Ursula Hiden

The role of oxidative stress in the pathophysiology of gestational diabetes mellitus

Normal human pregnancy is considered a state of enhanced oxidative stress. In pregnancy, it plays important roles in embryo development, implantation, placental development and function, fetal development, and labor. However, pathologic pregnancies, including gestational diabetes mellitus (GDM), are associated with a heightened level of oxidative stress, owing to both overproduction of free radicals and/or a defect in the antioxidant defenses. This has important implications on the mother, placental function, and fetal well-being. Animal models of diabetes have confirmed the important role of oxidative stress in the etiology of congenital malformations; the relative immaturity of the antioxidant system facilitates the exposure of embryos and fetuses to the damaging effects of oxidative stress. Of note, there are only a few clinical studies evaluating the potential beneficial effects of antioxidants in GDM. Thus, whether or not increased antioxidant intake can reduce the complications of GDM in both mother and fetus needs to be explored. This review provides an overview and updated data on our current understanding of the complications associated with oxidative changes in GDM.

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.

Insulin and the IGF system in the human placenta of normal and diabetic pregnancies

The insulin/insulin‐like growth factor (IGF) system regulates fetal and placental growth and development. In maternal diabetes, components of this system including insulin, IGF1, IGF2 and various IGF‐binding proteins are deregulated in the maternal or fetal circulation, or in the placenta. The placenta expresses considerable amounts of insulin and IGF1 receptors at distinct locations on both placental surfaces. This makes the insulin and the IGF1 receptor accessible to fetal and/or maternal insulin, IGF1 and IGF2. Unlike the receptor for IGF1, the insulin receptor undergoes a gestational change in expression site from the trophoblast at the beginning of pregnancy to the endothelium at term. Insulin and IGFs are implicated in the receptor‐mediated regulation of placental growth and transport, trophoblast invasion and placental angiogenesis. The dysregulation of the growth factors and their receptors may be involved in placental and fetal changes observed in diabetes, i.e. enhanced placental and fetal growth, placental hypervascularization and higher levels of fetal plasma amino acids.

Kisspeptins and the placenta: Regulation of trophoblast invasion

The invasion of extravillous trophoblasts into the uterine wall is of crucial importance for placental and fetal development, and its dysregulation has been implicated in a wide spectrum of abnormal pregnancies. Mechanistically, trophoblast invasion strongly resembles the invasion of tumour cells, but differs from it by tight regulation in time and space. This regulation is accomplished by different factors including cytokines and hormones, which are produced by both fetal as well as maternal tissues i.e., placenta and uterus, respectively. Recently, products of the KiSS-1 gene (kisspeptins) have been identified to not only inhibit metastasis in various tumours, but also to repress trophoblast invasion via binding to the G protein-coupled receptor KiSS-1R. In the placenta, expression levels of kisspeptins and their receptor are highest in the first trimester in humans and at day 12.5 in rats, respectively. This coincides with the time when invasiveness peaks and invasion regulation is of central importance. Human kisspeptins are predominantly produced by the syncytiotrophoblast, whereas KiSS-1R is additionally expressed on the invading extravillous trophoblasts indicating a paracrine regulation of extravillous trophoblast invasion by the syncytiotrophoblast. In the structurally different rat placenta both KiSS-1 and its receptor are predominantly expressed by the invasive trophoblast giant cells, thus establishing an autocrine system in the invasion regulation of this trophoblast subpopulation. Amongst all kisspeptins the highly conserved kisspeptin Kp-10 has strongest invasion inhibiting effects suggesting its major role in regulation of trophoblast invasion.

Identification of Novel Trophoblast Invasion-Related Genes: Heme Oxygenase-1 Controls Motility via Peroxisome Proliferator-Activated Receptor γ

Invasion of cytotrophoblasts (CTBs) into uterine tissues is essential for placental development. To identify molecules regulating trophoblast invasion, mRNA signatures of purified villous (CTB, poor invasiveness) and extravillous trophoblasts (EVTs) (high invasiveness) isolated from first trimester human placentae and villous explant cultures, respectively, were compared using GeneChip analyses yielding 991 invasion/migration-related transcripts. Several genes involved in physiological and pathological cell invasion, including A disintegrin and metalloprotease-12, -19, -28, as well as Spondin-2, were up-regulated in EVTs. Pathway prediction analyses identified several functional modules associated with either the invasive or noninvasive trophoblast phenotype. One of the genes that was down-regulated in the invasive mRNA pool, heme oxygenase-1 (HO-1), was selected for functional analyses. Real-time PCR analyses, Western blotting, and immunofluorescence of first trimester placentae and differentiating villous explant cultures demonstrated down-regulation of HO-1 in invasive EVTs as compared with CTBs. Modulation of HO-1 expression in loss-of as well as gain-of function cell models (BeWo and HTR8/SVneo, respectively) demonstrated an inverse relationship of HO-1 expression with trophoblast migration in transwell and wound healing assays. Importantly, HO-1 expression led to an increase in protein levels and activity of the nuclear hormone receptor peroxisome proliferator activated receptor (PPAR) gamma. Pharmacological inhibition of PPARgamma abrogated the inhibitory effects of HO-1 on trophoblast migration. Collectively, our results demonstrate that gene expression profiling of EVTs and CTBs can be used to unravel novel regulators of cell invasion. Accordingly, we identify HO-1 as a negative regulator of trophoblast motility acting via up-regulation of PPARgamma.

Human fetal placental endothelial cells have a mature arterial and a juvenile venous phenotype with adipogenic and osteogenic differentiation potential

Growing interest in the sources of origin of blood vessel related diseases has led to an increasing knowledge about the heterogeneity and plasticity of endothelial cells lining arteries and veins. So far, most of these studies were performed on animal models. Here, we hypothesized that the plasticity of human fetal endothelial cells depends on their vascular bed of origin i.e. vein or artery and further that the differences between arterial and venous endothelial cells would extend to phenotype and genotype. We established a method for the isolation of fetal arterial and venous endothelial cells from the human placenta and studied the characteristics of both cell types. Human placental arterial endothelial cells (HPAEC) and human placental venous endothelial cells (HPVEC) express classical endothelial markers and differ in their phenotypic, genotypic, and functional characteristics: HPAEC are polygonal cells with a smooth surface growing in loose arrangements and forming monolayers with classical endothelial cobblestone morphology. They express artery-related genes (hey-2, connexin 40, depp) and more endothelial-associated genes than HPVEC. Functional testing demonstrated that vascular endothelial growth factors (VEGFs) induce a higher proliferative response on HPAEC, whereas placental growth factors (PlGFs) are only effective on HPVEC. HPVEC are spindle-shaped cells with numerous microvilli at their surface. They grow closely apposed to each other, form fibroblastoid swirling patterns at confluence and have shorter generation and population doubling times than HPAEC. HPVEC overexpress development-associated genes (gremlin, mesenchyme homeobox 2, stem cell protein DSC54) and show an enhanced differentiation potential into adipocytes and osteoblasts in contrast to HPAEC. These data provide collective evidence for a juvenile venous and a more mature arterial phenotype of human fetal endothelial cells. The high plasticity of the fetal venous endothelial cells may reflect their role as tissue-resident endothelial progenitors during embryonic development with a possible benefit for regenerative cell therapy.

Insulin control of placental gene expression shifts from mother to foetus over the course of pregnancy

Aims/hypothesisThe human placenta is a complex organ situated at the interface between mother and foetus that separates maternal from foetal blood. The placental surfaces exposed to the two bloodstreams are different, i.e. trophoblasts and endothelial cells are in contact with the maternal and foetal circulation, respectively. Both cell types produce high insulin receptor levels. The aim of the present study was to test the hypothesis that spatio-temporal changes in insulin receptor expression in trophoblasts from first trimester to the endothelium at term shift the control of insulin-dependent processes from mother to foetus.MethodsGlobal microarray analysis of primary trophoblasts from first trimester and term human placentas and endothelial cells from term human placentas cultured under hyperinsulinaemic and control conditions identified different sets of regulated genes in trophoblasts and endothelial cells.ResultsInsulin effects on placental gene expression underwent developmental changes from trophoblasts in the first trimester to endothelial cells at term that were paralleled by changes in levels of activated insulin receptors. The changes in gene regulation were both quantitative (i.e. magnitude of effect) and qualitative (i.e. specific genes affected and direction of regulation).Conclusions/interpretationThis spatio-temporal shift in insulin sensitivity throughout pregnancy allows maternal and foetal insulin to regulate different processes within the placenta at different gestational stages, facilitated by compartmentalisation of the insulin response. Thus, by altering the levels and function of insulin receptors in space and time, control of insulin-dependent processes in the human placenta will change from mother to foetus throughout gestation. This will be of particular interest in conditions associated with altered maternal or foetal insulin levels, i.e. diabetes mellitus or intrauterine growth restriction.

The Placenta and Gestational Diabetes Mellitus

By its location between maternal and fetal bloodstreams the human placenta not only handles the materno-fetal transport of nutrients and gases, but may also be exposed to intrauterine conditions adversely affecting placental and fetal development. Such adverse conditions exist in pregnancies complicated by gestational diabetes mellitus (GDM), and have been associated with alterations in placental anatomy and physiology. These alterations are mainly based on changes on the micro-anatomical and/or even molecular level including aberrant villous vascularization, a disbalance of vasoactive molecules, and enhanced oxidative stress. The consequence thereof may be impaired fetal oxygenation and changes in transplacental nutrient supply. Although transplacental glucose flux is flow limited and independent of glucose transporter availability, transport of essential and nonessential amino acids and expression of genes involved in lipid transport and metabolism are significantly affected by GDM.

Dysregulation of Placental Endothelial Lipase in Obese Women With Gestational Diabetes Mellitus

OBJECTIVE This study addressed the hypothesis that placental endothelial lipase (EL) expression is affected by pregnancies complicated by obesity and gestational diabetes mellitus (GDM). RESEARCH DESIGN AND METHODS EL expression in placental tissues from pregnancies complicated by obesity, GDM, or obesity combined with GDM (obese-GDM) was analyzed by quantitative RT-PCR. Moreover, primary placental cells were isolated and treated with insulin, glucose, leptin, or tumor necrosis factor (TNF)-α, and EL expression was measured. Inhibitors of nuclear factor (NF)-κB or mitogen-activated protein kinase (MAPK) signaling were used to detect potential pathways of EL regulation in primary placental endothelial cells (ECs). RESULTS In placentas from obese-GDM pregnancies, EL expression was upregulated by 1.9-fold (P < 0.05) compared with lean pregnancies, whereas obesity or GDM alone had no significant effect. Analyses of metabolic parameters in maternal venous and umbilical venous plasma revealed significantly increased insulin and leptin as well as slightly increased glucose and TNF-α values in the obese and obese-GDM groups. Cell culture experiments identified TNF-α and leptin, but not glucose or insulin, as regulators of EL expression in ECs. Induction of EL expression by these mediators occurred in a para/endocrine manner, since only leptin and TNF-α receptors, but not the cytokines themselves, were expressed in ECs. Inhibitor experiments suggested that TNF-α and leptin-mediated upregulation of EL may occur via two different routes. Whereas TNF-α induced EL upregulation in ECs by activation of the NF-κB pathway, leptin did not stimulate NF-κB or MAPK signaling pathways in these cells. CONCLUSIONS Metabolic inflammation with high leptin and locally increased TNF-α concentrations at the fetal-placental interface regulates placental EL expression.

The first trimester human trophoblast cell line ACH-3P: A novel tool to study autocrine/paracrine regulatory loops of human trophoblast subpopulations – TNF-α stimulates MMP15 expression

BackgroundThe trophoblast compartment of the placenta comprises various subpopulations with distinct functions. They interact among each other by secreted signals thus forming autocrine or paracrine regulatory loops. We established a first trimester trophoblast cell line (ACH-3P) by fusion of primary human first trimester trophoblasts (week 12 of gestation) with a human choriocarcinoma cell line (AC1-1).ResultsExpression of trophoblast markers (cytokeratin-7, integrins, matrix metalloproteinases), invasion abilities and transcriptome of ACH-3P closely resembled primary trophoblasts. Morphology, cytogenetics and doubling time was similar to the parental AC1-1 cells. The different subpopulations of trophoblasts e.g., villous and extravillous trophoblasts also exist in ACH-3P cells and can be immuno-separated by HLA-G surface expression. HLA-G positive ACH-3P display pseudopodia and a stronger expression of extravillous trophoblast markers. Higher expression of insulin-like growth factor II receptor and human chorionic gonadotropin represents the basis for the known autocrine stimulation of extravillous trophoblasts.ConclusionWe conclude that ACH-3P represent a tool to investigate interaction of syngeneic trophoblast subpopulations. These cells are particularly suited for studies into autocrine and paracrine regulation of various aspects of trophoblast function. As an example a novel effect of TNF-α on matrix metalloproteinase 15 in HLA-G positive ACH-3P and explants was found.