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 ...
The effects of Fibroblast Growth Factor (FGF) and Epidermal Growth Factor (EGF) on the proliferation of bovine vascular endothelial cells has been examined. FGF induces the initiation of DNA synthesis and cell proliferation in cloned endothelial cells of fetal and adult origin at concentrations as low as 1 ng/ml and is saturating at 50 ng/ml. EGF had no effect over the same range of concentrations. The mitogenic effect of FGF is blocked by a crude extract of cartilage. Platelet extract is also mitogenic for vascular endothelial cells although to a lesser extent than the purified FGF. In contrast to vascular endothelial cells, both EGF and FGF are mitogenic for vascular smooth muscle cells although EGF is less mitogenic than FGF at 100 ng/ml. The mitogenic effect of EGF and FGF on vascular smooth muscle is not blocked by the addition of a crude extract of cartilage, thus demonstrating the specificity of the chalone like effect of cartilage crude extract for endothelial cells.
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