Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation

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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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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) doi:10.1038/nature07083

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