Model Of Human Trophoblast Research Articles

The immune checkpoint molecule, VTCN1/B7-H4, guides differentiation and suppresses proinflammatory responses and MHC class I expression in an embryonic stem cell-derived model of human trophoblast.

The placenta acts as a protective barrier to pathogens and other harmful substances present in the maternal circulation throughout pregnancy. Disruption of placental development can lead to complications of pregnancy such as preeclampsia, intrauterine growth retardation and preterm birth. In previous work, we have shown that expression of the immune checkpoint regulator, B7-H4/VTCN1, is increased upon differentiation of human embryonic stem cells (hESC) to an in vitro model of primitive trophoblast (TB), that VTCN1/B7-H4 is expressed in first trimester but not term human placenta and that primitive trophoblast may be uniquely susceptible to certain pathogens. Here we report on the role of VTCN1 in trophoblast lineage development and anti-viral responses and the effects of changes in these processes on major histocompatibility complex (MHC) class I expression and peripheral NK cell phenotypes.

In Vitro Model of Human Trophoblast in Early Placentation.

The complex process of placental implantation and development affects trophoblast progenitors and uterine cells through the regulation of transcription factors, cytokines, adhesion receptors and their ligands. Differentiation of trophoblast precursors in the trophectoderm of early ontogenesis, caused by the transcription factors, such as CDX2, TEAD4, Eomes and GATA3, leads to the formation of cytotrophoblast and syncytiotrophoblast populations. The molecular mechanisms involved in placental formation inside the human body along with the specification and differentiation of trophoblast cell lines are, mostly due to the lack of suitable cell models, not sufficiently elucidated. This review is an evaluation of current technologies, which are used to study the behavior of human trophoblasts and other placental cells, as well as their ability to represent physiological conditions both in vivo and in vitro. An in vitro 3D model with a characteristic phenotype is of great benefit for the study of placental physiology. At the same time, it provides great support for future modeling of placental disease.

Sex hormone-binding globulin regulates glucose metabolism in human placental trophoblasts via cAMP/PKA/CREB1.

AimThis study was conducted to investigate the effects of sex hormone‐binding globulin (SHBG) on glucose metabolism and insulin resistance in human placental trophoblasts and involvement of the cAMP/PKA/CREB1 signaling pathway in these effects.MethodsAn insulin resistance cell model of human trophoblasts was established. An SHBG‐overexpression plasmid was transfected into these cells, and the expression of glucose transporter 1 (GLUT1), CREB and p‐CREB was detected and analyzed in normal cells, model cells and all groups of transfected cells by real‐time PCR and western blotting; cAMP, PKA, glucose consumption and pyruvic acid levels were also detected.ResultsAmong the four groups, there was no significant difference in the expression of CREB mRNA or GLUT1 mRNA (P > 0.05); however, CREB, p‐CREB, GLUT1 protein, cAMP and PKA showed low expression (P < 0.05) and cell glucose consumption and pyruvate production were decreased (P < 0.05) in the model group, compared to the normal group. SHBG overexpression in insulin‐resistant cells partially increased the levels of p‐CREB, GLUT1, cAMP and PKA (P < 0.05). Intracellular glucose consumption and pyruvate production were nearly restored to the levels observed in cells from the normal group.ConclusionSex hormone‐binding globulin regulates GLUT1 expression via the cAMP/PKA/CREB1 pathway and affects glucose transport in the placenta, which can induce insulin resistance and gestational diabetes.

The BeWo cell line derived from a human placental choriocarcinoma is permissive for respiratory syncytial virus infection.

The respiratory syncytial virus (RSV) is the main pathogen associated with upper respiratory tract infections during early childhood. Vertical transmission of this virus has been suggested in humans, based on observations recorded during animal studies that revealed an association of RSV with persistent structural and functional changes in the developing lungs of the offspring. However, human placentas have not yet been evaluated for susceptibility to RSV infection. In this study, we examined the capacity of RSV to infect a human trophoblast model, the BeWo cell line. Our results suggest that BeWo cells are susceptible to RSV infection since they allow RNA viral replication, viral protein translation, leading to the production of infectious RSV particles. In this report, we demonstrate that a human placenta model system, consisting of BeWo cells, is permissive to RSV infection. Thus, the BeWo cell line may represent a useful model for studies that aim to characterize the events of a possible RSV infection at the human maternal-fetal interface.

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNFα), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNFα regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNFα signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNFα), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNFα regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNFα signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Characterization of a human trophoblast model to evaluate novel therapeutics for preeclampsia

Characterization of a human trophoblast model to evaluate novel therapeutics for preeclampsia

Development of Non-Viral, Trophoblast-Specific Gene Delivery for Placental Therapy.

Low birth weight is associated with both short term problems and the fetal programming of adult onset diseases, including an increased risk of obesity, diabetes and cardiovascular disease. Placental insufficiency leading to intrauterine growth restriction (IUGR) contributes to the prevalence of diseases with developmental origins. Currently there are no therapies for IUGR or placental insufficiency. To address this and move towards development of an in utero therapy, we employ a nanostructure delivery system complexed with the IGF-1 gene to treat the placenta. IGF-1 is a growth factor critical to achieving appropriate placental and fetal growth. Delivery of genes to a model of human trophoblast and mouse placenta was achieved using a diblock copolymer (pHPMA-b-pDMAEMA) complexed to hIGF-1 plasmid DNA under the control of trophoblast-specific promoters (Cyp19a or PLAC1). Transfection efficiency of pEGFP-C1-containing nanocarriers in BeWo cells and non-trophoblast cells was visually assessed via fluorescence microscopy. In vivo transfection and functionality was assessed by direct placental-injection into a mouse model of IUGR. Complexes formed using pHPMA-b-pDMAEMA and CYP19a-923 or PLAC1-modified plasmids induce trophoblast-selective transgene expression in vitro, and placental injection of PLAC1-hIGF-1 produces measurable RNA expression and alleviates IUGR in our mouse model, consequently representing innovative building blocks towards human placental gene therapies.

Interaction between human placental microvascular endothelial cells and a model of human trophoblasts: effects on growth cycle and angiogenic profile.

Intrauterine growth restriction (IUGR) is a leading cause of perinatal complications, and is commonly associated with reduced placental vasculature. Recent studies demonstrated over‐expression of IGF‐1 in IUGR animal models maintains placental vasculature. However, the cellular environment of the placental chorionic villous is unknown. The close proximity of trophoblasts and microvascular endothelial cells in vivo alludes to autocrine/paracrine regulation following Ad‐HuIGF‐1 treatment. We investigated the co‐culturing of BeWo Choriocarcinoma and Human Placental Microvascular Endothelial Cells (HPMVECs) on the endothelial angiogenic profile and the effect Ad‐HuIGF‐1 treatment of one cell has on the other. HPMVECs were isolated from human term placentas and cultured in EGM‐2 at 37°C with 5% CO2. BeWo cells were maintained in Ham's F12 nutrient mix with 10% FBS and 1% pen/strep. Co‐cultured HPMVECS+BeWo cells were incubated in serum‐free control media, Ad‐HuIGF‐1, or Ad‐LacZ at MOI 0 and MOI 100:1 for 48 h. Non‐treated cells and mono‐cultured cells were compared to co‐cultured cells. Angiogenic gene expression and proliferative and apoptotic protein expression were analysed by RT‐qPCR and immunocytochemistry, respectively. Statistical analyses was performed using student's t‐test with P <0.05 considered significant. Direct Ad‐HuIGF‐1 treatment increased HPMVEC proliferation (n =4) and reduced apoptosis (n =3). Co‐culturing HPMVECs+BeWo cells significantly altered RNA expression of the angiogenic profile compared to mono‐cultured HPMVECs (n =8). Direct Ad‐HuIGF‐1 treatment significantly increased Ang‐1 (n =4) in BeWo cells. Ad‐HuIGF‐1 treatment of HPMVECs did not alter the RNA expression of angiogenic factors. Trophoblastic factors may play a key role in placental vascular development and IGF‐1 may have an important role in HPMVEC growth.

Regulation of amino acid transporters by adenoviral-mediated human insulin-like growth factor-1 in a mouse model of placental insufficiency in vivo and the human trophoblast line BeWo in vitro

Previous work in our laboratory demonstrated that over-expression of human insulin-like growth factor-11 (hIGF-1) in the placenta corrects fetal weight deficits in mouse, rat, and rabbit models of intrauterine growth restriction without changes in placental weight. The underlying mechanisms of this effect have not been elucidated. To investigate the effect of intra-placental IGF-1 over-expression on placental function we examined amino acid transporter expression and localization in both a mouse model of placental Insufficiency (PI) and a model of human trophoblast, the BeWo Choriocarcinoma cell line.For in vitro human studies, BeWo Choriocarcinoma cells were maintained in F12 complete medium + 10%FBS. Cells were incubated in serum-free control media ± Ad-IGF-1 or Ad-LacZ for 48 h. MOIs of 10:1 and 100:1 were utilized. In BeWo, transfection efficiency was 100% at an MOI of 100:1 and Ad-IGF-1 significantly increased IGF-1 secretion, proliferation and invasion but reduced apoptosis compared to controls. In vitro, amino acid uptake was increased following Ad-IGF-1 treatment and associated with significantly increased RNA expression of SNAT1, 2, LAT1 and 4F2hc. Only SNAT2 protein expression was increased but LAT1 showed relocalization from a perinuclear location to the cytoplasm and cell membrane.For in vivo studies, timed-pregnant animals were divided into four groups on day 18; sham-operated controls, uterine artery branch ligation (UABL), UABL + Ad-hIGF-1 (108 PFU), UABL + Ad-LacZ (108 PFU). At gestational day 20, pups and placentas were harvested by C-section. Only LAT1 mRNA expression changed, showing that a reduced expression of the transporter levels in the PI model could be partially rectified with Ad-hIGF1 treatment. At the protein level, System L was reduced in PI but remained at control levels following Ad-hIGF1. The System A isoforms were differentially regulated with SNAT2 expression diminished but SNAT1 increased in PI and Ad-hIGF1 groups.Enhanced amino acid isoform transporter expression and relocalization to the membrane may be an important mechanism contributing to Ad-hIGF-1 mediated correction of placental insufficiency.

Adenoviral-Mediated Placental Gene Transfer of IGF-1 Corrects Placental Insufficiency via Enhanced Placental Glucose Transport Mechanisms

Previous work in our laboratory demonstrated that over-expression of human insulin-like growth factor -1 (hIGF-1) in the placenta corrects fetal weight deficits in mouse, rat, and rabbit models of intrauterine growth restriction without changes in placental weight. The underlying mechanisms of this effect have not been elucidated. To investigate the effect of intra-placental IGF-1 over-expression on placental function we examined glucose transporter expression and localization in both a mouse model of IUGR and a model of human trophoblast, the BeWo Choriocarcinoma cell line.MethodsAt gestational day 18, animals were divided into four groups; sham-operated controls, uterine artery branch ligation (UABL), UABL+Ad-hIGF-1 (108 PFU), UABL+Ad-LacZ (108 PFU). At gestational day 20, pups and placentas were harvested by C-section. For human studies, BeWo choriocarcinoma cells were grown in F12 complete medium +10%FBS. Cells were incubated in serum-free control media ±Ad-IGF-1 or Ad-LacZ for 48 hours. MOIs of 10∶1 and 100∶1 were utilized. The RNA, protein expression and localization of glucose transporters GLUT1, 3, 8, and 9 were analyzed by RT-PCR, Western blot and immunohistochemistry.ResultsIn both the mouse placenta and BeWo, GLUT1 regulation was linked to altered protein localization. GLUT3, localized to the mouse fetal endothelial cells, was reduced in placental insufficiency but maintained with Ad-I GF-1 treatment. Interestingly, GLUT8 expression was reduced in the UABL placenta but up-regulated following Ad-IGF-1 in both mouse and human systems. GLUT9 expression in the mouse was increased by Ad-IGF-1 but this was not reflected in the BeWo, where Ad-IGF-1 caused moderate membrane relocalization.ConclusionEnhanced GLUT isoform transporter expression and relocalization to the membrane may be an important mechanism in Ad-hIGF-1mediated correction of placental insufficiency.

Secreted cyclophilin A induction during embryo implantation in a model of human trophoblast–endometrial epithelium interaction

Embryo implantation in the human is a complex process that is crucial for a successful pregnancy. Implantation involves complex interactions between the embryo and the maternal endometrium. In order to understand the critical events involved in human embryo implantation, we established a human trophoblast-endometrial interaction model to study the putative alterations of gene expression during implantation. Comparative proteomic analysis was applied to the co-culture and separated culture systems of the trophoblast cell line BeWo and human endometrial epithelial cell (EEC) line RL95-2. Comparing the secreted molecules resulted in an interesting finding that secreted cyclophilin A (CypA) was increased in the co-culture system. To further verify our observation, human recombinant CypA was applied to endometrial cells to understand the downstream gene regulation. Cyclophilin A (CypA) was identified by MALDI-TOF Mass with higher expression levels in the co-cultured cells when compared to the endometrial cells alone. Western blot analysis further confirmed the increased expression of CypA in co-culture conditions. Immunoblotting analysis showed that both p38 MAPK and extracellular signal-regulated kinase (ERK) were activated in EEC. Our results suggest that the secreted CypA plays a specific role in the signaling pathway of embryo implantation. The identification of CypA opens a new avenue for early embryo implantation research.

Comparative Transcriptome Analysis of Human Trophectoderm and Embryonic Stem Cell-Derived Trophoblasts Reveal Key Participants in Early Implantation1

The implantation process begins with attachment of the trophectoderm (TE) of the blastocyst to the maternal endometrial epithelium. Herein we have investigated the transcriptome of mural TE cells from 13 human blastocysts and compared these with those of human embryonic stem cell (hESC)-derived-TE (hESC(troph)). The transcriptomes of hESC(troph) at Days 8, 10, and 12 had the greatest consistency with TE. Among genes coding for secreted proteins of the TE of human blastocysts and of hESC(troph) are several molecules known to be involved in the implantation process, as well as novel ones, such as CXCL12, HBEGF, inhibin A, DKK3, WNT5A, and follistatin. The similarities between the two lineages underscore some of the known mechanisms and offer discovery of new mechanisms and players in the process of the very early stages of human implantation. We propose that the hESC(troph) is a viable functional model of human trophoblasts to study trophoblast-endometrial interactions. Furthermore, the data derived herein offer the promise of novel diagnostics and therapeutics aimed at practical challenges in human infertility and pregnancy disorders associated with abnormal embryonic implantation.

449. Nanoparticle-Mediated Gene Transfer in an In Vitro Model of Human Trophoblasts

449. Nanoparticle-Mediated Gene Transfer in an In Vitro Model of Human Trophoblasts

Telomeric NAP1L4 and OSBPL5 of the KCNQ1 Cluster, and the DECORIN Gene Are Not Imprinted in Human Trophoblast Stem Cells

BackgroundGenomic imprinting of the largest known cluster, the Kcnq1/KCNQ1 domain on mChr7/hChr11, displays significant differences between mouse and man. Of the fourteen transcripts in this cluster, imprinting of six is ubiquitous in mice and humans, however, imprinted expression of the other eight transcripts is only found in the mouse placenta. The human orthologues of the latter eight transcripts are biallelically expressed, at least from the first trimester onwards. However, as early development is less divergent between species, placental specific imprinting may be present in very early gestation in both mice and humans.Methodology/Principal FindingsHuman embryonic stem (hES) cells can be differentiated to embryoid bodies and then to trophoblast stem (EB-TS) cells. Using EB-TS cells as a model of post-implantation invading cytotrophoblast, we analysed allelic expression of two telomeric transcripts whose imprinting is placental specific in the mouse, as well as the ncRNA KCNQ1OT1, whose imprinted expression is ubiquitous in early human and mouse development. KCNQ1OT1 expression was monoallelic in all samples but OSBPL5 and NAP1L4 expression was biallelic in EB-TS cells, as well as undifferentiated hES cells and first trimester human fetal placenta. DCN on hChr12, another gene imprinted in the mouse placenta only, was also biallelically expressed in EB-TS cells. The germline maternal methylation imprint at the KvDMR was maintained in both undifferentiated hES cells and EB-TS cells.Conclusions/SignificanceThe question of placental specific imprinting in the human has not been answered fully. Using a model of human trophoblast very early in gestation we show a lack of imprinting of two telomeric genes in the KCNQ1 region and of DCN, whose imprinted expression is placental specific in mice, providing further evidence to suggest that humans do not exhibit placental specific imprinting. The maintenance of both differential methylation of the KvDMR and monoallelic expression of KCNQ1OT1 indicates that the region is appropriately regulated epigenetically in vitro. Human gestational load is less than in the mouse, resulting in reduced need for maternal resource competition, and therefore maybe also a lack of placental specific imprinting. If genomic imprinting exists to control fetal acquisition of maternal resources driven by the placenta, placenta-specific imprinting may be less important in the human than the mouse.

Effects of low oxygen levels on the expression and function of transporter OCTN2 in BeWo cells

Although hypoxia is normal in early pregnancy, low placental oxygen concentrations later in pregnancy are often linked to complications such as pre-eclampsia and intrauterine growth restriction. The effects of low oxygen levels on drug and nutrient uptake via the organic cation transporter OCTN2 has been studied in BeWo cells, an in-vitro model of human trophoblast. BeWo cells were cultured under 20% (control) or 2% O(2) (hypoxia) for 48 h before each experiment. In-vitro hypoxia was also simulated by the addition of CoCl(2) to the cell culture medium. RT-PCR indicated increased transcription of OCTN2 in BeWo cells cultured under hypoxia, but Western blots did not show a corresponding increase in the amount of OCTN2 protein in the hypoxic cells compared with control. Hypoxia resulted in significant reductions in OCTN2-mediated carnitine uptake. Decreased placental transport of carnitine may lead to symptoms of carnitine deficiency in infants from hypoxic pregnancies, whether caused by high altitude, pre-eclampsia or other factors. The OCTN1 substrate ergothioneine reversed the effects of hypoxia on carnitine transport, but identical concentrations of N-acetylcysteine, another water-soluble intracellular antioxidant, did not have the same effect.

The Effect of Interleukin‐17 on the Proliferation and Invasion of JEG‐3 Human Choriocarcinoma Cells

As there has been a study in mice showing the expression of IL-17 by decidual cells and the status of IL-17 receptor expression in human pregnancy is not known, we hypothesized that IL-17 may regulate human trophoblast proliferation and invasion. JEG-3 cell line was used as a model for human trophoblast. Immunohistochemitry and reverse transcriptase polymerase chain reaction techniques were used to identify IL-17 receptor protein and mRNA, respectively. The effects of IL-17 on JEG-3 cell proliferation and invasion were tested using the BrdU incorporation and the Matrigel invasion assays, respectively. IL-17 increased the invasive capacity of JEG-3 cells but had no effect on the proliferation and multinucleated formation of JEG-3 cells. In this JEG-3 cell model of human trophoblast, the IL-17R and IL-17 may have a regulatory role in trophoblast invasion.

Expression of Cyclooxygenase Isoforms in Developing Rat Placenta, Human Term Placenta, and BeWo Human Trophoblast Model

Cyclooxygenase (COX) catalyzes the rate-limiting step in the conversion of essential fatty acids (EFAs) to bioactive molecules such as prostaglandins (PGs), which play critical roles in many aspects of female reproduction and in fetal development. There are two primary related COX isoforms, the constitutively expressed COX-1 and the inducible COX-2. Although the expression of COX-1 and COX-2 has been demonstrated in the amnion, chorion, and decidua, relatively little information exists with regard to their expression and physiological function in the placenta during gestation. In this study, we have elucidated the spatial and temporal patterns of COX-1 and COX-2 expression in the labyrinthine and junctional zones of the developing rat placenta, in the human term placenta, and in the BeWo human trophoblast model using semiquantitative RT-PCR, Western blot, and immunohistochemical analyses. The mRNA and protein expression of COX-1 and COX-2 were demonstrated in the developing rat placenta with increasing expression observed toward parturition. COX-2 exhibited greater expression than COX-1 after mid-gestation and had a corresponding shift in spatial expression from the labyrinthine to the junctional zone at term. COX-1 and -2 were also expressed in human term placenta, while BeWo cells exhibited moderate expression of COX-1 and weak expression of COX-2. The results demonstrate that COX-1 and COX-2 are expressed in the rat and human placentas. The differential expression patterns in the rat placenta, especially of COX-2, imply that there may be gestational changes in the biosynthesis of PGs and other potential bioactive EFA metabolites. Establishing the expression of the COX isoforms provides a framework for future investigations into the functional and physiological significance of COX-1 and COX-2 in the placenta, particularly with respect to influencing normal pregnancy and fetal development, and to provide insights into therapeutic utilization of COX inhibitors in pregnancy.

Carrier-mediated Transport of Folic Acid in BeWo Cell Monolayers as a Model of the Human Trophoblast

Using cultured BeWo cells as a model of human trophoblast, we investigated whether carrier-mediated transport of folic acid occurs. BeWo cells, which were derived from human choriocarcinoma, were cultured on a tissue culture plate or in a permeation chamber. When the cells reached confluence, drug uptake or transport experiments were performed. The uptake of [3H]folic acid by BeWo cells occurred at a much lower rate at 4°C than at 37°C. The uptake of [3H]folic acid was saturable at higher concentrations and inhibited by typical metabolic inhibitors, sodium azide and 2,4-dinitrophenol. The uptake of [3H]folic acid was significantly increased with decreasing pH of the incubation buffer and markedly inhibited by 4,4′-diidothiocyanostilbene-2,2′-disulfonic acid (DIDS). Analogs of folic acid, methotrexate and 5-methyltetrahydrofolate, inhibited the uptake of [3H]folic acid by BeWo cells. Kinetic analysis using Lineweaver-Burk plots revealed that methotrexate competitively inhibited the uptake of [3H]folic acid and folic acid competitively inhibited the uptake of [3H]methotrexate. In transport experiments, the permeation of [3H]folic acid from the apical-to-basal side was greater than that from the basal-to-apical side, and the transport of [3H]folic acid from the apical-to-basal side was inhibited by an excess of folic acid. The findings obtained in the present study confirm the existence of an asymmetric, carrier-mediated transport system for folic acid and its analog, methotrexate, across BeWo cells, a representative of the human trophoblast.

Carrier-mediated transport of valproic acid in BeWo cells, a human trophoblast cell line

The biochemical mechanisms mediating the rapid distribution of valproic acid across placenta are not precisely known. We have characterized valproic acid transport by the human trophoblast using the human choriocarcinoma cell line, BeWo. The uptake of [ 14C]valproic acid by BeWo cells was found to be saturable and blocked by pre-exposure to the metabolic inhibitors, sodium azide and 2,4-dinitrophenol. Valproic acid uptake by the BeWo cells was also inhibited by the protonophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone, but not anion exchange inhibitor. Selected monocarboxylic acids inhibited the uptake of [ 14C]valproic acid by BeWo cells, whereas dicarboxylic acids did not alter the uptake process. Analysis of Lineweaver–Burk plots of valproic acid uptake in the presence of benzoic acid, a marker for the monocarboxylic acid transporter, revealed a competitive process for uptake. In transcellular transport experiments, the permeation of [ 14C]valproic acid from the apical-to-basal side of the monolayers was significantly greater than the permeation from basal-to-apical side. Additionally, the permeation of [ 14C]valproic acid from apical-to-basal side was inhibited by monocarboxylic acids and not dicarboxylic acids. The results provide biochemical evidence of a proton-dependent, saturable, and asymmetric transport system, presumed to be a monocarboxylic acid transporter, for valproic acid in a human trophoblast model.