Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Suppressed NFAT-dependent VEGFR1 expression and constitutive VEGFR2 signaling in infantile hemangioma

Nature Medicine volume 14, pages 1236–1246 (2008)Cite this article

Abstract

Infantile hemangiomas are localized and rapidly growing regions of disorganized angiogenesis. We show that expression of vascular endothelial growth factor receptor-1 (VEGFR1) in hemangioma endothelial cells (hemECs) and hemangioma tissue is markedly reduced compared to controls. Low VEGFR1 expression in hemECs results in VEGF-dependent activation of VEGFR2 and downstream signaling pathways. In hemECs, transcription of the gene encoding VEGFR1 (FLT1) is dependent on nuclear factor of activated T cells (NFAT). Low VEGFR1 expression in hemECs is caused by reduced activity of a pathway involving β1 integrin, the integrin-like receptor tumor endothelial marker-8 (TEM8), VEGFR2 and NFAT. In a subset of individuals with hemangioma, we found missense mutations in the genes encoding VEGFR2 (KDR) and TEM8 (ANTXR1). These mutations result in increased interactions among VEGFR2, TEM8 and β1 integrin proteins and in inhibition of integrin activity. Normalization of the constitutive VEGFR2 signaling in hemECs with soluble VEGFR1 or antibodies that neutralize VEGF or stimulate β1 integrin suggests that local administration of these or similar agents may be effective in hemangioma treatment.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: VEGFR2-dependent signal transduction is upregulated in hemECs.
Figure 2: Low VEGFR1 expression in hemECs is caused by reduced activation of NFAT.
Figure 3: Reduced activation of β1 integrin in hemECs.
Figure 4: Mutations in ANTXR1 and KDR and their effects.
Figure 5: Mechanisms of repressed NFAT-mediated stimulation of VEGFR1 expression in hemECs.

Similar content being viewed by others

References

  1. Boye, E. et al. Clonality and altered behavior of endothelial cells from hemangiomas. J. Clin. Invest. 107, 745–752 (2001).

    Article  CAS  Google Scholar 

  2. Mulliken, J. & Young, A. Vascular Birthmarks: Hemangiomas and Malformations (W. B. Saunders Company, Philadelphia, 1988).

    Google Scholar 

  3. Mulliken, J.B. Cutaneous vascular anomalies. Semin. Vasc. Surg. 6, 204–218 (1993).

    CAS  PubMed  Google Scholar 

  4. Walter, J.W. et al. Somatic mutation of vascular endothelial growth factor receptors in juvenile hemangioma. Genes Chromosom. Cancer 33, 295–303 (2002).

    Article  CAS  Google Scholar 

  5. North, P.E., Waner, M., Mizeracki, A. & Mihm, M.C., Jr. GLUT1: a newly discovered immunohistochemical marker for juvenile hemangiomas. Hum. Pathol. 31, 11–22 (2000).

    Article  CAS  Google Scholar 

  6. Li, Q., Yu, Y., Bischoff, J., Mulliken, J.B. & Olsen, B.R. Differential expression of CD146 in tissues and endothelial cells derived from infantile hemangiomas and normal human skin. J. Pathol. 201, 296–302 (2003).

    Article  CAS  Google Scholar 

  7. Barnes, C.M. et al. Evidence by molecular profiling for a placental origin of infantile hemangioma. Proc. Natl. Acad. Sci. USA 102, 19097–19102 (2005).

    Article  CAS  Google Scholar 

  8. North, P.E. et al. A unique microvascular phenotype shared by juvenile hemangiomas and human placenta. Arch. Dermatol. 137, 559–570 (2001).

    Article  CAS  Google Scholar 

  9. Chiller, K.G., Passaro, D. & Frieden, I.J. Hemangiomas of infancy: clinical characteristics, morphologic subtypes, and their relationship to race, ethnicity and sex. Arch. Dermatol. 138, 1567–1576 (2002).

    Article  Google Scholar 

  10. Haggstrom, A.N. et al. Prospective study of infantile hemangiomas: demographic, prenatal and perinatal characteristics. J. Pediatr. 150, 291–294 (2007).

    Article  Google Scholar 

  11. Ferrara, N. The role of VEGF in the regulation of physiological and pathological angiogenesis. EXS 94, 209–231 (2005).

    Google Scholar 

  12. Roberts, D.M. et al. The vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR-1) modulates Flk-1 (VEGFR-2) signaling during blood vessel formation. Am. J. Pathol. 164, 1531–1535 (2004).

    Article  CAS  Google Scholar 

  13. Bradley, K.A., Mogridge, J., Mourez, M., Collier, R.J. & Young, J.A. Identification of the cellular receptor for anthrax toxin. Nature 414, 225–229 (2001).

    Article  CAS  Google Scholar 

  14. Werner, E., Kowalczyk, A.P. & Faundez, V. Anthrax toxin receptor 1/tumor endothelium marker 8 mediates cell spreading by coupling extracellular ligands to the actin cytoskeleton. J. Biol. Chem. 281, 23227–23236 (2006).

    Article  CAS  Google Scholar 

  15. Inoue, T. et al. Identification of a vascular endothelial growth factor (VEGF) antagonist, sFlt-1, from a human hematopoietic cell line NALM-16. FEBS Lett. 469, 14–18 (2000).

    Article  CAS  Google Scholar 

  16. Ito, N., Huang, K. & Claesson-Welsh, L. Signal transduction by VEGF receptor-1 wild type and mutant proteins. Cell. Signal. 13, 849–854 (2001).

    Article  CAS  Google Scholar 

  17. Wakiya, K., Begue, A., Stehelin, D. & Shibuya, M. A cAMP response element and an Ets motif are involved in the transcriptional regulation of flt-1 tyrosine kinase (vascular endothelial growth factor receptor 1) gene. J. Biol. Chem. 271, 30823–30828 (1996).

    Article  CAS  Google Scholar 

  18. Hogan, P.G., Chen, L., Nardone, J. & Rao, A. Transcriptional regulation by calcium, calcineurin and NFAT. Genes Dev. 17, 2205–2232 (2003).

    Article  CAS  Google Scholar 

  19. Hesser, B.A. et al. Down syndrome critical region protein 1 (DSCR1), a novel VEGF target gene that regulates expression of inflammatory markers on activated endothelial cells. Blood 104, 149–158 (2004).

    Article  CAS  Google Scholar 

  20. Hernandez, G.L. et al. Selective inhibition of vascular endothelial growth factor-mediated angiogenesis by cyclosporin A: roles of the nuclear factor of activated T cells and cyclooxygenase 2. J. Exp. Med. 193, 607–620 (2001).

    Article  CAS  Google Scholar 

  21. Satonaka, H. et al. Calcineurin promotes the expression of monocyte chemoattractant protein-1 in vascular myocytes and mediates vascular inflammation. Circ. Res. 94, 693–700 (2004).

    Article  CAS  Google Scholar 

  22. Schwartz, M.A. Spreading of human endothelial cells on fibronectin or vitronectin triggers elevation of intracellular free calcium. J. Cell Biol. 120, 1003–1010 (1993).

    Article  CAS  Google Scholar 

  23. Leavesley, D.I., Schwartz, M.A., Rosenfeld, M. & Cheresh, D.A. Integrin β1- and β3-mediated endothelial cell migration is triggered through distinct signaling mechanisms. J. Cell Biol. 121, 163–170 (1993).

    Article  CAS  Google Scholar 

  24. Jones, N.P., Peak, J., Brader, S., Eccles, S.A. & Katan, M. PLCγ1 is essential for early events in integrin signalling required for cell motility. J. Cell Sci. 118, 2695–2706 (2005).

    Article  CAS  Google Scholar 

  25. Aplin, A.E., Howe, A., Alahari, S.K. & Juliano, R.L. Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion molecules and selectins. Pharmacol. Rev. 50, 197–263 (1998).

    CAS  PubMed  Google Scholar 

  26. Sjaastad, M.D. & Nelson, W.J. Integrin-mediated calcium signaling and regulation of cell adhesion by intracellular calcium. Bioessays 19, 47–55 (1997).

    Article  CAS  Google Scholar 

  27. Luque, A. et al. Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common β1 chain. J. Biol. Chem. 271, 11067–11075 (1996).

    Article  CAS  Google Scholar 

  28. Schaller, M.D. & Parsons, J.T. Focal adhesion kinase and associated proteins. Curr. Opin. Cell Biol. 6, 705–710 (1994).

    Article  CAS  Google Scholar 

  29. Guan, J.-L. & Shalloway, D. Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation. Nature 358, 690–692 (1992).

    Article  CAS  Google Scholar 

  30. Abu-Ghazaleh, R., Kabir, J., Jia, H., Lobo, M. & Zachary, I. Src mediates stimulation by vascular endothelial growth factor of the phosphorylation of focal adhesion kinase at tyrosine 861 and migration and anti-apoptosis in endothelial cells. Biochem. J. 360, 255–264 (2001).

    Article  CAS  Google Scholar 

  31. Blei, F., Walter, J., Orlow, S.J. & Marchuk, D.A. Familial segregation of hemangiomas and vascular malformations as an autosomal dominant trait. Arch. Dermatol. 134, 718–722 (1998).

    Article  CAS  Google Scholar 

  32. Shinkai, A. et al. Mapping of the sites involved in ligand association and dissociation at the extracellular domain of the kinase insert domain-containing receptor for vascular endothelial growth factor. J. Biol. Chem. 273, 31283–31288 (1998).

    Article  CAS  Google Scholar 

  33. Kuriyama, M. et al. Activation and translocation of PKCδ is necessary for VEGF-induced ERK activation through KDR in HEK293T cells. Biochem. Biophys. Res. Commun. 325, 843–851 (2004).

    Article  CAS  Google Scholar 

  34. Sun, Y. et al. The kinase insert domain-containing receptor is an angiogenesis-associated antigen recognized by human cytotoxic T lymphocytes. Blood 107, 1476–1483 (2006).

    Article  CAS  Google Scholar 

  35. Shenoy, P.S. et al. beta1 Integrin-extracellular matrix protein interaction modulates the migratory response to chemokine stimulation. Biochem. Cell Biol. 79, 399–407 (2001).

    Article  CAS  Google Scholar 

  36. Vikkula, M. et al. Vascular dysmorphogenesis caused by an activating mutation in the receptor tyrosine kinase TIE2. Cell 87, 1181–1190 (1996).

    Article  CAS  Google Scholar 

  37. Brouillard, P. et al. Four common glomulin mutations cause two thirds of glomuvenous malformations (“familial glomangiomas”): evidence for a founder effect. J. Med. Genet. 42, e13 (2005).

    Article  CAS  Google Scholar 

  38. North, P.E., Waner, M., Buckmiller, L., James, C.A. & Mihm, M.C. Jr. Vascular tumors of infancy and childhood: beyond capillary hemangioma. Cardiovasc. Pathol. 15, 303–317 (2006).

    Article  Google Scholar 

  39. Ritter, M.R., Reinisch, J., Friedlander, S.F. & Friedlander, M. Myeloid cells in infantile hemangioma. Am. J. Pathol. 168, 621–628 (2006).

    Article  Google Scholar 

  40. Takahashi, K. et al. Cellular markers that distinguish the phases of hemangioma during infancy and childhood. J. Clin. Invest. 93, 2357–2364 (1994).

    Article  CAS  Google Scholar 

  41. Razon, M.J., Kräling, B.M., Mulliken, J.B. & Bischoff, J. Increased apoptosis coincides with onset of involution in infantile hemangioma. Microcirculation 5, 189–195 (1998).

    Article  CAS  Google Scholar 

  42. Akuzawa, N., Kurabayashi, M., Ohyama, Y., Arai, M. & Nagai, R. Zinc finger transcription factor Egr-1 activates Flt-1 gene expression in THP-1 cells on induction for macrophage differentiation. Arterioscler. Thromb. Vasc. Biol. 20, 377–384 (2000).

    Article  CAS  Google Scholar 

  43. Fragoso, R. et al. VEGFR-1 (FLT-1) activation modulates acute lymphoblastic leukemia localization and survival within the bone marrow, determining the onset of extramedullary disease. Blood 107, 1608–1616 (2006).

    Article  CAS  Google Scholar 

  44. Claesson-Welsh, L. Signal transduction by vascular endothelial growth factor receptors. Biochem. Soc. Trans. 31, 20–24 (2003).

    Article  CAS  Google Scholar 

  45. Liu, S. & Leppla, S.H. Cell surface tumor endothelium marker 8 cytoplasmic tail-independent anthrax toxin binding, proteolytic processing, oligomer formation and internalization. J. Biol. Chem. 278, 5227–5234 (2003).

    Article  CAS  Google Scholar 

  46. Kitamura, T. et al. Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics. Exp. Hematol. 31, 1007–1014 (2003).

    Article  CAS  Google Scholar 

  47. Khan, Z.A. et al. Endothelial progenitor cells from infantile hemangioma and umbilical cord blood display unique cellular responses to endostatin. Blood 108, 915–921 (2006).

    Article  CAS  Google Scholar 

  48. Picard, A. et al. IFG-2 and FLT-1/VEGF-R1 mRNA levels reveal distinctions and similarities between congenital and common infantile hemangioma. Pediatr. Res. 63, 263–267 (2008).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the John B. Mulliken Foundation and grants AR36820 and AR48564 from the US National Institutes of Health (to B.R.O.). We are indebted to J.B. Mulliken (Children's Hospital, Boston) and L. Boon (Universite Catholique de Louvain) for providing essential surgical material (control and hemangioma tissues and blood samples) for these studies. We thank N.A. Clipstone (Feinberg School of Medicine, Northwestern University) for providing the constitutively active form of NFATc1, M. Kurabayashi (Gunma University Graduate School of Medicine) for providing the promoter construct for FLT1, S. Dias (Instituto Portugues de Oncologia Francisco Gentil) for providing expression vectors for FLT1, L. Claesson-Welsh (Uppsala University) for providing expression vectors for KDR and mutant FLT1, S. Liu (US National Institute of Allergy and Infectious Diseases) for providing expression vector for ANTXR1 and T. Kitamura (University of Tokyo,) for the pMXs vector. We thank R. Ruijtenbeek and R. Houtman (both from PamGene Corporation) for providing reagents and equipment for kinase arrays, F. Naji and M. Dankers for kinase array bioinformatics assistance, T. Rector for assistance with protein multiplexing, J. Eastcott and J. Wylie-Sears for flow cytometry and technical assistance, S. Feske (New York University School of Medicine) for antibody to NFATc2 (clone 67.1) and advice and W. Kuo for technical advice. We thank Y. Pittel, S. Plotkina, N. Liu, A. Heilmann, Y. Ishida and Y. Yamamura for technical assistance and D. Glotzer for comments and advice on the manuscript.

Author information

Authors and Affiliations

  1. Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Masatoshi Jinnin, Damian Medici, Lucy Park, Yanqiu Liu, Eileen Boye & Bjorn R Olsen

  2. Human Molecular Genetics, de Duve Institute, Avenue Hippocrate 74, Universite Catholique de Louvain, Brussels, B-1200, Belgium

    Nisha Limaye & Miikka Vikkula

  3. Department of Surgery, Vascular Biology Program, Children's Hospital Boston, 300 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Elisa Boscolo & Joyce Bischoff

Authors
  1. Masatoshi Jinnin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Damian Medici
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lucy Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nisha Limaye
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yanqiu Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Elisa Boscolo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Joyce Bischoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Miikka Vikkula
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Eileen Boye
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Bjorn R Olsen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.J., cell signaling studies, immunoblotting and immunoprecipitation analyses, transcription and promoter studies, integrin studies; D.M., multiplex ELISA and kinase assays, proliferation and migration analyses; L.P., sequencing of candidate genes; N.L., allele-specific DNA sequencing; Y.L., initial signaling studies; E. Boscolo, hemangioma tissue characterization; J.B., isolation of hemEC lines, control cells and tissues; M.V., collection of control and hemangioma tissues and DNAs; E. Boye, planning, NFAT and Ca2+ studies; B.R.O., planning and directing the study, manuscript writing.

Note: Supplementary information is available on the Nature Medicine website.

Corresponding authors

Correspondence to Eileen Boye or Bjorn R Olsen.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–5, Supplementary Tables 1–3 and Supplementary Methods (PDF 833 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jinnin, M., Medici, D., Park, L. et al. Suppressed NFAT-dependent VEGFR1 expression and constitutive VEGFR2 signaling in infantile hemangioma. Nat Med 14, 1236–1246 (2008). https://doi.org/10.1038/nm.1877

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.1877

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing