Insulin-like growth factor-binding proteins from vascular endothelial cells: purification, characterization, and intrinsic biological activities.
Insulin-like growth factor (IGF)-binding proteins are produced by several cell types, including vascular endothelial cells. The production of IGF-binding proteins by endothelial cells is of particular interest, since these cells are directly bathed by the circulating IGFs and form the initial barrier to the passage of circulating IGFs from the bloodstream to subendothelial tissues. We have purified IGF-binding proteins from medium conditioned by cultured bovine endothelial cells by sequential passage over Bio-Gel P-60, multiplication-stimulating activity affinity, anion exchange (DEAE cellulose) and/or hydrophobic (phenyl-Sepharose) chromatography. Two peaks of IGF-binding activity were eluted from the phenyl-Sepharose column. After cross-linking, each peak contained two to five protein bands on gels that specifically bound IGF-I and -II with mol wt ranging from about 28-44K. Material in peak 1 bound IGF-I congruent to IGF-II and had no affinity for insulin and proinsulin. Peak 2 IGF-binding proteins bound IGF-II with substantially higher affinity than IGF-I and did not recognize insulin or proinsulin. Peak 1 material from phenyl-Sepharose chromatography was a potent stimulator of both glucose transport and aminoisobutyric acid (AIB) uptake in microvessel endothelial cells, with maximal stimulation of both processes being 300-400% of control values. In contrast, peak 2 material either had no intrinsic bioactivity or was slightly inhibitory or stimulatory, depending on the concentration of peak 2 material that was added. The bioactivity in peak 1 was not due to copurification of other endothelial proteins capable of stimulating glucose and AIB uptake, such as IGF-I/-II, platelet-derived growth factor, and basic fibroblast growth factor, since bioactivity was retained after acid treatment, antibody neutralization, and selective affinity chromatography to deplete these other factors. When peak 1 material was added to IGF-I the bioeffects (glucose and AIB uptake) of IGF and binding proteins were additive, and in some experiments the binding proteins potentiated the effect of IGF on endothelial cells, suggesting that the binding protein-IGF complex may retain the bioactivity of both the binding protein(s) and the IGF.
Bar
R S
RS
Department of Medicine, Veterans Administration Medical Center, Iowa City, Iowa.
Booth
B A
BA
Boes
M
M
Dake
B L
BL
D000818:Animals; D001667:Binding, Competitive; D001696:Biomechanical Phenomena; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000032:analysis* / Q000166:cytology; D007328:Insulin / Q000378:metabolism; D018969:Insulin-Like Growth Factor Binding Proteins; D011384:Proinsulin / Q000378:metabolism; D013002:Somatomedins / Q000378:metabolism / Q000502:physiology
D002352:Carrier Proteins; D007328:Insulin; D018969:Insulin-Like Growth Factor Binding Proteins; D013002:Somatomedins; D011384:Proinsulin