Transport of insulin-like growth factor-I across endothelial cell monolayers and its binding to the subendothelial matrix.
Cultured human umbilical vein endothelial cells (HUVEC) were used as a model to study transendothelial IGF-I transport, and its deposition into the extracellular matrix (ECM). Specific binding of (125)I-IGF-I to HUVEC monolayers was demonstrated, which was inhibited by aIR-3, a specific antibody directed against the IGF-I receptor. ECM-associated (125)I-IGF-I was approximately 10% of cell-bound IGF-I at 22 degrees C, and increased 4.5-fold at 37 degrees C, indicating that endothelial metabolism is required for the transport. However, neither monensin and cytochalasin B, both of which block endocytosis, nor aIR-3 did inhibit transport of (125)I-IGF-I into the ECM. In order to characterize IGF-I binding to the subendothelial ECM, HUVEC were removed nonenzymatically by treatment with Triton X-100 and ammonia. Specific, saturable binding of (125)I-IGF-I to the isolated ECM was observed, which was protease-sensitive. Antibodies directed against vitronectin inhibited IGF-I binding to the matrix by 35%, while antibodies directed against other ECM proteins had no significant influence on IGF-I binding. Using radioimmunoassays the IGF binding protein-2 was detected in the ECM, while IGFBP-1 and IGFBP-3 were below the detection limits. In order to evaluate functional aspects of IGF-I binding to the matrix, HUVEC were incubated under serum-free conditions in the absence and presence of IGF-I. Under serum-free conditions 48% of cells rounded up and started to detach after 2 hours incubation, while only 23% of the cells started to detach in the presence of IGF-I. These data indicate that IGF-I is transported via a paracellular route across endothelial cells, and becomes bound to the subendothelial ECM. Vitronection seems to be involved in binding of IGF-I to the ECM. ECM-associated IGF-I might play a role in endothelial cell survival and stability.
Grulich-Henn
J
J
Department of Pediatrics, University Children's Hospital, University of Heidelberg, Im Neuenheimer Feld 150, D-16920 Heidelberg, Germany. Juergen_Grulich-Henn@med.uni-heidelberg.de
Ritter
J
J
Mesewinkel
S
S
Heinrich
U
U
Bettendorf
M
M
Preissner
K T
KT
D000641:Ammonia / Q000494:pharmacology; D000906:Antibodies / Q000494:pharmacology; D001692:Biological Transport; D002448:Cell Adhesion; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004730:Endothelium, Vascular / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism*; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000032:analysis; D007334:Insulin-Like Growth Factor I / Q000378:metabolism* / Q000494:pharmacology; D007457:Iodine Radioisotopes; D017830:Octoxynol / Q000494:pharmacology; D014471:Umbilical Veins; D019096:Vitronectin / Q000276:immunology / Q000502:physiology
D000906:Antibodies; D016895:Culture Media, Serum-Free; D018971:Insulin-Like Growth Factor Binding Protein 2; D007457:Iodine Radioisotopes; D019096:Vitronectin; D007334:Insulin-Like Growth Factor I; D000641:Ammonia; D017830:Octoxynol