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In vitro angiogenesis by human umbilical vein endothelial cells (HUVEC) induced by three-dimensional co-culture with glioblastoma cells

  • Laboratory investigation - human/animal tissue
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Abstract

Glioblastoma multiforme (GBM) is one of the most highly vascularized of all human tumors. Our objective was to characterize a 3-dimensional (3-D) in vitro angiogenesis model by co-culturing HUVEC and GBM cells, and to study the role of VEGF in mediating capillary tubule formation in this model. HUVEC-coated dextran beads were suspended in fibrin gel with human glioma cells on top. The number of sprouts and the length of the processes were measured. HUVEC can be induced to form sprouts and longer processes with lumens, in co-culture with glioma cells that secrete VEGF. Addition of exogenous VEGF enhances this effect. In the absence of glioma cells, many single HUVEC migrate away from the beads, without significant tubule formation. Hypoxia further stimulated sprout formation by 50–100%. Anti-VEGF neutralizing antibody suppressed HUVEC sprouting by 75% in co-culture with glioma cells. This 3-D in vitro co-culture system provides a robust and useful model for analysis of the major steps of glioma-induced angiogenesis.

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References

  1. Plate KH, Risau W (1995) Angiogenesis in malignant glioma. Glia 15:339–347. doi:10.1002/glia.440150313

    Article  PubMed  CAS  Google Scholar 

  2. Zadeh G, Guha A (2003) Angiogenesis in nervous system disorders. Neurosurgery 53:1362–1376. doi:10.1227/01.NEU.0000093425.98136.31

    Article  PubMed  Google Scholar 

  3. Harrigan M (2003) Angiogenic factors in the central nervous system. Neurosurgery 53:639–661. doi:10.1227/01.NEU.0000079575.09923.59

    Article  PubMed  Google Scholar 

  4. Abbott A (2003) Cell culture: biology’s new dimension. Nature 424:870–872. doi:10.1038/424870a

    Article  PubMed  CAS  Google Scholar 

  5. Velazquez OC, Snyder R, Liu ZJ et al (2002) Fibroblast-dependent differentiation of human microvascular endothelial cells into capillary-like 3-dimensional networks. FASEB J 16:1316–1318

    PubMed  CAS  Google Scholar 

  6. Folkman J (2007) Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov 6:273–286. doi:10.1038/nrd2115

    Article  PubMed  CAS  Google Scholar 

  7. Oka N, Soeda A, Inagaki A et al (2007) VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells. Biochem Biophys Res Commun 360:553–559. doi:10.1016/j.bbrc.2007.06.094

    Article  PubMed  CAS  Google Scholar 

  8. Nakatsu MN, Sainson RC, Aoto JN et al (2003) Angiogenic sprouting and capillary lumen formation modeled by human umbilical vein endothelial cells (HUVEC) in fibrin gels: the role of fibroblasts and Angiopoietin–1. Microvasc Res 66:102–112. doi:10.1016/S0026-2862(03)00045-1

    Article  PubMed  CAS  Google Scholar 

  9. Juillerat-Jeanneret L, Monnet-Tschudi F, Zurich MG et al (2003) Regulation of peptidase activity in a three-dimensional aggregate model of brain tumor vasculature. Cell Tissue Res 311:53–59. doi:10.1007/s00441-002-0626-8

    Article  PubMed  CAS  Google Scholar 

  10. Wenger A, Kowalewski N, Stahl A et al (2005) Development and characterization of a spheroidal coculture model of endothelial cells and fibroblasts for improving angiogenesis in tissue engineering. Cells Tissues Organs 181:80–88. doi:10.1159/000091097

    Article  PubMed  Google Scholar 

  11. Hwa AJ, Fry RC, Sivaraman A et al (2007) Rat liver sinusoidal endothelial cells survive without exogenous VEGF in 3D perfused co-cultures with hepatocytes. FASEB J 21:2564–2579. doi:10.1096/fj.06-7473com

    Article  PubMed  CAS  Google Scholar 

  12. Horning JL, Sahoo SK, Vijayaraghavalu S et al (2008) 3-D tumor model for in vitro evaluation of anticancer drugs. Mol Pharm (Epub ahead of print)

  13. Nehls V, Schuchardt E, Drenckhahn D (1994) The effect of fibroblasts, vascular smooth muscle cells, and pericytes on sprout formation of endothelial cells in a fibrin gel angiogenesis system. Microvasc Res 48:349–363. doi:10.1006/mvre.1994.1061

    Article  PubMed  CAS  Google Scholar 

  14. McLaughlin N, Annabi B, Sik Kim K (2006) The response to brain tumor-derived growth factors is altered in radioresistant human brain endothelial cells. Cancer Biol Ther 5:1539–1545. doi:10.1158/1535-7163.MCT-06-0065

    Article  PubMed  CAS  Google Scholar 

  15. Nehls V, Drenckhahn D (1995) A novel, microcarrier-based in vitro assay for rapid and reliable quantification of three-dimensional cell migration and angiogenesis. Microvasc Res 50:311–322. doi:10.1006/mvre.1995.1061

    Article  PubMed  CAS  Google Scholar 

  16. Nehls V, Drenckhahn D (1995) A microcarrier-based cocultivation system for the investigation of factors and cells involved in angiogenesis in three-dimensional fibrin matrices in vitro. Histochem Cell Biol 104:459–466. doi:10.1007/BF01464336

    Article  PubMed  CAS  Google Scholar 

  17. Eyrich D, Brandl F, Appel B et al (2007) Long-term stable fibrin gels for cartilage engineering. Biomaterials 28:55–65. doi:10.1016/j.biomaterials.2006.08.027

    Article  PubMed  CAS  Google Scholar 

  18. Cao YJ, Shibata T, Rainov NG et al (2001) Hypoxia-inducible transgene expression in differentiated human NT2N neurons—a cell culture model for gene therapy of postischemic neuronal loss. Gene Ther 8:1357–1362. doi:10.1038/sj.gt.3301536

    Article  PubMed  CAS  Google Scholar 

  19. Khodarev NN, Yu J, Labay E et al (2003) Tumour-endothelium interactions in co-culture: coordinated changes of gene expression profiles and phenotypic properties of endothelial cells. J Cell Sci 116:1013–1022. doi:10.1242/jcs.00281

    Article  PubMed  CAS  Google Scholar 

  20. Brown CK, Khodarev NN, Yu J et al (2004) Glioblastoma cells block radiation-induced programmed cell death of endothelial cells. FEBS Lett 565:167–170. doi:10.1016/j.febslet.2004.03.099

    Article  PubMed  CAS  Google Scholar 

  21. Nakatsu MN, Sainson RC, Perez-del-Pulgar S et al (2003) VEGF(121) and VEGF(165) regulate blood vessel diameter through vascular endothelial growth factor receptor 2 in an in vitro angiogenesis model. Lab Invest 83:1873–1885. doi:10.1097/01.LAB.0000107160.81875.33

    Article  PubMed  CAS  Google Scholar 

  22. Sainson RC, Aoto J, Nakatsu MN et al (2005) Cell-autonomous notch signaling regulates endothelial cell branching and proliferation during vascular tubulogenesis. FASEB J 19:1027–1029

    PubMed  CAS  Google Scholar 

  23. Raza SM, Lang FF, Aggarwal BB et al (2002) Necrosis and glioblastoma: a friend or a foe? A review and a hypothesis. Neurosurg 51:2–12. doi:10.1097/00006123-200207000-00002

    Article  Google Scholar 

  24. Lund EL, Hog A, Olsen MW (2004) Differential regulation of VEGF, HIF1alpha and angiopoietin-1, -2 and -4 by hypoxia and ionizing radiation in human glioblastoma. Int J Cancer 108:833–838. doi:10.1002/ijc.11662

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The work at Virginia Commonwealth University was supported in part by the Hord Fund of the Medical College of Virginia Foundation. The work at the University of Virginia was supported in part by the Kopf Family Foundation, Inc.

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Authors and Affiliations

  1. Department of Neurosurgery, Medical College of Virginia Hospitals, Ambulatory Care Center, Virginia Commonwealth University, 417 North 11th Street, 6th Floor, Box 980631, Richmond, VA, 23298-0631, USA

    Zhijian Chen, Andre Htay, Wagner Dos Santos, George T. Gillies, Helen L. Fillmore & William C. Broaddus

  2. School of Engineering and Applied Science, University of Virginia, Box 400746, Charlottesville, VA, 22904, USA

    George T. Gillies

  3. Department of Anatomy and Neurobiology, Virginia Commonwealth University, Box 980709, Richmond, VA, 23298, USA

    Helen L. Fillmore & Milton M. Sholley

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  1. Zhijian Chen
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  2. Andre Htay
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  3. Wagner Dos Santos
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  4. George T. Gillies
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  7. William C. Broaddus
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Correspondence to William C. Broaddus.

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Chen, Z., Htay, A., Santos, W.D. et al. In vitro angiogenesis by human umbilical vein endothelial cells (HUVEC) induced by three-dimensional co-culture with glioblastoma cells. J Neurooncol 92, 121–128 (2009). https://doi.org/10.1007/s11060-008-9742-y

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  • DOI: https://doi.org/10.1007/s11060-008-9742-y

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