Trimester Trophoblast Cells Research Articles

First trimester trophoblast cell line ACH-3P as model to study invasion into arteries vs. veins

First trimester trophoblast cell line ACH-3P as model to study invasion into arteries vs. veins

The role of VEGF-A165b in trophoblast survival.

BackgroundPre-eclampsia remains a dominant cause of maternal and fetal mortality in developed countries. In a previous prospective study we identified a fall in the VEGF-A isoform VEGF-A165b in the plasma of patients in the first trimester to be a predictor of later pre-eclampsia. VEGF-A165b has been shown to have potent cytoprotective properties in many cell types. We therefore tested the hypothesis that VEGF-A165b may be cytoprotective for placental trophoblasts.MethodsWe used an immortalised first trimester trophoblast cell line exposed to chemical toxicity, and physiological (<2% O2) and atmospheric oxygen (21% O2) in the presence or absence of VEGF-A165b, angiogenic VEGF-A165a, a non-specific anti-VEGF-A blocking antibody (bevacizumab), or a specific anti-VEGF-A165b antibody. Cell viability and cytotoxicity were measured by trypan blue and LDH assay respectively.ResultsUnder high (21%) levels of oxygen, trophoblast viability was increased, and cytotoxicity reduced by exogenous recombinant VEGF-A165b (p < 0.05, n = 10) or VEGF-A165a. The cytoprotective effect was not seen under lower (<2%) oxygen conditions, where VEGF-A165b was upregulated. However inhibition of VEGF-A with blocking antibodies (bevacizumab or anti-VEGF-A165b) had marked cytotoxic effects under low oxygen conditions presumably through the blockade of autocrine survival pathways.ConclusionsThese results show that when trophoblasts are exposed to lower oxygen tensions (as they are early in the 1st trimester) endogenous VEGF-A165b contributes to their survival through an autocrine pathway. In contrast in high oxygen conditions exogenous VEGF-A isoforms have a greater effect on trophoblast survival.

Connective tissue growth factor induces tube formation and IL-8 production in first trimester human placental trophoblast cells

ObjectivesTo study the roles of connective tissue growth factor (CTGF) on tube formation (as a measure of cellular angiogenesis) and expression of angiogenesis-associated genes in first-trimester human extravillous trophoblast cells. Study designThe effects of recombinant human CTGF on tube forming ability were studied in the first trimester trophoblast cells using matrigel system. The expressions of genes involved in angiogenesis were studied using real time PCR microarray. ResultshCTGF (26nM) stimulated tube formation and enhanced tube length in these cells compared with control cells. However, hCTGF-stimulated tube formation was not inhibited by SU5416, a selective inhibitor of the membrane-bound tyrosine kinase activity of VEGF-2 receptor (Flk-1/KDR) indicating VEGF was not involved in the process. This was also supported by the observation that CTGF did not stimulate the expression of angiogenic factors VEGF or ANGPTL4 as determined by PCR array. To further elucidate roles of hCTGF in tube formation we then measured expression of several genes involved in angiogenesis signalling pathways. Among the genes, hCTGF increased expression of IL-8 mRNA by ∼6-fold and its protein secretion by ∼20-fold in these cells. Exogenously added IL-8 also stimulated the tube formation and proliferation in these cells. ConclusionshCTGF-induced tube formation is independent of VEGF. hCTGF stimulates production of IL-8, an important angiogenic factor in these cells. However their interrelationship in this process is yet to be known.

Vitamin D Reverses aPL‐induced Inflammation and LMWH‐induced sFlt‐1 Release by Human Trophoblast

Women with antiphospholipid syndrome (APS) are at increased risk of recurrent pregnancy loss (RPL) and preeclampsia. Antiphospholipid antibodies (aPL) directly alter trophoblast function. Treatment with low molecular weight heparin (LMWH) reduces the risk of RPL but not preeclampsia. Moreover, LMWH stimulates trophoblast sFlt-1 release, an anti-angiogenic factor associated with preeclampsia. Since vitamin D deficiency is associated with APS and preeclampsia, this study sought to determine the effect of vitamin D on trophoblast function in the setting of aPL and LMWH. A human first trimester trophoblast cell line (HTR8) and primary trophoblast cultures were treated with or without aPL in the presence and absence of vitamin D, LMWH or both. Trophoblast secretion of inflammatory cytokines and angiogenic factors were measured by ELISA. Vitamin D alone or in combination with LMWH attenuated the aPL-induced trophoblast inflammatory response in the HTR8 cells and primary cultures. While vitamin D did not have any impact on aPL-mediated modulation of angiogenic factors in the primary trophoblast, it significantly inhibited LMWH-induced sFlt-1 release. LMWH in combination with vitamin D may be more beneficial than single-agent therapy by preventing aPL-induced trophoblast inflammation and reversing LMWH-induced sFlt-1 secretion.

Trophoblast induces monocyte differentiation into CD14+/CD16+ macrophages.

During early pregnancy, macrophages and trophoblast come into close contact during placenta development, and regulated cross talk between these cellular compartments is crucial for maintaining a healthy pregnancy. As trophoblast cells constitutively secrete many chemokines and cytokines, we hypothesize that trophoblast-secreted factors can differentiate monocytes into a decidual phenotype. In this study, we describe a unique macrophage phenotype, following monocytes' exposure to trophoblast-soluble factors. Peripheral blood monocytes were treated with or without conditioned media (CM) from first trimester trophoblast cells. Phenotypic changes and phagocytic capacity were determined by flow cytometry. Cytokine and chemokine production was determined by multiplex analysis. Monocytes exposed to trophoblast factors undergo morphologic changes characterized by a gain in size and complexity and acquire a unique phenotype characterized by gain of CD14 surface expression as well as CD16. The presence of CD14+/CD16+ macrophages was confirmed in normal decidua. These cells secrete higher levels of IL-1b, IL-10, and IP-10 and have increased capacity for phagocytosis. We demonstrate that trophoblast-secreted factors can induce monocyte differentiation into a unique macrophage phenotype. These findings suggest that the microenvironment of the placenta can modulate the phenotype of macrophages present at the decidua.

Kisspeptin regulation of genes involved in cell invasion and angiogenesis in first trimester human trophoblast cells.

The precise regulation of extravillous trophoblast invasion of the uterine wall is a key process in successful pregnancies. Kisspeptin (KP) has been shown to inhibit cancer cell metastasis and placental trophoblast cell migration. In this study primary cultures of first trimester human trophoblast cells have been utilized in order to study the regulation of invasion and angiogenesis-related genes by KP. Trophoblast cells were isolated from first trimester placenta and their identity was confirmed by immunostaining for cytokeratin-7. Real-time quantitative RT-PCR demonstrated that primary trophoblast cells express higher levels of GPR54 (KP receptor) and KP mRNA than the trophoblast cell line HTR8Svneo. Furthermore, trophoblast cells also expressed higher GPR54 and KP protein levels. Treating primary trophoblast cells with KP induced ERK1/2 phosphorylation, while co-treating the cells with a KP antagonist almost completely blocked the activation of ERK1/2 and demonstrated that KP through its cognate GPR54 receptor can activate ERK1/2 in trophoblast cells. KP reduced the migratory capability of trophoblast cells in a scratch-migration assay. Real-time quantitative RT-PCR demonstrated that KP treatment reduced the expression of matrix metalloproteinase 1, 2, 3, 7, 9, 10, 14 and VEGF-A, and increased the expression of tissue inhibitors of metalloproteinases 1 and 3. These results suggest that KP can inhibit first trimester trophoblast cells invasion via inhibition of cell migration and down regulation of the metalloproteinase system and VEGF-A.

Differential migration and activation profile of monocytes after trophoblast interaction.

Macrophages at the maternal-placental interface coordinate opposite demands under the control of trophoblast cells such as the response against pathogens on one hand, and apoptotic cell clearance and wound healing with the production of suppressor cytokines. Here, we investigated whether trophoblast cells induce maternal monocyte activation towards an alternative activated macrophage profile and whether bacterial or viral stimuli modulate their migratory properties. We used an in vitro model of the maternal-placental interface represented by co-cultures of CD14+ cells isolated from fertile women with first trimester trophoblast cell line (Swan-71 cells) in the presence or absence of pathogen associated molecular pattern (PAMP) stimuli lipopolysaccharide (LPS), peptidoglycan (PGN) or poly [I:C]). Maternal CD14+ cells showed increased CD16 and CD39 expression, both markers associated to an alternative activation profile, with no changes in CD80 expression after trophoblast cell interaction. These changes were accompanied by increased IL-10 and decreased IL-12 production by CD14+ cells. After stimulation with LPS, PGN or poly [I:C], monocytes co-cultured with trophoblast cells had lower production of TNF-α and IL-1β compared with non co-cultured monocytes. Interestingly, monocyte migration towards trophoblast cells was prevented in the presence of LPS or PGN but not after 24h of stimulation with poly [I:C]. LPS or PGN also decreased CCR5, CXCL-8 and CCL5 expression. Finally, trophoblast cells co-cultured with monocytes in the presence of pathological stimuli failed to increase chemokine expression, indicating a bidirectional effect. In conclusion, trophoblast might ‘instruct’ maternal monocytes to express an alternative activation profile and restrain their early recruitment under pathological threats as one of the first strategies to avoid potential tissue damage at the maternal-placental interface.

Differential expression of ADAM (a disintegrin and metalloproteinase) genes between human first trimester villous and extravillous trophoblast cells.

A disintegrin and metalloproteinases (ADAMs) are members of the metzincin family of zinc-dependent metalloproteinases that play pivotal roles in the proteolytic degradation of the extracellular matrix for cell invasion. Few studies have investigated the ADAM subtypes that are expressed in first trimester trophoblast cells. The purpose of this study was to elucidate the differential expression profiles of ADAMs between first trimester villous trophoblast cells (VTs) and extravillous trophoblast cells (EVTs). We isolated EVTs from explanted human first trimester chorionic villi and investigated the mRNA expression levels of five members of the ADAM family (ADAMTS1, ADAMTS2, ADAM10, ADAM12, and ADAM17) using real-time PCR. Chorionic villous tips were defined as first trimester VTs. Of the differentially expressed ADAM genes between first trimester VTs and EVTs, ADAMTS1 was expressed at a significantly higher level in EVTs than in VTs. In contrast, both ADAM10 and ADAM12 were expressed at significantly higher levels in VTs than in EVTs. No differences were found in the mRNA levels of ADAMTS2 and ADAM17 between the two cell types. Moreover, we demonstrated that in VTs, the expression level of ADAM12 was significantly downregulated in the late first trimester (10-13 gestational weeks) compared to the middle first trimester (7-8 weeks). These results suggest that first trimester trophoblast cells express ADAM genes in cell type- and gestational age-dependent manners. Our data provide additional insight into the functions of ADAMs in the human placenta.

Effect of Hydroxychloroquine on Antiphospholipid Antibody‐Induced Changes in First Trimester Trophoblast Function

Women with antiphospholipid syndrome (APS) are at risk for pregnancy complications. Antiphospholipid antibodies (aPL) alter trophoblast function by triggering an inflammatory cytokine response; modulating angiogenic factor secretion; and inhibiting migration. While patients with APS are often treated with hydroxychloroquine (HCQ), its effect on trophoblast function is poorly understood. A human first trimester trophoblast cell line was treated with or without antihuman β2GPI mAbs in the presence or absence of HCQ. Supernatants were analyzed by ELISA. Cell migration was measured using a colormetric assay. Antiphospholipid antibodies-induced trophoblast IL-8, IL-1 β, PlGF, and sEndoglin secretion were not altered by HCQ. aPL-induced inhibition of trophoblast migration was partially reversed by HCQ, even though HCQ significantly increased secretion of pro-migratory IL-6 to greater than baseline. aPL-induced upregulation of TIMP2 appears to inhibit trophoblast migration; the inability of HCQ to prevent aPL-induced TIMP2 may explain why migration was only partially restored. Hydroxychloroquine reversed the aPL-inhibition of trophoblast IL-6 secretion and partially limited aPL-inhibition of cell migration. Thus, some form of combination therapy that includes HCQ may be beneficial to pregnant APS patients.

Identification of microRNAs That Regulate TLR2-Mediated Trophoblast Apoptosis and Inhibition of IL-6 mRNA

While infection-induced placental inflammation is a common mechanism of adverse pregnancy outcome, some pathogens can also trigger placental apoptosis, and Toll-like receptors (TLRs) mediate this response. Treatment of human first trimester trophoblast cells with bacterial peptidoglycan (PDG) reduces their constitutive secretion of IL-6 protein and induces apoptosis. This apoptotic response is dependent upon the cell’s expression of TLR1, TLR2 and TLR10, and their lack of TLR6, such that ectopic expression of TLR6 prevents PDG-induced apoptosis and restores IL-6 production. In this current study we have identified three microRNAs (miRs) that regulate TLR2-mediated responses in the human trophoblast. Herein we report that miR-329 plays a pivotal role in mediating PDG-induced trophoblast apoptosis and inhibition of IL-6 mRNA expression by targeting the NF-κB subunit, p65. TLR2 activation by PDG upregulates miR-329 expression and inhibits NF-κB p65 and IL-6 mRNA, and this is reversed by the presence of TLR6. Moreover, inhibition of miR-329 prevents PDG-induced inhibition of NF-κB p65 and IL-6 mRNA expression, and restores cell survival. In addition, we have found miR-23a and let-7c to directly regulate PDG-mediated inhibition of IL-6 mRNA. TLR2 activation by PDG upregulates miR23a and let-7c expression and this is reversed by the presence of TLR6. Furthermore, inhibition of both miR23a and let-7c prevents PDG-inhibition of trophoblast IL-6 mRNA expression. Together, our findings suggest that multiple miRs are involved in the molecular regulation of TLR2-mediated responses in the trophoblast towards gram-positive bacterial components.

NOD1 expression elicited by iE-DAP in first trimester human trophoblast cells and its potential role in infection-associated inflammation

ObjectivesThe underlying mechanisms of protective immunity of placental trophoblast cells against bacterial infection remain largely unknown. NOD1 are intracellular pattern recognition receptors that are activated by bacterial peptides and mediate innate immunity. This study aimed to investigate the expression and function of NOD1 in first trimester trophoblast cells, and evaluate the potential role of trophoblast cells in infection-associated inflammation. Study designHuman extravillous trophoblast cell line HTR8 cells were stimulated with various concentrations of iE-DAP for various periods of time. NOD1 expression was detected by immunofluorescence, and the changes in NOD1 and RICK mRNA and protein in H8 cells were determined by real-time polymerase chain reaction and Western blot analysis. The concentrations of interleukin (IL)-8 and IL-6 secreted by H8 cells were examined by enzyme-linked immunosorbent assay. NF-κB transcription activity and P65 expression were detected by electrophoretic mobility shift assay and Western blot analysis. ResultsH8 cells expressed NOD1, and the effects of iE-DAP on NOD1 were dose- and time-dependent. The concentration of IL-8 increased gradually with increasing concentration of iE-DAP, and the levels of IL-8 and IL-6 were associated with the duration of exposure to iE-DAP. The dose of iE-DAP was significantly associated with expression of RICK and P65, and stimulation of H8 cells by iE-DAP altered NF-κB transcription activity. ConclusionsNOD1 may have a role in mediating infection-associated inflammation. Once iE-DAP is recognized by NOD1, the inflammatory response may be induced via NOD1–RICK–NF-κB-mediated pathways.

Sildenafil prevents apoptosis of first trimester trophoblast cells exposed to environmental stress

Human trophoblast cells develop within a hypoxic environment during the first trimester of pregnancy. Oxygen fluctuations can create environmental stress and induce trophoblast apoptosis, leading to placental dysfunction. Sildenafil (Viagra) is a potent, selective inhibitor of phosphodiesterase type 5, which increases guanosine 3’,5’-cyclic monophosphate (cGMP). We evaluated whether Sildenafil could rescue human trophoblasts from apoptosis caused by ischemic reperfusion injury using first trimester chorionic villous explants and the cytotrophoblast cell line, HTR-8/SVneo.

Do decidual natural killer cells have a role in vascular smooth muscle cell dedifferentiation during spiral artery remodelling?

Adequate extravillous trophoblast (EVT) invasion is essential for successful placentation. Although miR-520c-3p plays an important role in CD44-mediated invasion in cancer cells, there is little information on whether miR-520c-3p is involved in the regulatory mechanisms of CD44-mediated EVT invasion.We screened first trimester trophoblast cells and trophoblast cell lines for expression of miR-520c-3p using real-time polymerase chain reaction. The cell invasion assay was performed using EVT cell lines, HTR8/SVneo and HChEpC1b, to investigate the capability of suppressing EVT invasion by miR-520c-3p. Laser microdissection analysis was then used to determine whether miR-520c-3p was present in the first trimester decidua. Finally, the possibility of chorionic villous trophoblast (CVT)-EVT communication via exosomal miR-520c-3p was determined using an in vitro model based on BeWo exosomes and the EVT cell lines as recipient cells.The miR-520c-3p level was significantly downregulated in EVT cell lines and EVTs. Cell invasion was significantly inhibited in miR-520c-3p-overexpressing cell lines, involving a significant reduction of CD44. Laser microdissection analysis showed that miR-520c-3p in the periarterial area of the decidua was significantly higher than that in the non-periarterial area. Using an in vitro model system, BeWo exosomal miR-520c-3p was internalized into the EVT cells with subsequently reduced cell invasion via CD44 repression.EVT invasion is synergistically enhanced by the reciprocal expression of endogenous miR-520c-3p and CD44. The present study supports a novel model involving a placenta-associated miRNA function in cell-cell communication in which CVT exosomal miR-520c-3p regulates cell invasion by targeting CD44 in EVTs.

Impact of human first trimester trophoblast cell line ACH-3P on the function of endothelial cells under different oxygen concentrations

Impact of human first trimester trophoblast cell line ACH-3P on the function of endothelial cells under different oxygen concentrations

Schistosoma japonicum Soluble Egg Antigens Attenuate Invasion in a First Trimester Human Placental Trophoblast Model

BackgroundSchistosomiasis affects nearly 40 million women of reproductive age, and is known to elicit a pro-inflammatory signature in the placenta. We have previously shown that antigens from schistosome eggs can elicit pro-inflammatory cytokine production from trophoblast cells specifically; however, the influence of these antigens on other characteristics of trophoblast function, particularly as it pertains to placentation in early gestation, is unknown. We therefore sought to determine the impact of schistosome antigens on key characteristics of first trimester trophoblast cells, including migration and invasion.MethodsFirst trimester HTR8/SVneo trophoblast cells were co-cultured with plasma from pregnant women with and without schistosomiasis or schistosome soluble egg antigens (SEA) and measured cytokine, cellular migration, and invasion responses.ResultsExposure of HTR8 cells to SEA resulted in a pro-inflammatory, anti-invasive signature, characterized by increased pro-inflammatory cytokines (IL-6, IL-8, MCP-1) and TIMP-1. Additionally, these cells displayed 62% decreased migration and 2.7-fold decreased invasion in vitro after treatment with SEA. These results are supported by increased IL-6 and IL-8 in the culture media of HTR8 cells exposed to plasma from Schistosoma japonica infected pregnant women.ConclusionsSoluble egg antigens found in circulation during schistosome infection increase pro-inflammatory cytokine production and inhibit the mobility and invasive characteristics of the first trimester HTR8/SVneo trophoblast cell line. This is the first study to assess the impact of schistosome soluble egg antigens on the behavior of an extravillous trophoblast model and suggests that schistosomiasis in the pre-pregnancy period may adversely impact placentation and the subsequent health of the mother and newborn.

A Role for Uric Acid and the Nalp3 Inflammasome in Antiphospholipid Antibody-Induced IL-1β Production by Human First Trimester Trophoblast

Women with antiphospholipid syndrome (APS) are at risk of recurrent pregnancy loss and obstetrical disorders, such as preeclampsia and intrauterine growth restriction (IUGR). Antiphospholipid antibodies (aPL) directly target the placenta by binding beta2-glycoprotein I (β2GPI) expressed on the trophoblast. We recently demonstrated in human first trimester trophoblast cells that anti-β2GPI antibodies (Abs) induce the secretion of IL-1β in a Toll-like receptor 4 (TLR4)-dependent manner. IL-1β secretion requires processing of pro-IL-1β and this is mediated by the inflammasome, a complex of Nalp3, apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1. The objective of this study was to determine if aPL induce IL-1β production in trophoblast via the inflammasome. Using a human first trimester trophoblast cell line, we demonstrated that a mouse anti-β2GPI mAb and human polyclonal aPL-IgG induce IL-1β processing and secretion, which was partially blocked upon caspase-1 inhibition. Nalp3 and ASC knockdown also attenuated anti-β2GPI Ab-induced IL-1β secretion. Furthermore, aPL stimulated the production of uric acid in a TLR4-dependent manner; and inhibition of uric acid prevented aPL-induced IL-1β production by the trophoblast. These findings demonstrate that aPL, via TLR4 activation, induce a uric acid response in human trophoblast, which in turn activates the Nalp3/ASC inflammasome leading to IL-1β processing and secretion. This novel mechanism may account for the inflammation at the maternal-fetal interface, which causes placental dysfunction and increases the risk of adverse pregnancy outcome in patients with APS.

5-Aza-dC treatment induces mesenchymal-to-epithelial transition in 1st trimester trophoblast cell line HTR8/SVneo

Placental trophoblast invasion involves a cellular transition from epithelial to mesenchymal phenotype. Cytotrophoblasts undergo epithelial to mesenchymal transition (EMT) when differentiating into extravillous trophoblasts and gaining the capacity of invasion. In this research, we investigated the role of DNA methylation in trophoblasts during this EMT. First, using BeWo and HTR8/SVneo cell lines as models of cytotrophoblasts and extravillous trophoblasts, respectively, we analyzed the gene expression and DNA methylation status of the known epithelial marker genes, E-Cadherin and Cytokeratin7. We found that, in HTR8/SVneo cells, both genes were silenced and their promoters hypermethylated, as compared with the high-level gene expression and promoter hypomethylation observed in BeWo cells. This result suggests that dynamic DNA methylation of epithelial marker genes plays a critical role in the trophoblast EMT process. To verify these results, we treated HTR8/SVneo cells with 5-aza-dC, a known inhibitor of DNA methyltransferase, for three days. Five-Aza-dC treatment significantly increased the expression of epithelial marker genes and slightly decreased the expression of mesenchymal genes, as detected by qRT-PCR, immunocytochemistry and Western blot. Furthermore, 5-aza-dC treated HTR8/SVneo cells changed their morphology from mesenchymal into epithelial phenotype, indicating that 5-aza-dC induced mesenchymal to epithelial transition. Lastly, we examined the effect of 5-aza-dC on trophoblast migration and invasion capacity. We applied 5-aza-dC to HTR8/SVneo cells in trans-well cell migration and invasion assays and found that 5-aza-dC treatment decreased trophoblast migration and invasion capacity. In conclusion, DNA methylation of epithelial marker genes represents a molecular mechanism for the process of trophoblast EMT.

The effects of cyclic AMP, sex steroids and global hypomethylation on the expression of genes controlling the activity of the renin–angiotensin system in placental cell lines

The placental renin–angiotensin system (RAS) is involved in placentation. We have shown that prorenin mRNA (REN) is expressed in a first trimester trophoblast cell line (HTR-8/SVneo) but not in a choriocarcinoma cell line (BeWo). We attempted to stimulate RAS expression in these cells by cAMP, 5′-aza-2′-deoxycytidine (AZA; an inhibitor of methylation), cAMP and AZA combined, and the sex steroids medroxyprogesterone acetate (MPA) and estradiol-17β (E2) with and without cAMP. RAS mRNAs were measured by qPCR and prorenin concentration in supernatants measured by an ELISA. In HTR-8/SVneo cells, all treatments increased REN expression compared to controls and cAMP + AZA combined was more effective than either treatment alone. Prorenin levels in supernatants were similarly upregulated. In HTR-8/SVneo cells, angiotensinogen (AGT) mRNA expression was increased by MPA + E2 either with or without cAMP. AGT expression was also significantly increased by AZA. BeWo cells did not express REN or prorenin and it was not inducible with any treatment. AGT expression was significantly increased with AZA, the combination of cAMP + AZA, and MPA + E2 + cAMP treatments. Since cAMP, AZA, cAMP and AZA combined, or MPA and E2 with and without cAMP in HTR-8/SVneo cells, a cell line most similar in its RAS expression to the in vivo placenta, these factors may affect placental RAS activity. Surprisingly, these treatments also induced AGT expression in BeWo cells. Whether they are involved in regulating AGT in choriocarcinomas in vivo remains to be determined.

Pravastatin does not prevent antiphospholipid antibody-mediated changes in human first trimester trophoblast function

What is the effect of pravastatin on antiphospholipid antibody (aPL) modulation of human first trimester trophoblast function? Pravastatin does not prevent the effects of aPL on human first trimester trophoblast cell function. Antiphospholipid syndrome (APS) is associated with recurrent pregnancy loss and late pregnancy complications, such as pre-eclampsia, owing to direct targeting of the placenta by aPL. While treatment with heparin reduces the rate of pregnancy loss, the risk for severe pre-eclampsia remains high. Thus, there is a need to find alternative treatments for the prenatal management of patients with APS. Statins have recently been shown to prevent aPL-mediated fetal loss in mice but their effects on a human pregnancy model of APS have not yet been studied. The human first trimester trophoblast cell line, HTR8, and human first trimester trophoblast primary cultures were incubated with or without a mouse anti-human beta 2 glycoprotein I (β(2)GPI) monoclonal antibody in the presence or absence of pravastatin. Cytokine and angiogenic factor secretion were measured by enzyme-linked immunosorbent assay and multiplex analysis. Cell migration was measured using a colorimetric two-chamber migration assay. Using the human first trimester trophoblast cell line, HTR8, pravastatin significantly augmented, compared with no treatment, aPL-dependent secretion of interleukin (IL)-8 (P< 0.05), IL-1β (P< 0.05) and soluble endoglin (P< 0.01) but had no effect on aPL-induced up-regulation of vascular endothelial growth factor, placenta growth factor or growth-related oncogene alpha secretion. Furthermore, pravastatin alone limited basal HTR8 cell migration (P< 0.01), and did not mitigate the adverse effect of aPL on trophoblast migration. Pravastatin also had no impact on the secretion of pro-inflammatory cytokines and angiogenic factors by primary human first trimester trophoblast cells exposed to aPL. While our in vitro findings suggest that pravastatin may not be effective in preventing pregnancy complications in patients with APS, the in vivo condition may be more complex, and thus, more studies are needed to determine the effectiveness of pravastatin in the prevention of aPL-associated pregnancy complications in humans. This work was supported by the American Heart Association.

MicroRNA expression profiles of trophoblastic cells

BackgroundMicroRNAs (miRNAs) are small single-stranded RNA molecules working as post-transcriptional modulators of gene expression. Trophoblast cells are a heterogenous group of fetal cells forming the feto–maternal interface and displaying a wide spectrum of functions. The regulation of their behavior may partly underly the control through miRNAs. Therefore, we aimed to compare the miRNA profile of primary first and third trimester trophoblast cells with that of different trophoblastic cell lines. Material and methodsTotal RNA was obtained from isolated cytotrophoblast cells from healthy term and first trimester placentae and the cell lines HTR-8/SVneo (immortalized trophoblast cells), JEG-3 (choriocarcinoma), ACH-3P and AC1-M59, which are choriocarcinoma cells fused with first and third trimester trophoblast cells, respectively. The expression level of 762 different miRNAs was quantitatively analyzed by using a TaqMan Human MicroRNA Array. For testing the reproducibility of the array technique, the expression of 9 selected miRNAs has been re-analyzed by individual qPCR. ResultsThe analyzed cell types share many similar patterns of miRNAs, but are significantly distinct in the expression of three miRNA clusters: chromosome 19 miRNA cluster (C19MC; containing 54 different miRNAs), C14MC (34 miRNAs) and a minor cluster (miRNA-371 to miRNA-373 cluster), also located on chromosome 19. Expression of miRNAs within C19MC increases significantly from first to third trimester trophoblast while that of C14MC members decreases. MiRNAs within the miR-371-3 cluster augment slightly. C19MC and the miR-371-3 cluster are not expressed by HTR-8/SVneo cells whilst C14MC is almost not detectable in the choriocarcinoma-derived cell lines complete array data available at NCBI Gene Expression Omnibus accession number GSE32346: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32346). Beside the miRNAs within the mentioned clusters, further 27 miRNAs are differentially expressed (>100 fold) between term and first trimester trophoblast cells. The placenta-specific miRNAs miR-141 and miR-21 as well as let-7g are expressed in all tested cells with the highest expression in primary trophoblast cells. ConclusionPrimary first trimester and term trophoblast cells and trophoblastic cell lines display major differences in their miRNA fingerprints which may be involved in their different behavior and characteristics.