Skip to main content
SpringerLink
Log in

A comparative molecular analysis of four rat smooth muscle cell lines

  • Cellular Models
  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Summary

Transcriptional regulation of smooth muscle cell (SMC) differentiation is a rapidly growing area of interest that has relevance for understanding intimal disease. Despite the wealth of data accumulating in vitro, however, no study has compared the cell-specific marker profile, transfectability, promoter activity, and growth characteristics among several SMC culture systems. Accordingly, we performed a comprehensive analysis of the marker profile, growth properties, transfectability, and SMC promoter activity in four rat SMC lines (A7r5, adult and pup aortic, and PAC1). Despite alterations in chromosomal number and structure, A7r5, adult aortic, and PAC1 cells express all SMC markers studied including SM α-actin, SM calponin, SM22, tropoelastin, and to a lesser extent, SM myosin heavy chain (SMMHC). In contrast, pup aortic cells express very low or undetectable levels of all the above markers except tropoelastin. Adult aortic, pup, and PAC1 cells display similar growth curves and levels of proto-oncogene transcripts, whereas those in the A7r5 line are comparatively less. All cell lines studied except pup cells show expression of SMC differentiation genes during active growth, indicating that growth and differentiation are not mutually exclusive in cultured smooth muscle. Transfection studies reveal dramatic differences in DNA uptake and SMC-restricted promoter activity between cell lines. Collectively, these results provide detailed information relating to SMC molecular biology in culture that should facilitate the selection of a cell line for studying the transcriptional regulatory mechanisms underlying SMC differentiation.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Belknap, J. K.; Grieshaber, N. A.; Schwartz, P. E., et al. Tropoelastin gene expression in individual vascular smooth muscle cells: relationship to DNA synthesis during vascular development and after arterial injury. Circ. Res. 78:388–394; 1996.

    PubMed  CAS  Google Scholar 

  2. Bochaton-Piallat, M. L.; Gabbiani, F.; Ropraz, P., et al. Cultured aortic smooth muscle cells from newborn and adult rats show distinct cytoskeletal features. Differentiation 49:175–185; 1992.

    Article  PubMed  CAS  Google Scholar 

  3. Byron, K. L. Vasopressin stimulates Ca2+ spiking activity in A7r5 vascular smooth muscle cells via activation of phospholipase A2. Circ. Res. 78:813–820; 1996.

    PubMed  CAS  Google Scholar 

  4. Campbell, G. R.; Chamley-Campbell, J. H. Smooth muscle phenotypic modulation: role in atherogenesis. Med. Hypotheses 7:729–735; 1981.

    Article  PubMed  CAS  Google Scholar 

  5. Chamley-Campbell, J.; Campbell, G. R.; Ross, R. The smooth muscle cell in culture. Physiol. Rev. 59:1–61; 1979.

    PubMed  CAS  Google Scholar 

  6. Chomczynski, P.; Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156–159; 1987.

    Article  PubMed  CAS  Google Scholar 

  7. Cook, C. L.; Weiser, M. C. M.; Schwartz, P. E., et al. Developmentally timed expression of an embryonic growth phenotype in vascular smooth muscle cells. Circ. Res. 74:189–196; 1994.

    PubMed  CAS  Google Scholar 

  8. de Lucca, E. J.; Dhaliwal, M. K.; Furlong, C. L., et al. A high resolution G-banding idiogram of rattus norvegicus chromosomes. Cytobios 62:153–160; 1990.

    PubMed  Google Scholar 

  9. Ehler, E.; Jat, P. S.; Noble, M. D., et al. Vascular smooth muscle cells of H-2Kb-tsA58 transgenic mice: characterization of cell lines with distinct properties. Circulation 92:3289–3296; 1995.

    PubMed  CAS  Google Scholar 

  10. Firulli, A. B.; Miano, J. M.; Bi, W., et al. Myocyte enhancer binding factor-2 expression and activity in vascular smooth muscle cells: association with the activated phenotype. Circ. Res. 78:196–204; 1996.

    PubMed  CAS  Google Scholar 

  11. Fritz, K. E.; Jarmolych, J.; Daoud, A. S. Association of DNA synthesis and apparent dedifferentiation of aortic smooth muscle cells in vitro. Exp. Mol. Pathol. 12:354–362; 1970.

    Article  PubMed  CAS  Google Scholar 

  12. Gotwals, P. J.; Chi-Rosso, G.; Lindner, V., et al. The α1β1 integrin is expressed during neointimal formation in rat arteries and mediates collagen matrix reorganization. J. Clin. Invest. 97:2469–2477; 1996.

    Article  PubMed  CAS  Google Scholar 

  13. Herring, B. P.; Smith, A. F. Telokin expression is mediated by a smooth muscle cell-specific promoter. Am. J. Physiol. 270:C1656-C1665; 1996.

    PubMed  CAS  Google Scholar 

  14. Holifield, B.; Helgason, T.; Jemelka, S., et al. Differentiated vascular myocytes: are they involved in neointimal formation? J. Clin. Invest. 97:814–825; 1995.

    Google Scholar 

  15. Huang, S.; Simonson, M. S.; Dunn, M. J. Manidipine inhibits endothelin-1-induced [Ca2+]1 signaling but potentiates endothelin’s effect on c-fos and c-jun induction in vascular smooth muscle and glomerular mesangial cells. Am. Heart J. 125:589–597; 1993.

    Article  PubMed  CAS  Google Scholar 

  16. Kimes, B. W.; Brandt, B. L. Characterization of two putative smooth muscle cell lines from rat thoracic aorta. Exp. Cell Res. 98:349–366; 1976.

    Article  PubMed  CAS  Google Scholar 

  17. Kokubu, T.; Pollak, O. J. In vitro cultures of aortic cells of untreated and of cholesterol-fed rabbits. J. Atherosclerosis Res. 1:229–239; 1961.

    Article  CAS  Google Scholar 

  18. Lemire, J. M.; Covin, C. W.; White, S., et al. Characterization of cloned aortic smooth muscle cells from young rats. Am. J. Pathol. 144:1068–1081; 1994.

    PubMed  CAS  Google Scholar 

  19. Li, L.; Miano, J. M.; Cserjesi, P., et al. SM22α, a marker of adult smooth muscle, is expressed in multiple myogenic lineages during embryogenesis. Circ. Res. 78:188–195; 1996.

    PubMed  CAS  Google Scholar 

  20. Li, L.; Miano, J. M.; Mercer, B., et al. Expression of the SM22α promoter in transgenic mice provides evidence for distinct transcriptional regulatory programs in vascular and visceral smooth muscle cells. J. Cell Biol. 132:849–859; 1996.

    Article  PubMed  CAS  Google Scholar 

  21. Longtine, J. A.; Pinkus, G. S.; Fujiwara, K., et al. Immunohistochemical localization of smooth muscle myosin in normal human tissues. J. Histochem. Cytochem. 33:179–184; 1985.

    PubMed  CAS  Google Scholar 

  22. Majesky, M. W.; Benditt, E. P.; Schwartz, S. M. Expression and developmental control of platelet-derived growth factor A-chain and B-chain/Sis genes in rat aortic smooth muscle cells. Proc. Natl. Acad. Sci. USA 85:1524–1528; 1988.

    Article  PubMed  CAS  Google Scholar 

  23. Majesky, M. W.; Giachelli, C. M.; Reidy, M. A., et al. Rat carotid neointimal smooth muscle cells reexpress a developmentally regulated mRNA phenotype during repair of arterial injury. Circ. Res. 71:759–768; 1992.

    PubMed  CAS  Google Scholar 

  24. Miano, J. M.; Cserjesi, P.; Ligon, K. L., et al. Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis. Circ. Res. 75:803–812; 1994.

    PubMed  CAS  Google Scholar 

  25. Miano, J. M.; Olson, E. N. Expression of the smooth muscle cell calponin gene marks the early cardiac and smooth muscle cell lineages during mouse embryogenesis. J. Biol. Chem. 271:7095–7103; 1996.

    Article  PubMed  CAS  Google Scholar 

  26. Miano, J. M.; Tota, R. R.; Vlasic, N., et al. Early proto-oncogene expression in rat aortic smooth muscle cells following endothelial removal. Am. J. Pathol. 137:761–765; 1990.

    PubMed  CAS  Google Scholar 

  27. Miano, J. M.; Vlasic, N.; Tota, R. R., et al. Localization of Fos and Jun proteins in rat aortic smooth muscle cells after vascular injury. Am. J. Pathol. 142:715–724; 1993.

    PubMed  CAS  Google Scholar 

  28. Moessler, H.; Mericskay, M.; Li, Z., et al. The SM22 promoter directs tissue-specific expression in arterial but not in venous or visceral smooth muscle cells in transgenic mice. Development 122:2415–2425; 1996.

    PubMed  CAS  Google Scholar 

  29. Moss, N. S.; Benditt, E. Spontaneous and experimentally induced arterial lesions I. An ultrastructural survey of the normal chicken aorta. Lab. Invest. 22:166–183; 1970.

    PubMed  CAS  Google Scholar 

  30. Nachtigal, M.; Nagpal, M. L.; Greenspan, P., et al. Characterization of a continuous smooth muscle cell line derived from rabbit aorta. In Vitro Cell. Dev. Biol. 25:892–898; 1989.

    Article  PubMed  CAS  Google Scholar 

  31. Orlandi, A.; Ehrlich, H. P.; Ropraz, P., et al. Rat aortic smooth muscle cells isolated from different layers and at different times after endothelial denudation show distinct biological features in vitro. Arterioscler. Thromb. 14:982–989; 1994.

    PubMed  CAS  Google Scholar 

  32. Owens, G. K. Regulation of differentiation of vascular smooth muscle cells. Physiol. Rev. 75:487–517; 1995.

    PubMed  CAS  Google Scholar 

  33. Perez-Reyes, N.; Halbert, C. L.; Smith, P. P., et al. Immortalization of primary human smooth muscle cells. Proc. Natl. Acad. Sci. USA 89:1224–1228; 1992.

    Article  PubMed  CAS  Google Scholar 

  34. Reilly, C. Rat vascular smooth muscle cells immortalized with SV40 large T antigen possess defined smooth muscle cell characteristics including growth inhibition by heparin. J. Cell. Physiol. 142:342–357; 1990.

    Article  PubMed  CAS  Google Scholar 

  35. Rosenquist, T. H.; Beall, A. C. Elastogenic cells in the developing cardiovascular system: smooth muscle, nonmuscle, and cardiac neural crest. Ann. NY Acad. Sci. 588:106–119; 1990.

    Article  PubMed  CAS  Google Scholar 

  36. Rothman, A.; Kulik, T. J.; Taubman, M. B., et al. Development and characterization of a cloned rat pulmonary arterial smooth muscle cell line that maintains differentiated properties through multiple subcultures. Circ. Res. 86:1977–1986; 1992.

    CAS  Google Scholar 

  37. Rothman, A.; Wolner, B.; Button, D., et al. Immediate-early gene expression in response to hypertrophic and proliferative stimuli in pulmonary arterial smooth muscle cells. J. Biol. Chem. 269:6399–6404; 1994.

    PubMed  CAS  Google Scholar 

  38. Samaha, F. F.; Ip, H. S.; Morrisey, E. E., et al. Developmental pattern of expression and genomic organization of the calponin-h1 gene: a contractile smooth muscle cell marker. J. Biol. Chem. 271:395–403; 1996.

    Article  PubMed  CAS  Google Scholar 

  39. Schwartz, S. M.; deBlois, D.; O’Brien, E. R. M. The intima: soil for atherosclerosis and restenosis. Circ. Res. 77:445–465; 1995.

    PubMed  CAS  Google Scholar 

  40. Seifert, R. A.; Schwartz, S. M.; Bowen-Pope, D. F. Developmentally regulated production of platelet-derived growth factor-like molecules. Nature 311:669–671; 1984.

    Article  PubMed  CAS  Google Scholar 

  41. Solway, J.; Seltzer, J.; Samaha, F. F., et al. Structure and expression of a smooth muscle cell-specific gene, SM22α. J. Biol. Chem. 270:13460–13469; 1995.

    Article  PubMed  CAS  Google Scholar 

  42. Thyberg, J.; Blomgren, K.; Hedin, U., et al. Phenotypic modulation of smooth muscle cells during the formation of neointimal thickenings in the rat carotid artery after balloon injury: an electron-microscopic and stereological study. Cell Tissue Res. 281:421–433; 1995.

    PubMed  CAS  Google Scholar 

  43. Thyberg, J.; Palmberg, L.; Nilsson, J., et al. Phenotype modulation in primary cultures of arterial smooth muscle cells: on the role of platelet-derived growth factor. Differentiation 25:156–167; 1983.

    Article  PubMed  CAS  Google Scholar 

  44. Walker, L. N.; Bowen-Pope, D. F.; Ross, R., et al. Production of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury. Proc. Natl. Acad. Sci. USA 83:7311–7315; 1986.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Hamon Center for Basic Research, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, 75235-9148, Dallas, Texas

    Anthony B. Firulli & Eric N. Olson

  2. Department of Pathology, Vascular Biology, University of Washington School of Medicine, Box 357335, 98195-7335, Seattle, Washington

    David Han & Stephen M. Schwartz

  3. Department of Physiology, Cardiovascular Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, 53226, Milwaukee, Wisconsin

    Louise Kelly-Roloff & Joseph M. Miano

  4. Biogen, Inc., 02142, Cambridge, Massachusetts

    Victor E. Koteliansky

Authors
  1. Anthony B. Firulli
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Louise Kelly-Roloff
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Victor E. Koteliansky
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Stephen M. Schwartz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eric N. Olson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Joseph M. Miano
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Firulli, A.B., Han, D., Kelly-Roloff, L. et al. A comparative molecular analysis of four rat smooth muscle cell lines. In Vitro Cell.Dev.Biol.-Animal 34, 217–226 (1998). https://doi.org/10.1007/s11626-998-0127-5

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-998-0127-5

Key words

Search

Navigation