An assay measuring the stimulation of several types of bovine endothelial cells by growth factor(s) derived from cultured human tumor cells

Abstract

Endothelial cell growth factor(s) from several previously untested human tumor cell lines (i.e., SK-HEP-1, MG63, A375, TE671-C1, RD) were detected using a low cell inoculum growth assay. The final cell density in the 2-cm2 wells was determined by a highly sensitive DNA content measurement performed directly in the tissue culture plates. The sensitivity of the assay to human tumor cell growth factors depended critically on the low cell inocula, 2,000 to 5,000 cells/well. Most of the bovine endothelial cells used were cloned from primary cultures; all the cell lines obtained from various fetal and nonfetal sources responded to the growth factor(s) (up to a 16x stimulation) as well as to endothelial cell growth supplement. Dose response curves showing the cell specific response of bovine endothelial cells were obtained. The growth stimulatory activity and the in vivo chick embryo chorioallantoic membrane assay responses correlated sufficiently to imply that the assay is detecting tumor angiogenesis factor or some closely related activity. This in vitro assay should prove useful in the identification and purification of tumor-derived factors and in the elucidation of the role of these factors in the events comprising angiogenesis.

Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.

“…Abbreviations used in this paper: BAE, bovine aortic endothelial cells; EGF, epidermal growth factor; FBS, fetal bovine serum; FGF, fibroblast growth factor; HUE, human umbilical vein endothelial cells; NEpHGE, nonequilibrium pH gradient electrophoresis; PDGF, platelet-derived growth factor; TGF, transforming growth factor; TNF, tumor necrosis factor; VPF, vascular permeability factor. fied from the conditioned medium ofguinea pig line 10 tumor cells grown in vitro (6, 7, 7a). The NH2-terminal amino acid sequence of VPF has been shown to be unrelated to other proteins for which sequence information is available, including those other proteins that are known to affect vessel permeability (7a).…”

Purification from a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis, and migration.

“…In particular, the cell lysate of the human hepatoma cell line SK-Hep-1 has been shown to be strongly angiogenic when assayed on the chorioallantoic membrane (18,24). The same preparation has also been shown to stimulate PA and collagenase production (12), growth (18), and motility (16,24) in cultured endothelial cells. These data therefore suggest the presence of a basic MATERIALS AND METHODS Cell culture.…”

Increased capillary endothelial cell protease activity in response to angiogenic stimuli in vitro.

“…We have proposed that neovascularization requires the elaboration of proteases by capillary endothelial cells in order to degrade the proteins of the capillary basal lamina and the surrounding stroma (2, 3). Cultured bovine capillary endothelial (BCE) cells produce increased amounts of the proteases plasminogen activator (PA) and latent collagenase in response to nanomolar concentrations of the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) (2,3 (11,12).We have investigated whether the levels of PA and latent collagenase in BCE cells can also be stimulated by crude preparations known to contain angiogenic activity and have compared the response of BCE cells to these preparations to the response of bovine endothelial cells derived from aortae (BAE cells).MATERIALS AND METHODS Cell Culture. BCE cells were isolated from the adrenals of freshly slaughtered calves, as described by Folkman et aL (13).…”

“…We have proposed that neovascularization requires the elaboration of proteases by capillary endothelial cells in order to degrade the proteins of the capillary basal lamina and the surrounding stroma (2, 3). Cultured bovine capillary endothelial (BCE) cells produce increased amounts of the proteases plasminogen activator (PA) and latent collagenase in response to nanomolar concentrations of the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) (2,3 (11,12).…”

“…(iii) Extracts of tumors promote angiogenesis in vivo (8)(9)(10) and stimulate the proliferation and motility of cultured BCE cells (10)(11)(12). One of the most active angiogenic preparations is derived from lysates of a human hepatoma cell line (11,12).…”