Hypoxia regulates insulin-like growth factor-binding protein 1 in human fetal hepatocytes in primary culture: Suggestive molecular mechanisms for in utero fetal growth restriction caused by uteroplacental insufficiency

Intrauterine growth restriction (IUGR) can be a consequence of decreased ut erine blood flow (uteroplacental insufficiency) and maternal and fetal hypo xia. Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are key elements in fetal growth. IGF-I is a major growth promoter in uter o. IGFBP-1 is primarily made in the Liver, and it mostly inhibits IGF actio ns at the cellular level. IGFBP-1 is elevated in the fetal circulation of h uman and animal pregnancies complicated by IUGR caused by placental insuffi ciency and in utero hypoxia and is believed to restrict fetal growth by seq uestering IGFs. In this study, we developed a protocol to establish highly pure primary cultures of human fetal hepatocytes in vitro and investigated their expression of IGFBP-1 messenger RNA (mRNA) and protein and the effect s of hypoxia on their expression of IGFBP-1 mRNA and protein. Hepatocytes w ere isolated from second-trimester human fetal livers (n = 7) and purified by Percoll gradient centrifugation. Hepatocyte cultures were characterized by immunocytochemistry and were compared with hepatocytes in situ in human fetal liver tissue, by immunohistochemistry,using specific antibodies and i ndirect immunofluorescence. Cultures consisted primarily (>90%) of cells po sitive for cytokeratin 18, fibrinogen, and IGFBP-1, with less than 2% vascu lar cells and less than 8% macrophages. Identification of isolated hepatocy tes was further confirmed by morphology. Hepatocytes were cultured in defin ed medium, and Northern analysis revealed expression of a 1.5-kb IGFBP-1 mR NA transcript in hepatocytes cultured under normoxic conditions, for 24 h, that did not increase in steady-state levels after 48 h in culture. Under h ypoxic conditions (2% O-2), IGFBP-1 mRNA expression increased 3- to 4-fold, compared with normoxic controls. Cells cultured under 10% O-2 did not demo nstrate an increase in IGFBP-1 mRNA levels. IGFBP-1 protein in conditioned medium (CM) was measured by immunoradiometric assay and increased 3- to 4-f old under hypoxic (2% O-2), compared with normoxic, conditions. Western lig and blot analysis of CM revealed the presence of IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4. IGFBP-1 was the most abundant IGFBP in CM, and densitometric analysis revealed a 2.5-fold increase in IGFBP-1 under hypoxic, compared wi th normoxic, conditions, supporting the immunoradiometric assay results. A 3-fold increase in IGFBP-3 mRNA, but not other IGFBPs, was noted under hypo xic, compared with normoxic, conditions. This study demonstrates that human fetal hepatocytes can be cultured in defined medium, as primary cultures w ith high purity, and that they express IGFBP-1 mRNA and secrete IGFBP-1 pro tein in vitro. In addition, the data demonstrate that hypoxia up-regulates fetal hepatocyte IGFBP-1 mRNA steady-state levels and protein, with this be ing the major IGFBP derived from the fetal hepatocyte. The data support a r ole for the fetal liver as a source of elevated circulating levels of IGFBP -1 in fetuses with in utero hypoxia and IUGR.