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Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3

Nature Medicine volume 7, pages 199–205 (2001)Cite this article

Abstract

The lymphatic vasculature transports extravasated tissue fluid, macromolecules and cells back into the blood circulation. Recent reports have focused on the molecular mechanisms regulating the lymphatic vessels. Vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to stimulate lymphangiogenesis and their receptor, VEGFR-3, has been linked to human hereditary lymphedema. Here we show that a soluble form of VEGFR-3 is a potent inhibitor of VEGF-C/VEGF-D signaling, and when expressed in the skin of transgenic mice, it inhibits fetal lymphangiogenesis and induces a regression of already formed lymphatic vessels, though the blood vasculature remains normal. Transgenic mice develop a lymphedema-like phenotype characterized by swelling of feet, edema and dermal fibrosis. They survive the neonatal period in spite of a virtually complete lack of lymphatic vessels in several tissues, and later show regeneration of the lymphatic vasculature, indicating that induction of lymphatic regeneration may also be possible in humans.

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Figure 1: Soluble VEGFR-3 inhibits ligand-dependent receptor signaling.
Figure 2: K14-VEGFR-3–Ig mice lack dermal lymphatic vessels.
Figure 3: Soluble VEGFR-3 inhibits lymphangiogenesis specifically.
Figure 4: Circulating levels of soluble VEGFR-3 inhibit lymphatic vessel development in internal organs.
Figure 5: Features of human lymphedema in the K14-VEGFR-3–Ig mice.
Figure 6: Soluble VEGFR-3 leads to a regression of the lymphatic vessels in embryonic skin.

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References

  1. Ferrara, N. Molecular and biological properties of vascular endothelial growth factor. J. Mol. Med. 77, 527–543 (1999).

    Article  CAS  Google Scholar 

  2. Carmeliet, P. Mechanisms of angiogenesis and arteriogenesis. Nature Med. 6, 389–395 (2000).

    Article  CAS  Google Scholar 

  3. Eriksson, U. & Alitalo, K. Structure, expression and receptor-binding properties of novel vascular endothelial growth factors. Curr. Top. Microbiol. Immunol. 237, 41–57 (1999).

    CAS  Google Scholar 

  4. Veikkola, T., Karkkainen, M.J., Claesson-Welsh, L. & Alitalo, K. Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res. 60, 203–212 (2000).

    CAS  Google Scholar 

  5. Fong, G.-H., Zhang, L., Bryce, D.-M. & Peng, J. Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice. Development 126, 3015–3025 (1999).

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Kaipainen, A. et al. Expression of the fms-like tyrosine kinase FLT4 gene becomes restricted to lymphatic endothelium during development. Proc. Natl. Acad. Sci. USA 92, 3566–3570 (1995).

    Article  CAS  Google Scholar 

  9. Partanen, T.A. et al. VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues. FASEB J. 14, 2087–2096 (2000).

    Article  CAS  Google Scholar 

  10. Jeltsch, M. et al. Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science 276, 1423–1425 (1997).

    Article  CAS  Google Scholar 

  11. Oh, S.J. et al. VEGF and VEGF-C: specific induction of angiogenesis and lymphangiogenesis in the differentiated avian chorioallantoic membrane. Dev. Biol. 188, 96–109 (1997).

    Article  CAS  Google Scholar 

  12. Karkkainen, M.J. et al. Missense mutations interfere with VEGFR-3 signaling in primary lymphoedema. Nature Genet. 25, 153–159 (2000).

    Article  CAS  Google Scholar 

  13. Irrthum, A., Karkkainen, M.J., Devriendt, K., Alitalo, K. & Vikkula, M. Congenital hereditary lymphedema caused by a mutation that inactivates VEGFR3 tyrosine kinase. Am. J. Hum. Gen. 67, 295–301 (2000).

    Article  CAS  Google Scholar 

  14. Kendall, R.L. & Thomas, K.A. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc. Natl. Acad. Sci. USA 90, 10705–10709 (1993).

    Article  CAS  Google Scholar 

  15. Aiello, L.P. et al. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc. Natl. Acad. Sci. USA 92, 10457–10461 (1995).

    Article  CAS  Google Scholar 

  16. Ferrara, N. et al. Vascular endothelial growth factor is essential for corpus luteum angiogenesis. Nature Med. 4, 336–340 (1998).

    Article  CAS  Google Scholar 

  17. Lin, P. et al. Inhibition of tumor growth by targeting tumor endothelium using a soluble vascular endothelial growth factor receptor. Cell Growth Differ. 9, 49–58 (1998).

    CAS  Google Scholar 

  18. Kong, H.L. et al. Regional suppression of tumor growth by in vivo transfer of a cDNA encoding a secreted form of the extracellular domain of the flt-1 vascular endothelial growth factor receptor. Hum. Gene Ther. 9, 823–833 (1998).

    Article  CAS  Google Scholar 

  19. Goldman, C.K. et al. Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate. Proc. Natl. Acad. Sci. USA 95, 8795–8800 (1998).

    Article  CAS  Google Scholar 

  20. Takayama, K. et al. Suppression of tumor angiogenesis and growth by gene transfer of a soluble form of vascular endothelial growth factor receptor into a remote organ. Cancer Res. 60, 2169–2177 (2000).

    CAS  Google Scholar 

  21. Achen, M.G. et al. Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3. Eur. J. Biochem. 267, 2505–2515 (2000).

    Article  CAS  Google Scholar 

  22. 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).

    CAS  Google Scholar 

  23. Kubo, H. et al. Involvement of vascular endothelial growth factor receptor-3 in maintenance of integrity of endothelial cell lining during tumor angiogenesis. Blood 96, 546–553 (2000).

    CAS  Google Scholar 

  24. Banerji, S. et al. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J. Cell Biol. 144, 789–801 (1999).

    Article  CAS  Google Scholar 

  25. Thurston, G. et al. Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science 286, 2511–2514 (1999).

    Article  CAS  Google Scholar 

  26. Hakumaki, J.M., Poptani, H., Sandmair, A.M., Yla-Herttuala, S. & Kauppinen, R.A. 1H MRS detects polyunsaturated fatty acid accumulation during gene therapy of glioma: implications for the in vivo detection of apoptosis. Nature Med. 5, 1323–1327 (1999).

    Article  CAS  Google Scholar 

  27. Byrne, C., Tainsky, M. & Fuchs, E. Programming gene expression in developing epidermis. Development 120, 2369–2383 (1994).

    CAS  Google Scholar 

  28. Korhonen, J. et al. Endothelial-specific gene expression directed by the tie gene promoter in vivo. Blood 86, 1828–1835 (1995).

    CAS  Google Scholar 

  29. Gerber, H.P. et al. VEGF is required for growth and survival in neonatal mice. Development 126, 1149–1159 (1999).

    CAS  Google Scholar 

  30. Witte, M.H., Way, D.L., Witte, C.L. & Bernas, M. Lymphangiogenesis: Mechanisms, significance and clinical implications. in Regulation of Angiogenesis (eds. Goldberg, I.D. & Rosen, E.M.) 65–112 (Birkhäuser Verlag, Basel, Switzerland, 1997).

    Chapter  Google Scholar 

  31. Mortimer, P.S. The pathophysiology of lymphedema. Cancer 83, 2798–2802 (1998).

    Article  CAS  Google Scholar 

  32. Karkkainen, M.J., Jussila, L., Ferral, R. E., Finegold, D.N. & Alitalo, K. Molecular regulation of lymphangiogenesis and targets for tissue oedema. Trends Mol. Med. 7, 18–22 (2000)

    Article  Google Scholar 

  33. Benjamin, L.E., Hemo, I. & Keshet, E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development 125, 1591–1598 (1998).

    CAS  Google Scholar 

  34. Joukov, V. et al. Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J. 16, 3898–3911 (1997).

    Article  CAS  Google Scholar 

  35. Achen, M.G. et al. Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4). Proc. Natl. Acad. Sci. USA 95, 548–553 (1998).

    Article  CAS  Google Scholar 

  36. Cao, Y. et al. Vascular endothelial growth factor C induces angiogenesis in vivo. Proc. Natl. Acad. Sci. USA 95, 14389–14392 (1998).

    Article  CAS  Google Scholar 

  37. Witzenbichler, B. et al. Vascular endothelial growth factor-C (VEGF-C/VEGF-2) promotes angiogenesis in the setting of tissue ischemia. Am. J. Pathol. 153, 381–394 (1998).

    Article  CAS  Google Scholar 

  38. Marconcini, L. et al. c-fos-induced growth factor/vascular endothelial growth factor D induces angiogenesis in vivo and in vitro. Proc. Natl. Acad. Sci. USA 96, 9671–9676 (1999).

    Article  CAS  Google Scholar 

  39. Joukov, V. et al. A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities. J. Biol. Chem. 273, 6599–6602 (1998).

    Article  CAS  Google Scholar 

  40. Lymboussaki, A. et al. Expression of the vascular endothelial growth factor C receptor VEGFR-3 in lymphatic endothelium of the skin and in vascular tumors. Am. J. Pathol. 153, 395–403 (1998).

    Article  CAS  Google Scholar 

  41. 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).

    Article  CAS  Google Scholar 

  42. Salven, P. et al. Vascular endothelial growth factors VEGF-B and VEGF-C are expressed in human tumors. Am. J. Pathol. 153, 103–108 (1998).

    Article  CAS  Google Scholar 

  43. Yonemura, Y. et al. Role of vascular endothelial growth factor C expression in the development of lymph node metastasis in gastric cancer. Clin. Cancer Res. 5, 1823–1829 (1999).

    CAS  Google Scholar 

  44. Tsurusaki, T. et al. Vascular endothelial growth factor-C expression in human prostatic carcinoma and its relationship to lymph node metastasis. Br. J. Cancer 80, 309–313 (1999).

    Article  CAS  Google Scholar 

  45. Mandriota, S.J. et al. Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. EMBO J. in the press (2001).

  46. Skobe., M. et al. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nature Med. this issue (2001).

  47. Stacker, S.A. et al. VEGF-D promotes the metastatic spread of cancer by lymphatics. Nature Med. this issue (2001).

Download references

Acknowledgements

We thank G. Thurston and D. McDonald for teaching the lectin staining. J Jänne, L. Alhonen, M. Jänne and H. Rauvala for help in the generation of the transgenic mice; I. Seppälä for advice on ELISA; and T. Tainola, P. Ylikantola, S. Karttunen, R. Kivirikko and K. Makkonen for technical assistance. The K14 vector was a gift from E. Fuchs. This study was supported by grants from the Finnish Cancer Organization, Ida Montini Foundation, Finnish Cultural Foundation and Paulo Foundation.

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Authors and Affiliations

  1. Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Haartman Institute, University of Helsinki, Helsinki, Finland

    Taija Mäkinen, Lotta Jussila, Tanja Veikkola, Terhi Karpanen & Kari Alitalo

  2. National Bio-NMR Facility, A.I. Virtanen Institute, University of Kuopio, Kuopio, Finland

    Mikko I. Kettunen & Risto Kauppinen

  3. Department of Molecular Medicine, A.I. Virtanen Institute, University of Kuopio, Kuopio, Finland

    Kalevi J. Pulkkanen & Seppo Ylä-Herttuala

  4. Department of Oncology, Kuopio University Hospital, Kuopio, Finland

    Kalevi J. Pulkkanen

  5. Nuffield Department of Medicine, University of Oxford, Molecular Immunology Group, Headington, Oxford, United Kingdom

    David G. Jackson

  6. Department of Molecular Genetics, Kyoto University, Japan

    Hajime Kubo & Shin-Ichi Nishikawa

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  1. Taija Mäkinen
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Correspondence to Kari Alitalo.

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Mäkinen, T., Jussila, L., Veikkola, T. et al. Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3. Nat Med 7, 199–205 (2001). https://doi.org/10.1038/84651

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