Skip to main content
SpringerLink
Log in

Improved quantification of angiogenesis in the rat aortic ring assay

  • Published:
Angiogenesis Aims and scope Submit manuscript

Abstract

In vitro angiogenesis assays are essential for the identification of potential angiogenic agents and screening for pharmacological inhibitors. Among these assays, the rat aortic ring model developed by Nicosia bridges the gap between in vivo and in vitro models. The quantification of angiogenesis on this system must be applicable to characterise vascular networks of various states of complexity. We present here an improved computer-assisted image analysis which allows: (1) the determination of the aortic ring area and its factor shape; (2) the number of microvessels, the total number of branchings, the maximal microvessel length and the microvessel distribution; (3) the total number of isolated fibroblast-like cells and their distribution. We show that this method is suitable to quantify spontaneous angiogenesis as well as to analyse a complex microvascular network induced by various concentrations of vascular endothelial growth factor (VEGF). In addition, by evaluating a new parameter, the fibroblast-like cell distribution, our results show that: (1) during spontaneous angiogenic response, maximal fibroblast-like cell migration delimits microvascular outgrowth; and (2) the known angiogenic inhibitor Batimastat prevents endothelial cell sprouting without completely blocking fibroblast-like cell migration. Finally, this new method of quantification is of great interest to better understand angiogenesis and to test pro- or anti-angiogenic agents.

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

Similar content being viewed by others

References

  1. Folkman J, Brem H. Angiogenesis and inflammation. In Gallin JI, Goldstein IM, Snyderman RS (eds): Inflammation: Basic Principles and Clinical Correlates. New York: Raven Press 1996; 809–39.

    Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  3. Folkman J, Klagsbrun M. Angiogenic factors. Science 1987; 235: 442–7.

    PubMed  CAS  Google Scholar 

  4. Auerbach R, Auerbach W, Polakowski I. Assays of angiogenesis: A review. Pharmacol Ther 1991; 51: 1–11.

    Article  PubMed  Google Scholar 

  5. Nicosia RF, Ottinetti A. Growth of microvessels in serum-free matrix culture of rat aorta: A quantitative assay of angiogenesis in vitro. Lab Invest 1990; 63: 115–22.

    PubMed  CAS  Google Scholar 

  6. Kobayashi S, Fukuta M, Kontani H et al. A quantitative assay for angiogenesis of cultured choroidal tissues in streptozotocin-diabetic wistar and spontaneously diabetic GK rats. Jpn J Pharmacol 1998; 78: 471–8.

    Article  PubMed  CAS  Google Scholar 

  7. Brown KJ, Maynes SF, Bezos A et al. A novel in vitro assay for human angiogenesis. Lab Invest 1996; 75: 539–55.

    PubMed  CAS  Google Scholar 

  8. Bocci G, Danesi R, Benelli U et al. Inhibitory effect of suramin in rat models of angiogenesis in vitro and in vivo. Cancer Chemother Pharmacol 1999; 43: 205–12.

    Article  PubMed  CAS  Google Scholar 

  9. Kruger EA, Duray PH, Tkosos MG et al. Endostatic inhibits microvessel formation in the ex vivo rat aortic ring angiogenesis assay. Biochem Biophys Res Commun 2000; 268: 183–91.

    Article  PubMed  CAS  Google Scholar 

  10. Nissanov J, Tuman RW, Gruver LM, Fortunato JM. Automatic vessel segmentation and quantification of the rat aortic ring assay of angiogenesis. Lab Invest 1995; 73: 734–9.

    PubMed  CAS  Google Scholar 

  11. Montesano R, Orci L, Vassali P. In vitro rapid organisation of endothelial cells into capillary-like networks is promoted by collagen matrices. J Cell Biol 1983; 97: 1648–52.

    Article  PubMed  CAS  Google Scholar 

  12. Serra J. Image Analysis and Mathematical Morphology, Vol 1. New York: Academic Press 1982.

    Google Scholar 

  13. Voyta JC, Via DP, Butterfield CE, Zetter BR. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol 1984; 99: 2034–40.

    Article  PubMed  CAS  Google Scholar 

  14. Himmelblau DM. Process Analysis by Statistical Methods. New York: Wiley 1970.

    Google Scholar 

  15. Belotti D, Paganoni P, Giavazzi R. MMP inhibitors: Experimental and clinical studies. Int J Biol Markers 1999; 14: 232–8.

    PubMed  CAS  Google Scholar 

  16. Moses MA. The regulation of neovascularization by matrix metalloproteinases and their inhibitors. Stem Cells 1997; 15: 180–9.

    Article  PubMed  CAS  Google Scholar 

  17. Botos I, Scappozza L, Zhang D. Batimastat, a potent matrix metalloproteinase inhibitor, exhibits an unexpected mode of binding. Proc Natl Acad Sci USA 1996; 39: 2749–54.

    Article  Google Scholar 

  18. Carmeliet P, Collen D. Role of vascular endothelial growth factor and vascular endothelial growth factor receptors in vascular development. Curr Top Microbiol Immunol 1999; 237: 133–58.

    PubMed  CAS  Google Scholar 

  19. Benelli R, Albini A. In vitro models of angiogenesis: The use of Matrigel. Int J Biol Markers 1999; 14: 243–6.

    PubMed  CAS  Google Scholar 

  20. Ribatti D, Vacca A, Wild R et al. Models for studying angiogenesis in vivo. Int J Biol Markers 1999; 14: 207–13.

    PubMed  CAS  Google Scholar 

  21. Chambers AF, MacDonald IC, Schmidt EE et al. Clinical targets for anti-metastasis therapy. Adv Cancer Res 2000; 79: 91–121.

    Article  PubMed  CAS  Google Scholar 

  22. 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 

  23. Carmeliet P, Moons L, Luttun A et al. Synergism between VEGF and Placental Growth Factor (PIGF) contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med 2001; 5: 75–83.

    Google Scholar 

  24. Nicosia RF, Villaschi S. Rat aortic smooth muscle cells become pericytes during angiogenesis in vitro. Lab Invest 1995; 73: 658–66.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory of Tumor and Developmental Biology, University of Liège, Tour de Pathologie +3, CHU, Sart-Tilman, Liège, Belgium

    Silvia Blacher, Laetitia Devy, Guy Roland & Jean-Michel Foidart

  2. Experimental Oncology Division, Institut de Recherches Servier, Suresnes, France

    Mike F. Burbridge & Gordon Tucker

  3. Laboratory of Tumor and Developmental Biology, University of Liège, Tour de Pathologie +3, CHU, Sart-Tilman, Liège, Belgium Fax:

    Agnes Noël

Authors
  1. Silvia Blacher
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Laetitia Devy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mike F. Burbridge
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Guy Roland
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gordon Tucker
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Agnes Noël
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jean-Michel Foidart
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Blacher, S., Devy, L., Burbridge, M.F. et al. Improved quantification of angiogenesis in the rat aortic ring assay. Angiogenesis 4, 133–142 (2001). https://doi.org/10.1023/A:1012251229631

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1012251229631

Search

Navigation