Methods for Isolation of Endothelial and Smooth Muscle Cells and In Vitro Proliferation Assays

  • Ganapati H. Mahabeleshwar
  • Payaningal R. Somanath
  • Tatiana V. Byzova
Part of the Methods in Molecular Medicine book series (MIMM, volume 129)


Angiogenesis, the formation of new blood vessel from pre-existing blood vessel, occurs in a variety of normal and pathological conditions. It is complex morphogenetic process involving the coordinate migration, invasion, and reorganization of several cell types including endothelial cells, pericytes, smooth muscle cells, and stromal fibroblasts. The angiogenic response begins with excess protease secretion to facilitate basement membrane remodeling, proliferation of endothelial cells, and endothelial cell migration to form capillary network and lumen closure. In this chapter, we describe the methods to isolate mouse and human endothelial cells, smooth muscle cells, which will provide abundant, convenient and useful tool for the investigation of many aspects of endothelial cell biology. The high degrees of functional diversity have been observed from endothelial cells derived from different organs, and within the different vascular beds of a given organ. Therefore, this apparent heterogeneity has highlighted the requirement for the endothelial cell isolation and culture from a variety of tissues of different species in order to establish more realistic in vitro angiogenic models.

Key Words

Endothelial cells angiogenesis aorta matrigel VEGF smooth muscle cell MTT human umbilical vein endothelial cells HUVEC 


  1. 1.
    Reynolds, L. E., Wyder, L., Lively, J. C., et al. (2002) Enhanced pathological angiogenesis in mice lacking β3 integrin or β3 and β5 integrins. Nat. Med. 8, 27–34.CrossRefPubMedGoogle Scholar
  2. 2.
    Magid, R., Martinson, D., Hwang, J., Jo, H., and Galis, Z. S. (2003) Optimization of isolation and functional characterization of primary murine aortic endothelial cells. Endothelium 10, 103–109.CrossRefPubMedGoogle Scholar
  3. 3.
    Xu, H. Q., Hao, H. P., Zhang, X., and Pan, Y. (2004) Morroniside protects cultured human umbilical vein endothelial cells from damage by high ambient glucose. Acta Pharmacol. Sin. 25, 412–415.PubMedGoogle Scholar
  4. 4.
    Ewing, P., Wilke, A., Brockhoff, G., et al. (2003) Isolation and transplantation of allogeneic pulmonary endothelium derived from GFP transgenic mice. J. Immunol. Methods 283, 307–315.CrossRefPubMedGoogle Scholar
  5. 5.
    Dong, Q. G., Bernasconi, S., Lostaglio, S., et al. (1997) A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants. Arterioscler. Thromb. Vasc. Biol. 17, 1599–1604.PubMedGoogle Scholar
  6. 6.
    Ray, J. L., Leach, R., Herbert, J. M., and Benson, M. (2001) Isolation of vascular smooth muscle cells from a single murine aorta. Methods Cell Sci. 23, 185–188.CrossRefPubMedGoogle Scholar
  7. 7.
    Byzova, T. V., Goldman, C. K., Pampori, N., et al. (2000) A mechanism for modulation of cellular responses to VEGF: activation of the integrins. Mol. Cell. 6, 851–860.PubMedGoogle Scholar
  8. 8.
    Xiang, Y., Ma, B., Li, T., Gao, J. W., Yu, H. M., and Li, X. J. (2004) Acetazolamide inhibits aquaporin-1 protein expression and angiogenesis. Acta Pharmacol. Sin. 25, 812–816.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Ganapati H. Mahabeleshwar
    • 1
  • Payaningal R. Somanath
    • 1
  • Tatiana V. Byzova
    • 1
  1. 1.Department of Molecular CardiologyThe Cleveland Clinic FoundationCleveland

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