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Rapid Insulin-Like Growth Factor (IGF)-Independent Effects of IGF Binding Protein-3 on Endothelial Cell Survival

Division of Pediatric Endocrinology and Diabetes (S.L.F., P.C.), Mattel Children’s Hospital, University of California, Los Angeles, California 90095-1752; and Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics (R.J.F.), The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900

Address all correspondence and requests for reprints to: Robert J. Ferry, Jr., M.D., Director of Research and Training, Division of Pediatric Endocrinology, Department of Pediatrics, The University of Texas Health Science Center at San Antonio, 528L-2 MSC 7806, 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900. E-mail: bob{at}uthscsa.edu.

Angiogenic factors, such as vascular endothelial-derived growth factor (VEGF) and IGF-I, play pivotal roles in endothelial proliferation and migration. IGF binding protein-3 (IGFBP-3) is emerging as a key regulator of cell growth and apoptosis, both as an IGF antagonist and as an independent molecule. We investigated the role of IGFBP-3 in VEGFmediated survival of human macrovascular umbilical vein endothelial cells (HUVEC). Specific commercial ELISAs quantified cell proliferation and apoptosis, and Akt phosphorylation was assessed by immunoblots and confocal microscopy. IGF-I and VEGF significantly stimulated HUVEC proliferation and survival. Addition of IGFBP-3 reversed both IGF- and VEGF-induced proliferation and prevented the survival induced by these factors. The antiproliferative and proapoptotic effects of exogenous IGFBP-3 upon VEGF-induced HUVEC survival were not inhibited by blockade of the type 1 IGF receptor with IR-3 immunoglobulin, which fully prevented IGF actions. An IGFBP-3 mutant, which binds IGFs normally but has a substituted mid-region domain, lost the ability to inhibit VEGF actions. VEGF-induced phosphorylation of Akt, as evident by both specific immunoblots and confocal microscopy, was significantly and rapidly reduced in the presence of IGFBP-3, as well as wortmannin.

This work was supported in part by NIH Grants 2R01-DK-47591, 1RO1-AI-40203, 1R01-AG-20954, and 1UO1-CA-84128 (to P.C.); 5T32-DK-007688 (to S.L.F.); K08-DK-02876 and 5P30-HD-34610 (to R.F.); grants from the Juvenile Diabetes Foundation; and a Pharmacia GEM grant (to P.C.) as well as a fellowship award from Eli Lilly (to S.L.F.).

Abbreviations: DAPI, 4',6-Diamidino-2-phenylindole; EGM-2-MV Bullet Kit, Microvascular Endothelial Cell Growth Medium Bullet Kit-2; FITC, fluorescein isothiocyanate; HBD, heparin-binding domain; HUVEC, human macrovascular umbilical vein endothelial cells; IGF1R, type 1 IGF receptor; IGFBP-3, IGF binding protein-3; PI3-kinase, phosphatidylinositol 3'-kinase; SDS, sodium dodecyl sulfate; SFM, serum-free medium; VEGF, vascular endothelial-derived growth factor.

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