Cultured Endothelial Cells Regulate Platelet Adhesion to their Extracellular Matrix by Regulating its von Willebrand Factor Content

Summary

Endothelial cells and their extracellular matrix formed in vitro are often used as a model for subendothelium in studies on platelet-vessel wall interaction. We have characterized the influence of culture conditions of endothelial cells on the formation of extracellular matrix and on the interaction of the matrix with platelets. Passage number, time of confluence, serum concentration and the addition of heparin, growth factors and antibiotics to the culture medium were varied and the extracellular matrices were isolated. The amount of fibronectin and von Willebrand factor present in the matrix were measured and the number of platelets adhering to these matrices after perfusion with citrated whole blood at a shear rate of 1000 s^-l was determined. A three times increase of the amount of von Willebrand factor in the matrix was found when the serum concentration was increased from 2.5% to 30%. When the passage number of the cells was increased or the period during which the cells were at confluence was extended, the amount of von Willebrand factor in the matrix was decreased up to 50%. Addition of heparin or ECGS (endothelial cell growth supplement) decreased the von Willebrand factor content in the matrix. Addition of penicillin or streptomycin to the culture medium had no influence on the amount of von Willebrand factor deposited in the matrix or secreted into the medium, however, other antibiotics such as gentamycin and neomycin decrease the amount of von Willebrand factor in the matrix. No influence on the amount of fibronectin in the matrix was found under all conditions tested.

There was a strong correlation between the amount of von Willebrand factor in the matrix and the number of platelets adhering to the matrix. A decrease or increase of the amount of von Willebrand factor was always correlated with a decrease or increase of the number of platelets adhering to the matrix. These results indicate that the synthesis and deposition of von Willebrand factor in the extracellular matrix by cultured endothelial cells is very sensitive to variations in culture conditions and that the amount of von Willebrand factor in the matrix predominantly determines the reactivity of the matrix for platelets.

• References

• 1 Harker LA, Schwartz SM, Ross R. Endothelium and arteriosclerosis. Clin Haematol 1991; 10: 283-296
• 2 Ross RR. The pathogenesis of atherosclerosis. An update N Eng J Med 1986; 314: 488-500
• 3 De Groot PhG, Sixma JJ. Platelet-vessel wall interaction. Vase Med Rev 1993; 3: 83-93
• 4 Sixma JJ, Pronk A, Nievelstein PF E M, Zwaginga JJ, Hindriks GA, Tijburg PN M, Banga JD, De Groot PhG. Platelet adhesion to extracellular matrices of cultured cells. Ann New York Acad Sei 1991; 614: 181-92
• 5 Booyse FM, Quarfoot AJ, Feder S. Culture-produced subendothelium. I. Platelet interaction and properties. Haemostasis 1982; 11: 49-61
• 6 Vlodavski IA, Eldor A, Hyam R, Atzmon R, Fuks Z. Platelet interaction with the extracellular matrix produced by cultured endothelial cells: a model to study the thrombogenicity of isolated subendothelial basal lamina. Thromb Res 1982; 28: 179-191
• 7 Sakariassen KS, Banga JD, De Groot PhG, Sixma JJ. Comparison of platelet interaction of subendothelium of human renal and umbilical arteries and the extracellular matrix produced by human venous endothelial cells. Thromb Haemost 1984; 52: 60-65
• 8 Aznar-Salatti J, Bastida E, Haas TA, Escolar G, Ordinas A, de Groot PhG, Buchanan MR. Platelet adhesion to exposed endothelial cell extracellular matrixes is influenced by the method of preparation. Arterioscler Thromb 1991; 11: 436-442
• 9 Gabrowski EF. Platelet aggregation in flowing blood at a site of injury to an endothelial cell monolayer: quantitation and real time imaging with a TAB monoclonal antibody. Blood 1990; 75: 390-398
• 10 Van Hinsbergh VW M, Scheffer MA, Langeler EG. Macro and micro endothelial cells from human tissue. In: Cell culture techniques in heart and vessel research. Piper HM. (ed) Berlin, Heidelberg, New York, London, Paris, Tokyo, Hong Kong: Springer Verlag; 1990: 178-204
• 11 Jaffe EA. Culture of human endothelial cells. Transplant Proc 1980; 12: 49-53
• 12 Iruela-Arispe ML, Diglio CA, Sage EH. Modulation of extracellular matrix proteins by endothelial cells undergoing angiogenesis in vitro. Arterioscler Thromb 1991; 11: 805-815
• 13 Carey DJ. Control of growth and differentiation of vascular cells by extracellular matrix proteins. Annu Rev Physiol 1991; 53: 161-177
• 14 Ingber DE, Folkman J. Inhibition of angiogenesis through modulation of collagen metabolism. Lab Invest 1988; 59: 44-51
• 15 Maciag T, Hoover GA, Stemerman MB, Weinstein R. Serial propagation of human endothelial cells in vitro. J Cell Biol 1981; 91: 420-426
• 16 Maciag T, Cerundolo J, Ilsley S, Kelley PR, Forand R. An endothelial cell growth factor from bovine hypothalamus: identification and partial characterization. Proc Natl Acad Sci USA 1979; 76: 5674-5678
• 17 Willems CH, Astaldi GC B, De Groot PhG, Janssen MC, Gonsalves 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
• 18 Sixma JJ, Nievelstein PF E M, Zwaginga JJ, De Groot PhG. Adhesion of blood platelets to the extracellular matrix of cultured human endothelial cells. Ann New York Acad Sei 1987; 516: 22-38
• 19 Gospodarowicsz III DC. Extracellular matrix and control of proliferation of vascular endothelial cells. J Clin Invest 1980; 65: 1351-1364
• 20 Gospodarowicsz D, Gonsalez R, Fuji DK. Are factors originating from serum, plasma or cultured cells involved in the growth-promoting effect of the extracellular matrix produced by cultured bovine corneal endothelial cells. J Cell Physiol 1983; 144: 191-202
• 21 Sakariassen KS, Aarts PA M M, De Groot PhG, Houdijk WP, Sixma JJ. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. J Lab Clin Med 1983; 102: 522-536
• 22 Sixma JJ, Nievelstein PF E M, Houdijk W PM, Van Breugel HH F I, Hindriks GA, De Groot PhG. Adhesion of blood platelets to isolated components of the vessel wall. Ann New York Acad Sci 1987; 509: 103-117
• 23 Stel HV, Sakariassen KS, Scholte BJ, Veerman EC I, Van der Kwast ThJ, De Groot PhG, Sixma JJ, Van Mourik JA. Characterization of 25 monoclonal antibodies to factor VUI-von Willebrand factor: relationship between ristocetin-induced platelet aggregation and platelet adherence to subendothelium. Blood 1984; 63: 1408-1416
• 24 De Groot PhG, Reinders JH, Sixma JJ. Pertubation of human endothelilal cells by thrombin or PMA changes the reactivity of their extracellular matrix towards platelets. J Cell Biol 1987; 104: 697-704
• 25 Stel HV, Sakariassen KS, De Groot PhG, Van MourikJ, Sixma JJ. von Willebrand factor in the vessel wall mediates platelet adhesion. Blood 1985; 65: 85-90
• 26 Houdijk WP, De Groot PhG, Nievelstein PF E M, Sakariassen KS, Sixma JJ. Subendothelial proteins and platelet adhesion: von Willebrand factor and fibronectin not thrombospondin are involved in platelet adhesion to extracellular matrix of human vascular endothelial cells. Arteriosclerosis 1986; 6: 24-33
• 27 Hindriks GA, Sixma JJ, De Groot PhG. Ascorbic acid increases the thrombogenicity of cellular matrices. Thromb and Haemost 1991; 66: 505-509
• 28 Hindriks GA, IJseldijk MJ W, Sonnenberg A, Sixma JJ, De Groot PhG. Platelet adhesion to laminin: Role of Ca²⁺and Mg²⁺ions, shear rate and platelet membrane glycoproteins. Blood 1992; 79: 928-935
• 29 Nievelstein PF E M, De Groot PhG, D’Alessio P, Heijnen HF, Orlando E, Sixma JJ. Platelet adhesion to vascular cells: The matrix of cultured fibroblasts as a model to study the role of exogenous von Willebrand factor in platelet adhesion. Arteriosclerosis 1990; 10: 462-469
• 30 Aznar-Salatti J, Diaz-Ricart M, Anton P, Bastida E, Escolar G, Ordinas A. Variaciones en las propiedades thrombogenicas de la matriz extracellular producida por celulas endoteliales en el curso de siembras conecutivas. Sangre Bare 1990; 35: 173-177
• 31 Johnson TE, Umbenhauer DR, Hill R, Bradt C, Mueller SN, Levine EM, Nichols WW. Karyotypic and phenotypic changes during in vitro aging of human endothelial cells. J Cell Physiol 1992; 150: 17-27
• 32 Canfield AE, Wren FE, Schor SL, Grant ME, Schor AM. Aortic endothelial cell heterogeneity in vitro. Lack of association between morphological phenotype and collagen synthesis J Cell Sci 1993; 102: 807-814
• 33 Blann AD. Increased release of von Willebrand factor antigen by endothelial cells whilst in active growth phase. Cell Biol Int Rep 1991; 15: 493-498
• 34 Chersh DA. Human endothelial cells synthesize and express an RGd directed adhesion receptor involved in attachment to fibrinogen and von Willebrand factor. Proc Natl Acad Sci USA 1987; 84: 6471-6475
• 35 Ondrik K, Samojla BC. Angiogenesis. Clin Pediatr Med Surg 1992; 9: 185-202
• 36 Ingber D, Fujita T, Kishimoto S, Sudo K, Kanamaro T, Bren H, Folkman J. Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumor growth. Nature 1990; 348: 555-557
• 37 Brown ML, Deykin D. Passage state affects arachidonic acid content and eicosanoid release in porcine aortic endothelial cells. Arterioscler Thromb 1991; 11: 167-173
• 38 Ryan US, Clements E, Habliston D, Ryan JW. Isolation and culture of pulmonary artery endothelial cells. Tissue Cell 1978; 10: 535-554
• 39 Adragna NC. Changes in cation composition of aortic endothelial cells in culture. Tissue Cell 1988; 20: 313-329
• 40 Gerlach H, Lieberman H, Bach R, Godman G, Brett J, Stern D. Enhanced responsiveness of endothelium in the growin/motile state to tumor necrosis factor/cachetin. J Exp Med 1989; 170: 913-931
• 41 Shimokawa H, Flavahan NA, Shepherd JT, Vanhoutte PM. Endothelium dependent inhibition of ergonovine induced contraction is impaired in porcine coronary endothelium with regenerating endothelium. Circulation 1989; 80: 643-650
• 42 Folkman J, Moscona A. Role of cell shape in growth control. Nature 1978; 273: 345-349