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 vascu lar endothelial cell growth inhibitors including leukemia inhibitory factor , oncostatin M, tumor necrosis factor-alpha, and transforming growth factor -beta 1. Characterization of partially purified fractions from the conditio ned media of phorbol ester-treated U-937 cells suggested the existence of u nknown endothelial growth inhibitors. Using a combination of copper affinit y, heparin affinity, cation exchange, and reversed phase liquid chromatogra phies, 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 non reducing conditions. Microsequencing analyses of the tryptic fragments of t he growth inhibitor and a BLAST search analysis revealed a unique sequence showing no homology to known proteins, The purified protein inhibited endot helial 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 cell 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 s tudy.