Cell Biology and Toxicology

, Volume 35, Issue 1, pp 49–58 | Cite as

Impact of homocysteine on vasculogenic factors and bone formation in chicken embryos

  • Gilian Fernando Bourckhardt
  • Manuela Sozo Cecchini
  • Maria Luísa da Silveira Hahmeyer
  • Aline Pertile Remor
  • Alexandra Latini
  • Dib Ammar
  • Yara Maria Rauh Müller
  • Evelise Maria NazariEmail author
Original Article


Developmental endochondral ossification requires constant blood supply, which is provided by the embryonic vascular network. High levels of homocysteine (Hcy) have vasculotoxic properties, but it remains unclear how Hcy disrupts blood vessel formation in endochondral ossification. Thus, we investigated the toxicity of Hcy on contents of vasculogenic factors (VEGF, VCAM-1, NOS3) and osteocalcin, using developing limbs as model. Chicken embryos were submitted to treatment with 20 μmol D-L Hcy at 12H&H and the analyses occur at 29H&H and 36H&H. We did not identify differences in the area of limb ossification in Hcy-treated (7.5 × 105 μm2 ± 3.9 × 104) and untreated embryos (7.6 × 105 μm2 ± 3.3 × 104) at 36H&H. In Hcy-treated embryos, we observed a significantly decrease of 46.8% at 29H&H and 26.0% at 36H&H in the number of VEGF-reactive cells. Also, treated embryos showed decrease of 98.7% in VCAM-1-reactive cells at 29H&H and 34.6% at 36H&H. The number of NOS3-reactive cells was reduced 54.0% at 29H&H and 91.5% at 36H&H, in the limbs of Hcy-treated embryos. Finally, in Hcy-treated embryos at 36H&H, we observed a reduction of 58.86% in the number of osteocalcin-reactive cells. Here, we demonstrated for the first time that the toxicity of Hcy is associated with a reduction in the contents of proteins involved in blood vessel formation and bone mineralization, which interferes with endochondral ossification of the limb during embryonic development.

Graphical abstract


Hyperhomocysteinemia VEGF/VCAM-1/NOS3 proteins Osteocalcin protein Endothelial cells Osteoblasts Chicken embryo 



This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil, and Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC), Brazil.

Compliance with ethical standards

All experiments were performed according to the Ethics Committee of Federal University of Santa Catarina, Brazil (175/CEUA/PROPESQ/2014).


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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Gilian Fernando Bourckhardt
    • 1
  • Manuela Sozo Cecchini
    • 1
  • Maria Luísa da Silveira Hahmeyer
    • 1
  • Aline Pertile Remor
    • 2
  • Alexandra Latini
    • 2
  • Dib Ammar
    • 3
  • Yara Maria Rauh Müller
    • 1
  • Evelise Maria Nazari
    • 1
    Email author
  1. 1.Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, UFSCCampus UniversitárioFlorianópolisBrazil
  2. 2.Departamento de Bioquímica, Centro de Ciências Biológicas, UFSCCampus UniversitárioFlorianópolisBrazil
  3. 3.Centro Universitário Católica de Santa CatarinaJoinvilleBrazil

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