Compartmentalized coculture of porcine arterial endothelial and smooth muscle cells on a microporous membrane

  • Fabienne Kinard
  • Thérése Sergent-Engelen
  • André Trouet
  • Claude Remacle
  • Yves-Jacques Schneider
Cellular Models
Received:
Accepted:

Summary

Endothelial and smooth muscle cells were harvested from porcine pulmonary arteries and grown to two passages from primary culture in serum-containing medium. Thereafter, the cells were plated on the opposite sides of a microporous poly-(ethylene terephthalate) membrane and cultivated in a chemically defined, serum-free medium. The membrane with pores of 1 µm diameter allowed the passage of molecules and the extension of cell processes, while maintaining separate homogeneous cell populations. Pores of 3 µm diameter permitted the crossing of smooth muscle cells through the membrane. The coating of the polymer with constituents of the extracellular matrix optimized cell adhesion. Morphological analysis of the model showed typical cobblestone pattern and ultrastructure of endothelial cells, which lost rapidly the expression of von Willebrand factor but kept that of angiotensin-converting enzyme. Smooth muscle cells were spindle shaped and specific α-actin was revealed by immunochemistry and quantitated by enzyme-linked immunosorbent assay (ELISA). Their ultrastructure featured an intermediate contractile-synthetic phenotype. Permeability studies to different molecules showed a marked reduction of the albumin clearance. Finally, in coculture in the presence of endothelial cells, the smooth muscle cells proliferation was increased, whereas it was not the case in autologous cocultures. In conclusion, such a coculture model may help to a better understanding of the interactions between endothelial and smooth muscle cells that may be important in the pathogenesis of vascular diseases.

Key words

endothelium smooth muscle cells coculture microporous membrane blood vessel 
This is a preview of subscription content, log in to check access.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Albelda, S. M.; Sampson, P. M.; Haselton, F. R., et al. Permeability characteristics of cultured endothelial cell monolayers. J. Appl. Physiol. 64:308–322; 1988.PubMedGoogle Scholar
  2. Buul-Wortelboer, M. F.; Brinkman, H. J. M.; Dingemans, K. P., et al. Reconstitution of the vascular wall in vitro: a novel model to study interactions between endothelial and smooth muscle cells. Exp. Cell Res. 162:151–158; 1986.PubMedCrossRefGoogle Scholar
  3. Casnocha, S. A.; Eskin, S. G.; Hall, E. R., et al. Permeability of human endothelial monolayers: effect of vasoactive agonist and cAMP. J. Appl. Physiol. 67(5):1997–2005; 1989.PubMedGoogle Scholar
  4. Casnocha, S. A.; McIntire, L. V.; Eskin, S. G., et al. Measurement of endothel ial monolayer permeability and expression of endothelial cell products. Abstract, American Institute of Chemical Engineers, Annual Meeting. 173g; 1987.Google Scholar
  5. Castellot, J. J., Jr.; Addonizio, M. L.; Rosenberg, R., et al. Cultured endothelial cells produce a heparinlike inhibitor of smooth muscle cell growth. J. Cell Biol. 90:372–379; 1981.PubMedCrossRefGoogle Scholar
  6. Chamley, J. H.; Campbell, G. R. Endothelial cell influences on vascular smooth muscle phenotype. Annu. Rev. Physiol. 48:295–306; 1986.CrossRefGoogle Scholar
  7. Chamley, J. H.; Gordon, R.; Campbell, G. R., et al. The smooth muscle cell in culture. Physiol. Rev. 59:1–51; 1979.Google Scholar
  8. Ching, S. F.; Hayes, L. W.; Slakey, L. L. Angiotensin-converting enzyme in cultured endothelial cells: synthesis, degradation and transfer to culture medium. Atherosclerosis 3:581–588; 1983.Google Scholar
  9. Cooper, J. A.; Del Vecchio, P. J.; Minnear, F. L., et al. Measurement of albumin permeability across endothelial monolayers in vitro. J. Appl. Physiol 62:1076–1083; 1987.PubMedGoogle Scholar
  10. Davies, P. F.; Truskey, G. A.; Warren, H. B., et al. Metabolic cooperation between vascular endothelial cells and smooth muscle cells in coculture: changes in low density lipoprotein metabolism. J. Cell Biol. 101:871–879; 1985.PubMedCrossRefGoogle Scholar
  11. Downie, G. H.; Ryan, U. S.; Hayes, B. A., et al. Interleukin-2 directly increases albumin permeability of bovine and human vascular endothelium in vitro. Am. J. Respir. Cell Mol. Biol. 7:58–65; 1992.PubMedGoogle Scholar
  12. Fass, D.; Knutson, G. J.; Katzmann, J. Monoclonal antibodies to porcine factor VIII coagulation and their use in the isolation of active coagulation protein. Blood 59:594–600; 1982.PubMedGoogle Scholar
  13. Fillinger, M. F.; O’Connor, S. E.; Wagner, R. J., et al. The effect of endothelial cell coculture on smooth muscle cell proliferation. J. Vasc. Surg. 17:1058–1068; 1993.PubMedCrossRefGoogle Scholar
  14. Gajdusek, C.; Di Corleto, P. E.; Ross, R., et al. An endothelial cell derived growth factor. J. Cell Biol. 85:467–472; 1980.PubMedCrossRefGoogle Scholar
  15. Ganz, P.; Davies, P. F.; Leopold, J. A., et al. Short and long term interaction of endothelium and vascular smooth muscle in coculture: effects on cyclic GMP production. Proc. Natl. Acad. Sci. USA 83:3552–3556; 1986.PubMedCrossRefGoogle Scholar
  16. Garcia, J. G.; Siflinger-Birnboim, A.; Bizios, R., et al. Thrombin-induced increase in albumin permeability across the endothelium. J. Cell. Physiol. 128:96–104; 1986.PubMedCrossRefGoogle Scholar
  17. Garg, U. C.; Hassid, A. Nitric oxide-generating vasodilators and 8-bromocyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J. Clin. Invest. 83(5):1774–1777; 1989.PubMedGoogle Scholar
  18. Graham, D. J.; Alexander, J. J.; Miguel, R. Aortic endothelial and smooth muscle cell co-culture: an in vitro model of the arterial wall. J. Invest. Surg. 4:487–494; 1991.PubMedGoogle Scholar
  19. Halleux, C.; Schneider, Y.-J. Iron absorption by intestinal epithelial cells: 1. CaCo2 cells cultivated in serum-free medium, on polyethylene terephtalate microporous membranes, as an in vitro model. In Vitro Cell. Dev. Biol. 27A:293–302; 1991.PubMedCrossRefGoogle Scholar
  20. Hayes, L. W.; Goguen, C. A.; Ching, S. F., et al. Angiotensin-converting enzyme: accumulation in medium from cultured endothelial cells. Biochem. Biophys. Res. Commun. 82:1147–1153; 1978.PubMedCrossRefGoogle Scholar
  21. Hickey, K. A.; Rubanyi, G. M.; Paul, R. J., et al. Characterization of a coronary vasoconstrictor produced by cultured endothelial cells. Am. J. Physiol. 248:C550–556; 1985.PubMedGoogle Scholar
  22. Jaffe, E. A. Endothelial cells. Ann. NY Acad. Sci. 401:163–170; 1982.PubMedCrossRefGoogle Scholar
  23. Jones, P. A. Construction of an artificial blood vessel wall cultured endothelial and smooth muscle cells. Proc. Natl. Acad. Sci. USA 76:1882–1886; 1979.PubMedCrossRefGoogle Scholar
  24. Kapuscinski, J.; Skoczylas, B. Simple and rapid fluorimetric method for DNA microassay. Anal. Biochem. 83:252–257; 1977.PubMedCrossRefGoogle Scholar
  25. Larson, D. M.; Sheridan, J. D. Intercellular junctions and transfer of small molecules in primary vascular endothelial cultures. J. Cell Biol. 92:183–191; 1982.PubMedCrossRefGoogle Scholar
  26. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L., et al. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951.PubMedGoogle Scholar
  27. Merrilees, M. J.; Lesley, S. Culture of rat and pig aortic endothelial cells. Atherosclerosis 38:19–26; 1981.CrossRefGoogle Scholar
  28. Navab, M. G.; Hama, S. Y.; Van Lenten, B. J., et al. A new antiinflammatory compound, leumedin, inhibits modification of low density lipoprotein and the resulting monocyte transmigration into the subendothelial space of cocultures of human aortic wall cells. J. Clin. Invest. 91:1225–1230; 1993.PubMedGoogle Scholar
  29. Navab, M. G.; Hough, G. P.; Stevenson, L. W., et al. Monocyte migration into the subendothelial space of a coculture of adult human aortic endothelial and smooth muscle cells. J. Clin. Invest. 82:1853–1863; 1988.PubMedCrossRefGoogle Scholar
  30. Pederson, D. C.; Fass, D. N. Failure to detect Von Willebrand Factor in cultured porcine endothelial cells. Circulation 62 (suppl. III) 169; 1980.Google Scholar
  31. Ryan, U. S.; Ryan, J. W. Vital and functional activities of endothelial cells: In: Nossell, H. L.; Vogel, H. J., eds. Pathobiology of the endothelial cell. New York: Academic Press; 1985:455–469.Google Scholar
  32. Saunders, K. B.; D’Amore, P. A. An in vitro model for cell-cell interactions. In Vitro Cell. Dev. Biol. 28A:521–528; 1992.PubMedCrossRefGoogle Scholar
  33. Schneider, Y.-J. Optimization of hybridoma cell growth and monoclonal antibody secretion in a chemically defined, serum- and protein-free culture medium. J. Immunol. Methods 116:65–77; 1989.PubMedCrossRefGoogle Scholar
  34. Sergent-Engelen, T.; Halleux, C.; Ferain, E., et al. Improved cultivation of polarized animal cells on culture inserts with new transparent polyethylene terephtalate or polycarbonate microporous membranes. Biotechnol. Tech. 4:89–96; 1990.CrossRefGoogle Scholar
  35. Siflinger-Birnboim, A.; Del Vecchio, P. J.; Cooper, J. A., et al. Molecular sieving characteristics of the cultured endothelial monolayer. J. Cell. Physiol. 132:111–117; 1987.PubMedCrossRefGoogle Scholar
  36. Stoll, L. L.; Spector, A. A. Lipid transfer between endothelial and smooth muscle cells in coculture. J. Cell. Physiol. 133:103–110; 1987.PubMedCrossRefGoogle Scholar
  37. Thomae, K. R.; Nakayama, D. K.; Billiar, T. R., et al. The effect of nitric oxide on fetal pulmonary artery smooth muscle growth. J. Surg. Res. 59(3):337–343; 1995.PubMedCrossRefGoogle Scholar
  38. Vanhoutte, P. M.; Rubanyi, G. M.; Miller, V. M., et al. Modulation of vascular smooth muscle contraction by endothelium. Annu. Rev. Physiol. 48:307–320; 1986.PubMedCrossRefGoogle Scholar
  39. Xu, C. B.; Stavenow, L.; Pessah-Rasmussen, H. Interactions between cultured bovine arterial endothelial and smooth muscle cells: further studies on the effects of injury and modification of the consequences of injury. Artery 20(3):163–179; 1993.PubMedGoogle Scholar

Copyright information

© Society for In Vitro Biology 1997

Authors and Affiliations

  • Fabienne Kinard
    • 1
  • Thérése Sergent-Engelen
    • 2
  • André Trouet
    • 1
  • Claude Remacle
    • 1
  • Yves-Jacques Schneider
    • 2
  1. 1.Laboratoire de Biologie CellulaireUniversité Catholique de LouvainLouvain-la-NeuveBelgium
  2. 2.Laboratoire de Biochimie CellulaireUniversité Catholique de LouvainLouvain-la-NeuveBelgium

Personalised recommendations