Skip to main content
SpringerLink
Log in

A Modified Chorioallantoic Membrane Assay Allows for Specific Detection of Endothelial Apoptosis Induced by Antiangiogenic Substances

  • Published:
Angiogenesis Aims and scope Submit manuscript

Abstract

Current in vivo angiogenesis assays allow for the assessment of vascular growth inhibition induced by a test substance, but they usually do not provide information about the mechanisms underlying such an inhibition. A potential antiangiogenic mechanism is the triggering of endothelial apoptosis in the growing vessels. Apoptogenic substances can be of interest for antiangiogenic therapy specially if they specifically perform their action on the angiogenic endothelium. We have developed a modification of the chorioallantoic membrane (CAM) assay using embryos of quail (Coturnix coturnix japonica). This novel assay allows to elucidate whether an antiangiogenic substance is specifically triggering an apoptotic response in endothelial cells. We have used a quail-specific monoclonal endothelial marker (QH1), a standard TUNEL technique of apoptotic cell labelling together with a general nuclear counterstaining with propidium iodide. Through laser confocal microscopy, paraffin sections of chorioallantoic membranes treated with test substances are stained in three colours: red for normal cell nuclei, yellow—green for apoptotic nuclei and blue for endothelial cells and endothelial progenitors. In a test experience, our assay showed significant differences in the apoptogenic properties of two antiangiogenic substances, camptothecin and aeroplysinin-1.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

References

  1. Folkman J. Angiogenesis in cancer, vascular rheumatoid and other disease. Nat Med 1995; 1: 27–31.

    Article  PubMed  CAS  Google Scholar 

  2. Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors. Nature Rev Cancer 2002; 2: 727–39.

    Article  CAS  Google Scholar 

  3. Jain RK, Schlenger K, Höckel M, Yuan F. Quantitative angiogenesis assays: Progress and problems. Nat Med 1997; 3: 1203–8.

    Article  PubMed  CAS  Google Scholar 

  4. Auerbach R, Lewis R, Shinners B et al. Angiogenesis assays: A critical overview. Clin Chem 2003; 49: 32–40.

    Article  PubMed  CAS  Google Scholar 

  5. Parsons-Wingerter P, Lwai B, Yang MC et al. A novel assay of angiogenesis in the quail chorioallantoic membrane: Stimulation by bFGF and inhibition by angiostatin according to fractal dimension and grid intersection. Microvasc Res 1998; 55: 201–14.

    Article  PubMed  CAS  Google Scholar 

  6. Oh SJ, Jeltsch MM, Birkenhager R et al. VEGF and VEGF-C: Specific induction of angiogenesis and lymphangiogenesis in the differentiated avian chorioallantoic membrane. De Biol 1997; 188: 96–109.

    Article  PubMed  CAS  Google Scholar 

  7. Pardanaud L, Altmann C, Kitos P et al. Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells. Development 1987; 100: 339–49.

    PubMed  CAS  Google Scholar 

  8. Kollmannsberger C, Mross K, Jakob A et al. Topotecan – A novel topoisomerase I inhibitor: Pharmacology and clinical experience. Oncology 1999; 56(1): 1–12.

    Article  PubMed  CAS  Google Scholar 

  9. Kaufmann SH. Cell death induced by topoisomerase-targeted drugs: More questions than answers. Biochim Biophys Acta 1998; 1400: 195–211.

    PubMed  CAS  Google Scholar 

  10. Jung LL, Zamboni WC. Cellular, pharmacokinetic, and pharmacodynamic aspects of response to camptothecins: Can we improve it? Drug Resist Update 2001; 4: 273–88.

    Article  CAS  Google Scholar 

  11. Clements MK, Jones CB, Cumming M, Daoud SS. Antiangiogenic potential of camptothecin and topotecan. Cancer Chemother Pharmacol 1999; 44: 411–6.

    Article  PubMed  CAS  Google Scholar 

  12. Xiao D, Tan W, Li M, Ding J. Antiangiogenic potential of 10-hydroxycamptothecin. Life Sci 2001; 69: 1619–28.

    Article  PubMed  CAS  Google Scholar 

  13. Nakashio A, Fujita N, Tsuruo T. Topotecan inhibits VEGF and bFGF induced vascular endothelial cell migration via downregulation of the PI3K-Akt signaling pathway. Int J Cancer 2002; 98: 36–41.

    Article  PubMed  CAS  Google Scholar 

  14. Rodríguez-Nieto S, González-Iriarte M, Carmona R et al. Antiangiogenic activity of aeroplysinin-1, a brominated compound isolated from a marine sponge. FASEB J 2002; 16: 261–3.

    PubMed  Google Scholar 

  15. Kreuter MH, Leake RE, Rinaldi F et al. Inhibition of intrinsic protein tyrosine kinase activity of EGF-receptor kinase complex from human breast cancer cells by the marine sponge metabolite (+)-aeroplysinin-1. Comp Biochem Physiol 1990; 97: 151–8.

    CAS  Google Scholar 

  16. Ribatti D, Urbinati C, Nico B et al. Endogenous basic fibroblast growth factor is implicated in the vascularization of the chick embryo chorioallantoic membrane. Biol 1995; 170: 39–49.

    CAS  Google Scholar 

  17. Kreuter MH, Bernd A, Holzmann H et al. Cytostatic activity of aeroplysinin-1 against lymphoma and epithelioma cells. Z Naturforsch [C] 1989; 44(7–8): 680–8.

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Animal Biology, University of Málaga, Málaga, Spain

    Mauricio González-Iriarte, Rita Carmona, José María Pérez-Pomares, David Macías & Ramón Muñoz-Chápuli

  2. Department of Molecular Biology and Biochemistry, University of Málaga, Málaga, Spain

    Miguel Ángel Medina & Ana R. Quesada

Authors
  1. Mauricio González-Iriarte
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rita Carmona
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. José María Pérez-Pomares
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David Macías
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Miguel Ángel Medina
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ana R. Quesada
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ramón Muñoz-Chápuli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ramón Muñoz-Chápuli.

Rights and permissions

Reprints and permissions

About this article

Cite this article

González-Iriarte, M., Carmona, R., Pérez-Pomares, J.M. et al. A Modified Chorioallantoic Membrane Assay Allows for Specific Detection of Endothelial Apoptosis Induced by Antiangiogenic Substances. Angiogenesis 6, 251–254 (2003). https://doi.org/10.1023/B:AGEN.0000021388.59617.6b

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/B:AGEN.0000021388.59617.6b

Search

Navigation