The antithrombin binding regions of heparin mediate fetal growth and reduced placental damage in the RUPP model of preeclampsia†.

Preeclampsia is a serious hypertensive disorder of pregnancy, which is only cured with delivery of the placenta, thereby commonly necessitating preterm birth of the fetus. Low-molecular-weight heparin (LMWH) has demonstrated potential to reduce the incidence of preeclampsia in high-risk pregnant women, although the underlying mechanism by which LMWH protects against preeclampsia is unknown. Given the complex structure and biologic actions of heparin, we tested the hypothesis that heparin can mediate preeclampsia prevention via nonanticoagulant pathways. We compared the effects of a nonanticoagulant, glycol-split LMWH (gsHep)-rendered nonanticoagulant through disruption of the antithrombin binding regions-with the LMWH dalteparin in the rat reduced uterine perfusion pressure (RUPP) surgical model of preeclampsia. Although RUPP animals exhibit significantly elevated blood pressure and reduced plasma levels of placental growth factor (PGF) compared to sham, neither dalteparin nor gsHep treatment significantly impacted these parameters. However, the observed positive correlation between PGF levels and number of viable fetuses in RUPP-induced animals suggests that reduced PGF levels were predominately due to placental loss. Daily subcutaneous injections of low-dose dalteparin but not gsHep significantly restored fetal growth that was impaired by RUPP surgery. Placentas from RUPP animals exhibited an abnormal labyrinth structure, characterized by expanded sinusoidal blood spaces, relative to sham-operated animals. Morphometric analysis demonstrated that dalteparin but not gsHep treatment normalized development of the labyrinth in RUPP-exposed conceptuses. These data suggest that the antithrombin-binding regions of LMWH are required to confer its protective effects on fetal growth and placental development.

Effects of glycol-split low molecular weight heparin on placental, endothelial, and anti-inflammatory pathways relevant to preeclampsia.

Low molecular weight heparin (LMWH) is being investigated as a potential preventative therapy against preeclampsia. There is evidence suggesting that LMWH may prevent preeclampsia through anticoagulation-independent mechanisms. In this study, we compared the in vitro placental, endothelial, and anti-inflammatory effects of an LMWH (dalteparin) with a nonanticoagulant, glycol-split heparin derivative (gsHep). In contrast with dalteparin, gsHep did not interact with antithrombin III, possess significant anti-Factor Xa activity, or significantly prolong in vitro plasma clotting time. However, dalteparin and gsHep were otherwise mechanistically similar, both interacting with soluble fms-like tyrosine kinase-1 (sFlt1) and promoting release of the pro-angiogenic protein placental growth factor, but not the antiangiogenic sFlt1, from healthy placental villous explants. Placental explant media pretreated with dalteparin or gsHep significantly stimulated endothelial cell tube formation compared to untreated explants. Lastly, dalteparin and gsHep both significantly suppressed inflammation by inhibiting complement activation and leukocyte adhesion to endothelial cells that were activated using serum from preeclamptic women. Our data suggest that nonanticoagulant heparin derivatives may be utilized as a tool to distinguish the anticoagulation-independent mechanisms of LMWH, and provide insight into the role of anticoagulation in the prevention of preeclampsia.

Endothelial Dysfunction in Severe Preeclampsia is Mediated by Soluble Factors, Rather than Extracellular Vesicles.

In severe early-onset preeclampsia (sPE) the placenta releases soluble angiogenesis-regulating proteins, trophoblast-derived fragments, and extracellular vesicles (EVs). Their relative importance in disease pathogenesis is not presently understood. We explanted placental villi from healthy and sPE women then separated the media into: total-conditioned, EV-depleted and EV-enriched media. Three fractions were compared for; angiogenic protein secretion by ELISA, angiogenic and inflammation gene mRNA expression and leukocyte adhesion assay. sPE placental villi secreted significantly less PlGF (70 ± 18 pg/mL) than preterm controls (338 ± 203; p = 0.03). sFlt-1:PlGF ratios in total-conditioned (115 ± 29) and EV-depleted media (136 ± 40) from sPE placental villi were significantly higher than in EV-enriched media (42 ± 12; p < 0.01) or any preterm or term media. Fluorescent-labeled EVs derived across normal gestation, but not from sPE, actively entered HUVECs. From sPE placental villi, the soluble fraction, but not EV-enriched fraction, significantly repressed angiogenesis (0.83 ± 0.05 fold, p = 0.02), induced HO-1 mRNA (15.3 ± 5.1 fold, p < 0.05) and induced leukocyte adhesion (2.2 ± 0.4 fold, p = 0.04). Soluble media (total-conditioned and EV-depleted media) from sPE placental villi induced endothelial dysfunction in HUVEC, while the corresponding EV-enriched fraction showed no such effects. Our data suggest that soluble factors including angiogenesis-regulating proteins, dominate the vascular pathology of this disease.

Low Molecular Weight Heparin Improves Endothelial Function in Pregnant Women at High Risk of Preeclampsia.

Low molecular weight heparin (LMWH) has been investigated for the prevention of severe preeclampsia, although the mechanisms of action are unknown. The objective of this study was to investigate the cardiovascular effects of LMWH in pregnant women at high risk of preeclampsia. Pregnant women at high risk of preeclampsia (n=25) and low-risk pregnant controls (n=20) at 22 to 26 weeks' gestation underwent baseline cardiovascular assessments. High-risk women were then randomized to LMWH or saline placebo (30 mg IV bolus and 1 mg/kg subcutaneous dose). Cardiovascular function was assessed 1 and 3 hours post randomization. The in vitro endothelial effects of patient serum and exogenous LMWH on human umbilical venous endothelial cells were determined. High-risk women demonstrated a reduced cardiac output, high resistance hemodynamic profile with impaired radial artery flow-mediated dilation compared with controls. LMWH increased flow-mediated dilation in high-risk women 3 hours after randomization compared with baseline and increased plasma levels of placental growth factor, soluble fms-like tyrosine kinase-1, and myeloperoxidase. Serum from high-risk women impaired endothelial cell angiogenesis and increased PlGF-1 and PlGF-2 transcription compared with serum from low-risk controls. Coexposure of high-risk serum with LMWH improved the in vitro angiogenic response such that it was equivalent to that of low-risk serum and promoted placental growth factor secretion. LMWH improves maternal endothelial function in pregnant women at high risk of developing preeclampsia, possibly mediated through increased placental growth factor bioavailability.

PPAR- γ Regulates Trophoblast Differentiation in the BeWo Cell Model.

Common pregnancy complications, such as severe preeclampsia and intrauterine growth restriction, disrupt pregnancy progression and impair maternal and fetal wellbeing. Placentas from such pregnancies exhibit lesions principally within the syncytiotrophoblast (SCT), a layer in direct contact with maternal blood. In humans and mice, glial cell missing-1 (GCM-1) promotes differentiation of underlying cytotrophoblast cells into the outer SCT layer. GCM-1 may be regulated by the transcription factor peroxisome proliferator-activated receptor-gamma (PPAR- γ ); in mice, PPAR- γ promotes labyrinthine trophoblast differentiation via Gcm-1, and, as we previously demonstrated, PPAR- γ activation ameliorates disease features in rat model of preeclampsia. Here, we aimed to characterize the baseline activity of PPAR- γ in the human choriocarcinoma BeWo cell line that mimics SCT formation in vitro and modulate PPAR- γ activity to study its effects on cell proliferation versus differentiation. We report a novel negative autoregulatory mechanism between PPAR- γ activity and expression and show that blocking PPAR- γ activity induces cell proliferation at the expense of differentiation, while these remain unaltered following treatment with the agonist rosiglitazone. Gaining a deeper understanding of the role and activity of PPAR- γ in placental physiology will offer new avenues for the development of secondary prevention and/or treatment options for placentally-mediated pregnancy complications.

Preeclampsia is associated with alterations in the p53-pathway in villous trophoblast.

BACKGROUND: Preeclampsia (PE) is characterized by exaggerated apoptosis of the villous trophoblast of placental villi. Since p53 is a critical regulator of apoptosis we hypothesized that excessive apoptosis in PE is mediated by abnormal expression of proteins participating in the p53 pathway and that modulation of the p53 pathway alters trophoblast apoptosis in vitro. METHODS: Fresh placental villous tissue was collected from normal pregnancies and pregnancies complicated by PE; Western blotting and real-time PCR were performed on tissue lysate for protein and mRNA expression of p53 and downstream effector proteins, p21, Bax and caspases 3 and 8. To further assess the ability of p53 to modulate apoptosis within trophoblast, BeWo cells and placental villous tissue were exposed to the p53-activator, Nutlin-3, alone or in combination with the p53-inhibitor, Pifithrin-α (PFT-α). Equally, Mdm2 was knocked-down with siRNA. RESULTS: Protein expression of p53, p21 and Bax was significantly increased in pregnancies complicated by PE. Conversely, Mdm2 protein levels were significantly depleted in PE; immunohistochemistry showed these changes to be confined to trophoblast. Reduction in the negative feedback of p53 by Mdm2, using siRNA and Nutlin-3, caused an imbalance between p53 and Mdm2 that triggered apoptosis in term villous explants. In the case of Nutlin, this was attenuated by Pifithrin-α. CONCLUSIONS: These data illustrate the potential for an imbalance in p53 and Mdm2 expression to promote excessive apoptosis in villous trophoblast. The upstream regulation of p53 and Mdm2, with regard to exaggerated apoptosis and autophagy in PE, merits further investigation.

DREAM mediated regulation of GCM1 in the human placental trophoblast.

The trophoblast transcription factor glial cell missing-1 (GCM1) regulates differentiation of placental cytotrophoblasts into the syncytiotrophoblast layer in contact with maternal blood. Reduced placental expression of GCM1 and abnormal syncytiotrophoblast structure are features of hypertensive disorder of pregnancy--preeclampsia. In-silico techniques identified the calcium-regulated transcriptional repressor--DREAM (Downstream Regulatory Element Antagonist Modulator)--as a candidate for GCM1 gene expression. Our objective was to determine if DREAM represses GCM1 regulated syncytiotrophoblast formation. EMSA and ChIP assays revealed a direct interaction between DREAM and the GCM1 promoter. siRNA-mediated DREAM silencing in cell culture and placental explant models significantly up-regulated GCM1 expression and reduced cytotrophoblast proliferation. DREAM calcium dependency was verified using ionomycin. Furthermore, the increased DREAM protein expression in preeclamptic placental villi was predominantly nuclear, coinciding with an overall increase in sumolylated DREAM and correlating inversely with GCM1 levels. In conclusion, our data reveal a calcium-regulated pathway whereby GCM1-directed villous trophoblast differentiation is repressed by DREAM. This pathway may be relevant to disease prevention via calcium-supplementation.

The molecular role of connexin 43 in human trophoblast cell fusion.

Connexin expression and gap junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43)/gap junction A1 (GJA1) are required for cytotrophoblast fusion into the syncytium, the outer functional layer of the human placenta. Cx43 also impacts intracellular signaling through protein-protein interactions. The transcription factor GCM1 and its downstream target ERVW-1/SYNCYTIN-1 are key players in trophoblast fusion and exert their actions through the ERVW-1 receptor SLC1A5/ASCT-2/RDR/ATB(0). To investigate the molecular role of the Cx43 protein and its interaction with this fusogenic pathway, we utilized stable Cx43-transfected cell lines established from the choriocarcinoma cell line Jeg3: wild-type Jeg3, alphahCG/Cx43 (constitutive Cx43 expression), JpUHD/Cx43 (doxycyclin-inducible Cx43 expression), or JpUHD/trCx43 (doxycyclin-inducible Cx43 carboxyterminal deleted). We hypothesized that truncation of Cx43 at its C-terminus would inhibit trophoblast fusion and protein interaction with either ERVW-1 or SLC1A5. In the alphahCG/Cx43 and JpUHD/Cx43 lines, stimulation with cAMP caused 1) increase in GJA1 mRNA levels, 2) increase in percentage of fused cells, and 3) downregulation of SLC1A5 expression. Cell fusion was inhibited by GJIC blockade using carbenoxylone. Neither Jeg3, which express low levels of Cx43, nor the JpUHD/trCx43 cell line demonstrated cell fusion or downregulation of SLC1A5. However, GCM1 and ERVW-1 mRNAs were upregulated by cAMP treatment in both Jeg3 and all Cx43 cell lines. Silencing of GCM1 prevented the induction of GJA1 mRNA by forskolin in BeWo choriocarcinoma cells, demonstrating that GCM1 is upstream of Cx43. All cell lines and first-trimester villous explants also demonstrated coimmunoprecipitation of SLC1A5 and phosphorylated Cx43. Importantly, SLC1A5 and Cx43 gap junction plaques colocalized in situ to areas of fusing cytotrophoblast, as demonstrated by the loss of E-cadherin staining in the plasma membrane in first-trimester placenta. We conclude that Cx43-mediated GJIC and SLC1A5 interaction play important functional roles in trophoblast cell fusion.

Translational analysis of mouse and human placental protein and mRNA reveals distinct molecular pathologies in human preeclampsia.

Preeclampsia (PE) adversely impacts ~5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent molecular mechanisms in the origins of this disease.

Cell specific patterns of methylation in the human placenta.

Epigenetic processes, such as DNA methylation, are known to regulate tissue specific gene expression. We explored this concept in the placenta to define whether DNA methylation is cell-type specific. Cytotrophoblasts and fibroblasts were isolated from normal midtrimester placentas. Using immunocytochemistry, we demonstrated 95% purity for cytotrophoblasts and 60-70% for fibroblasts. We compared DNA methylation profiles from cytotrophoblasts, fibroblasts and whole placental villi using bisulfite modified genomic DNA hybridized to the Illumina Methylation27 array. Euclidean cluster analysis of the DNA methylation profiles showed 2 main clusters, one containing cytotrophoblasts and placenta, the other fibroblasts. Differential methylation analysis identified 442 autosomal CpG sites that differed between cytotrophoblasts and fibroblasts, 315 between placenta and fibroblasts and 61 between placenta and cytotrophoblasts. Three candidate methylation differences were validated by targeted pyrosequencing assays. Pyrosequencing assays were developed for CpG sites less methylated in cytotrophoblasts than fibroblasts mapping to the promoter region of the beta subunit of human chorionic gonadotropin 5 (CGB5), as well as 2 CpG sites mapping to each of 2 tumor suppressor genes. Our data suggest that epigenetic regulation of gene expression is likely to be a key factor in the functional specificity of cytotrophoblasts. These data are proof of principle for cell-type specific epigenetic regulation in placenta and demonstrate that the methylation profile of placenta is mainly driven by cytotrophoblasts.

Fusion assays and models for the trophoblast.

A healthy syncytium in the placenta is vital to a successful pregnancy. The trophoblast builds up the natural barrier between the mother and the developing fetus and is the site of gas, nutrition, and waste exchange. An inadequate formation of this tissue leads to several pathologies of pregnancy, which may result in fetal death during the second trimester or iatrogenic preterm delivery due to intrauterine growth restriction, preeclampsia, or abruption.Cytotrophoblastic cells fuse constantly with the overlying syncytiotrophoblast/syncytium to maintain the function of the trophoblast. Syncytin-1 is the only molecule known to directly induce fusion in the placental trophoblast. Many other proteins, such as gap junctions (e.g., connexin 40) and transcription factors, play a role in the molecular pathways directing the trophoblast turn over. Despite the significance of this process for successful placentation, the mechanisms regulating its activity remain poorly understood.In this chapter we present several different model systems that can be utilized to investigate the regulation of the cell fusion process in the trophoblast. We describe cell-based assays as well as tissue-related protocols. We show how fusion can be monitored in (1) BeWo cells as a trophoblast cell line model, (2) HEK239 using syncytin-1 as a fusion molecule, and (3) a floating villi explant model. Furthermore, we will present strategies to inhibit fusion in the different models. These techniques represent powerful tools to study the molecular mediators of cell fusion in the trophoblast.