Background: The 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). Results: Expression 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. Conclusion: We 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-alpha on matrix metalloproteinase 15 in HLA-G positive ACH-3P and explants was found.
Objective To examine the expression pattern of biomarker proteins in extravillous trophoblast (EVT) cells obtained noninvasively by trophoblast retrieval and isolation from the cervix (TRIC) in patients with early pregnancy loss compared with control patients with uncomplicated term delivery. Design Case-control study. Setting Academic medical center. Patient(s) Women with either early pregnancy loss (EPL, n = 10) or an uncomplicated term delivery (N = 10). Intervention(s) Endocervical specimens obtained from ongoing pregnancies at gestational ages of 5–10 weeks to generate an archive of EVT cells isolated by TRIC, with medical records examined to select specimens matched for gestational age at the time of endocervical sampling. Main Outcome Measure(s) Known serum biomarkers for adverse pregnancy outcome that are expressed by EVT cells were evaluated by semiquantitative immunocytochemistry, using antibodies against endoglin (ENG), FMS-like tyrosine kinase-1 (FLT-1), α-fetoprotein (AFP), pregnancy-associated plasma protein-A (PAPP-A), galectin-13 (LGALS13), galectin-14 (LGALS14), and placental growth factor (PGF). Result(s) The EVT purity was over 95% in all specimens, based on chorionic gonadotropin expression; however, the number of EVT cells obtained was significantly lower in women with EPL than the control group. There was a statistically significant elevation of AFP, ENG, and FLT-1, and statistically significant reduction of PAPP-A, LGALS14, and PGF in the EPL group compared with controls. Conclusion(s) In this pilot study, EVT cells isolated by TRIC early in gestation exhibited altered protein expression patterns before an EPL compared with uncomplicated term pregnancies.
In pregnancy, trophoblast invasion and uterine spiral artery remodelling are important for lowering maternal vascular resistance and increasing uteroplacental blood flow. Impaired spiral artery remodelling has been implicated in pre-eclampsia, a major complication of pregnancy, for a long time but the underlying mechanisms remain unclear(1,2). Corin (also known as atrial natriuretic peptide-converting enzyme) is a cardiac protease that activates atrial natriuretic peptide (ANP), a cardiac hormone that is important in regulating blood pressure(3). Unexpectedly, corin expression was detected in the pregnant uterus(4). Here we identify a new function of corin and ANP in promoting trophoblast invasion and spiral artery remodelling. We show that pregnant corin-or ANP-deficient mice developed high blood pressure and proteinuria, characteristics of pre-eclampsia. In these mice, trophoblast invasion and uterine spiral artery remodelling were markedly impaired. Consistent with this, the ANP potently stimulated human trophoblasts in invading Matrigels. In patients with pre-eclampsia, uterine Corin messenger RNA and protein levels were significantly lower than that in normal pregnancies. Moreover, we have identified Corin gene mutations inpre-eclamptic patients, which decreased corin activity in processing pro-ANP. These results indicate that corin and ANP are essential for physiological changes at the maternal-fetal interface, suggesting that defects in corin and ANP function may contribute to pre-eclampsia.
During early placentation the trophoblast of the human placenta differentiates to the villous and extravillous types of trophoblast. Villous trophoblast provides the epithelial cover of the placental villous trees in direct contact to maternal blood. Extravillous trophoblast invades maternal uterine tissues thus directly contacting maternal stromal and immune cells. A subset of extravillous trophoblast, endovascular trophoblast initially occludes the lumen of spiral arteries and comes into direct contact with maternal blood. In recent years apoptosis has been described in both types of trophoblast and the importance of this cascade for the normal function of the trophoblast has become obvious. One feature of serious conditions such as preeclampsia or intrauterine growth restriction is changes in apoptosis regulation in villous and/or extravillous trophoblast resulting in altered trophoblast invasion and/or shedding into the maternal circulation. This review summarizes recent findings on trophoblast apoptosis in normal and pathologic pregnancies.
Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot (R)) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte (TM)) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (< 18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-alpha released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications.
Preeclampsia (PE) is one of the leading causes of maternal and fetal mortality globally. The imbalance of trophoblast homeostasis is closely linked with the pathogenesis of PE. MicroRNA-423–5p (miR-423–5p) has been reported to be abnormally expressed in placenta and blood plasma of pregnant women with PE. In the present study, miR-423–5p expression in blood plasma of pregnant women with PE and healthy pregnant women was detected. Also, the roles and molecular mechanisms of miR-423–5p in the development of trophoblast cells were further investigated. Expression of miR-423–5p and insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) mRNA was detected by RT-qPCR assay. Protein expression of IGF2BP1, Bcl-2 and Bax was determined using western blot assay. Cell migratory and invasive capacities were assessed by transwell migration and invasion assay. Cell apoptotic rate was determined using flow cytometry via the double-staining of Annexin V-FITC/Propidium Iodide. The interaction between miR-423–5p and IGF2BP1 was demonstrated by bioinformatics analysis and luciferase reporter assay. MiR-423–5p was highly expressed in blood plasma of pregnant women with PE. MiR-423–5p inhibited migration, invasion and proliferation as well as induced apoptosis in HTR-8/SVneo cells. Further investigation revealed that IGF2BP1 was a target of miR-423–5p. Moreover, IGF2BP1 overexpression promoted migration, invasion and proliferation, suppressed apoptosis, and weakened miR-423–5p function in HTR-8/SVneo cells. MiR-423–5p inhibited migration, invasion and proliferation as well as induced apoptosis by targeting IGF2BP1 in HTR-8/SVneo cells, presenting a novel molecular basis implicated in PE pathogenesis.
Preeclampsia (PE), a condition during pregnancy that involves high blood pressure and proteinuria, is potentially fatal to both mother and child. PE currently has no known etiology or cure but has been tied to poor placental trophoblast cell migration. Increased levels of the toxic metal cadmium (Cd) have been associated with increased risk of developing PE, as well as miRNA-associated regulation of the transforming growth factorbeta (TGF-β) pathway. Signal reprogramming of the TGF-β pathway via epigenetic mechanisms is hypothesized to modify placental trophoblast function. In the present study we investigated the role of increased and decreased signaling of the TGF-β pathway in relation to Cd-induced reduction in cellular migration in JEG3 trophoblast cells. Furthermore, the role of a miR-26a as a molecular mediator of placental trophoblast migration was confirmed. The results demonstrate that increased expression of miR-26a and decreased signaling of the TGF-β pathway increase placental cell migration. These findings have relevance for mechanistic understanding of the underpinnings of poor placentation associated with PE.
Background Maternal alcohol abuse leading to fetal alcohol spectrum disorder (FASD) includes fetal growth restriction (FGR). Ethanol (EtOH) induces apoptosis of human placental trophoblast cells, possibly disrupting placentation and contributing to FGR in FASD. EtOH facilitates apoptosis in several embryonic tissues, including human trophoblasts, by raising intracellular Ca2+. We previously found that acute EtOH exposure increases trophoblast apoptosis due to signaling from both intracellular and extracellular Ca2+. Therefore, nifedipine, a Ca2+ channel blocker that is commonly administered to treat preeclampsia and preterm labor, was evaluated for cytoprotective properties in trophoblast cells exposed to alcohol. Methods Human first‐trimester chorionic villous explants and the human trophoblast cell line HTR‐8/SVneo (HTR) were pretreated with 12.5 to 50 nM of the Ca2+ channel blocker nifedipine for 1 hour before exposure to 50 mM EtOH for an additional hour. Intracellular Ca2+ concentrations were monitored in real time by epifluorescence microscopy, using fluo‐4‐AM. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), accumulation of cytoplasmic cytochrome c, and cleavage rates of caspase 3 and caspase 9. Results The increase in intracellular Ca2+ upon exposure to EtOH in both villous explants and HTR cells was completely blocked (p < 0.05) when pretreated with nifedipine, accompanied by inhibition of EtOH‐induced release of cytochrome c, caspase activities, and TUNEL. Conclusions This study indicates that nifedipine can interrupt the apoptotic pathway downstream of EtOH exposure and could provide a novel strategy for future interventions in women with fetuses at risk for FASD.
Several aquaporins (AQPs) are expressed in extravillous (EVT) and villous trophoblast cells. Among them, AQP3 is the most abundant AQP expressed in chorionic villi samples from first trimester, followed by AQP1 and AQP9. Although AQP3 expression persists in term placentas, it is significantly decreased in placentas from preeclamptic pregnancies. AQP3 is involved in the migration of different cell types, however its role in human placenta is still unknown. Here, we evaluated the role of AQP3 in the migration of EVT cells during early gestation. Our results showed that Swan 71 cells expressed AQP1, AQP3 and AQP9 but only the blocking of AQP3 by CuSO or the silencing of its expression by siRNA significantly attenuates EVT cell migration. Our work provides evidence that AQP3 is required for EVT cell migration and suggests that an altered expression of placental AQP3 may produce failures in placentation such as in preeclampsia.
Abstract Introduction Incomplete human extravillous trophoblast (EVT) invasion of the decidua and maternal spiral arteries is characteristic of pre-eclampsia, a condition linked to low maternal vitamin D status. It is hypothesized that dysregulated vitamin D action in uteroplacental tissues disrupts EVT invasion leading to malplacentation. Methods This study assessed the effects of the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3 ), and its precursor, 25-hydroxyvitamin D3 (25-D3 ), on primary human EVT isolated from first trimester pregnancies. Expression of EVT markers (cytokeratin-7, HLA-G), the vitamin D-activating enzyme (CYP27B1) and 1,25-D3 receptor (VDR) was assessed by immunocytochemistry. EVT responses following in vitro treatment with 1,25-D3 (0–10 nM) or 25-D3 (0–100 nM) for 48–60 h were assessed using quantitative RT-PCR (qRT-PCR) analysis of key target genes. Effects on EVT invasion through Matrigel® were quantified alongside zymographic analysis of secreted matrix metalloproteinases (MMPs). Effects on cell viability were assessed by measurement of MTT. Results EVT co-expressed mRNA and protein for CYP27B1 and VDR, and demonstrated induction of mRNA encoding vitamin D-responsive genes, 24-hydroxylase (CYP24A1) and cathelicidin following 1,25-D3 treatment. EVT could respond to 1,25-D3 and 25-D3 , both of which significantly increased EVT invasion, with maximal effect at 1 nM 1,25-D3 (1.9-fold; p < 0.01) and 100 nM 25-D3 (2.2-fold; p < 0.05) respectively compared with untreated controls. This was accompanied by increased pro-MMP2 and pro-MMP9 secretion. The invasion was independent of cell viability, which remained unchanged. Discussion These data support a role for vitamin D in EVT invasion during human placentation and suggest that vitamin D-deficiency may contribute to impaired EVT invasion and pre-eclampsia.