Quantitation by Radioimmunoassay of Thrombospondin Synthesised and Secreted by Human Endothelial Cells

Summary

Synthesis and secretion of thrombospondin by cultured human endothelial cells was quantified using a sensitive radioimmunoassay. Two endothelial lines were examined; those from umbilical veins and those from saphenous veins. The foetal line secreted ̴17 μg/10⁶ cells of the antigen at 24 hr, whereas the saphenous line secreted five times less in the same period. Intracellular values were similar in both lines.

Thrombin and calcium ionophore A23187 were added to umbilical vein endothelial cultures. In contrast to platelets where both agents cause a rapid release reaction, no such thrombospondin release was observed in endothelial cells.

Key words

Radioimmunoassay - Thrombospondin - Endothelial cells

• References

• 1 Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. J Clin Invest 1973; 52: 2745-2756
• 2 Baezniger NL, Brodie GN, Majerus PW. Isolation and properties of a thrombin sensitive protein of human platelets. J Biol Chem 1972; 247: 2723-2731
• 3 Jaffe EA, Leung LL K, Nachman RL, Levin RI, Mosher DF. Thrombospondin is the endogenous lectin of human platelets. Nature 1982; 295: 246-248
• 4 Gartner TK, Dockter ME. Secreted platelet thrombospondin binds monovalently to platelets and erythrocytes in the absence of free Ca⁺⁺ . Thromb Res 1984; 33: 19-31
• 5 Lawler JW, Slayter HS, Coligan JE. Isolation and characterisation of a high molecular weight glycoprotein from human blood platelets. J Biol Chem 1978; 253: 8609-8616
• 6 Margossian SS, Lawler JW, Slayter HS. Physical characterisation of platelet thrombospondin. J Biol Chem 1981; 256: 7495-7500
• 7 McPherson J, Sage H, Bornstein P. Isolation and characterisation of a glycoprotein secreted by aortic endothelial cells in culture - apparent identity with platelet thrombospondin. J Biol Chem 1981; 256: 11330-11336
• 8 Sage H, Pritz LP, Bomstein P. Secretory phenotypes of endothelial cells in culture; comparison of aortic, venous, capillary and corneal endothelium. Arteriosclerosis 1981; 1: 427-442
• 9 Mosher DF, Doyle MJ, Jaffe EA. Synthesis and secretion of thrombospondin by cultured human endothelial cells. J Biol Chem 1982; 93: 343-348
• 10 Dawes J, Clemetson KJ, Gogstad GO, McGregor J, Clezardin P, Prowse CV, Pepper DS. A radioimmunoassay for thrombospondin, used in a comparative study of thrombospondin, β-thromboglobulin and platelet factor 4 in healthy volunteers. Thromb Res 1983; 29: 569-581
• 11 Loesberg C, Gonsalves MD, Zandbergen J, Willems C, Van Aken WG, Stel HV, Van Mourik JA, de Groot PGxs. The effect of calcium on the secretion of factor VIII related antigen by cultured human endothelial cells. Biochim Biophys Acta 1983; 763: 160-168
• 12 Levine JD, Harlan JM, Harker LA, Joseph ML, Count RB. Thrombin mediated release of factor VIII related antigen from human umbilical vein endothelial cells in culture. Blood 1982; 60: 531-534
• 13 Maciag T, Cerundolo J, Ilsley S, Kelley PR, Forand R. An endothelial cell growth factor from bovine hypothalamus: identification and partial characterisation. Proc Natl Acad Sci USA 1979; 76: 5674-5678
• 14 Glassberg MK, Bern MM, Coughlin SR, Haudenschild CC, Hoyer LW, Antoniades HN, Zetter BR. Cultured endothelial cells derived from human iliac arteries. In Vitro 1982; 18: 859-866
• 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 Naito S, Sueishi K, Hattori F, Tanaka K. Immunological analysis of plasminogen activators from cultured human cancer cells. Virchow Arch Acta 1980; 387: 251-257
• 17 Johnson AR. Human pulmonary endothelial cells in culture. J Clin Invest 1980; 65: 841-850
• 18 Ritchie AW S, James K, Micklem HS. The distribution and possible significance of cells identified in human lymphoid tissue by monoclonal antibody HNK-1. Clin Exp Immunol 1983; 51: 439-477
• 19 Hampson IN. Production of proteoglycans by cultured human neuroblastoma cells. PhD Thesis 1982. University of Manchester;
• 20 De Groot PG, Strijland A, Kalbeek R, Meera Khan P, Westerveld A, Hammers NN, Tager JM. Effect of 2-deoxyglucose on lysosomal enzyme in cultured human skin fibroblasts. Exp Cell Res 1980; 126: 207-216
• 21 Raugi GJ, Mumby SM, Abbott-Brown D, Bornstein P. Thrombospondin: synthesis and secretion by cells in culture. J Cell Biol 1982; 95: 351-354
• 22 Hunter NR, Dawes J, MacGregor IR, Pepper DS. Synthesis and secretion of thrombospondin, plasminogen activators, and heparan SO₄ by endothelial and other cell types. Thromb Haemostas 1983; 50: 296 (Abstr.)
• 23 Fry G, Parsons T, Hoak J, Sage H, Gingrich RD, Ercolani L, Nghiem D, Czervionke R. Properties of cultured endothelium from adult human vessels. Arteriosclerosis 1984; 4: 4-13
• 24 Ager A, Gordon JL, Moncada S, Pearson JD, Salmon JA, Trevethick MA. Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial cells and smooth muscle cells. J CeU Physiol 1982; 110: 9-16
• 25 Hunter NR, MacGregor IR, McArthur MM, Taylor PM, Prowse CV, Pepper DS. Tissue plasminogen activator measured by radioimmunoassay in human endothelial cell cultures. In: Progress in Fibrinolysis. Davidson J. (Ed) Churchill Livingstone; 1984. 7: (in press)
• 26 Del Vecchio PJ, Smith J. Ageing of endothelium in culture: decrease in angiotensin converting-enzyme activity. Cell Biol Int Rep 1982; 6: 379-384