Summary
Work on endothelial cells has been limited by the availability of procedures for obtaining such cells in quantities adequate for direct in vitro analysis. The present paper describes a method for the isolation of endothelial cells from bovine cavernous bodies. A number of cells ranging from 2.5 to 4 × 108 per animal has been obtained. The cells were identified as follows 1) presence of the “Weibel and Palade” bodies in the isolated cells, 2) “cobblestone” appearance of cell cultures, and 3) presence of factor VIII, as demonstrated by immunofluorescence assays. The cell viability at the end of the purification procedure was tested 1) by dye-exclusion tests and 2) by metabolic assays. Features of this preparation are 1) the very high yield of viable endothelial cells, 2) the absence of contamination by fibroblasts and smooth muscle cells and a very low contamination by erythrocytes and 3) the fine dispersion of the isolated cells. These properties allow functional and subcellular fractionation studies on freshly isolated endothelial cells of microvascular origin.
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Ager A, Gordon JL, Moncada S, Pearson JD, Salmon JA, Trevethick MA (1982) Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial and smooth muscle cells. J Cell Physiol 110:9–16
Beesley JE, Pearson JD, Hutchings A, Carleton JS, Gordon JL (1979) Granulocyte migration through endothelium in culture. J Cell Sci 38:237–245
Blose SH, Chacko S (1975) In vitro behavior of guinea pig arterial and venous endothelial cells. Dev Growth Differ 17:153–161
Booyse FM, Sedlak BG, Rafelson ME Jr (1975) Culture of arterial endothelial cells: characterization and growth of bovine aortic cells. Thromb Diath Hemorrh 34:825–839
Brendel K, Meezan E, Carlson EC (1974) Isolated brain microvessels: a purified, metabolically active preparation from bovine cerebral cortex. Science 185:953–955
Buonassisi V, Venter JC (1976) Hormone and neurotransmitter receptors in an established vascular endothelial cell line. Proc Natl Acad Sci USA 73:1612–1616
Cotta-Pereira G, Sage H, Bornstein P, Ross R, Schwartz S (1980) Studies of morphologically atypical (“Sprouting”) cultures of bovine aortic endothelial cells. Growth characteristics and connective tissue protein synthesis. J Cell Physiol 102:183–191
Davison PM, Bensch K, Karasek MA (1980) Growth and morphology of rabbit marginal vessel endothelium in cell culture. J Cell Biol 85:187–198
De Leeuw AM, Barelds RJ, de Zanger R, Knook DL (1982) Primary cultures of endothelial cells of the rat liver. A model for ultrastructural and functional studies. Cell Tissue Res 223:201–215
Denny JB, Johnson AR (1979) Uptake of 125I-labelled C3a by cultured human endothelial cells. Immunology 36:169–177
Dosne AM, Dupuy E, Bodevin E (1978) Production of a fibrinolytic inhibitor by cultured endothelial cells derived from human umbilical vein. Thromb Res 12:377–387
Dym M (1977) Male genital system. In: Weiss L, Greep RO (eds) Histology. 4th edn, McGraw-Hill Inc., New York, p 1033–1035
Gimbrone MA Jr (1976) Culture of vascular endothelium. In: Spaet TH (ed) Progress in hemostasis and thrombosis Vol III. Grune & Stratton Inc., New York, p 1–28
Gimbrone MA Jr, Cotran RS, Folkman J (1974) Human vascular endothelial cells in culture. Growth and DNA synthesis. J Cell Biol 60:673–684
Gospodarowicz D, Brown KD, Birdwell LR, Zetter BR (1978) Control of proliferation of human vascular endothelial cells. Characterization of the response of human unbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin. J Cell Biol 77:774–788
Haudenschild CC, Cotran RS, Gimbrone MA Jr, Folkman J (1975) Fine structure of vascular endothelium in culture. J Ultrastr Res 50:22–32
Hoover RL, Folger R, Haering WA, Ware BR, Karnovsky MJ (1980) Adhesion of leukocytes to endothelium: roles of divalent cations, surface charge, chemotactic agents and substrate. J Cell Sci 45:73–86
Jaffe EA, Nachman RL, Becker CG, Minick CR (1973) Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52:2745–2756
Jaffe EA, Hoyer LW, Nachmann RL (1973) Synthesis of antihemophilic factor antigen by cultured human endothelial cells. J Clin Invest 52:2757–2764
Johnson AR (1980) Human pulmonary endothelial cells in culture. Activities of cells from arteries and cells from veins. J Clin Invest 65:841–850
Lai FM, Udenfriend A, Spector S (1975) Presence of nonepinephrine and related enzymes in isolated brain microvessels. Proc Natl Acad Sci USA 72:4622–4625
Linder E (1981) Binding of C1a and complement activation by vascular endothelium. J Immunol 126:648–658
Lockwood WR (1964) A reliable and easily sectioned epoxy embedding medium. Anatomical Record 150:129–140
MacIntyre DE, Pearson JD, Gordon JL (1978) Localization and stimulation of prostacyclin production in vascular cells. Nature 271:549–551
MacGregor R, Macarack E, Kefalides N (1978) Comparative adherence of granulocytes to endothelial monolayers and nylon fiber. J Clin Invest 61:697–702
Magargal WW, Dickinson ES, Slakey LL (1978) Distribution of membrane marker enzymes in cultured arterial and smooth muscle cells. The subcellular location of oleoyl-CoA: 1-acyl- sn-glycero-3-phosphocholine acyltransferase. J Biol Chem 253:8311–8318
Murata K, Quilligan JJ Jr, Morrison LM (1965) Growth of chicken aortic endothelial cells: incorporation of tritiated uridine and thymidine. Experientia 21:637–638
Nemecek GM (1980) Properties of adenylate cyclase and cyclic nucleotide phosphodiesterase in hamster isolated capillary preparations. Biochim Biophys Acta 628:125–135
Pearson JD, Gordon JL (1979) Vascular endothelial and smooth muscle cells in culture selectively release adenine nucleotides. Nature 281:384–386
Romeo D, Zabucchi G, Marzi T, Rossi F (1973) Kinetic and enzymatic features of metabolic stimulation of alveolar and peritoneal macrophages challenged with bacteria. Exp Cell Res 78:423–432
Ryan US, Clements E, Habliston D (1978) Isolation and culture of pulmonary artery endothelial cells. Tissue Cell 10:535–554
Schwartz SM, Benditt EP (1972) Studies on aortic intima. I. Structure and permeability of rat thoracic aortic intima. Am J Pathol 66:241–264
Shepro D, Schleef R, Hechtman HB (1980) Plasminogen activator activity by cultured bovine aortic endothelial cells. Life Sci 26:415–422
Silver MG (1981) Mechanisms of hemostasis and therapy of thrombosis: new concepts based on the metabolism of arachidonic acid by platelets and endothelial cells. Adv Pharmacol Chemother 18:1–48
Slater DN, Sloan JM (1975) The porcine endothelial cell in tissue culture. Atherosclerosis 21:259–272
Venables JH, Coggeshall R (1965) A simplified lead citrate stain for use in electron microscopy. J Cell Biol 25:407–408
Wagner RC, Kreiner P, Barnett RJ, Bitensky MW (1972) Biochemical characterization and cytochemical localization of a catecholamine-sensitive adenylate cyclase in isolated capillary endothelium. Proc Natl Acad Sci USA 69:3175–3179
Wechezak AR, Mansfield PB (1973) Isolation and growth characteristics of cell lines from bovine venous endothelium. In Vitro 9:39–45
Weibel ER, Palade GE (1964) New cytoplasmic components in arterial endothelia. J Cell Biol 23:101–112
Weksler BB, Marcus AJ, Jaffe EA (1977) Synthesis of prostaglandin I2 (PGI2 or prostacyclin) by cultured human and bovine endothelial cells. Proc Natl Acad Sci USA 74:3922–3926
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Dobrina, A., Soranzo, M.R. & Rossi, F. Isolation of metabolically active endothelial cells in high yield from bovine cavernous bodies. Cell Tissue Res. 232, 579–591 (1983). https://doi.org/10.1007/BF00216430
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DOI: https://doi.org/10.1007/BF00216430