The first human tumor derived protein with in vivo angiogenic activity to be obtained in pure form has been isolated from serum-free supernatants of an established human adenocarcinoma cell line (HT-29) and named angiogenin. It was purified by cation-exchange and reversed-phase high-performance liquid chromatography; the yield was approximately 0.5 microgram/L of medium. Biological activity of angiogenin was monitored throughout purification by using the chick embryo chorioallantoic membrane assay. Statistical evaluation demonstrates that it displays activity in this system with as little as 35 fmol per egg. Moreover, only 3.5 pmol is required to induce extensive blood vessel growth in the rabbit cornea. The amino acid composition of this basic (isoelectric point greater than 9.5), single-chain protein of molecular weight approximately 14 400 has been determined. The amino terminus is blocked, and the carboxyl-terminal residue is proline.

The results of previous preclinical and clinical studies have identified angiogenin (ANG) as a potentially important target for anticancer therapy. Here we report the design and implementation of a high-throughput screening assay to identify small molecules that bind to the ribonucleolytic active site of ANG, which is critically involved in the induction of angiogenesis by this protein.

The 42-kDa angiogenin binding protein isolated previously has been purified to electrophoretic homogeneity. It has been identified as a member of the actin family by peptide mapping and partial amino acid sequencing. The interaction of bovine muscle actin with angiogenin is similar to that of the angiogenin binding protein. Angiogenin induces the polymerization of actin below the critical concentration for spontaneous polymerization. The interaction occurs both in solution and on a poly(vinylidene difluoride) membrane. It is inhibited by excess unlabeled angiogenin and also by platelet factor 4 and protamine, which are known inhibitors of angiogenesis. Two other angiogenic molecules, basic fibroblast growth factor and tumor necrosis factor a, bind to 125I-labeled actin and can be crosslinked by a water-soluble carbodilmide. Both actin and an anti-actin antibody inhibit the angiogenic activity of angiogenin in the chicken embryo chorioallantoic membrane assay. The results indicate that the angiogenin binding protein is a cell surface actin and suggest that the reaction between angiogenin and this actin is an essential step in the angiogenesis process induced by angiogenin.Angiogenin is a 14-kDa protein purified initially from serumfree supernatants of an established human adenocarcinoma cell line, HT-29 (1). It was the first human tumor-derived angiogenic protein to be isolated based on its in vivo activity. It stimulates endothelial cells to produce diacylglycerol (2) and secrete prostacyclin (3) by phospholipase activation. It supports endothelial and fibroblast cell adhesion (4) and modulates a mitogenic effect in certain cells (5). An angiogenin binding protein (AngBP), which has properties consistent with its being a component of a cellular receptor for angiogenin, has been identified and isolated from a transformed endothelial cell line, GM7373 (6). It is a cell-surface protein with an apparent molecular mass of 42 kDa and is released from endothelial cells by exposure to heparnn, heparan sulfate, or angiogenin itself.We report here the further purification and characterization of AngBP. Tryptic peptide mapping and amino acid sequence analysis indicate that AngBP is a member of the actin family. The binding of angiogenin to bovine muscle actin and the inhibitory effect of actin and anti-actin antibodies on angiogenin-induced neovascularization on the chicken embryo chorioallantoic membrane (CAM) are also reported. The mechanism by which AngBP is released from the cell surface and the physiological significance of its presence and displacement remain to be elucidated.

No abstract