Angiogenic activity from bovine retina: partial purification and characterization

Abstract

Bovine retinas contain a factor that stimulates proliferation of aortic endothelial cells in culture as well as neovascularization on the chicken chorioallantoic membrane. The stimulatory activity has been partially purified from a balanced salt solution extract of bovine retinas. The stability of the activity to acid pH was utilized as the first step in purification. The acid-treated material was subjected to ion-exchange chromatography on DEAE Bio-Gel A. The material that was eluted with 0.1 M NaCl/50 mM Tris.HCl (pH 7.6) stimulated both the proliferation of vascular endothelial cells in culture and angiogenesis in vivo. A molecular weight of between 50,000 and 100,000 for the native molecule is suggested by ultrafiltration and gel chromatography; gel electrophoresis of partially purified material under reducing and denaturing conditions revealed the presence of two major components with apparent molecular weights of 50,000 and 70,000. The endothelial cell stimulatory activity was stable to pH 4-9 and to heating at up to 60 degrees C for 30 min, to incubating for 1 hr with DNase or RNase, to incubating for 2 hr with immobilized Pronase or immobilized or soluble trypsin, and to treating with 1 mM 2-mercaptoethanol, 4 M urea, or 2 M guanidine. Heating at 65-75 degrees C for 30 min, boiling for 2 min, extreme acidic (pH 2) or basic (pH 12) conditions, treating with 0.02% NaDodSO4, or incubating (5 hr) with soluble Pronase destroyed the activity. The significance of an angiogenic factor derived from retina stems from the fact that neovascularization is a serious complication in a number of ocular diseases.