Skip to main content
SpringerLink
Log in

Effects of collagen gel configuration on behavior of vascular smooth muscle cells in vitro: Association with vascular morphogenesis

  • Cell Growth/Differentiation/Apoptosis
  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Summary

The growth, behavior, and contractile protein expression of rabbit aortic smooth muscle cells (SMC) grown on, between layers, or within a collagen gel was investigated by confocal laser scanning fluorescence microscopy and Western analysis. SMC grown on collagen gel behaved similarly to those on conventional culture dishes. However, when a second layer of collagen was overlaid, cells underwent an elongated quiescent phase before onset of proliferation and a more than threefold lower logarithmic growth rate was observed. These cells self-organized into a network with ring-like structures. With increasing culture time, some of the rings developed into funnel-like, incomplete or complete tubular structures. If a tubular template preexisted within the gel, the SMC established a cylinder-shaped tube with several circularly arranged muscular layers (similar to an artery wall). This behavior mimicked endothelial cells during angiogenesis in vitro. A similar phenomenon occurred in cultures in which SMC were randomly mixed in a collagen gel, but here their behavior and morphology varied with their position within the gel. Western blot analysis showed that the SMC differentiation marker, smooth muscle myosin heavy chain-2 (SM-2), rapidly decreased, disappearing by day 10 in SMC grown on collagen, but was still detectable until day 25 in cells cultured between or within the same gel. These findings indicate that like endothelial cells, vascular SMC can display blood vessel formation behavior in vitro when an appropriate three-dimensional matrix environment is provided to keep them in a relatively higher-differentiated and low-proliferative state.

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

  • Akita, M.; Murata, E.; Merker, H. J.; Kaneko, K. Morphology of capillary-like structures in a three-dimensional aorta/collagen gel culture. Anat. Anz. 179:127–136; 1997.

    CAS  Google Scholar 

  • Arciniegas, E. A.; Mota, M. A.; Castillo, M. C. Behavior of chick embryo aortic cells obtained through nonenzymatic means cultured onto collagen gels. Anat. Embryol. 182:569–582; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Biegel, D.; Pachter, J. S. Growth of brain microvessel endothelial cells on collagen gels: applications to the study of blood-brain barrier physiology and CNS inflammation. In Vitro Cell Dev. Biol. 30A: 581–588; 1994.

    Article  CAS  Google Scholar 

  • Blau, H.; Guzowski, D. E.; Siddiqi, Z. A.; Scarpelli, E. M.; Bienkowski, R. S. Fetal type 2 pneumocytes from alveolar-like structures and maintain long-term differentiation on extracellular matrix. J. Cell Physiol. 136:203–214; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Campbell, G. R.; Campbell, J. H. Smooth muscle cell diversity. Implications for the question: what is a smooth muscle cell? Biomed. Res. 8:81–125; 1997.

    Google Scholar 

  • Campbell, J. H.; Kocher, O.; Skalli, O.; Gabbiani, G.; Campbell, G. R. Cytodifferentiation and expression of α-smooth muscle actin mRNA and protein during primary culture of aortic smooth muscle cells: correlation with cell density and proliferative state. Arteriosclerosis 9:633–643; 1989.

    PubMed  CAS  Google Scholar 

  • Delvos, U.; Gajdusek, C.; Sage, H.; Harker, L. A.; Schwartz, S. M. Interactions of vascular wall cells with collagen gels. Lab. Invest. 46:61–72; 1982.

    PubMed  CAS  Google Scholar 

  • Drake, C. J.; Hungeford, J. E.; Little, C. D. Morphogenesis of the first blood vessels. Ann. NY Acad. Sci. 857:155–179; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Elsdale, T.; Bard, J. Collagen substrata for studies on cell behavior. J. Cell Biol. 54:626–637; 1972.

    Article  PubMed  CAS  Google Scholar 

  • Ezzell, R. M.; Toner, M.; Hendricks, K.; Dunn, J. C. Y.; Tompkins, R. G.; Yarmush, M. L. Effect of collagen gel configuration on the cytoskeleton in cultured rat hepatocytes. Exp. Cell Res. 208:442–452; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Folkman, J.; D'Amore, P.A. Blood vessel formation: what is its molecular basis?. Cell 87:1153–1155; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Folkman, J.; Handenschild, C. Angiogenesis in vitro. Nature 288:551–556; 1980.

    Article  PubMed  CAS  Google Scholar 

  • Goto, F.; Goto, K.; Weindel, K.; Folkman, J. Synergistic effects of vascular endothelial growth factor and basic fibroblast growth factor on proliferation and cord formation of bovine capillary endothelial cells within collagen gel. Lab. Invest. 69:491–493; 1993.

    Google Scholar 

  • Grant, D. S.; Tashiro, K. I.; Segui-Real, B.; Yamada, Y.; Martin, G. R.; Kleinman, H. K. Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro. Cell 58:933–943; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Hadley, M. A.; Byers, S. W.; Suarcz-Quian, C. A.; Kleinman, H. K.; Dym, M. Extracellular matrix regulates Sertoli cell differentiation, testicular cord formation and germ cell development in vitro. J. Cell Biol. 101: 1511–1522; 1985.

    Article  PubMed  CAS  Google Scholar 

  • Hikichi, Y.; Sugihara, H.; Sugimoto, E. Differentiation of brown adipose cells in three-dimensional collagen gel culture. Pathol. Res. Pract. 189:73–82; 1993.

    PubMed  CAS  Google Scholar 

  • Hoying, J. B.; Boswell, C. A.; Williams, S. K. Angiogenic potential of microvessel fragments established in three-dimensional collagen gels. In Vitro Cell Dev. Biol. 32A:409–419; 1996.

    Google Scholar 

  • Ingber, D. E.; Dike, L.; Hansen, L., et al. Cellular tensegrity: exploring how mechanical changes in the cytoskeleton regulate cell growth, migration, and tissue pattern during morphogenesis. Int. Rev. Cytol. 150:173–224; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Iwig, M.; Glaesser D. Cell-substratum interactions and the cytoskeleton in cell shape-mediated growth regulation of lens epithelial cells. Lens Eye Toxic Res. 8:281–309; 1991.

    PubMed  CAS  Google Scholar 

  • Kennedy, A.; Frank, R. N.; Sotolongo, L. B.; Das, A.; Zhang, N. L. Proliferative response and macromolecular synthesis by ocular cells cultured on extracellular matrix materials. Curr. Eye Res. 9:307–322; 1990.

    PubMed  CAS  Google Scholar 

  • Kleinman, H. K.; Klebe, R. J.; Martin, G. R. Role of collagenous matrices in the adhesion and growth of cells. J. Cell Biol. 88:473–485; 1981.

    Article  PubMed  CAS  Google Scholar 

  • Kohler, A.; Jostarndt-Fögen, K.; Rottner, K.; Alliegro, M. C.; Draeger, A. Intima-like smooth muscle cells: developmental link between endothelium and media? Anat. Embryol. 200:313–323; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685; 1970.

    Article  PubMed  CAS  Google Scholar 

  • Lindahl, P.; Hellstrom, M.; Kalen, M.; Betsholtz, C. Endothelial-perivascular cell signaling in vascular development: lessons from knockout mice. Curr. Opin. Lipidol. 9:407–411; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Lukashev, M. E.; Werb, Z. ECM signalling: orchestrating cell behaviour and misbehaviour. Trends Cell Biol 8:437–441; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Maurizio, P.; Mario, M. Development changes in glycosaminoglycans during skeletal muscle cell differentiation in culture. Exp. Cell Res. 126:143–152; 1980.

    Article  Google Scholar 

  • Montesano, R.; Orci, L.; Vassalli, P. In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices. J. Cell Biol. 97:1648–1652; 1983.

    Article  PubMed  CAS  Google Scholar 

  • Nagai, R.; Kuro-o M.; Babij, P.; Periasamy, M. Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis. J. Biol. Chem. 264:9734–9737; 1989.

    PubMed  CAS  Google Scholar 

  • Nehls, V.; Drenckhahn, D. The versatility of microvascular pericytes: from mesenchyme to smooth muscle? Histochemistry 99:1–12; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Nehls, V.; Herrmann, R.; Palmetshofer, A. Contact-dependent inhibition of angiogenesis by cardiac fibroblasts in three-dimensional fibrin gels in vitro: implications for microvascular network remodeling and coronary collateral formation. Cell Tissue Res. 293:479–488; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Nehls, V.; Schuchardt, E.; Drenckhahn, D. The effect of fibroblasts, vascular smooth muscle cells and pericytes on sprout formation of endothelial cells in a fibrin gel angiogenesis system. Microvasc. Res. 48:349–363; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Nicosia, R. F.; Bonanno, E.; Villaschi, S. Large-vessel endothelium switches to a microvascular phenotype during angiogenesis in collagen gel culture of rat aorta. Atherosclerosis 95:191–199; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Nicosia, R. F.; Villaschi, S. Rat aortic smooth muscle cells become pericytes during angiogenesis in vitro. Lab. Invest. 73:658–666; 1995.

    PubMed  CAS  Google Scholar 

  • Niiya, A.; Matsumoto, Y.; Ishibashi, T.; Matsumoto, K.; Kinoshita, S. Collagen gel-embedding culture of conjunctival epithelial cells. Graefes Arch Clin. Exp. Opathalmol. 235:32–40; 1997.

    Article  CAS  Google Scholar 

  • Nishikawa, Y.; Tokusashi, Y.; Kadohama, T.; Nishimori, H.; Ogawa, K. Hepatocytic cells form bile duct-like structures within a three-dimensional collagen gel matrix. Exp. Cell Res. 223:357–371; 1996.

    Article  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • Parry, G.; Cullen, B.; Kaetzel, C. S.; Kramer, R.; Moss, L. Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences. J. Cell Biol. 105:2043–2051; 1987.

    Article  PubMed  CAS  Google Scholar 

  • Poole, V.; Coffin, J. D. Vasculogenesis and angiogenesis: two distinct morphogenetic mechanisms establish embryonic vascular pattern. J. Exp. Zool. 251:224–231; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Schapter, W.; Ito, W. D. Molecular mechanisms of coronary collateral vessel growth. Circ. Res. 79: 911–919; 1996.

    Google Scholar 

  • Song, J.; Rolfe, B. E.; Campbell, J. H.; Campbell, G. R. Changes in three-dimensional architecture of microfilaments in cultured vascular smooth muscle cells during phenotypic modulation. Tissue Cell. 39:324–333; 1998.

    Article  Google Scholar 

  • Stadler, E.; Campbell, J. H.; Campbell, G. R. Do cultured vascular smooth muscle cells resemble those of the artery? If not, why not?. J. Cardiovasc. Pharmacol. 14(Suppl. 6):S1-S8; 1989.

    CAS  Google Scholar 

  • Tokunaga, O.; Morimatsu, M.; Nakashima, T. Ring formation by human variant endothelial cells in vitro. Br. J. Exp. Pathol. 65:165–170; 1984.

    PubMed  CAS  Google Scholar 

  • Towbin, H.; Staehelin, T.; Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proc. Natl. Acad. Sci. USA 76:4350–4354; 1979.

    Article  PubMed  CAS  Google Scholar 

  • Vernon, R. B.; Angello, J. C.; Luisa Iruela-Arispe, M.; Lane, T. F.; Helene Sage, E. Reorganisation of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro. Lab. Invest. 66:536–547; 1992.

    PubMed  CAS  Google Scholar 

  • Vukicevic, S.; Luyten, F. P.; Kleinman, H. K.; Reddi, A. H. Differentiation of canalicular cell processes in bone cells by basement membrane matrix components: regulation by domains of laminin. Cell 63:437–445; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Yamamoto, M.; Nakamura, H.; Yamato, M.; Aoyagi, M.; Yamamoto, K. Retardation of phenotypic transition of rabbit arterial smooth muscle cells in three-dimensional primary culture. Exp. Cell. Res. 225:12–21; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Yamamoto, M.; Yamamoto, K.; Noumura, T. Type I collagen promotes modulation of cultured rabbit arterial smooth muscle cells from a contractile to a synthetic phenotype. Exp. Cell Res. 204:121–129; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Yamamoto, M.; Yamato, M.; Aoyagi, M.; Yamamoto, K. Identification of integrins involved in cell adhesion to native and denatured type I collagen and the phenotypic transition of rabbit arterial smooth muscle cells. Exp. Cell Res. 219:249–256; 1995.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Center for Research in Vascular Biology, Department of Anatomical Sciences, University of Queensland, 4072, Brisbane, Queensland, Australia

    Jian Song, Barbara E. Rolfe, Ian P. Hayward, Gordon R. Campbell & Julie H. Campbell

Authors
  1. Jian Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Barbara E. Rolfe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ian P. Hayward
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gordon R. Campbell
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Julie H. Campbell
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Julie H. Campbell.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Song, J., Rolfe, B.E., Hayward, I.P. et al. Effects of collagen gel configuration on behavior of vascular smooth muscle cells in vitro: Association with vascular morphogenesis. In Vitro Cell.Dev.Biol.-Animal 36, 600–610 (2000). https://doi.org/10.1007/BF02577528

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02577528

Key words

Search

Navigation