In Vitro Assay of Angiogenesis: Inhibition of Capillary Tube Formation

Sharon McGonigle1, Victor Shifrin2

1 Eisai Research Institute, Andover, Massachusetts, 2 International Consulting, Inc., Auburndale, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.12
DOI:  10.1002/0471141755.ph1212s43
Online Posting Date:  December, 2008
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The growth of new blood vessels, or angiogenesis, is a naturally occurring process in both health and disease states. An area of active research, regulation of angiogenesis, is being studied as an approach for the treatment of cancer and a range of other disorders having vascular proliferation as a component. The process of angiogenesis is very complex and occurs in multiple steps, with a major involvement of endothelial cells. Various in vivo models have been developed to assess inhibitors of angiogenesis. As these are generally technically difficult and labor intensive, with observed effects difficult to quantify, they do not lend themselves to compound screening. Rather they are used for confirmatory studies. In contrast, in vitro assays developed to model various steps in the angiogenesis process are easy to perform and lend themselves to high‐throughput analysis. Described in this unit is an in vitro assay that can be employed to investigate endothelial differentiation inhibitors through assessment of their effects on capillary tube formation by endothelial cells on Matrigel. Curr. Protoc. Pharmacol. 43:12.12.1‐12.12.7. © 2008 by John Wiley & Sons, Inc.

Keywords: angiogenesis; capillary tube formation; endothelial cells; inhibitors; Matrigel

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Table of Contents

  • Introduction
  • Basic Protocol 1: Capillary Tube Formation Assay
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Capillary Tube Formation Assay

  • Human umbilical vascular endothelial cells (HUVEC; Lonza)
  • Phosphate buffered saline (PBS)
  • 0.05%/0.53 mM trypsin/EDTA in HBSS
  • EGM‐2 complete medium (EBM basal with growth factors, cytokines, supplements; Lonza)
  • Matrigel, phenol red–free (BD Biosciences)
  • Angiogenesis inhibitors in DMSO or other suitable diluent
  • Green Cell Tracker (CMFDA, Molecular Probes cat. no. C‐2925)
  • 75‐ and 150‐cm2 tissue culture flasks
  • 37°C incubator
  • 15‐ml centrifuge tubes
  • Hemacytometer
  • 24‐well tissue culture plates (BD Falcon cat. no. 353047), 4°C
  • 1‐ml pipet tips, 4°C
  • 37°C, 5% CO 2 humidified incubator
  • 5‐ml pipets
  • Phase contrast microscope
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Literature Cited

Literature Cited
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   Auerbach, R., Lewis, R., Shinners, B., Kubai, L., and Akhtar, N. 2003. Angiogenesis assays: A critical overview. Clin. Chem. 49:32‐40.
   Carpenter, A.E., Jones, T.R., Lamprecht, M.R., Clarker, C., Kang, I.H., Friman, O., Guertin, D.A., Chang, J.H., Lindquist, R.A., Moffat, J., Golland, P., and Sabatini, D.M. 2006. CellProfiler: Image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7:R100.
   Folkman, J. 1971. Tumor angiogenesis: Therapeutic implications. N. Engl. J. Med. 285:1182‐1186.
   Folkman, J. 1985. Toward an understanding of angiogenesis: Search and discovery. Perspect. Biol. Med. 29:10‐36.
   Folkman, J. 1995. Seminars in medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N. Engl. J. Med. 333:1757‐1763.
   Gastl, G., Hermann, T., Steurer, M., Zmija, J., Gunsilius, E., Unger, C., and Kraft, A. 1997. Angiogenesis as a target for tumor treatment. Oncology 54:177‐184.
   Hanahan, D. and Folkman, J. 1996. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86:353‐364.
   Kubota, Y., Kleinman, H.K., Martin, G.R., and Lawley, T.J. 1988. Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary‐like structures. J. Cell Biol. 107:1589‐1598.
   Staton, C.A., Stribbling, S.M., Tazzyman, S., Hughes, R., Brown, N.J., and Lewis, C.E. 2004. Current methods for assaying angiogenesis in vitro and in vivo. Int. J. Exp. Path. 85:233‐248.
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