Tumor angiogenic factor. Purification from the Walker 256 rat tumor

We report here the purification of an angiogenic substance from the Walker 256 rat ascites tumor which is mitogenic for fetal bovine aortic endothelial cells in culture and which also stimulates new blood vessel growth in vivo. Purification was monitored in endothelial cell cultures by cell counting or by [3H]thymidine incorporation into DNA. Lyophilized crude tumor cell homogenate was extracted with absolute ethanol, and the extract was further purified by silica gel column chromatography. The most purified material eluted with ethyl acetate:methanol (25:1) and behaved as a single component when analyzed either by thin layer chromatography with both silica gel and polyethylene-imine-cellulose systems or by reversed phase (C18) high pressure liquid chromatography. Bio-Gel P-30 chromatography indicates a Mr 400-800 for the active material. The mitogenic activity of the purified material was observed with the vascular endothelial cells cultured in serum-less as well as serum-containing media. The angiogenic activity of this material was revealed in both the chicken chorioallantoic membrane and rat corneal micropocket bioassays. Our results demonstrate that a factor with mitogenic and angiogenic activities can be purified from the Walker rat tumor by a novel, facile, and high yield process.