    Alitalo, K., Tammela, T. & Petrova, T. V. Lymphangiogenesis in development and human disease. Nature 438, 946–953 (2005)

  19. 19

    Dixelius, J. et al. Ligand-induced vascular endothelial growth factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites. J. Biol. Chem. 278, 40973–40979 (2003)

  20. 20

    Dumont, D. J. et al. Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science 282, 946–949 (1998)

  21. 21

    Covassin, L. D., Villefranc, J. A., Kacergis, M. C., Weinstein, B. M. & Lawson, N. D. Distinct genetic interactions between multiple Vegf receptors are required for development of different blood vessel types in zebrafish. Proc. Natl Acad. Sci. USA 103, 6554–6559 (2006)

  22. 22

    Laakkonen, P. et al. Vascular endothelial growth factor receptor 3 is involved in tumor angiogenesis and growth. Cancer Res. 67, 593–599 (2007)

  23. 23

    Veikkola, T. et al. Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function. FASEB J. 17, 2006–2013 (2003)

  24. 24

    Goldman, J. et al. Cooperative and redundant roles of VEGFR-2 and VEGFR-3 signaling in adult lymphangiogenesis. FASEB J. 21, 1003–1012 (2007)

  25. 25

    Makinen, T. et al. Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3. Nature Med. 7, 199–205 (2001)

  26. 26

    Baldwin, M. E. et al. The specificity of receptor binding by vascular endothelial growth factor-D is different in mouse and man. J. Biol. Chem. 276, 19166–19171 (2001)

  27. 27

    Shawber, C. J. et al. Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression. J. Clin. Invest. 117, 3369–3382 (2007)

  28. 28

    Ferrara, N. & Kerbel, R. S. Angiogenesis as a therapeutic target. Nature 438, 967–974 (2005)

  29. 29

    Baluk, P. et al. Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation. J. Clin. Invest. 115, 247–257 (2005)

  30. 30

    Stacker, S. A., Achen, M. G., Jussila, L., Baldwin, M. E. & Alitalo, K. Lymphangiogenesis and cancer metastasis. Nature Rev. Cancer 2, 573–583 (2002)

  31. 31

    Karkkainen, M. J. et al. Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nature Immunol. 5, 74–80 (2004)

  32. 32

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

  33. 33

    Kiba, A., Sagara, H., Hara, T. & Shibuya, M. VEGFR-2-specific ligand VEGF-E induces non-edematous hyper-vascularization in mice. Biochem. Biophys. Res. Commun. 301, 371–377 (2003)

  34. 34

    Zheng, Y. et al. Chimeric VEGF-ENZ7/PlGF promotes angiogenesis via VEGFR-2 without significant enhancement of vascular permeability and inflammation. Arterioscler. Thromb. Vasc. Biol. 26, 2019–2026 (2006)

  35. 35

    Hanahan, D. Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature 315, 115–122 (1985)

  36. 36

    Esni, F. et al. Neural cell adhesion molecule (N-CAM) is required for cell type segregation and normal ultrastructure in pancreatic islets. J. Cell Biol. 144, 325–337 (1999)

  37. 37

    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)

  38. 38

    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)

  39. 39

    Dovey, H. F. et al. Functional gamma-secretase inhibitors reduce β-amyloid peptide levels in brain. J. Neurochem. 76, 173–181 (2001)

  40. 40

    Weijzen, S. et al. The Notch ligand Jagged-1 is able to induce maturation of monocyte-derived human dendritic cells. J. Immunol. 169, 4273–4278 (2002)

  41. 41

    Enholm, B. et al. Adenoviral expression of vascular endothelial growth factor-C induces lymphangiogenesis in the skin. Circ. Res. 88, 623–629 (2001)

  42. 42

    Wirzenius, M. et al. Distinct vascular endothelial growth factor signals for lymphatic vessel enlargement and sprouting. J. Exp. Med. 204, 1431–1440 (2007)

  43. 43

    Tammela, T. et al. Angiopoietin-1 promotes lymphatic sprouting and hyperplasia. Blood 105, 4642–4648 (2005)

  44. 44

    Kozaki, K. et al. Establishment and characterization of a human lung cancer cell line NCI-H460–LNM35 with consistent lymphogenous metastasis via both subcutaneous and orthotopic propagation. Cancer Res. 60, 2535–2540 (2000)

  45. 45

    Sakai, K. et al. Expression and function of class II antigens on gastric carcinoma cells and gastric epithelia: differential expression of DR, DQ, and DP antigens. J. Natl Cancer Inst. 79, 923–932 (1987)

  46. 46

    Karpanen, T. et al. Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res. 61, 1786–1790 (2001)

  47. 47

    Jussila, L. et al. Lymphatic endothelium and Kaposi’s sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3. Cancer Res. 58, 1599–1604 (1998)

  48. 48

    Kriehuber, E. et al. Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages. J. Exp. Med. 194, 797–808 (2001)

  49. 49

    Karpanen, T. et al. Lymphangiogenic growth factor responsiveness is modulated by postnatal lymphatic vessel maturation. Am. J. Pathol. 169, 708–718 (2006)

  50. 50

    Saaristo, A. et al. Lymphangiogenic gene therapy with minimal blood vascular side effects. J. Exp. Med. 196, 719–730 (2002)

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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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Correspondence to Kari Alitalo.

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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) doi:10.1038/nature07083

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