Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Use of the mouse aortic ring assay to study angiogenesis

Abstract

Here we provide a protocol for quantitative three-dimensional ex vivo mouse aortic ring angiogenesis assays, in which developing microvessels undergo many key features of angiogenesis over a timescale similar to that observed in vivo. The aortic ring assay allows analysis of cellular proliferation, migration, tube formation, microvessel branching, perivascular recruitment and remodeling—all without the need for cellular dissociation—thus providing a more complete picture of angiogenic processes compared with traditional cell-based assays. Our protocol can be applied to aortic rings from embryonic stage E18 through to adulthood and can incorporate genetic manipulation, treatment with growth factors, drugs or siRNA. This robust assay allows assessment of the salient steps in angiogenesis and quantification of the developing microvessels, and it can be used to identify new modulators of angiogenesis. The assay takes 6–14 d to complete, depending on the age of the mice, treatments applied and whether immunostaining is performed.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Flow diagram of the general procedure.
Figure 2: Dissection of mouse aorta from the thoracic cavity.
Figure 3: Optimization of the mouse ex vivo aortic ring assay.
Figure 4: Collecting aortic ring data.
Figure 5: Immunofluorescence staining of aortic rings.
Figure 6: Microvessel sprout imaging: visualizing supporting cell coverage.
Figure 7: Cellular proliferation in aortic rings.
Figure 8: RNAi in aortic rings.

Similar content being viewed by others

References

  1. Hodivala-Dilke, K.M., Reynolds, A.R. & Reynolds, L.E. Integrins in angiogenesis: multitalented molecules in a balancing act. Cell Tissue Res. 314, 131–144 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Ferrara, N. & Kerbel, R.S. Angiogenesis as a therapeutic target. Nature 438, 967–974 (2005).

    CAS  PubMed  Google Scholar 

  3. Hodivala-Dilke, K.M. αvβ3 integrin and angiogenesis: a moody integrin in a changing environment. Curr. Opin. Cell Biol. 20, 514–519 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Nicosia, R.F. & Ottinetti, A. Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro. Lab. Invest. 63, 115–122 (1990).

    CAS  PubMed  Google Scholar 

  5. Carmeliet, P. Angiogenesis in life, disease and medicine. Nature 438, 932–936 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Reynolds, L.E. et al. Tumour angiogenesis is reduced in the Tc1 mouse model of Down's syndrome. Nature 465, 813–817 (2010). Erratum in Nature 466, 398 (15 July 2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Reynolds, A.R. et al. Stimulation of tumor growth and angiogenesis by low concentrations of RGD-mimetic integrin inhibitors. Nat. Med. 4, 392–400 (2009).

    Article  Google Scholar 

  8. Reynolds, L.E. et al. Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins. Nat. Med. 8, 27–34 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Ebos, J.M. et al. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell 15, 232–239 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Falcon, B.L. et al. Increased vascular delivery and efficacy of chemotherapy after inhibition of platelet-derived growth factor-B. Am. J. Pathol. 178, 2920–2930 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Oehler, M.K., Hague, S., Rees, M.C. & Bicknell, R. Adrenomedullin promotes formation of xenografted endometrial tumors by stimulation of autocrine growth and angiogenesis. Oncogene 21, 2815–2821 (2002).

    Article  CAS  PubMed  Google Scholar 

  12. Tavora, B. et al. Endothelial FAK is required for tumour angiogenesis. EMBO Mol. Med. 2, 516–528 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. da Silva, R.G. et al. Endothelial α3β1-integrin represses pathological angiogenesis and sustains endothelial-VEGF. Am. J. Path. 177, 1534–1548 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lee, S. et al. Autocrine VEGF signaling is required for vascular homeostasis. Cell 130, 691–703 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Seo, D.W. et al. TIMP-2 mediated inhibition of angiogenesis: an MMP-independent mechanism. Cell 114, 171–180 (2003).

    Article  CAS  PubMed  Google Scholar 

  16. Ribatti, D. Chick embryo chorioallantoic membrane as a useful tool to study angiogenesis. Int. Rev. Cell Mol. Biol. 270, 181–224 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Gale, N.W. et al. Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1. Dev. Cell 3, 411–423 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Silvestre, J.S. et al. Antiangiogenic effect of interleukin-10 in ischemia-induced angiogenesis in mice hindlimb. Circ. Res. 87, 448–452 (2000).

    Article  CAS  PubMed  Google Scholar 

  19. Pitulescu, M.E., Schmidt, I., Benedito, R. & Adams, R.H. Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice. Nat. Protoc. 5, 1518–1534 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Lawson, N.D. & Weinstein, B.M. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 248, 307–318 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Rouhi, P. et al. Hypoxia-induced metastasis model in embryonic zebrafish. Nat. Protoc. 5, 1911–1918 (2010).

    Article  CAS  PubMed  Google Scholar 

  22. Norrby, K. In vivo models of angiogenesis. J. Cell Mol. Med. 10, 588–612 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Zou, L. et al. Rapid xenograft tumor progression in beta-arrestin1 transgenic mice due to enhanced tumor angiogenesis. FASEB J. 22, 355–364 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Koblizek, T.I., Weiss, C., Yancopoulos, G.D., Deutsch, U. & Risau, W. Angiopoietin-1 induces sprouting angiogenesis in vitro. Curr. Biol. 8, 529–532 (1998).

    Article  CAS  PubMed  Google Scholar 

  25. Mavria, G. et al. ERK-MAPK signaling opposes Rho-kinase to promote endothelial cell survival and sprouting during angiogenesis. Cancer Cell 9, 33–44 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Nicosia, R.F., Lin, Y.J., Hazelton, D. & Qian, X. Endogenous regulation of angiogenesis in the rat aorta model—role of vascular endothelial growth factor. Am. J. Path. 151, 1379–1386 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Aplin, A.C., Fogel, E., Zorzi, P. & Nicosia, R.F. The aortic ring model of angiogenesis. Methods Enzymol. Chapter 7 443, 119–136 (2008).

    Article  CAS  PubMed  Google Scholar 

  28. Nicosia, R.F. The aortic ring model of angiogenesis: a quarter century of search and discovery. J. Cell Mol. Med. 13, 4113–4136 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Scott, A.N. et al. Farnesyltransferase inhibitors target multiple endothelial cell functions in angiogenesis. Angiogenesis 11, 337–346 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Piqueras, L. et al. Activation of PPARbeta/delta induces endothelial cell proliferation and angiogenesis. Arterioscler. Thromb. Vasc. Biol. 27, 63–69 (2007).

    Article  CAS  PubMed  Google Scholar 

  31. Kruger, E.A. et al. Endostatin inhibits microvessel formation in the ex vivo rat aortic ring angiogenesis assay. Biochem. Biophys. Res. Comm. 268, 183–191 (2000).

    Article  CAS  PubMed  Google Scholar 

  32. Salcedo, R. et al. Human endothelial cells express CCR2 and respond to MCP-1: direct role of MCP-1 in angiogenesis and tumor progression. Blood 96, 34–40 (2000).

    CAS  PubMed  Google Scholar 

  33. Sounni, N.E. et al. MT1-MMP expression promotes tumor growth and angiogenesis through an upregulation of vascular endothelial growth factor expression. FASEB J. 16, 555–564 (2002).

    Article  CAS  PubMed  Google Scholar 

  34. Pan, Q. et al. Deficiency induced by tetrathiomolybdate suppresses tumor growth and angiogenesis. Cancer Res. 62, 4854 (2002).

    CAS  PubMed  Google Scholar 

  35. Stiffey-Wilusz, J., Boice, J.A., Ronan, J., Fletcher, A.M. & Anderson, M.S. An ex vivo angiogenesis assay utilizing commercial porcine carotidartery: modification of the rat aortic ring assay. Angiogenesis 4, 3–9 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Masson, V. et al. Mouse aortic ring assay: a new approach of the molecular genetics of angiogenesis. Biol. Proced. Online 4, 24–31 (2002).

    Article  CAS  PubMed Central  Google Scholar 

  37. Devy, L. et al. The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent. FASEB J. 16, 147–154 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Gelati, M., Aplin, A.C., Fogel, E., Smith, K.D. & Nicosia, R.F. The angiogenic response of the aorta to injury and inflammatory cytokines requires macrophages. J. Immunol. 181, 5711–5719 (2008).

    Article  CAS  PubMed  Google Scholar 

  39. Robinson, S.D. et al. Alphav beta3 integrin limits the contribution of neuropilin-1 to vascular endothelial growth factor-induced angiogenesis. J. Biol. Chem. 284, 33966–33968 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. D'Amico, G. et al. Endothelial-Rac1 is not required for tumor angiogenesis unless αvβ3-integrin is absent. PLoS ONE 5, e9766 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Reynolds, A.R. et al. Enhanced VEGF receptor 2 mediated responses in β3-integrin deficient endothelial cells in vivo and in vitro. Cancer Res. 64, 8643–8650 (2004).

    Article  CAS  PubMed  Google Scholar 

  42. Silva, R., D'Amico, G., Hodivala-Dilke, K.M. & Reynolds, L.E. Integrins: the keys to unlocking angiogenesis. Arterioscler. Thromb. Vasc. Biol. 28, 1703–1713 (2008).

    Article  CAS  PubMed  Google Scholar 

  43. Germain, M.A. et al. Genetic ablation of the alpha 6-integrin subunit in Tie1Cre mice enhances tumour angiogenesis. J. Pathol. 220, 370–381 (2010).

    CAS  PubMed  Google Scholar 

  44. Claxton, S. et al. Efficient, inducible Cre-recombinase activation in vascular endothelium. Genesis 46, 74–80 (2008).

    Article  CAS  PubMed  Google Scholar 

  45. Zhu, W.H., Iurlaro, M., MacIntyre, A., Fogel, E. & Nicosia, R.F. The mouse aorta model: influence of genetic background and aging on bFGF- and VEGF-induced angiogenic sprouting. Angiogenesis 6, 193–199 (2003).

    Article  CAS  PubMed  Google Scholar 

  46. Ventura, A. et al. Cre-lox-regulated conditional RNA interference from transgenes. Proc. Natl. Acad. Sci. USA 101, 10380–10385 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Alian, A., Eldor, A., Falk, H. & Panet, A. Viral mediated gene transfer to sprouting blood vessels during angiogenesis. J. Virol. Meth. 105, 1–11 (2002).

    Article  CAS  Google Scholar 

  48. Hajitou, A . et al. The antitumoral effect of endostatin and angiostatin is associated with a down-regulation of vascular endothelial growth factor expression in tumor cells. FASEB J. 16, 1802–1804 (2002).

    Article  CAS  PubMed  Google Scholar 

  49. Zhu, W.H. & Nicosia, R.F. The thin prep rat aortic ring assay: a modified method for the characterization of angiogenesis in whole mounts. Angiogenesis 5, 81–86 (2002).

    Article  CAS  PubMed  Google Scholar 

  50. Bruyère, F. et al. Modeling lymphangiogenesis in a three-dimensional culture system. Nat. Protoc. 5, 431–437 (2008).

    Google Scholar 

  51. Krilleke, D. et al. Molecular mapping and functional characterization of the VEGF164 heparin-binding domain. J. Biol. Chem. 282, 28045–28056 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Blatt, R.J., Clark, A.N., Courtney, J., Tully, C. & Tucker, A.L. Automated quantitative analysis of angiogenesis in the rat aorta model using Image-Pro Plus 4.1. Comput. Meth. Prog. Bio. 75, 75–79 (2004).

    Article  Google Scholar 

Download references

Acknowledgements

We thank all the members of our laboratory and collaborators who have contributed time, effort, expertise, reagents and equipment to the optimization of these protocols. This work was sponsored by Cancer Research UK, Breast Cancer Campaign and the Medical Research Council (grant no. 93277).

Author information

Authors and Affiliations

Authors

Contributions

M.B. compiled the protocol with input from the authors, collected collagen time-course and control data and developed the proliferation assay adaptation. S.D.R. performed the optimization experiments, developed the siRNA and lentivirus protocols and provided Matrigel data. T.L. carried out the Imaris analysis and optimized simultaneous protein/RNA extraction from rings using the Nucleospin kit. P.R.B. developed the TRI2 program and wrote the automated microvessel area quantification protocol. B.T. optimized the fibrin protocol and provided confocal images and data. G.D. provided Rac1 knockdown data and D.T.J. provided Flk1 knockdown data. B.V. oversaw the design and application of the automated vessel-counting system. K.H.-D. contributed to the development of the methods and oversaw the writing of the paper.

Corresponding author

Correspondence to Marianne Baker.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Automatic quantification of microvessel area in TRI2. An image of an aortic ring embedded in collagen, stained with BS1 lectin-FITC and imaged on an epifluorescence microscope after 9 days has been loaded into the TRI2 program (Original). The final binary image is shown (Segmented) in addition to being used as an overlay over the original data (Masked). The resulting statistics for total and average intensity with standard deviation plus the total vessel area in pixels are highlighted in the red box. See BOX 2. (TIFF 4489 kb)

Supplementary Video 1

Dissection of the murine thoracic aorta. After cleaning the fur with 70% ethanol, a single cut and blunt dissection are used to expose the rib cage. The thoracic cavity is opened and the heart and lungs removed. The aorta is indicated with white arrowheads in the still image. The aorta is carefully dissected as described in Figure 2 and the text and transferred to Opti-MEM®. Images of an aorta before and after cleaning are shown with the fatty layer (arrowheads) and aortic vessel (arrow) indicated. The approximate size of rings to be cut is indicated alongside the cleaned aorta with a scale bar. (MOV 16258 kb)

Supplementary Video 2

Embedding aortic rings in a collagen matrix. Materials required for embedding are shown: the 96-well plate and sterile forceps. The unpolymerised collagen mixture consisting of 10x DMEM, sterile distilled water and type 1 rat tail collagen is visible as a yellow liquid. After adding a small drop of 5N NaOH to the mixture and mixing well, the pH increases and the mixture turns pink. 50 µl aliquots of this mixture are transferred to the 96-well plate and aortic rings carefully embedded in them, with care taken to remove as much Opti-MEM® from the ring as possible so as not to dilute the collagen matrix excessively (as this would impair polymerization). (MOV 13427 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Baker, M., Robinson, S., Lechertier, T. et al. Use of the mouse aortic ring assay to study angiogenesis. Nat Protoc 7, 89–104 (2012). https://doi.org/10.1038/nprot.2011.435

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.435

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing