Bovine vascular endothelial cells do not grow when cultured at low density unless fibroblast growth factor is included in the culture medium. When endothelial cells obtained from the intimal surface of fetal and adult aortas were seeded at low density (8 cells per cm2), they formed small colonies of large, irregular, vacuolated cells. At very low density (0.3 cells per cm2) they did not survive. The addition of fibroblast growth factor to endothelial cells maintained at such low densities resulted in the formation of vigorously growing colonies of small, uniform cells. Electron microscopy showed that the cultured endothelial cells had the fine structure characteristics of endothelial cells. Immunofluorescence microscopy revealed antihemophilic factor (Factor VIII) antigen in the cells. Our results demonstrated that fibroblast growth factor permits the survival of endothelial cells plated at extremely low cell density. With the use of fibroblast growth factor, endothelial cell clones are easily produced. Endothelial cells constitute the inner lining of the blood vascular system. Because of their location at the interface between blood and tissue, they are the chief elements involved in the permeability of blood vessels (1, 2). Abnormalities of endothelial cell structure and function are prominent in the pathology of a number of diseases of blood vessel walls such as thromboangiitis (3) and microangiopathy (4).Since the continuity of the vascular endothelium is essential for the survival of the organism, the elucidation of the factors involved in endothelial cell survival and proliferation is important. Their survival and proliferation can be examined most easily in tissue culture.Recently, several factors that promote the growth of cells in culture have been identified. One of the most potent is the fibroblast growth factor (FGF) which stimulates the growth of a variety of mesoderm-derived cells (5).Three observations have led us to examine the effect of FGF on vascular endothelial cells: (i) FGF is a potent mitogen for Balb/c 3T3 cells (6). Although these cells are commonly referred to as fibroblasts, their morphology and the fact that they can produce vasoformative sarcomas in vivo (7) Dulbecco's modified Eagle's medium (DME) supplemented with 10% calf serum (Gibco) and 100 ng/ml of FGF in an atmosphere of 12% CO2 in air to maintain the pH of the medium between pH 7.3 and 7.5. Bovine fetuses of 4 months' gestation (45 cm, crown to rump), bovine adult aortic arches, and bovine umbilical cords were obtained from a local slaughterhouse.Endothelial Cell Culture. The aortic arches were opened lengthwise with a scalpel and the intimal surface was washed with Ca++-free phosphate-buffered saline to remove blood. The endothelial cell layer was removed by gently scraping the intimal surface with a grooved director. The grooved director was dipped in 10 ml of DME with 10% calf serum. This technique gave cell populations composed of 99% endothelial cells.For purer populations, a cotton swab was used instead of a ...

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We have examined bovine aortic endothelial cell cultures for the presence of fibronectin, a high molecular weight cell-surface glycoprotein. Sparse cultures contain fibronectin only on dorsal cell surfaces at regions of cell-cell contact, as detected by immunofluorescence. In contrast, when the endothelial cells reached confluence as a highly contactinhibited monolayer, fibronectin was detected in an extracellular matrix underneath the cell monolayer but not on top of the monolayer. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of isolated extracellular matrix revealed that a predominant component of the matrix is a protein of approximately 2.3 X 105 molecular weight,.which has been identified as fibronectin.The vascular endothelium exists as a monolayer of highly flattened and contact-inhibited cells; because of their location at the interface between blood and tissue, endothelial cells are the chief elements involved in the permeability of blood vessels (1, 2). Although the side of the vascular endothelium exposed to the bloodstream is nonthrombogenic, the subendothelial matrix on which the cells rest is very thrombogenic (3,4). Thus, a disruption of the vascular endothelium can expose the underlying basement membrane, resulting.in the aggregation of platelets and thrombus formation. Therefore, factors that are involved in the attachment of endothelial cells to the basement membrane are very important for the proper functioning of the vascular endothelium.Biochemical analyses have established that basement membranes possess a highly cross-linked form of collagen (5), rendering basement membranes completely insoluble under physiological conditions. Although most biochemical studies of basement membranes have dealt with the structure and synthesis of collagen, noncollagenous matrix glycoproteins have also been found in some basement membranes (6). Recent studies indicate that one of these glycoproteins may be identical to fibronectin (7), a major cell-surface glycoprotein that is immunologically identical to cold-insoluble globulin (8), a plasma protein which is presumably the plasma form of fibronectin that is shed from cells into the blood. Fibronectin appears to be similar, if not identical, to the large external transformationsensitive (LETS) protein, which exists on the surface of certain untransformed cells, but not on the surface of transformed cells (9)(10)(11) (21), and we also show that fibronectin is a major component of the extracellular matrix produced by ABAE cells. METHODSCell Culture. The cloning, culturing, and characterization of ABAE cells have been described (21). ABAE cells are identified as endothelial cells on the basis of their morphology and ultrastructure and on their ability to synthesize Factor VIII (antihemophilic factor antigen), a marker for endothelial cells (22). These cells are routinely maintained in fibroblast growth factor (23).Electron Microscopy. Cells were prepared for electron microscopy by described methods (24), and then examined in a Hitachi S-500 ...

Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium. When adsorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion; they also stimulate the rate of cell-cell aggregation. Adheron-stimulated adhesion is tissue specific, and the spontaneous aggregation of neural retina cells is inhibited by monovalent Fab' fragments prepared from an antiserum against neural retina adherons. Therefore cell surface antigenic determinants shared with adherons are involved in normal cell-cell adhesions. The particles from the heterogeneous neural retina population contain many proteins and several glycosaminoglycans. The adherons migrate as a symmetrical 12S peak on sucrose gradients and are predominantly 15-nm spheres when examined by electron microscopy. Finally, the specific activity of neural retina adherons increases from embryonic days 7 through 12 and then declines. These results suggest that glycoprotein particles may be involved in some of the adhesive interactions between neural retina cells and between the cells and their environment.