AN ENDOTHELIAL CELL-DERIVED GROWTH FACTOR Cells

Cell-free plasma-derived serum (PDS) is deficient in the platelet-derived growth factor and will not support the growth of 3T3 cells, fibroblasts, or smooth muscle cells. However, when PDS-containing medium is preincubated with endothelial cells, the medium becomes modified so that it will support growth. The activity produced by the endothelial cells results from a polypeptide of 10,000 to 30,000 daltons which has several features that differ from those of the platelet-derived growth factor, including heat instability and lack of adsorption to CM Sephadex . Growth of nontransformed cell strains in culture requires one or more exogenous growth factors . The availability of these polypeptides appears to determine the extent of growth, and account, at least in part, for the phenomenon of density-dependent inhibition of growth (12, 28, 31-33) . In contrast, studies of endothelial cell strains have failed to demonstrate a requirement for plateletderived growth factor, epidermal growth factor, fibroblast growth factor, or insulin (3, 4, 10, 14, 25, 26, 30, 34). These observations led us to examine the effects of endothelial cells on growth of other cell types in medium lacking the plateletderived growth factor. We report here the presence of a potent growth factor produced by endothelial cells . MATERIALS AND METHODS Primary cultures of bovine aortic endothelial cells were isolated (27) in 15% cell-free plasma-derived serum (PDS) . PDS was prepared from citrated bovine whole blood serum by the CM-Sephadex chromatography method of Vogel et al . (33) and heat inactivated for 30 min at 56 °C. Bovine PDS contains 10% of the growth-promoting activity originally present in homologous whole blood serum . Under these conditions, it was possible to select for cultures with little or no evidence of smooth muscle overgrowth (3) . Subcultures were grown in 75-cm= flasks (Corning Glass Works, Science Products Div ., Corning, N. Y .) in 10 ml of Waymouth's medium containing 5% PDS . Duplicate experiments were performed with endothelial cells isolated by the RAPID COMMUNICATIONS thymidine selection technique (27). Results were identical using both isolation methods, including thymidine-selected cells grown in whole blood serum or PDS . Cultures of Swiss and BALB/c-3T3 A cells, originally obtained from Dr. Stewart Aaronson, were maintained in 10% fetal calf serum (Grand Island Biological Co ., Grand Island, N . Y .) in Dulbecco-Vogt modification of Eagle's medium . Human newborn skin diploid fibroblasts, in the 4th passage, were grown in Waymouth's medium containing 10% fetal calf serum . Bovine smooth muscle cells, isolated by the explant technique (20), were maintained as described for fibroblasts and used from passages 2-7 .