Purification and characterization of a novel vascular endothelial cell growth inhibitor secreted by macrophage-like U-937 cells.
A human histiocytic lymphoma cell line, U-937 cells, secretes several vascular endothelial cell growth inhibitors including leukemia inhibitory factor, oncostatin M, tumor necrosis factor-alpha, and transforming growth factor-beta1. Characterization of partially purified fractions from the conditioned media of phorbol ester-treated U-937 cells suggested the existence of unknown endothelial growth inhibitors. Using a combination of copper affinity, heparin affinity, cation exchange, and reversed phase liquid chromatographies, a growth inhibitor for endothelial cells was purified to homogeneity from conditioned media. The purified growth inhibitor migrated as a 65 kDa band on SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Microsequencing analyses of the tryptic fragments of the growth inhibitor and a BLAST search analysis revealed a unique sequence showing no homology to known proteins. The purified protein inhibited endothelial cell growth in a dose-dependent manner, but had no effect on smooth muscle cell growth. The factor also blocked endothelial cell growth induced by both fibroblast growth factor-2 and vascular endothelial growth factor, and was additively effective in inhibiting the growth of endothelial cells by U-937 cell-derived endothelial cell growth inhibitors. Thus, this factor appears to be a novel inhibitor with specificity for vascular endothelial cells, and is tentatively called endothelial cell inhibitor (ECI) in this study.
Nakano
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N
Department of Biochemistry, Osaka University Medical School, Yamadaoka,Suita, Osaka, Japan.
Higashiyama
S
S
Takashima
S
S
Tsuruoka
N
N
Klagsbrun
M
M
Taniguchi
N
N
D000818:Animals; D002417:Cattle; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000502:physiology; D006131:Growth Inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D008264:Macrophages; D018919:Neovascularization, Physiologic; D017421:Sequence Analysis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020298:U937 Cells / Q000187:drug effects* / Q000378:metabolism