Skip to main content

Human Vascular Smooth Muscle Cell Culture

  • Protocol
  • First Online:
Human Cell Culture Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 806))

Abstract

Human vascular smooth muscle cells (VSMCs) in culture are an important tool in understanding how VSMCs function and contribute to vessel wall contraction as well as disease. In this chapter, we describe methodologies that enable the investigator to culture large numbers of proliferative VSMCs. These VSMCs are heterogeneous and vary in size, shape, and proliferative capacity depending on the disease state and location of the vessel of origin. Therefore, we also describe techniques to validate their identity as bone fide VSMCs. Briefly, the methods include information on how to dissect the blood vessel to remove the medial layer containing VSMCs, as well as methods on how to propagate these cells, by either allowing VSMCs to migrate from the explanted medial tissue or by enzymatically dispersing the cells from the tissue. Both methods are suitable for culturing VSMCs derived from most vessel types with modifications of the enzyme dispersal method suitable for the isolation of microvessel VSMCs. An important feature of VSMCs in culture is that they lose many of their in vivo contractile properties and so model disease-associated VSMCs in the vessel wall rather than a non-proliferative contractile cell. To overcome this limitation, we also describe alternate methods that enable the study of cultured VSMCs in their contractile state by allowing the VSMCs to remain within an intact vessel ring. Overall, these procedures enable the investigator to undertake a diverse array of experimental assays on cultured VSMCs.

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

Access this chapter

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Chamley-Campbell, J., Campbell, G. R., and Ross, R. (1979) The smooth muscle cell in culture. Physiol Rev 59, 1–61.

    PubMed  CAS  Google Scholar 

  2. Ross, R. (1986) The pathogenesis of atherosclerosis – an update. N Engl J Med 314, 488–500.

    Article  PubMed  CAS  Google Scholar 

  3. Schwartz, S. M., deBlois, D., and O’Brien, E. R. (1995) The intima. Soil for atherosclerosis and restenosis. Circ Res 77, 445–65.

    CAS  Google Scholar 

  4. Jonasson, L., Holm, J., Skalli, O., Gabbiani, G., and Hansson, G. K. (1985) Expression of class II transplantation antigen on vascular smooth muscle cells in human atherosclerosis. J Clin Invest 76, 12531.

    Article  PubMed  CAS  Google Scholar 

  5. Hansson, G. K., Jonasson, L., Holm, J., and Claesson-Welsh, L. (1986) Class II MHC antigen expression in the atherosclerotic plaque: smooth muscle cells express HLA-DR, HLA-DQ and the invariant gamma chain. Clin Exp Immunol 64, 261–8.

    PubMed  CAS  Google Scholar 

  6. Ross, R., and Glomset, J. A. (1976) The pathogenesis of atherosclerosis (first of two parts). N Engl J Med 295, 369–77.

    Article  PubMed  CAS  Google Scholar 

  7. Ross, R., and Glomset, J. A. (1976) The pathogenesis of atherosclerosis (second of two parts). N Engl J Med 295, 420–5.

    Article  PubMed  CAS  Google Scholar 

  8. Libby, P. (2000) Coronary artery injury and the biology of atherosclerosis: inflammation, thrombosis, and stabilization. Am J Cardiol 86, 3J–8J; discussion 8J–9J.

    Google Scholar 

  9. Davies, M. J. (1995) Acute coronary thrombosis – the role of plaque disruption and its initiation and prevention. Eur Heart J 16 Suppl L, 3–7.

    Google Scholar 

  10. Iyemere, V. P., Proudfoot, D., Weissberg, P. L., and Shanahan, C. M. (2006) Vascular smooth muscle cell phenotypic plasticity and the regulation of vascular calcification. J Intern Med 260, 192–210.

    Article  PubMed  CAS  Google Scholar 

  11. Thyberg, J., Hedin, U., Sjolund, M., Palmberg, L., and Bottger, B. A. (1990) Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arteriosclerosis 10, 966–90.

    Article  PubMed  CAS  Google Scholar 

  12. Babaev, V. R., Bobryshev, Y. V., Stenina, O. V., Tararak, E. M., and Gabbiani, G. (1990) Heterogeneity of smooth muscle cells in atheromatous plaque of human aorta. Am J Pathol 136, 1031–42.

    PubMed  CAS  Google Scholar 

  13. Glukhova, M. A., Kabakov, A. E., Frid, M. G., Ornatsky, O. I., Belkin, A. M., Mukhin, D. N., Orekhov, A. N., Koteliansky, V. E., and Smirnov, V. N. (1988) Modulation of human aorta smooth muscle cell phenotype: a study of muscle-specific variants of vinculin, caldesmon, and actin expression. Proc Natl Acad Sci USA 85, 9542–6.

    Article  PubMed  CAS  Google Scholar 

  14. Frid, M. G., Dempsey, E. C., Durmowicz, A. G., and Stenmark, K. R. (1997) Smooth muscle cell heterogeneity in pulmonary and systemic vessels. Importance in vascular disease. Arterioscler Thromb Vasc Biol 17, 1203–9.

    Article  CAS  Google Scholar 

  15. Bochaton-Piallat, M. L., Ropraz, P., Gabbiani, F., and Gabbiani, G. (1996) Phenotypic heterogeneity of rat arterial smooth muscle cell clones. Implications for the development of experimental intimal thickening. Arterioscler Thromb Vasc Biol 16, 815–20.

    Article  CAS  Google Scholar 

  16. Dartsch, P. C., Weiss, H. D., and Betz, E. (1990) Human vascular smooth muscle cells in culture: growth characteristics and protein pattern by use of serum-free media supplements. Eur J Cell Biol 51, 285–94.

    PubMed  CAS  Google Scholar 

  17. Fujita, H., Shimokado, K., Yutani, C., Takaichi, S., Masuda, J., and Ogata, J. (1993) Human neonatal and adult vascular smooth muscle cells in culture. Exp Mol Pathol 58, 25–39.

    Article  PubMed  CAS  Google Scholar 

  18. Bennett, M. R., Littlewood, T. D., Schwartz, S. M., and Weissberg, P. L. (1997) Increased sensitivity of human vascular smooth muscle cells from atherosclerotic plaques to p53-mediated apoptosis. Circ Res 81, 591–9.

    PubMed  CAS  Google Scholar 

  19. Proudfoot, D., Skepper, J. N., Shanahan, C. M., and Weissberg, P. L. (1998) Calcification of human vascular cells in vitro is correlated with high levels of matrix Gla protein and low levels of osteopontin expression. Arterioscler Thromb Vasc Biol 18, 379–88.

    Article  PubMed  CAS  Google Scholar 

  20. Shanahan, C. M., Weissberg, P. L., and Metcalfe, J. C. (1993) Isolation of gene markers of differentiated and proliferating vascular smooth muscle cells. Circ Res 73, 193204.

    PubMed  CAS  Google Scholar 

  21. Newman, C. M., Bruun, B. C., Porter, K. E., Mistry, P. K., Shanahan, C. M., and Weissberg, P. L. (1995) Osteopontin is not a marker for proliferating human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 15, 2010–8.

    Article  PubMed  CAS  Google Scholar 

  22. Rauch, B. H., Millette, E., Kenagy, R. D., Daum, G., Fischer, J. W., and Clowes, A. W. (2005) Syndecan-4 is required for thrombin-induced migration and proliferation in human vascular smooth muscle cells. J Biol Chem 280, 17507–11.

    Article  PubMed  CAS  Google Scholar 

  23. Cheng, G. C., Briggs, W. H., Gerson, D. S., Libby, P., Grodzinsky, A. J., Gray, M. L., and Lee, R. T. (1997) Mechanical strain tightly controls fibroblast growth factor-2 release from cultured human vascular smooth muscle cells. Circ Res 80, 28–36.

    PubMed  CAS  Google Scholar 

  24. Gittenberger-de Groot, A. C., DeRuiter, M. C., Bergwerff, M., and Poelmann, R. E. (1999) Smooth muscle cell origin and its relation to heterogeneity in development and disease. Arterioscler Thromb Vasc Biol 19, 1589–94.

    Article  PubMed  CAS  Google Scholar 

  25. Lee, C. S., Patton, W. F., Chung-Welch, N., Chiang, E. T., Spofford, K. H., and Shepro, D. (1998) Selective propagation of retinal pericytes in mixed microvascular cell cultures using L-leucine-methyl ester. Biotechniques 25(3), 482–8, 490–2, 494.

    Google Scholar 

  26. Shroff, R. C., Shah, V., Hiorns, M. P., Schoppet, M., Hofbauer, L. C., Hawa, G., Schurgers, L. J., Singhal, A., Merryweather, I., Brogan, P., Shanahan, C., Deanfield, J., and Rees, L. (2008) The circulating calcification inhibitors, fetuin-A and osteoprotegerin, but not matrix Gla protein, are associated with vascular stiffness and calcification in children on dialysis. Nephrol Dial Transplant 23, 3263–71.

    Article  PubMed  CAS  Google Scholar 

  27. Shroff, R. C., McNair, R., Skepper, J. N., Figg, N., Schurgers, L. J., Deanfield, J., Rees, L., and Shanahan, C. M. (2010)Chronic mineral dysregulation promotes vascular smooth muscle cell adaptation and extracellular matrix calcification. J Am Soc Nephrol 21(1): 103–12.

    Article  PubMed  CAS  Google Scholar 

  28. Skalli, O., Ropraz, P., Trzeciak, A., Benzonana, G., Gillessen, D., and Gabbiani, G. (1986) A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 103, 2787–96.

    Article  PubMed  CAS  Google Scholar 

  29. Arciniegas, E., Sutton, A. B., Allen, T. D., and Schor, A. M. (1992) Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro. J Cell Sci 103 (Pt 2), 521–9.

    PubMed  CAS  Google Scholar 

  30. Shi, Y., O’Brien, J. E., Jr., Fard, A., and Zalewski, A. (1996) Transforming growth factor-beta 1 expression and myofibroblast formation during arterial repair. Arterioscler Thromb Vasc Biol 16, 1298–305.

    Article  PubMed  CAS  Google Scholar 

  31. Nayak, R. C., Berman, A. B., George, K. L., Eisenbarth, G. S., and King, G. L. (1988) A monoclonal antibody (3 G5)-defined ganglioside antigen is expressed on the cell surface of microvascular pericytes. J Exp Med 167, 1003–15.

    Article  PubMed  CAS  Google Scholar 

  32. Shepro, D., and Morel, N. M. (1993) Pericyte physiology. Faseb J 7, 1031–8.

    PubMed  CAS  Google Scholar 

  33. Schlingemann, R. O., Rietveld, F. J., de Waal, R. M., Ferrone, S., and Ruiter, D. J. (1990) Expression of the high molecular weight melanoma-associated antigen by pericytes during angiogenesis in tumors and in healing wounds. Am J Pathol 136, 1393–405.

    PubMed  CAS  Google Scholar 

  34. Maguire, J. J., and Davenport, A. P. (1999) Endothelin receptor expression and pharmacology in human saphenous vein graft. Br J Pharmacol 126, 443–50.

    Article  PubMed  CAS  Google Scholar 

  35. Shirinsky, V. P., Birukov, K. G., Sobolevsky, A. V., Vedernikov, Y. P., Posin, E., and Popov, E. G. (1992) Contractile rabbit aortic smooth muscle cells in culture. Preparation and characterization. Am J Hypertens 5, 124S–130S.

    CAS  Google Scholar 

  36. Li, S., Sims, S., Jiao, Y., Chow, L. H., and Pickering, J. G. (1999) Evidence from a novel human cell clone that adult vascular smooth muscle cells can convert reversibly between noncontractile and contractile phenotypes. Circ Res 85, 338–48.

    PubMed  CAS  Google Scholar 

  37. L’Heureux, N., Stoclet, J. C., Auger, F. A., Lagaud, G. J., Germain, L., and Andriantsitohaina, R. (2001) A human tissue-engineered vascular media: a new model for pharmacological studies of contractile responses. Faseb J 15, 515–24.

    Article  PubMed  Google Scholar 

  38. Ewence, A. E., Bootman, M., Roderick, H. L., Skepper, J. N., McCarthy, G., Epple, M., Neumann, M., Shanahan, C. M., and Proudfoot, D. (2008) Calcium phosphate crystals induce cell death in human vascular smooth muscle cells: a potential mechanism in atherosclerotic plaque destabilization. Circ Res 103, e28–34.

    Article  PubMed  CAS  Google Scholar 

  39. Gimbrone, M. A., Jr., and Cotran, R. S. (1975) Human vascular smooth muscle in culture. Growth and ultrastructure. Lab Invest 33, 16–27.

    Google Scholar 

  40. Schor, A. M., Allen, T. D., Canfield, A. E., Sloan, P., and Schor, S. L. (1990) Pericytes derived from the retinal microvasculature undergo calcification in vitro. J Cell Sci 97 (Pt 3), 449–61.

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Diane Proudfoot .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Proudfoot, D., Shanahan, C. (2012). Human Vascular Smooth Muscle Cell Culture. In: Mitry, R., Hughes, R. (eds) Human Cell Culture Protocols. Methods in Molecular Biology, vol 806. Humana Press. https://doi.org/10.1007/978-1-61779-367-7_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-367-7_17

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-366-0

  • Online ISBN: 978-1-61779-367-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics