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 Insufficiency1
Intrauterine growth restriction (IUGR) can be a consequence of decreased uterine blood flow (uteroplacental insufficiency) and maternal and fetal hypoxia. 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...
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Veröffentlicht in: | The journal of clinical endocrinology and metabolism 2001-06, Vol.86 (6), p.2653-2659 |
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Zusammenfassung: | Intrauterine growth restriction (IUGR) can be a consequence of
decreased uterine blood flow (uteroplacental insufficiency) and
maternal and fetal hypoxia. Insulin-like growth factors (IGFs) and
their binding proteins (IGFBPs) are key elements in fetal growth. IGF-I
is a major growth promoter in utero. IGFBP-1 is
primarily made in the liver, and it mostly inhibits IGF actions at the
cellular level. IGFBP-1 is elevated in the fetal circulation of human
and animal pregnancies complicated by IUGR caused by placental
insufficiency and in utero hypoxia and is believed to
restrict fetal growth by sequestering 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 effects of hypoxia
on their expression of IGFBP-1 mRNA and protein. Hepatocytes were
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 indirect
immunofluorescence. Cultures consisted primarily (>90%) of cells
positive for cytokeratin 18, fibrinogen, and IGFBP-1, with less than
2% vascular cells and less than 8% macrophages. Identification of
isolated hepatocytes was further confirmed by morphology. Hepatocytes
were cultured in defined medium, and Northern analysis revealed
expression of a 1.5-kb IGFBP-1 mRNA transcript in hepatocytes cultured
under normoxic conditions, for 24 h, that did not increase in
steady-state levels after 48 h in culture. Under hypoxic
conditions (2% O2), IGFBP-1 mRNA expression increased
3- to 4-fold, compared with normoxic controls. Cells cultured under
10% O2 did not demonstrate an increase in IGFBP-1 mRNA
levels. IGFBP-1 protein in conditioned medium (CM) was measured by
immunoradiometric assay and increased 3- to 4-fold under hypoxic (2%
O2), compared with normoxic, conditions. Western ligand
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 with normoxic, conditions, supporting the
immunoradiometric assay results. A 3-fold increase in IGFBP-3 mRNA, but
not other IGFBPs, was noted under hypoxic, compared with normoxic |
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ISSN: | 0021-972X 1945-7197 |
DOI: | 10.1210/jcem.86.6.7526 |