Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1981 Jun 1;89(3):568–578. doi: 10.1083/jcb.89.3.568

Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture

PMCID: PMC2111818  PMID: 6454694

Abstract

Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml). The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell growth. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell growth but also in increasing the longevity of the cultures.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barnes D., Sato G. Serum-free cell culture: a unifying approach. Cell. 1980 Dec;22(3):649–655. doi: 10.1016/0092-8674(80)90540-1. [DOI] [PubMed] [Google Scholar]
  2. Bottenstein J., Hayashi I., Hutchings S., Masui H., Mather J., McClure D. B., Ohasa S., Rizzino A., Sato G., Serrero G. The growth of cells in serum-free hormone-supplemented media. Methods Enzymol. 1979;58:94–109. doi: 10.1016/s0076-6879(79)58127-0. [DOI] [PubMed] [Google Scholar]
  3. Brown B. G., Mahley R., Assmann G. Swine aortic smooth muscle in tissue culture. Some effects of purified swine lipoproteins on cell growth and morphology. Circ Res. 1976 Sep;39(3):415–424. doi: 10.1161/01.res.39.3.415. [DOI] [PubMed] [Google Scholar]
  4. Cohen S., Taylor J. M. Epidermal growth factor: chemical and biological characterization. Recent Prog Horm Res. 1974;30(0):533–550. doi: 10.1016/b978-0-12-571130-2.50017-1. [DOI] [PubMed] [Google Scholar]
  5. 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 Mol Pathol. 1976 Jun;24(3):346–359. doi: 10.1016/0014-4800(76)90070-8. [DOI] [PubMed] [Google Scholar]
  6. Fowler S., Shio H., Wolinsky H. Subcellular fractionation and morphology of calf aortic smooth muscle cells. Studies on whole aorta, aortic explants, and subcultures grown under different conditions. J Cell Biol. 1977 Oct;75(1):166–184. doi: 10.1083/jcb.75.1.166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gospodarowicz D., Bialecki H., Greenburg G. Purification of the fibroblast growth factor activity from bovine brain. J Biol Chem. 1978 May 25;253(10):3736–3743. [PubMed] [Google Scholar]
  8. Gospodarowicz D., Delgado D., Vlodavsky I. Permissive effect of the extracellular matrix on cell proliferation in vitro. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4094–4098. doi: 10.1073/pnas.77.7.4094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gospodarowicz D., Greenburg G. The coating of bovine and rabbit corneas denuded of their endothelium with bovine corneal endothelial cells. Exp Eye Res. 1979 Mar;28(3):249–265. doi: 10.1016/0014-4835(79)90087-3. [DOI] [PubMed] [Google Scholar]
  10. Gospodarowicz D., Greenburg G., Vlodavsky I., Alvarado J., Johnson L. K. The identification and localization of fibronectin in cultured corneal endothelial cells: cell surface polarity and physiological implications. Exp Eye Res. 1979 Nov;29(5):485–509. doi: 10.1016/0014-4835(79)90151-9. [DOI] [PubMed] [Google Scholar]
  11. Gospodarowicz D., Ill C. R. Do plasma and serum have different abilities to promote cell growth? Proc Natl Acad Sci U S A. 1980 May;77(5):2726–2730. doi: 10.1073/pnas.77.5.2726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gospodarowicz D., Ill C. Extracellular matrix and control of proliferation of vascular endothelial cells. J Clin Invest. 1980 Jun;65(6):1351–1364. doi: 10.1172/JCI109799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gospodarowicz D., Mescher A. L., Birdwell C. R. Control of cellular proliferation by the fibroblast and epidermal growth factors. Natl Cancer Inst Monogr. 1978 May;(48):109–130. [PubMed] [Google Scholar]
  14. Gospodarowicz D., Mescher A. L., Birdwell C. R. Stimulation of corneal endothelial cell proliferations in vitro by fibroblast and epidermal growth factors. Exp Eye Res. 1977 Jul;25(1):75–89. doi: 10.1016/0014-4835(77)90248-2. [DOI] [PubMed] [Google Scholar]
  15. Gospodarowicz D., Moran J. S., Braun D. L. Control of proliferation of bovine vascular endothelial cells. J Cell Physiol. 1977 Jun;91(3):377–385. doi: 10.1002/jcp.1040910307. [DOI] [PubMed] [Google Scholar]
  16. Gospodarowicz D., Moran J. S. Mitogenic effect of fibroblast growth factor on early passage cultures of human and murine fibroblasts. J Cell Biol. 1975 Aug;66(2):451–457. doi: 10.1083/jcb.66.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gospodarowicz D., Moran J., Braun D., Birdwell C. Clonal growth of bovine vascular endothelial cells: fibroblast growth factor as a survival agent. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4120–4124. doi: 10.1073/pnas.73.11.4120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. HAVEL R. J., EDER H. A., BRAGDON J. H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955 Sep;34(9):1345–1353. doi: 10.1172/JCI103182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Henriksen T., Evensen S. A., Carlander B. Injury to human endothelial cells in culture induced by low density lipoproteins. Scand J Clin Lab Invest. 1979 Jun;39(4):361–368. doi: 10.3109/00365517909106120. [DOI] [PubMed] [Google Scholar]
  20. Hessler J. R., Robertson A. L., Jr, Chisolm G. M., 3rd LDL-induced cytotoxicity and its inhibition by HDL in human vascular smooth muscle and endothelial cells in culture. Atherosclerosis. 1979 Mar;32(3):213–229. doi: 10.1016/0021-9150(79)90166-7. [DOI] [PubMed] [Google Scholar]
  21. Jonas A. Physicochemical properties of bovine serum high density lipoprotein. J Biol Chem. 1972 Dec 10;247(23):7767–7772. [PubMed] [Google Scholar]
  22. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  23. Layman D. L., Jelen B. J., Illingworth D. R. Inability of serum from abetalipoproteinemic subjects to stimulate proliferation of human smooth muscle cells and dermal fibroblasts in vitro. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1511–1515. doi: 10.1073/pnas.77.3.1511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  25. Phillips J. L. "Specific" binding of radioiodinated transferrin to polypropylene culture tubes. Biochem Biophys Res Commun. 1976 Aug 9;71(3):726–732. doi: 10.1016/0006-291x(76)90891-3. [DOI] [PubMed] [Google Scholar]
  26. Ross R., Glomset J. A. Atherosclerosis and the arterial smooth muscle cell: Proliferation of smooth muscle is a key event in the genesis of the lesions of atherosclerosis. Science. 1973 Jun 29;180(4093):1332–1339. doi: 10.1126/science.180.4093.1332. [DOI] [PubMed] [Google Scholar]
  27. Ross R., Glomset J., Kariya B., Harker L. A platelet-dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1207–1210. doi: 10.1073/pnas.71.4.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ross R., Nist C., Kariya B., Rivest M. J., Raines E., Callis J. Physiological quiescence in plasma-derived serum: influence of platelet-derived growth factor on cell growth in culture. J Cell Physiol. 1978 Dec;97(3 Pt 2 Suppl 1):497–508. doi: 10.1002/jcp.1040970325. [DOI] [PubMed] [Google Scholar]
  29. Savage C. R., Jr, Cohen S. Epidermal growth factor and a new derivative. Rapid isolation procedures and biological and chemical characterization. J Biol Chem. 1972 Dec 10;247(23):7609–7611. [PubMed] [Google Scholar]
  30. Scher C. D., Shepard R. C., Antoniades H. N., Stiles C. D. Platelet-derived growth factor and the regulation of the mammalian fibroblast cell cycle. Biochim Biophys Acta. 1979 Aug 10;560(2):217–241. doi: 10.1016/0304-419x(79)90020-9. [DOI] [PubMed] [Google Scholar]
  31. Tauber J. P., Cheng J., Gospodarowicz D. Effect of high and low density lipoproteins on proliferation of cultured bovine vascular endothelial cells. J Clin Invest. 1980 Oct;66(4):696–708. doi: 10.1172/JCI109907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Van Wyk J. J., Svoboda M. E., Underwood L. E. Evidence from radioligand assays that somatomedin-C and insulin-like growth factor-I are similar to each other and different from other somatomedins. J Clin Endocrinol Metab. 1980 Jan;50(1):206–208. doi: 10.1210/jcem-50-1-206. [DOI] [PubMed] [Google Scholar]
  33. Van Wyk J. J., Underwood L. E., Hintz R. L., Clemmons D. R., Voina S. J., Weaver R. P. The somatomedins: a family of insulinlike hormones under growth hormone control. Recent Prog Horm Res. 1974;30(0):259–318. [PubMed] [Google Scholar]
  34. Vlodavsky I., Lui G. M., Gospodarowicz D. Morphological appearance, growth behavior and migratory activity of human tumor cells maintained on extracellular matrix versus plastic. Cell. 1980 Mar;19(3):607–616. doi: 10.1016/s0092-8674(80)80037-7. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES