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 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% O(2)), IGFBP-1 mRNA expression increased 3- to 4-fold, compared with normoxic controls. Cells cultured under 10% O(2) 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% O(2)), 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, conditions. This study demonstrates that human fetal hepatocytes can be cultured in defined medium, as primary cultures with high purity, and that they express IGFBP-1 mRNA and secrete IGFBP-1 protein in vitro. In addition, the data demonstrate that hypoxia up-regulates fetal hepatocyte IGFBP-1 mRNA steady-state levels and protein, with this being the major IGFBP derived from the fetal hepatocyte. The data support a role for the fetal liver as a source of elevated circulating levels of IGFBP-1 in fetuses with in utero hypoxia and IUGR.

Insulin-like growth factor (IGF)-II inhibition of endometrial stromal cell tissue inhibitor of metalloproteinase-3 and IGF-binding protein-1 suggests paracrine interactions at the decidua:trophoblast interface during human implantation.

In human pregnancy, insulin-like growth factor (IGF)-II messenger RNA (mRNA) is expressed at the maternal-fetal interface exclusively by the placental trophoblast. Highest levels are expressed by the invading extravillous trophoblasts, which also secrete matrix metalloproteinases as they degrade the decidual extracellular matrix. In contrast, the maternal decidua expresses high levels of IGF-binding protein (IGFBP)-1 and tissue inhibitors of matrix metalloproteinase (TIMPs), both of which inhibit trophoblast invasiveness in vitro. The present study investigated the hypothesis that IGF-II may serve as a paracrine modulator of maternal restraints on invasion, by examining its effects on TIMP-3 and IGFBP-1 expression by decidualized endometrial stromal cells. Human endometrial stromal cells were decidualized in vitro with progesterone (P), after which 0-130 nM IGF-II and IGF analogs were added. IGFBP-1 in conditioned medium was assayed by immunoradiometric assay. In addition, Northern analyses were conducted using a PCR-generated 421-bp complementary DNA (cDNA) fragment corresponding to nucleotides 132-553 of the human TIMP-3 cDNA, and a 934-bp EcoRI fragment of the human IGFBP-1 cDNA. TIMP-3 mRNA transcripts of 2.2, 2.5, and 4.4 kilobases were detected in decidualized stromal cells not treated with IGF-II, but not detected in nondecidualized stromal cells, consistent with its known induction upon decidualization and in response to P. In decidualized stromal cells, IGF-II and Des(1-6) IGF-II, an analog with reduced affinity for IGFBPs, caused a dose-dependent inhibition of TIMP-3 mRNA expression. Long R(3) IGF-I, an IGF analog with minimal affinity for IGFBPs, also significantly inhibited (79 +/- 0.3%) TIMP-3 mRNA expression in these cells at 6 nM. Decidualized stromal cells secreted IGFBP-1 and expressed a 1.5-kilobase IGFBP-1 transcript, which was not detected in nondecidualized cells, consistent with its known induction upon decidualization and in response to P. IGF-II caused a dose-dependent inhibition of IGFBP-1 mRNA expression and protein secretion in decidualized stromal cells when added in molar excess of endogenous IGFBP-1 levels, with virtually complete inhibition at higher concentrations of IGF-II (65 and 130 nM). By comparison, Long R(3) IGF-I inhibited IGFBP-1 expression with a 50% effective dose of 0.2-0.4 nM. These data suggest that the invading trophoblast has the capacity, via IGF-II, to inhibit maternal restraints on trophoblast invasiveness by regulating decidual TIMP-3 and IGFBP-1.

Discovery of new inducible genes in in vitro decidualized human endometrial stromal cells using microarray technology.

A prerequisite for implantation in humans is differentiation (decidualization) of stromal cells in the endometrium, believed to be stimulated by progesterone (P) and/or cAMP. In the current study, advances in microarray technology have allowed us to investigate genes differentially expressed in human endometrial stromal cells decidualized in vitro in response to P or cAMP, compared to nondecidualized cells. Endometrial stromal cells were isolated from endometrial biopsy tissue and cultured without steroid hormones, with 1 microM P (after E2 priming), or 1 mM 8-bromo-cAMP. Total RNA was isolated and reverse transcribed to synthesize 32P-labeled cDNA probes using primers corresponding to genes represented on the Clontech Human Atlas cDNA Expression Array. After hybridization, signals were quantified by phosphor imaging densitometry and were normalized to GAPDH and ubiquitin. Of the 588 genes screened, marked upregulation was observed of cytokines, growth factors, nuclear transcription factors, members of the cyclin family, and mediators of the cAMP signal transduction pathway. Additional mRNAs expressed unexpectedly and regulated by P and cAMP, include the insulin receptor, some neurotransmitter receptors, neuromodulators, the FSH receptor, inhibin/activin betaA subunit, inhibin alpha, and TNF-related apoptosis-inducing ligand (TRAIL). Expression of previously unrecognized genes regulated in decidualized human endometrial stromal cells suggests mechanisms not yet appreciated in the endometrium during decidualization. In addition, marked upregulation of cytokines, chemokines, growth factors, apoptosis modulators, and their receptors in decidualized stromal cells supports a major role for paracrine interactions between the stroma and other endogenous and transient cell populations within the endometrium and during early pregnancy.

Hypoxia and cAMP stimulate vascular endothelial growth factor (VEGF) in human endometrial stromal cells: potential relevance to menstruation and endometrial regeneration.

The human female reproductive tract shows unique cycle-specific changes in vascularization. Vascular endothelial growth factor (VEGF) is a specific vascular endothelial mitogen which is produced by human endometrium and is known to be regulated by steroid hormones. Vasoconstriction during menstruation leads to endometrial hypoxia, a possible stimulus for angiogenesis. In the current study we tested the hypothesis that hypoxia and cAMP, a known stimulus for endometrial decidualization, can induce VEGF in human endometrial stromal cells. Decidualized as well as non decidualized stromal cells from 6 patients were exposed to normoxia (20% oxygen) and hypoxia (2% oxygen) for up to 72h. VEGF levels were assessed by Northern analysis using a 605 bp BamHI fragment of the human VEGF cDNA, and hybridization signals were normalized to levels of 18S RNA. VEGF protein was determined by ELISA. Hypoxia stimulated VEGF mRNA in decidualized stromal cells by 10.2 fold at 48h compared to normoxic controls. VEGF protein increased 10 fold by 48h and increased further to 13 fold at 72h. In the presence of 2% oxygen VEGF mRNA in nondecidualized endometrial stromal cells was increased 1.2-8 fold between 2 and 72h of treatment. VEGF protein also increased 1.2-9 fold in this time period. cAMP regulated both VEGF mRNA and protein in non decidualized stromal cells. VEGF mRNA increased 2-4 fold in 2-72h and protein production showed a 2-6 fold increase. VEGF was seen to be regulated by both cAMP and hypoxia in an additive manner. These results demonstrate that both non-decidualized and decidualized endometrial stromal cells respond to hypoxia with increasing levels of VEGF. 8Br-cAMP, which is shown to increase VEGF levels in endometrial cells per se, has an additive effect on VEGF production under hypoxic conditions. This effect may have physiologic and pathophysiologic relevance during the process of menstruation and in post menstrual endometrial repair and angiogenesis.