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

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Abstract

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.

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References

  • Azizi ZA, Zamani A, Omrani LR, Omrani L, Dabaghmanesh MH, Mohammadi A, et al. Effects of hyperhomocysteinemia during the gestational period on ossification in rat embryo. Bone. 2009;46:1344–8.

    Article  CAS  PubMed  Google Scholar 

  • Ben Shoham A, Rot C, Stern T, Krief S, Akiva A, Dadosh T, et al. Deposition of collagen type I onto skeletal endothelium reveals a new role for blood vessels in regulating bone morphology. Dev. 2016;143:3933–43.

    Article  CAS  Google Scholar 

  • Bourckhardt GF, Cecchini MS, Ammar D, Kobus-Bianchini K, Müller YMR, Nazari EM. Effects of homocysteine on mesenchymal cell proliferation and differentiation during chondrogenesis on limb development. J Appl Toxicol. 2015;35:1390–7.

    Article  CAS  PubMed  Google Scholar 

  • Brauer PR, Tierney BJ. Consequences of elevated homocysteine during embryonic development and possible modes of action. Curr Pharm Des. 2004;10:2719–32.

    Article  CAS  PubMed  Google Scholar 

  • Chim SM, Tickner J, Chow ST, Kuek V, Guo B, Zhang G, et al. Angiogenic factors in bone local environment. Cytokine and Growth Factor Rev. 2013;24:297–310.

    Article  CAS  Google Scholar 

  • Ganguly P, Alam SF. Role of homocysteine in the development of cardiovascular disease. Nutr J. 2015;14

  • Hall BK, Miyake T. All for one and one for all: condensations and the initiation of skeletal development. BioEssays. 2000;22:138–47.

    Article  CAS  PubMed  Google Scholar 

  • Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. Dev Dyn. 1951;195:231–72.

    Article  Google Scholar 

  • Hankenson KD, Dishowitz M, Gray C, Schenker M. Angiogenesis in bone regeneration. Injury. 2011;42:556–61.

    Article  PubMed  PubMed Central  Google Scholar 

  • Hannibal L, Blom HJ. Homocysteine and disease: causal associations or epiphenomenons? Mol Asp Med. 2017;53:36–42.

    Article  CAS  Google Scholar 

  • Herrmann M, Umanskaya N, Wildemann B, Colaianni G, Widmann T, Zallone A, et al. Stimulation of osteoblast activity by homocysteine. J Cell Mol Med. 2008;12:1205–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hinchliffe JR. Developmental basis of limb evolution. Int J Dev Biol. 2002;46:835–45.

    CAS  PubMed  Google Scholar 

  • Hofmann MA, Lalla E, Lu Y, Gleason MR, Wolf BM, Tanji N, et al. Hyperhomocysteinemia enhances vascular inflammation and accelerates atherosclerosis in a murine model. J Clin Invest. 2001;107:675–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hohsfield LA, Humpel C. Homocysteine enhances transmigration of rat monocytes through a brain capillary endothelial cell monolayer via ICAM-1. Curr Neurovasc Res. 2010;7:192–200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hou HH, Hammock BD, Su KH, Morisseau C, Kou YR, Imaoka S, et al. N-terminal domain of soluble epoxide hydrolase negatively regulates the VEGF-mediated activation of endothelial nitric oxide synthase. Cardiovasc Res. 2012;93:120–9.

    Article  CAS  PubMed  Google Scholar 

  • Huhta JC, Hernandez-Robles JA. Homocysteine, folate, and congenital heart defects. Fetal Pediatr Pathol. 2005;24:71–9.

    Article  CAS  PubMed  Google Scholar 

  • Iademarco MF, Barks JL, Dean DC. Regulation of vascular cell adhesion molecule-1 expression by IL-4 and TNF-alpha in cultured endothelial cells. J Clin Invest. 1995;95:264–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kain KH, Miller JW, Jones-Paris CR, Thomason RT, Lewis JD, Bader DM, et al. The chick embryo as an expanding experimental model for cancer and cardiovascular research. Dev Dyn. 2014;243:216–28.

    Article  PubMed  Google Scholar 

  • Karaöz E, Okçu A, Gacar G, Sağlam Ö, Yürüker S, Kenar H. A comprehensive characterization study of human bone marrow mscs with an emphasis on molecular and ultrastructural properties. J Cell Physiol. 2011;226:1367–82.

    Article  CAS  PubMed  Google Scholar 

  • Kobus K, Ammar D, Nazari EM, Rauh Muller YM. Homocysteine causes disruptions in spinal cord morphology and changes the expression of Pax 1/9 and Sox 9 gene products in the axial mesenchyme. Birth Defects Res A Clin Mol Teratol. 2013;97:386–97.

    Article  CAS  PubMed  Google Scholar 

  • Kular J, Tickner J, Chim SM, Xu J. An overview of the regulation of bone remodelling at the cellular level. Clin Biochem. 2012;45:863–73.

    Article  CAS  PubMed  Google Scholar 

  • Latacha KS, Rosenquist TH. Homocysteine inhibits extra-embryonic vascular development in the avian embryo. Dev Dyn. 2005;234:323–31.

    Article  CAS  PubMed  Google Scholar 

  • Levasseur R. Bone tissue and hyperhomocysteinemia. Joint Bone Spine. 2009;76:234–40.

    Article  CAS  PubMed  Google Scholar 

  • Lizio M, Deviatiiarov R, Nagai H, Galan L, Arner E, Itoh M, et al. Systematic analysis of transcription start sites in avian development. PLoS Biol. 2017;15(9):e2002887.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–75.

    CAS  PubMed  Google Scholar 

  • Ma Y, Peng D, Liu C, Huang C, Luo J. Serum high concentrations of homocysteine and low levels of folic acid and vitamin B(12) are significantly correlated with the categories of coronary artery diseases. BMC Cardiovasc Disord. 2017;17:37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mackie EJ, Tatarczuch L, Mirams M. The skeleton: a multi-functional complex organ. The growth plate chondrocyte and endochondral ossification. J Endocrinol. 2011;211:109–21.

    Article  CAS  PubMed  Google Scholar 

  • Maes C, Carmeliet P, Moermans K, Stockmans I, Smets N, Collen D, et al. Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Mech Dev. 2002;111:61–73.

    Article  CAS  PubMed  Google Scholar 

  • Oosterbaan AM, Steegers EA, Ursem NT. The effects of homocysteine and folic acid on angiogenesis and VEGF expression during chicken vascular development. Microvasc Res. 2012;83:98–104.

    Article  CAS  PubMed  Google Scholar 

  • Rosenquist TH, Ratashak SA, Selhub J. Homocysteine induces congenital defects of the heart and neural tube: effect of folic acid. Proc Natl Acad Sci U S A. 1996;93:15227–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sakamoto W, Isomura H, Fujie K, Deyama Y, Kato A, Nishihira J, et al. Homocysteine attenuates the expression of osteocalcin but enhances osteopontin in MC3T3-E1 preosteoblastic cells. Biochim Biophys Acta. 2005;1740:12–6.

    Article  CAS  PubMed  Google Scholar 

  • Salazar VS, Gamer LW, Rosen V. BMP signalling in skeletal development, disease and repair. Nat Rev Endocrinol. 2016;12:203–21.

    Article  CAS  PubMed  Google Scholar 

  • Schoenwolf GC. 28—The avian embryo: a model for descriptive and experimental embryology A2 - Moody, Sally A. Cell lineage and fate determination. San Diego: Academic Press; 1999. p. 429–36.

    Google Scholar 

  • van Mil NH, Oosterbaan AM, Steegers-Theunissen RP. Teratogenicity and underlying mechanisms of homocysteine in animal models: a review. Reprod Toxicol. 2010;30:520–31.

    Article  CAS  PubMed  Google Scholar 

  • Zelzer E, Glotzer DJ, Hartmann C, Thomas D, Fukai N, Soker S, et al. Tissue specific regulation of VEGF expression during bone development requires Cbfa1/Runx2. Mech Dev. 2001;106:97–106.

    Article  CAS  PubMed  Google Scholar 

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Funding

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.

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Authors and Affiliations

  1. Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, UFSC, Campus Universitário, Trindade, Florianópolis, SC, 88040-900, Brazil

    Gilian Fernando Bourckhardt, Manuela Sozo Cecchini, Maria Luísa da Silveira Hahmeyer, Yara Maria Rauh Müller & Evelise Maria Nazari

  2. Departamento de Bioquímica, Centro de Ciências Biológicas, UFSC, Campus Universitário, Trindade, Florianópolis, SC, 88040-900, Brazil

    Aline Pertile Remor & Alexandra Latini

  3. Centro Universitário Católica de Santa Catarina, Joinville, SC, 89203-005, Brazil

    Dib Ammar

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  1. Gilian Fernando Bourckhardt
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  2. Manuela Sozo Cecchini
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  3. Maria Luísa da Silveira Hahmeyer
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  4. Aline Pertile Remor
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  5. Alexandra Latini
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  6. Dib Ammar
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  7. Yara Maria Rauh Müller
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  8. Evelise Maria Nazari
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Corresponding author

Correspondence to Evelise Maria Nazari.

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

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Bourckhardt, G.F., Cecchini, M.S., da Silveira Hahmeyer, M.L. et al. Impact of homocysteine on vasculogenic factors and bone formation in chicken embryos. Cell Biol Toxicol 35, 49–58 (2019). https://doi.org/10.1007/s10565-018-9436-y

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  • DOI: https://doi.org/10.1007/s10565-018-9436-y

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