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.

Adipose angiogenesis: quantitative methods to study microvessel growth, regression and remodeling in vivo

Nature Protocols volume 5, pages 912–920 (2010)Cite this article

Subjects

Abstract

Genetic and diet-induced rodent obesity models provide outstanding opportunities to study the role of angiogenesis and vascular remodeling in modulation of adipogenesis and obesity. In this study, we describe methods to quantitatively study adipose angiogenesis and vascular remodeling on the basis of immunohistochemical analyses. Fresh white adipose tissue or brown adipose tissue are prepared for whole mount, cryosectioned and paraffin-embedded samples, followed by staining with specific markers such as platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31, CD34, isolectin B4 or α-smooth muscle actin. Adipocytes are visualized by staining lipid droplets with 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY) 558/568 C12. This protocol may take 2–5 d to obtain results. In the view of the crucial roles of vasculature in modulation of adipogenesis and obesity, this protocol is valuable for studying the molecular mechanisms of angiogenesis in obese adipose tissues and for assessing the anti-obesity activity of angiogenesis modulators.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Comparison of various blood and lymphatic vascular endothelial cell markers in WAT.
Figure 2: Comparative evaluation of vascular structure and density in WAT and BAT.
Figure 3: Analysis of vascular remodeling and functions in adipose tissue.

References

  1. Carmeliet, P. & Jain, R.K. Angiogenesis in cancer and other diseases. Nature 407, 249–257 (2000).

    Article  CAS  PubMed  Google Scholar 

  2. Cao, Y. Angiogenesis and lymphangiogenesis in common diseases. Editorial. Curr. Mol. Med. 9, 928 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Lim, S.D. et al. Expression of the neural stem cell markers NG2 and L1 in human angiomyolipoma: are angiomyolipomas neoplasms of stem cells? Mol. Med. 13, 160–165 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Folkman, J. Angiogenesis: an organizing principle for drug discovery? Nat. Rev. Drug. Discov. 6, 273–286 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Cao, Y., Zhong, W. & Sun, Y. Improvement of antiangiogenic cancer therapy by understanding the mechanisms of angiogenic factor interplay and drug resistance. Semin. Cancer Biol. 19, 338–343 (2009).

    Article  CAS  PubMed  Google Scholar 

  6. Hurwitz, H. et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N. Engl. J. Med. 350, 2335–2342 (2004).

    Article  CAS  PubMed  Google Scholar 

  7. Kerbel, R.S. Tumor angiogenesis. N. Engl. J. Med. 358, 2039–2049 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rosenfeld, P.J. et al. Ranibizumab for neovascular age-related macular degeneration. N. Engl. J. Med. 355, 1419–1431 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Gragoudas, E.S., Adamis, A.P., Cunningham, E.T. Jr., Feinsod, M. & Guyer, D.R. Pegaptanib for neovascular age-related macular degeneration. N. Engl. J. Med. 351, 2805–2816 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Brakenhielm, E. et al. Angiogenesis inhibitor, TNP-470, prevents diet-induced and genetic obesity in mice. Circ. Res. 94, 1579–1588 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Cao, Y. Angiogenesis modulates adipogenesis and obesity. J. Clin. Invest. 117, 2362–2368 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Rupnick, M.A. et al. Adipose tissue mass can be regulated through the vasculature. Proc. Natl. Acad. Sci. USA 99, 10730–10735 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Xue, Y. et al. FOXC2 controls Ang-2 expression and modulates angiogenesis, vascular patterning, remodeling, and functions in adipose tissue. Proc. Natl. Acad. Sci. USA 105, 10167–10172 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Xue, Y. et al. Hypoxia-independent angiogenesis in adipose tissues during cold acclimation. Cell. Metab. 9, 99–109 (2009).

    Article  CAS  PubMed  Google Scholar 

  15. Planat-Benard, V. et al. Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation 109, 656–663 (2004).

    Article  PubMed  Google Scholar 

  16. Dobson, D.E. et al. 1-Butyryl-glycerol: a novel angiogenesis factor secreted by differentiating adipocytes. Cell 61, 223–230 (1990).

    Article  CAS  PubMed  Google Scholar 

  17. Brown, J.Q. et al. Quantitative optical spectroscopy: a robust tool for direct measurement of breast cancer vascular oxygenation and total hemoglobin content in vivo. Cancer Res. 69, 2919–2926 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Vineberg, A.M. et al. Myocardial revascularization by Omental graft without pedicle: experimental background and report on 25 cases followed 6 to 16 months. J. Thorac. Cardiovasc. Surg 49, 103–129 (1965).

    CAS  PubMed  Google Scholar 

  19. Cao, R., Brakenhielm, E., Wahlestedt, C., Thyberg, J. & Cao, Y. Leptin induces vascular permeability and synergistically stimulates angiogenesis with FGF-2 and VEGF. Proc. Natl. Acad. Sci. USA 98, 6390–6395 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Brakenhielm, E. et al. Adiponectin-induced antiangiogenesis and antitumor activity involve caspase-mediated endothelial cell apoptosis. Proc. Natl. Acad. Sci. USA 101, 2476–2481 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bjorndahl, M. et al. Insulin-like growth factors 1 and 2 induce lymphangiogenesis in vivo. Proc. Natl. Acad. Sci. USA 102, 15593–15598 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Cao, R. et al. Hepatocyte growth factor is a lymphangiogenic factor with an indirect mechanism of action. Blood 107, 3531–3536 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Wicki, A. et al. Tumor invasion in the absence of epithelial-mesenchymal transition: podoplanin-mediated remodeling of the actin cytoskeleton. Cancer. Cell. 9, 261–272 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Cho, C.H. et al. Angiogenic role of LYVE-1-positive macrophages in adipose tissue. Circ. Res. 100, e47–57 (2007).

    CAS  PubMed  Google Scholar 

  25. Hedlund, E.M., Hosaka, K., Zhong, Z., Cao, R. & Cao, Y. Malignant cell-derived PlGF promotes normalization and remodeling of the tumor vasculature. Proc. Natl. Acad. Sci. USA. 106, 17505–17510 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Yihai Cao's laboratory is supported through research grants from the Swedish Research Council, the Swedish Cancer Foundation, the Karolinska Institute Foundation, the Karolinska Institute Distinguished Professor Award, the European Union Integrated Project of Metoxia (Project no. 222741) and the European Research Council (ERC) advanced grant ANGIOFAT (Project no. 250021).

Author information

Author notes

  1. Yuan Xue and Sharon Lim: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden

    Yuan Xue, Sharon Lim & Yihai Cao

  2. Institute for Biomedical Research, Rouen Medical University, Rouen, France

    Ebba Bråkenhielm

Authors
  1. Yuan Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sharon Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ebba Bråkenhielm
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yihai Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.C. designed the study. Y.X. and S.L. performed and analyzed experiments. E.B. contributed to the development of these protocols. Y.X., S.L. and Y.C. wrote the paper.

Corresponding author

Correspondence to Yihai Cao.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Xue, Y., Lim, S., Bråkenhielm, E. et al. Adipose angiogenesis: quantitative methods to study microvessel growth, regression and remodeling in vivo. Nat Protoc 5, 912–920 (2010). https://doi.org/10.1038/nprot.2010.46

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.46

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

Advanced 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