The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model

M. Lourdes Ponce1, Hynda K. Kleinmann1

1 National Institutes of Health Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.5
DOI:  10.1002/0471143030.cb1905s18
Online Posting Date:  May, 2003
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The chick chorioallantoic membrane (CAM) assay for angiogenic activity is a model originally developed to study the angiogenic activity of tumor samples. It is an in vivo assay that can be readily performed in any laboratory setting. The effects of a test compound on angiogenesis are tested by exposing day 10 embryos to the compound and following the patterns of blood vessel development, scoring the appearance of the CAM at day 12 or 13.

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • 3‐day‐old embryonated chicken eggs (∼20 eggs/condition), maintained in 37°C incubator (an egg incubator is not necessary)
  • 70% ethanol
  • Test substance(s)
  • Positive control: bFGF (PeproTech) or another angiogenic stimulus
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Heavy cream
  • Sterile dissecting implements:
  •  Scalpel
  •  Curved tweezers
  •  Smooth‐tipped (nonserrated) tweezers
  •  Scissors
  • 6‐ml syringes, sterile
  • 20‐G, 1.5‐in. (3.5‐cm) needles, sterile
  • 1‐in. (2.5‐cm) wide clear adhesive tape
  • Single‐edged razor blades
  • 13‐mm‐diameter Thermanox coverslips (Nunc), sterile
  • Stereoscopic microscope of variable magnification, equipped with camera and light source
  • NOTE: As an additional, optional sterility precaution, this protocol may be performed in a sterile laminar‐flow hood (also see unit 1.3).
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Literature Cited

Literature Cited
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  Auerbach, W. and Auerbach, R. 1994. ANgiogenesis inhibition: A review. Pharmacol. Ther. 63: 265‐311.
  Auerbach, R., Kubai, L., Knighton, D., and Folkman, J. 1974. A simple procedure for the long‐term cultivation of chicken embryos. Dev. Biol. 41: 1391‐394.
  Ausprunk, D.H., Knighton, D.R., and Folkman, J. 1974. Differentiation of vascular endothelium in the chick chorioallantois: A structural and autoradiographic study. Dev. Biol. 38: 237‐248.
  Brenchley, P.E. 2001. Antagonising angiogenesis in rheumatoid arthritis. Ann. Rheum. Dis. 60 Suppl 3: iii71‐74.
  Campochiaro, P.A. 2000. Retinal and choroidal neovascularization. J. Cell Physiol. 184:301‐310.
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  Gho, Y.S., Kim, P.N., Li, H.C., Elkin, M., and Kleinman, H.K. 2001. Stimulation of tumor growth by human soluble intercellular adhesion molecule‐1. Cancer Res. 61:4253‐4257.
  Majesky, M.W. 1996. A little VEGF goes a long way: Therapeutic angiogenesis by direct injection of vascular endothelial growth factor‐encoding plasmid DNA. Circulation 94:3062‐3064.
  Ponce, M.L., Nomizu, M., and Kleinman, H.K. 2001. An angiogenic laminin site and its antagonist bind through the alpha(v)beta3 and alpha5beta1 integrins. FASEB J. 15:1389‐1397.
  Reynolds, L.P. and Redmer, D.A. 2001. Angiogenesis in the placenta. Biol. Reprod. 64:1033‐1040.
  Ribatti, D., Nico, B., Vacca, A., Roncali, L., Burri, P.H., and Djonov, V. 2001. Chorioallantoic membrane capillary bed: A useful target for studying angiogenesis and anti‐angiogenesis in vivo. Anat. Rec. 264:317‐324.
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  Vale, P.R., Isner, J.M., and Rosenfield, K. 2001. Therapeutic angiogenesis in critical limb and myocardial ischemia. J. Interv. Cardiol. 14:511‐528.
  Yoshida, A., Yoshida, S., Ishibashi, T., and Inomata, H. 1999. Intraocular neovascularization. Histol. Histopathol. 14:1287‐1294.
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