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:

VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation

Nature Medicine volume 5, pages 623–628 (1999)Cite this article

Abstract

Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Histological sections of proximal tibia of mice treated for 2 weeks with control IgG (cont) or mFlt(1-3)IgG (mFlt).
Figure 2: The changes in the growth plate are reversible.
Figure 3: Expression of collagen 2 and 10 in the growth plate.
Figure 4: Reduction of TRAP-positive chondroclasts expressing GelB mRNA.
Figure 5: Expression of Flt-1 and Flk-1 in the growth plate.

Similar content being viewed by others

References

  1. Poole, A.R. in Cartilage: Molecular Aspects (eds. Hall, B.K. & Newman, S.A.) 179–211 (CRC Press, Boca Raton, Florida, 1991).

    Google Scholar 

  2. Jee, W.S.S. in Cell and Tissue Biology (ed. Weiss, L.) 213–253 (Urban & Schwarzemberg, New York, 1988).

    Google Scholar 

  3. Baron J. et al. Induction of growth plate cartilage ossification by basic fibroblast growth factor. Endocrinology 135, 2790– 2793 (1994).

    Article  CAS  Google Scholar 

  4. Babic A.M., Kireeva M.L., Kolesnikova T.V., & Lau L.F. CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth. Proc. Natl. Acad. Sci. USA 95, 6355–6360 (1998).

    Article  CAS  Google Scholar 

  5. Shinar, D.M., Endo, N., Halperin, D., Rodan, G.A, & Weinreb, M. Differential expression of insulin-like growth factor-I (IGF-I) and IGF-II messenger ribonucleic acid in growing rat bone. Endocrinology 132, 1158–1167 (1993).

    Article  CAS  Google Scholar 

  6. Jingushi, S, Scully, S.P., Joyce, M.E., Sugioka, Y., & Bolander, M.E. Transforming growth factor-beta 1 and fibroblast growth factors in rat growth plate. J. Orthop. Res. 13, 761–768 ( 1995).

    Article  CAS  Google Scholar 

  7. Carlevaro, M.F. et al. Transferrin promotes endothelial cell migration and invasion: implication in cartilage neovascularization J. Cell Biol. 136, 1375–1384 (1997).

    Article  CAS  Google Scholar 

  8. Ferrara, N. & Davis-Smyth, T. The biology of vascular endothelial growth factor. Endocr. Rev. 18, 4– 25 (1997).

    Article  CAS  Google Scholar 

  9. Fong, G. H., Rossant, J., Gertsenstein, M., & Breitman, M. L. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 376, 66– 70 (1995).

    Article  CAS  Google Scholar 

  10. 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 

  11. Waltenberger, J., Claesson-Welsh, L., Siegbahn, A., Shibuya, M. & Heldin, C.-H. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor J. Biol. Chem. 268, 26988– 26995 (1994).

    Google Scholar 

  12. Keyt, B.A. et al. Identification of VEGF determinants for binding Flt-1 and KDR receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis. J. Biol. Chem. 271, 5638– 5646 (1996).

    Article  CAS  Google Scholar 

  13. Gerber, H.P. et al. VEGF regulates endothelial cell survival by the PI3-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J. Biol. Chem. 273, 30336–30345 (1998).

    Article  CAS  Google Scholar 

  14. Hiratsuka, S., Minowa, O., Kuno, J., Noda, T., and Shibuya, M. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc. Natl. Acad. Sci. USA 95, 9349–9354 ( 1998).

    Article  CAS  Google Scholar 

  15. Barleon, B. et al.Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood 87 3336–3343 ( 1996).

    CAS  PubMed  Google Scholar 

  16. Soker, S., Takashima, S., Miao, H. Q., Neufeld, G., & Klagsbrun, M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 92 735–745 (1998).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Davis-Smyth, T., Chen, H., Park, J., Presta, L.G., & Ferrara, N. The second immunoglobulin-like domain of the VEGF tyrosine kinase receptot Flt-1 determines ligand binding and may initiate a signal transduction cascade. EMBO J. 15 4919– 4927 (1996).

    Article  CAS  Google Scholar 

  22. Ala-Kokko, L., & Prockop, D. J. Completion of the intron-exon structure of the gene for human type II procollagen (COL2A1): variations in the nucleotide sequences of the alleles from three chromosomes. Genomics 8, 454–460 (1990).

    Article  CAS  Google Scholar 

  23. Elima, K. et al. The mouse collagen X gene: complete nucleotide sequence, exon structure and expression pattern. Biochem. J. 289, 247–253 (1993).

    Article  CAS  Google Scholar 

  24. Farnum, C.E. & Wilsman, N.J. Condensation of hypertrophic chondrocytes at the chondro-osseous junction of growth plate cartilage in Yucatan swine: relationship to long bone growth. Am. J. Anat. 186, 346–358 (1989).

    Article  CAS  Google Scholar 

  25. Lewinson, D., & Silberman, M. Chondroclasts and endothelial cells collaborate in the process of cartilage resorption. Anat. Rec. 233, 504–514 ( 1992).

    Article  CAS  Google Scholar 

  26. Vu, T.H. et al.MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93 , 411–422 (1998).

    Article  CAS  Google Scholar 

  27. Shen, B.Q. et al. Homologous up-regulation of KDR/Flk-1 receptor expression by vascular endothelial growth factor in vitro. J. Biol. Chem. 273, 29979–29985 ( 1998).

    Article  CAS  Google Scholar 

  28. Barleon, B. et al. Vascular endothelial growth factor up-regulates its receptor fms- like tyrosine kinase 1 (FLT-1) and a soluble variant of FLT-1 in human vascular endothelial cells. Cancer Res. 57, 5421–5425 (1997).

    CAS  PubMed  Google Scholar 

  29. Alon, T. et al. Vascular endothelial growth factor acts a s survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nature Med. 1, 1024–1028 (1995).

    Article  CAS  Google Scholar 

  30. Yuan, F. et al. Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by anti-VEGF/VPF antibody Proc. Nat. Acad. Sci. USA 93, 14765– 14770 (1996).

    Article  CAS  Google Scholar 

  31. Simionescu, N. & Simionescu, M. in Endothelial Cell Dysfunctions (Plenum, New York, 1992).

    Book  Google Scholar 

  32. Midy, V. & Plouet, J. Vasculotropin/vascular endothelial growth factor induces differentiation in cultured osteoblasts. Biochem. Biophys. Res. Commun. 199 380– 386 (1994).

    Article  CAS  Google Scholar 

  33. Carmeliet, P., & Collen, D. Vascular development and disorders: molecular analysis and pathogenic insights. Kidney Internat. 53, 1519–1549 (1998).

    Article  CAS  Google Scholar 

  34. Enholm, B., Jussila, L., Karkkainen, M., & Alitalo, K. Vascular endothelial growth factor-C: A growth factor for lymphatic and blood vessel endothelial cells. Trends Cardiovasc. Med. 8 , 292–296 (1998).

    Article  CAS  Google Scholar 

  35. Ryan, A.M. et al.Preclinical safety evaluation of rhuMAbVEGF, an antiangiogenic humanized monoclonal antibody. Toxicol. Pathol. 27, 78–86 (1999).

    Article  CAS  Google Scholar 

  36. Barness, L.A. in Potter's Pathology of the Fetus and Infant (ed. Gilbert-Barness, E.) 561–563 (Mosby, St. Louis, Missouri, 1997).

    Google Scholar 

  37. Schlaeppi, J.M., Gutzwiller, S., Finkenzeller, G., & Fournier, B. 1,25- Dihydroxyvitamin D3 induces the expression of vascular endothelial growth factor in osteoblastic cells. Endocr. Res. 23, 213–229 (1997).

    Article  CAS  Google Scholar 

  38. Maroteaux, P. et al. Opsismodysplasia: a new type of chondrodysplasia with predominant involvement of the bones of the hand and the vertebrae. Am. J. Med. Genet. 19, 171–182 (1984).

    Article  CAS  Google Scholar 

  39. Shehan, D.C. & Hrapchak, B.B. in Theory and Practice of Histotechnology (Lipshaw, 1980).

    Google Scholar 

  40. Albrecht, U., Eichele, G., Helms, J. A., & Lu, H. in Molecular and Cellular Methods in Developmental Toxicology (ed. Daston, G.P.) 23–48 (CRC Press, Boca Raton, Florida, 1997).

    Google Scholar 

  41. Metsaranta, M., Toman, D., De Crombrugghe, B., & Vuorio, E. Specific hybridization probes for mouse type I, II, III and IX collagen mRNAs. Biochim. Biophys. Acta 1089, 241– 243 (1991).

    Article  CAS  Google Scholar 

  42. Reponen, P., Sahlberg, C., Munaut, C., Thesleff, I., & Tryggvason, K. High expression of 92-kD type IV collagenase (gelatinase B) in the osteoclast lineage during mouse development. J. Cell Biol. 124, 1091–1102 ( 1994).

    Article  CAS  Google Scholar 

  43. Finnerty, H. et al. Molecular cloning of murine FLT and FLT4. Oncogene 8, 2293–2298 ( 1993).

    CAS  PubMed  Google Scholar 

  44. Quinn, T.P., Peters, K.G., deVries, C., Ferrara, N. & Williams, L.T. Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium. Proc. Natl. Acad. Sci. USA 90, 7533–7537 (1993).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Latvala, J. Ross, H. Steinmetz, W. Wong and M. Van Hoy for help with animal work. We also thank D. Peers and P. Lester for purification of mFlt(1-3)-IgG, J. Burr and N. Lin for cell culture, P. Tobin and K. Hagler for histology and immunohistochemistry, M. Lew for in situ hybridization and D. Wood and L. Tamayo for graphic artwork. We are grateful to E. Filvaroff and K. Hillan for helpful discussions and advice. This work was also supported by grants from the National Institutes of Health (DE10306 to Z.W.) and a Mentored Clinician Scientist Award (HL03880 to T.H.V.).

Author information

Author notes

  1. Hans-Peter Gerber and Thiennu H. Vu: H.-P.G and T.H.V. contributed equally.

Authors and Affiliations

  1. Departments of Cardiovascular Research, Genentech 1 DNA Way, South San Francisco, 94080, California, USA

    Hans-Peter Gerber, Joe Kowalski & Napoleone Ferrara

  2. Pathology, Genentech, 1 DNA Way, South San Francisco, 94080, California, USA

    Anne M. Ryan

  3. Department of Anatomy, Box 0452, University of California Medical Center, San Francisco, 94143, California, USA

    Thiennu H. Vu & Zena Werb

Authors
  1. Hans-Peter Gerber
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thiennu H. Vu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anne M. Ryan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Joe Kowalski
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zena Werb
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Napoleone Ferrara
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Napoleone Ferrara.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gerber, HP., Vu, T., Ryan, A. et al. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med 5, 623–628 (1999). https://doi.org/10.1038/9467

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/9467

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