Intrinsic Bioactivity of Insulin-Like Growth Factor-Binding Proteins from Vascular Endothelial Cells*

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Abstract
Conditioned medium from cultured vascular endothelial cells contains material capable of stimulating acute metabolic processes in endothelial cells. The bioactivity of the conditioned medium is not caused by the copurification of known growth factors produced by the cells, in particular platelet-derived growth factor, basic fibroblast growth factor, or insulin-like growth factor (IGF)-I/II. We no demonstrate that the bioactivity is directly due to an IGF-binding protein(s) (ECBP) and, further, that the bioactive domain of the binding protein differs from the IGF-binding domain. Binding proteins (BPs) from cultured pulmonary artery endothelial cells were purified by sequential passage over sizing, multiplication-stimulating activity affinity, and hydrophobic columns. BP fractions were separated into those with and those without biological activity. The bioactive binding protein(s) was cross-linked with disuccinimidyl suberate to IGF-I or the recombinant IGF analog [1-27,Gly4,38-70]IGF-I (Analog). The IGF-I Analog, by itself, had minimal interaction with the type I IGF receptor in cultured microvessel endothelial cells and no intrinsic bioactivity, but did bind with high affinity to ECBP. All free BP and free IGF-I/Analog were removed from the cross-linked mixture by passage over gel filtration and IGF affinity columns. The cross-linked BP-IGF-I complex did not bind to the type I receptor of cultured endothelial cells, but did stimulate glucose and .alpha.-aminoisobutyric acid uptake in endothelial cells (.apprx.2-fold increase); the magnitude of the response was nearly equal to the effect of ECBP or IGF-I alone. The BP-Analog complex also stimulated glucose and .alpha.-aminoisobutyric acid uptake, with the magnitude of the response approaching the effect of ECBP alone. The BP-Analog complex also did not react with type I IGF receptors on the cultured endothelial cells. We conclude 1) IGF-BP produced by endothelial cells possess intrinsic biological activity; 2)bioactivity of the BP(s) is retained when the IGF-binding domain of the BP is occupied by IGF-I or an inactive IGF-I analog; and 3) IGF-I bound to the bioactive BP does not react with its receptor and possesses minimal, if any, bioactivity in vitro.

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