Download PDF
Thromb Haemost 2006; 95(01): 182-189
DOI: 10.1160/TH05-05-0357
New Technologies, Diagnostic Tools and Drugs
Schattauer GmbH

A novel in vitro model for the study of plaque development in atherosclerosis

Bernhard Dorweiler   #
1   Department of Cardiothoracic and Vascular Surgery
,
Michael Torzewski   #
2   Institute of Clinical Chemistry and Laboratory Medicine, University of Mainz, Mainz, Germany
,
Manfred Dahm
1   Department of Cardiothoracic and Vascular Surgery
,
Viola Ochsenhirt
2   Institute of Clinical Chemistry and Laboratory Medicine, University of Mainz, Mainz, Germany
,
Hans-Anton Lehr
3   Institute Universitaire de Pathologie, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland
,
Karl J. Lackner
2   Institute of Clinical Chemistry and Laboratory Medicine, University of Mainz, Mainz, Germany
,
Christian-Friedrich Vahl
1   Department of Cardiothoracic and Vascular Surgery
› Author AffiliationsFinancial support: This work was supported in part by the University of Mainz (MAIFOR A11 to B.D.) and the German Research Foundation (SFB 432, Z3 to H.A.L.).
Further Information

Publication History

Received 24 May 2005

Accepted after revision 28 October 2005

Publication Date:
28 November 2017 (online)

Also available at
    Permissions and Reprints

Summary

For the study of atherogenesis in vitro, coculture systems have been devised, in which two or more cell types can be cultured in close contact to each other. Herein, we describe a novel in vitro model that aims at the simulation of the morphology ofa normal muscular artery allowing for the study of the initial events in atherosclerosis. Usinga modified fibrin gel as a scaffold for the coculture of endothelial cells (ECs) and smooth muscle cells (SMCs), we generated an autologous in vitro model with a multilayer growth of SMCs (intima-like structure) covered by an endothelium. The production of extracellular matrix (ECM) could be visualized histologically and verified by (i) ascorbic-acid dependent secretion of procollagenI into the supernatant and (ii) deposition of collagens I and III as well as laminin in the gel as assessed by immunohistochemistry. By BrdU-incorporation and Ki67 expression, the SMCs exhibited minimal proliferative activity, even when the culture period was extended to6 weeks. Lipoprotein insudation was investigated under simulated hypo-, normo-and hypercholesterolemic conditions through addition of 0.5, 1 or2 mg/mL LDL to the medium with subsequent time and dose dependend insudation of LDL. When human monocytes were added to the culture medium, infiltration and foam cell formation of macrophages and SMCs as well as expression of interleukin-8 (IL-8) was demonstrated. The in vitro model of the human vascular wall described herein appears to be suitable for the study of pivotal events in atherosclerotic plaque development. The applicability for long-term culture, the ability to study cell-matrix interactions and the opportunities for histomorphological and immunohistochemical examinations represent additional advantages of this model.

Keywords

In vitro model - endothelium - atherosclerosis - lipoprotein - foam cell

# Both authors contributed equally to this work.


 

  • References

  • 1 Stary HC. Natural history and histological classification of atherosclerotic lesions: an update. Arterioscler Thromb Vasc Biol 2000; 20: 1177-8.
    CrossrefPubMedSearch in Google Scholar
  • 2 Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med 1999; 340: 115-26.
    CrossrefPubMedSearch in Google Scholar
  • 3 Williams KJ, Tabas I. The response-to-retention hypothesis of atherogenesis reinforced. Curr Opin Lipidol 1998; 09: 471-4.
    CrossrefPubMedSearch in Google Scholar
  • 4 Bhakdi S, Lackner KJ, Han SR. et al. Beyond cholesterol: the enigma of atherosclerosis revisited. Thromb Haemost 2004; 91: 639-45.
    Thieme ConnectPubMedSearch in Google Scholar
  • 5 Navab M, Hough GP, Stevenson LW. et al. Monocyte migration into the subendothelial space of a coculture of adult human aortic endothelial and smooth muscle cells. J Clin Invest 1988; 82: 1853-63.
    CrossrefPubMedSearch in Google Scholar
  • 6 Takaku M, Wada Y, Jinnouchi K. et al. An in vitro coculture model of transmigrant monocytes and foam cell formation. Arterioscler Thromb Vasc Biol 1999; 19: 2330-39.
    CrossrefPubMedSearch in Google Scholar
  • 7 Babaei S, Stewart DJ. Overexpression of endothelial NO synthase induces angiogenesis in a co-culture model. Cardiovasc Res 2002; 55: 190-200.
    CrossrefPubMedSearch in Google Scholar
  • 8 Ross R. The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol 1971; 50: 172-86.
    CrossrefPubMedSearch in Google Scholar
  • 9 Jaffe EA, Nachman RL, Becker CG. et al. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973; 52: 2745-56.
    CrossrefPubMedSearch in Google Scholar
  • 10 Bhakdi S, Dorweiler B, Kirchmann R. et al. On the pathogenesis of atherosclerosis: enzymatic transformation of human low density lipoprotein to an atherogenic moiety. J Exp Med 1995; 182: 1959-71.
    CrossrefPubMedSearch in Google Scholar
  • 11 Young SG, Witztum JL, Casal DC. et al. Conservation of the low density lipoprotein receptor-binding domain of apoprotein B. Demonstration by a new monoclonal antibody, MB47. Arteriosclerosis 1986; 06: 178-88.
    CrossrefPubMedSearch in Google Scholar
  • 12 Ziegler-Heitbrock HW, Thiel E, Futterer A. et al. Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes. Int J Cancer 1988; 41: 456-61.
    CrossrefPubMedSearch in Google Scholar
  • 13 Lipman JM. Fluorophotometric quantitation of DNA in articular cartilage utilizing Hoechst 33258. Anal Biochem 1989; 176: 128-31.
    CrossrefPubMedSearch in Google Scholar
  • 14 Labarca C, Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 1980; 102: 344-52.
    CrossrefPubMedSearch in Google Scholar
  • 15 Yu CC, Woods AL, Levison DA. The assessment of cellular proliferation by immunohistochemistry: a review of currently available methods and their applications. HistochemJ 1992; 24: 121-31.
    PubMedSearch in Google Scholar
  • 16 Jones PA. Construction of an artificial blood vessel wall from cultured endothelial and smooth muscle cells. Proc Natl Acad Sci U S A 1979; 76: 1882-6.
    CrossrefPubMedSearch in Google Scholar
  • 17 Weinberg CB, Bell E. A blood vessel model constructed from collagen and cultured vascular cells. Science 1986; 231: 397-400.
    CrossrefPubMedSearch in Google Scholar
  • 18 Axel DI, Brehm BR, Wolburg-Buchholz K. et al. Induction of cell-rich and lipid-rich plaques in a transfilter coculture system with human vascular cells. J Vasc Res 1996; 33: 327-39.
    CrossrefPubMedSearch in Google Scholar
  • 19 Lafleur MA, Handsley MM, Knauper V. et al. Endothelial tubulogenesis within fibrin gels specifically requires the activity of membrane-type-matrix metalloproteinases (MT-MMPs). J Cell Sci 2002; 115: 3427-38.
    PubMedSearch in Google Scholar
  • 20 Teichert-Kuliszewska K, Maisonpierre PC, Jones N. et al. Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis is associated with activation of Tie2. Cardiovasc Res 2001; 49: 659-70.
    CrossrefPubMedSearch in Google Scholar
  • 21 Ikari Y, Fujikawa K, Yee KO. et al. Alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, or alpha(2)-macroglobulin is required for vascular smooth muscle cell spreading in three-dimensional fibrin gel. J Biol Chem 2000; 275: 12799-805.
    CrossrefPubMedSearch in Google Scholar
  • 22 Grassl ED, Oegema TR, Tranquillo RT. Fibrin as an alternative biopolymer to type-I collagen for the fabrication of a media equivalent. J Biomed Mater Res 2002; 60: 607-12.
    CrossrefPubMedSearch in Google Scholar
  • 23 Larson DM, Haudenschild CC, Beyer EC. Gap junction messenger RNA expression by vascular wall cells. Circ Res 1990; 66: 1074-80.
    CrossrefPubMedSearch in Google Scholar
  • 24 Navab M, Liao F, Hough GP. et al. Interaction of monocytes with cocultures of human aortic wall cells involves interleukins 1 and 6 with marked increases in connexin43 message. J Clin Invest 1991; 87: 1763-72.
    CrossrefPubMedSearch in Google Scholar
  • 25 Laird DW, Puranam KL, Revel JP. Turnover and phosphorylation dynamics of connexin43 gap junction protein in cultured cardiac myocytes. Biochem J 1991; 273: 67-72.
    CrossrefPubMedSearch in Google Scholar
  • 26 Gospodarowicz D, Greenburg G, Foidart JM. et al. The production and localization of laminin in cultured vascular and corneal endothelial cells. J Cell Physiol 1981; 107: 171-83.
    CrossrefPubMedSearch in Google Scholar
  • 27 Raines EW. The extracellular matrix can regulate vascular cell migration, proliferation, and survival: relationships to vascular disease. Int J Exp Pathol 2000; 81: 173-82.
    PubMedSearch in Google Scholar
  • 28 Davidson JM, LuValle PA, Zoia O. et al. Ascorbate differentially regulates elastin and collagen biosynthesis in vascular smooth muscle cells and skin fibroblasts by pretranslational mechanisms. J Biol Chem 1997; 272: 345-52.
    CrossrefPubMedSearch in Google Scholar
  • 29 Yoshida T, Owens GK. Molecular determinants of vascular smooth muscle cell diversity. Circ Res 2005; 96: 280-91.
    CrossrefPubMedSearch in Google Scholar
  • 30 Smith EB, Staples EM. Distribution of plasma proteins across the human aortic wall--barrier functions of endothelium and internal elastic lamina. Atherosclerosis 1980; 37: 579-90.
    CrossrefPubMedSearch in Google Scholar
  • 31 Chobanian AV, Menzoian JO, Shipman J. et al. Effects of endothelial denudation and cholesterol feeding on in vivo transport of albumin, glucose, and water across rabbit carotid artery. Circ Res 1983; 53: 805-14.
    CrossrefPubMedSearch in Google Scholar
  • 32 Ramirez CA, Colton CK, Smith KA. et al. Transport of 125I-albumin across normal and deendothelialized rabbit thoracic aorta in vivo . Arteriosclerosis 1984; 04: 283-91.
    CrossrefPubMedSearch in Google Scholar
  • 33 Cristofalo VJ, Sharf BB. Cellular senescence and DNA synthesis. Thymidine incorporation as a measure of population age in human diploid cells. Exp Cell Res 1973; 76: 419-27.
    CrossrefPubMedSearch in Google Scholar
  • 34 Nicholson DW, Ali A, Thornberry NA. et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 1995; 376: 37-43.
    CrossrefPubMedSearch in Google Scholar
  • 35 Gerszten RE, Garcia-Zepeda EA, Lim YC. et al. MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature 1999; 398: 718-23.
    CrossrefPubMedSearch in Google Scholar
  • 36 Ryoo SW, Kim DU, Won M. et al. Native LDL induces interleukin-8 expression via H2O2, p38 Kinase, and activator protein-1 in human aortic smooth muscle cells. Cardiovasc Res 2004; 62: 185-93.
    CrossrefPubMedSearch in Google Scholar
  • 37 Steinberg D. Atherogenesis in perspective: hypercholesterolemia and inflammation as partners in crime. Nat Med 2002; 08: 1211-7.
    CrossrefPubMedSearch in Google Scholar