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Comparison of vascular smooth muscle cells from adult human, monkey and rabbit in primary culture and in subculture

Cell and Tissue Research volume 177, pages 503–522 (1977)Cite this article

Summary

A method is presented for growing large numbers of pure isolated smooth muscle cells from adult human, monkey, and rabbit blood vessels in primary culture.

In the first few days in culture these cells closely resembled those in vivo and could be induced to contract with angiotensin II, noradrenaline and mechanical stimulation. They stained intensely with antibodies against smooth muscle actin and myosin. Fibroblasts and endothelial cells did not stain with these antibodies thereby allowing the purity of each batch of cultures to be monitored. This was consistently found to be better than 99%. The smooth muscle cells modified or “dedifferentiated” after about 9 days in culture to morphologically resemble fibroblasts. At this stage cells could no longer be induced to contract and did not stain with the myosin antibodies. Intense proliferation of these cells soon resulted in a confluent monolayer being formed at which stage some differentiated characteristics returned. The modification or “dedifferentiation” process could be inhibited by the presence of a feeder layer of fibroblasts or endothelial cells, or the addition of cAMP to the culture medium.

Smooth muscle cells which had migrated from explants in primary culture, and cells in subculture, had morphological and functional properties of “dedifferentiated” cells at all times.

The advantages of differentiated rather than “dedifferentiated” smooth muscle cells in culture for the study of mitogenic agents in atherosclerosis is discussed.

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References

  • Anderson, P.J.: The actin of muscle and fibroblasts. Biochem. J. 155, 297–316 (1976)

    Google Scholar 

  • Blose, S.H., Lazarides, E., Chacko. S.K.: Loss of differentiated phenotype in the progeny of aortic smooth muscle cells. J. Cell Biol. 67, 34a (1975)

  • Booyse, F.M., Hoveke, T., Rafelson, M.: Human platelet actin, isolation and properties. J. biol. Chem. 248, 4083–4091 (1973)

    Google Scholar 

  • Burridge, K.: A comparison of fibroblast and smooth muscle myosins. Febs Letters 45, 14–17 (1974)

    Google Scholar 

  • Campbell, G.R., Chamley, J.H.: Thick filaments in vertebrate smooth muscle. Cell Tiss. Res. 156, 201–216 (1975)

    Google Scholar 

  • Campbell, G.R., Chamley, J.H.: Ultrastructure of differentiating vascular smooth muscle. Vascular neuroeffector mechanisms. 2nd Int. Symp., Odense 1975, pp. 1–9. Basel: Karger 1976

    Google Scholar 

  • Campbell, G.R., Chamley, J.H., Burnstock, G.: Differentiation and dedifferentiation of arterial smooth muscle cells. In: Functional and comparative anatomy of the artery (C.J. Schwartz, ed.). (In press) 1977

  • Campbell, G.R., Uehara, Y., Malmfors, T., Burnstock, G.: Degeneration and regeneration of smooth muscle transplants in the anterior eye chamber. An ultrastructural study. Z. Zellforsch. 117, 155–175 (1971b)

    Google Scholar 

  • Campbell, G.R., Uehara, Y., Mark, G., Burnstock, G.: Fine structure of smooth muscle cells grown in tissue culture. J. Cell Biol. 49, 21–34 (1971a)

    Google Scholar 

  • Caro, L.G., Palade, G.E.: Protein synthesis, storage, and discharge in the pancreatic exocrine cell. An autoradiographic study. J. Cell Biol. 20, 473–495 (1964)

    Google Scholar 

  • Chamley, J.H., Campbell, G.R.: Mitosis of contractile smooth muscle cells in tissue culture. Exp. Cell Res. 84, 105–109 (1974)

    Google Scholar 

  • Chamely, J.H., Campbell, G.R.: Trophic influences of sympathetic nerves and cyclic AMP on differentiation and proliferation of single smooth muscle cells in culture. Cell Tiss. Res. 161, 497–510 (1975a)

    Google Scholar 

  • Chamley, J.H., Campbell, G.R.: Isolated ureteral smooth muscle cells in culture. Including their interaction with intrinsic and extrinsic nerves. Cytobiol. 11, 358–365 (1975b)

    Google Scholar 

  • Chamley, J.H., Campbell, G.R.: Tissue culture: Interaction between sympathetic nerves and vascular smooth muscle. Vascular neuroeffector mechanisms. 2nd Int. Symp. Odense 1975, pp. 10–18. Basel: Karger 1976

    Google Scholar 

  • Chamley, J.H., Campbell, G.R., Burnstock, G.: An analysis of the interactions between sympathetic nerve fibers and smooth muscle cells in tissue culture. Develop. Biol. 33, 344–361 (1973)

    Google Scholar 

  • Chamley, J.H., Campbell, G.R., Burnstock, G.: Dedifferentiation, redifferentiation and bundle formation of smooth muscle cells in tissue culture: The influence of cell number and nerve fibres. J. Emb. exp. Morph. 32, 297–323 (1974)

    Google Scholar 

  • Chamley, J.H., Gröschel-Stewart, U., Campbell, G.R., Burnstock, G.: Distinction between smooth muscle, fibroblasts and endothelial cells in culture by the use of fluoresceinated antibodies against smooth muscle actin. Cell Tiss. Res., 177, 445–457 (1977)

    Google Scholar 

  • Cliff, W.J.: The aortic tunica media in growing rats studied with the electron microscope. Lab. Invest. 17, 599–615 (1967)

    Google Scholar 

  • Cliff, W.J.: Blood vessels. London: Cambridge University Press 1976

    Google Scholar 

  • Elzinga, M., Maron, B.J., Adelstein, R.S.: Human heart and platelet actins are products of different genes. Science 191, 94–95 (1976)

    Google Scholar 

  • Fischer-Dzoga, K., Fraser, R., Wissler, R.W.: Stimulation of proliferation in stationary primary cultures of monkey and rabbit aortic smooth muscle cells. I. Effects of lipoprotein fractions of hyperlipemic serum and lymph. Exp. molec. Path. 24, 346–359 (1976)

    Google Scholar 

  • Fritz, K.E., Jarmolych, J., Daoud, A.S.: Association of DNA synthesis and apparent dedifferentiation of aortic smooth muscle cells in vitro. Exp. molec. Path. 12, 354–362 (1970)

    Google Scholar 

  • Gabella, G.: Fine structure of smooth muscle. I. Cellular structures and electrophysiological behaviour. Phil. Trans. B 265, 7–16 (1973)

    Google Scholar 

  • Geer, J.C., Haust, M.D.: Smooth muscle cells in atherosclerosis. Monogr. Atheroscler. 2, 1–88 (1972)

    Google Scholar 

  • Gerrity, R.G., Adams, E.P., Cliff, W.J.: The aortic tunica media of the developing rat. II. Incorporation by medial cells of 3H-proline into collagen and elastin: autoradiographic and chemical studies. Lab. Invest. 32, 601–609 (1975)

    Google Scholar 

  • Glagov, S., Ts'ao, C.: Restitution of aortic wall after sustained necrotizing transmural ligation injury: Role of blood cells and artery cells. Amer. J. Path. 79, 7–30 (1975)

    Google Scholar 

  • Gröschel-Stewart, U., Ceurremans, S., Lehr, I., Mahlmeister, Ch., Paar, E.: Production of specific antibodies to contractile proteins, and their use in immunofluorescence microscopy. II. Speciesspecific and species-non-specific antibodies to smooth and striated chicken muscle actin. Histochemistry, in press (1976)

  • Gröschel-Stewart, U., Chamley, J.H., Campbell, G.R., Burnstock, G.: Changes in myosin distribution in dedifferentiating and redifferentiating smooth muscle cells in tissue culture. Cell Tiss. Res. 165, 13–22 (1975a)

    Google Scholar 

  • Gröschel-Stewart, U., Chamley, J.H., McConnell, J.D., Burnstock, G.: Comparison of the reaction of cultured smooth and cardiac muscle cells and fibroblasts to specific antibodies to myosin. Histochemistry 43, 215–224 (1975b)

    Google Scholar 

  • Gröschel-Stewart, U., Schreiber, J., Mahlmeister, Ch., Weber, K.: Production of specific antibodies to smooth and striated chicken muscle myosin. Histochemistry 46, 229–236 (1976)

    Google Scholar 

  • Gruenstein, E., Rich, A.: Non-identity of muscle and non-muscle actins. Biochem. biophys. Res. Commun. 64, 472–477 (1975)

    Google Scholar 

  • Hinek, A., Konwiński, M.: Electron microscopic studies on cells isolated from aorta of newborn and adult rabbits. Pol. med. Sci. Hist. Bull. 15, 9–22 (1975)

    Google Scholar 

  • Imai, H., Lee, K.J., Lee, S.K., Lee, K.T., O'Neal, R.M., Thomas, W.A.: Ultrastructural features of aortic cells in mitosis in control and cholesterol-fed swine. Lab. Invest. 23, 401–415 (1970)

    Google Scholar 

  • Jamieson, J.D., Palade, G.E.: Role of the Golgi complex in the intracellular transport of secretory proteins. Proc. natl. Acad. Sci. (Wash.) 55, 424–431 (1966)

    Google Scholar 

  • Jamieson, J.D., Palade, G.E.: Intracellular transport of secretory proteins in the pancreatic exocrine cell. I. Role of the peripheral elements of the Golgi complex. J. Cell Biol. 34, 577–596 (1967)

    Google Scholar 

  • Jurukova, Z., Rohr, H.P.: Beitrag zur Bildung bindegewebiger Matrix in glatten Muskelzellen. Elektronenmikroskopisch-autoradiographische Untersuchungen mit 35S-Sulfat an glatten Muskelzellen nach Doppelligatur der Arteria carotis. Path. europ. 3, 551–570 (1968)

    Google Scholar 

  • Karrer, H.E.: An electron microscope study of the aorta in young and aging mice. J. Ultrastruct. Res. 5, 1–27 (1961)

    Google Scholar 

  • Lazarides, E.: Immunofluorescence studies on the structure of actin filaments in tissue culture cells. J. Histochem. Cytochem. 23, 507–528 (1975)

    Google Scholar 

  • Mauger, J.P., Worcel, M., Tassin, J., Courtois, Y.: Contractility of smooth muscle cells of rabbit aorta in tissue culture. Nature (Lond.) 255, 337–338 (1975)

    Google Scholar 

  • Paule, W.J.: Electron microscopy of the newborn rat aorta J. Ultrastruct. Res. 8, 219–235 (1963)

    Google Scholar 

  • Pease, D.C., Paule, W.J.: Electron microscopy of elastic arteries. The thoracic aorta of the rat. J. Ultrastruct. Res. 3, 469–483 (1960)

    Google Scholar 

  • Poole, J.C.F., Cromwell, S.B., Benditt, E.P.: Behaviour of smooth muscle cells and formation of extracellular structures in the reaction of arterial walls to injury. Amer. J. Path. 62, 391–414 (1971)

    Google Scholar 

  • Rohr, H.P., Jurukova, Z.: Beitrag zur Bildung bindegewebiger Matrix in glatten Muskelzellen. (Elektronenmikroskopisch-autoradiographische Untersuchungen mit 3H-Prolin und 35S-Sulfat am Oestrogen-stimulierten Mäuseuterus.) Path. europ. 3, 571–579 (1968)

    Google Scholar 

  • Ross, R.: The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. J. Cell Biol. 50, 172–186 (1971)

    Google Scholar 

  • Ross, R., Glomset, J.A.: Atherosclerosis and the arterial smooth muscle cell. Science 180, 1332 -1339 (1973)

    Google Scholar 

  • Ross, R., Glomset, J.A.: The pathogenesis of atherosclerosis. New Engl. J. Med. 295, 420–425 (1976)

    Google Scholar 

  • Ross, R., Klebanoff, S.J.: The smooth muscle cell. I. In vivo synthesis of connective tissue proteins. J. Cell Biol. 50, 159–171 (1971)

    Google Scholar 

  • Rutherford, R.B., Ross, R.: Platelet factors stimulate fibroblasts and smooth muscle cells quiescent in plasma serum to proliferate. J. Cell Biol. 69, 196–203 (1976)

    Google Scholar 

  • Scott, R.F., Jones, R., Daoud, A.S., Zumbo, O., Coulston, F., Thomas, W.A.: Experimental atherosclerosis in Rhesus monkeys. II. Cellular elements of proliferative lesions and possible role of cytoplasmic degeneration in pathogenesis as studied by electron microscopy. Exp. molec. Path. 7, 34–57 (1967)

    Google Scholar 

  • Stary, H.C.: Proliferation of arterial cells in atherosclerosis Adv. exp. Med. Biol. 43, 59–65 (1974)

    Google Scholar 

  • Storti, R.V., Rich, A.: Chick cytoplasmic actin and muscle actin have different structural genes. Proc. nat. Acad. Sci. (Wash.) 73, 2346–2350 (1976)

    Google Scholar 

  • Thomas, W.A., Florentin, R.A., Nam, S.C., Kim, D.N., Jones, R.M., Lee, K.T.: Preproliferative phase of atherosclerosis in swine fed cholesterol. Arch. Path. 86, 621–643 (1968)

    Google Scholar 

  • Thomas, W.A., Florentin, R.A., Nam, S.C., Reiner, J.M., Lee, K.T.: Alterations in population dynamics of arterial smooth muscle cells during atherogenesis I. Activation of interphase cells in cholesterol-fed swine prior to gross atherosclerosis demonstrated by “postpulse salvage labelling”. Exp. molec. Path. 15, 245–267 (1971)

    Google Scholar 

  • Thomas, W.A., Jones, R., Scott, R.F., Morrison, E., Goodale, F., Imai, H.: Production of early atherosclerotic lesions in rats characterised by proliferation of “modified smooth muscle cells”. Exp. molec. Path., Suppl. 1, 40–61 (1963)

    Google Scholar 

  • Tokuoka, S.: Response of the uterine smooth muscle cell to oestrogen stimulation: an electron microscopic and autoradiographic study. Acta path. jap. 18, 417–423 (1968)

    Google Scholar 

  • Uehara, Y., Campbell, G.R., Burnstock, G.: Cytoplasmic filaments in developing and adult vertebrate smooth muscle. J. Cell Biol. 50, 484–497 (1971)

    Google Scholar 

  • Warshawsky, H., Leblond, C.P., Droz, B.: Synthesis and migration of proteins in the cells of the exocrine pancreas as revealed by specific activity determination from radioautographs. J. Cell Biol. 16, 1–28 (1963)

    Google Scholar 

  • Zeigel, R.F., Dalton, A.J.: Speculations based on the morphology of the Golgi systems in several types of protein secreting cells. J. Cell Biol. 15, 45–54 (1962)

    Google Scholar 

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Author information

Affiliations

  1. Department of Anatomy and Embryology, University College, London, England

    Julie H. Chamley, Gordon R. Campbell & Janet D. McConnell

  2. Institut für Zoologie, Technische Hochschule, Darmstadt, Germany

    Ute Gröschel-Stewart

Authors
  1. Julie H. Chamley
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  2. Gordon R. Campbell
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  3. Janet D. McConnell
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  4. Ute Gröschel-Stewart
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Additional information

The authors wish to thank Professor H.H. Bentall of the Royal Postgraduate Medical School, Hammersmith Hospital, London, for making available human material, and Dr. S. Zeki of Department of Anatomy, University College London for material from monkeys. We are also extremely grateful to Professor G. Burnstock for the use of his laboratory facilities

Holder of a “John Halliday Travelling Fellowship” from the Life Insurance Medical Research Fund of Australia and New Zealand

Research Fellow with the National Heart Foundation of Australia

Supported by the Deutsche Forschungsgemeinschaft

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Chamley, J.H., Campbell, G.R., McConnell, J.D. et al. Comparison of vascular smooth muscle cells from adult human, monkey and rabbit in primary culture and in subculture. Cell Tissue Res. 177, 503–522 (1977). https://doi.org/10.1007/BF00220611

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  • DOI: https://doi.org/10.1007/BF00220611

Key words

  • Smooth muscle
  • Blood vessels
  • Tissue culture
  • Antibodies
  • Immunofluorescence