A Simple In Vitro Model of Mechanical Injury of Confluent Cultured Endothelial Cells to Study Quantitatively the Repair Process

Summary

A model of in vitro mechanical injury of confluent human endothelial cells (EC) in culture was developed. Human EC were obtained from umbilical veins and grown to confluence. Application on the EC monolayer of a calibrated disk of cellulose poly acetate paper resulted in removal of the EC, leaving a continuous subendothelial extracellular matrix (ECM) on the culture dish. The regeneration time depended on the original size of the lesion. Regeneration was similar with EC grown on different substrates such as human fibronectin, human subendothelial ECM, bovine collagen type I or surfaces coated with Transglutine®, a surgical glue containing adhesive proteins. A human brain extract containing growth factor activity accelerated significantly the repair of the lesion, especially at low serum concentration. This simple in vitro model of mechanical injury allows the quantitative study of the effects of matrices, growth factors and pharmacological agents on the repair process.

Key words

Endothelium - Injury - Growth factor - Extracellular matrix-Pharmacology

• References

• 1 Cazenave JP, Klein-Soyer C, Beretz A. The vascular endothelium. An important site of blood-tissue exchanges. Inter Angio 1984; 3: 27-32
• 2 Haudenschild C. Growth control of endothelial cells in atherogenesis and tumor angiogenesis. Adv Microcric 1980; 9: 226-251
• 3 Reidy MA, Schwartz SM. Recent advances in molecular pathology. Arterial endothelium. Assessment of in vivo injury. Exp Molec Pathol 1984; 41: 419-434
• 4 Schwartz SM, Selden SC III, Bowman P. Growth control in aortic endothelium at wound edges. In: Hormones and Cell Culture Sato GH, Ross R. (Eds). Cold Spring Harbor Conference on Cell Proliferation Cold Spring Harbor Laboratory; 1979. 6A 593-610
• 5 Sholley MM, Gimbrone MA, Cotran RB. Cellular migration and replication in endothelial regeneration. Lab Invest 1977; 36: 18-25
• 6 Maca RD, Fry GL, Hoak JC, Loh PT. The effect of intact platelets on cultured human endothelial cells. Thrombos Res 1977; 11: 715-727
• 7 Madri JA, Stenn KS. Aortic endothelial cell migration. I. Matrix requirements and composition. Am J Pathol 1982; 106: 180-185
• 8 Gospodarowicz D, Cheng J, Hirabayashi K, Tauber JP. The extracellular matrix and the control of vascular endothelial and smooth muscle cell proliferation. In: Cellular Interactions. Dingle & Gordon (Eds) Elsevier/North-Holland Biomedical Press,; 1981: 135-165
• 9 Klein-Soyer C, Stierlé A, Bouderbala B, Cazenave JP. Effects of an extract of human brain containing growth factor activity on the proliferation of human vascular endothelial cells in primary culture. Biol Cell 1984; 52: 9-20
• 10 Jaffe EA. Culture of human endothelial cells. Transplant Proc 1980; 12 Suppl (Suppl. 01) 49-53
• 11 Willems C, Astaldi GC B, De Groot PG, Janssen MC, Gonsalvez MD, Zeijlemaker WP, van Mourik JA, van Aken WG. Media conditioned by cultured human vascular endothelial cells inhibit the growth of vascular smooth muscle cells. Exp Cell Res 1982; 139: 191-197
• 12 Ruoslahti E, Vuento M, Engvall E. Interaction of fibronectin with antibodies and collagen in radioimmunoassay. Biochem Biophys Acta 1978; 534: 210-218
• 13 Gospodarowicz D. 111 C. Extracellular matrix and control of proliferation of vascular endothelial cells. J Clin Invest 1980; 65: 1351-1364
• 14 Greenburg G, Gospodarowicz D. Inactivation of a basement membrane component responsible for cell proliferation but not for cell attachment. Exp Cell Res 1982; 140: 1-14
• 15 Kramer RH, Vogel KG, Nicolson GL. Solubilization and degradation of subendothelial matrix glycoproteins and proteoglycans by metastatic tumor cells. J Biol Chem 1982; 257: 2678-2686
• 16 Goetschy JF, Ulrich G, Aunis D, Ciesielski-Treska J. The organization and solubility properties of intermediate filaments and microtubules of cortical astrocytes in culture. J Neurocytol 1986; 15: 375-387
• 17 Pugatch EM J, Saunders AM. A new technique for making haiitchen preparations of unfixed aortic endothelium. J Atheroscler Res 1968; 8: 735-738
• 18 Pugatch EM J, Foster EA, MacFarlane DE, Poole JC F. The extraction and separation of activators and inhibitors of fibrinolysis from bovine endothelium and mesothelium. Brit J Haemat 1970; 18: 669-681
• 19 Buchanan MR, Dejana E, Cazenave JP, Richardson M, Mustard JF, Hirsh J. Differences in inhibition of PGI₂ production by aspirin in rabbit artery and vein segments. Thrombos Res 1980; 20: 447-460
• 20 Smith U, Ryan JW. Electron microscopy of endothelial cells collected on cellulose acetate-paper. Tissue and Cell 1973; 5: 333-336
• 21 Wittendorp-Rechenmann E, Klein-Soyer C, Cazenave JP, Rechenmann RV. A high efficiency autoradiographic method to study “in vitro” regeneration of human endothelial cell lesions implicated in thrombosis and atherosclerosis. 12th Int. Conf. Solid State Nuclear Track Detectors. Mexico, Sept 4-10 1983;
• 22 Ross R. Atherosclerosis: a problem of the biology of arterial wall cells and their interactions with blood components. Atherosclerosis 1981; 1: 293-311
• 23 McGill Jr HC. Persistent problems in the pathogenesis of atherosclerosis. Atherosclerosis 1984; 4: 443-451
• 24 Schwartz SM. Role of endothelial integrity in atherosclerosis. Artery 1980; 8: 305-314
• 25 Schwartz SM. Vascular integrity. In: Biologic and Synthetic Vascular Prostheses Stanley JC. (Ed) New York: Grune & Stratton; 1982: 27-36
• 26 Cazenave JP, Klein-Soyer C, Ebel C, Beretz A, Hemmendinger S, Grunebaum L. Etude quantitative de l’interaction des plaquettes humaines avec les compos-ants de la paroi vasculaire en culture: cellules endotheliales (CE), sous endothelium (SE) et cellules musculaires lisses (CML). Arch Mai Coeur Vais 1983; 76: 80
• 27 Sakariassen KS, Aarts PA M M, De Groot PG, Houdijk WP M, Sixma JJ. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix and purified component. J Lab Clin Med 1983; 102: 522-535