Labyrinthine Trophoblast Research Articles

Tipping the balance toward stemness in trophoblast: Metabolic programming by Cox6B2.

Metabolic effector(s) driving cell fate is an emerging concept in stem cell biology. Here we showed that Cytochrome C Oxidase Subunit 6B2 (Cox6B2) is essential to maintain the stemness of trophoblast stem (TS) cells. RNA interference of Cox6b2 resulted in decreased mitochondrial Complex IV activity, ATP production, and oxygen consumption rate in TS cells. Furthermore, depletion of Cox6b2 in TS cells led to decreased self-renewal capacity indicated by compromised BrdU incorporation, Ki67 staining, and decreased expression of TS cell genetic markers. As expected, the consequence of Cox6b2 knockdown was the induction of differentiation. TS cell stemness factor CDX2 transactivates Cox6b2 promoter in TS cells. In differentiated cells, Cox6b2 is post-transcriptionally regulated by two microRNAs, miR-322-5p and miR-503-5p,leading to its downregulation as demonstrated by the gain-in or loss of function of these miRNAs. Cox6b2 transcripts gradually rise in placental trophoblast gestation progresses in both mice and rats with predominant expression in labyrinthine trophoblast. Cox6b2 expression is compromised in the growth-restricted placenta of rats with reciprocal up-regulation of miR-322-5p and miR-503-5p. These data highlight the importance of Cox6B2 in the regulation of TS cell state and uncompromised placental growth.

Pregnancy, but not dietary octanoic acid supplementation, stimulates the ghrelin-pituitary growth hormone axis in mice.

Circulating growth hormone (GH) concentrations increase during pregnancy in mice and remain pituitary-derived. Whether abundance or activation of the GH secretagogue ghrelin increase during pregnancy, or in response to dietary octanoic acid supplementation, is unclear. We therefore measured circulating GH profiles in late pregnant C57BL/6J mice and in aged-matched non-pregnant females fed with standard laboratory chow supplemented with 5% octanoic or palmitic (control) acid (n = 4-13/group). Serum total and acyl-ghrelin concentrations, stomach and placenta ghrelin mRNA and protein expression, Pcsk1 (encoding prohormone convertase 1/3) and Mboat4 (membrane bound O-acyl transferase 4) mRNA were determined at zeitgeber (ZT) 13 and ZT23. Total and basal GH secretion were higher in late pregnant than non-pregnant mice (P < 0.001), regardless of diet. At ZT13, serum concentrations of total ghrelin (P = 0.004), but not acyl-ghrelin, and the density of ghrelin-positive cells in the gastric antrum (P = 0.019) were higher, and gastric Mboat4 and Pcsk1 mRNA expression were lower in pregnant than non-pregnant mice at ZT23. In the placenta, ghrelin protein was localised mostly to labyrinthine trophoblast cells. Serum acyl-, but not total, ghrelin was lower at mid-pregnancy than in non-pregnant mice, but not different at early or late pregnancy. In conclusion, dietary supplementation with 5% octanoic acid did not increase activation of ghrelin in female mice. Our results further suggest that increases in maternal GH secretion throughout murine pregnancy are not due to circulating acyl-ghrelin acting at the pituitary. Nevertheless, time-dependent increased circulating total ghrelin could potentially increase ghrelin action in tissues that express the acylating enzyme and receptor.

TGF-β induces Smad2 Phosphorylation, ARE Induction, and Trophoblast Differentiation

BackgroundTransforming growth factor beta (TGF-β) signaling has been shown to control a large number of critical cellular actions such as cell death, differentiation, and development and has been implicated as a major regulator of placental function. SM10 cells are a mouse placental progenitor cell line, which has been previously shown to differentiate into nutrient transporting, labyrinthine-like cells upon treatment with TGF-β. However, the signal transduction pathway activated by TGF-β to induce SM10 progenitor differentiation has yet to be fully investigated.Materials and MethodsIn this study the SM10 labyrinthine progenitor cell line was used to investigate TGF-β induced differentiation. Activation of the TGF-β pathway and the ability of TGF-β to induce differentiation were investigated by light microscopy, luciferase assays, and Western blot analysis.Results and ConclusionsIn this report, we show that three isoforms of TGF-β have the ability to terminally differentiate SM10 cells, whereas other predominant members of the TGF-β superfamily, Nodal and Activin A, do not. Additionally, we have determined that TGF-β induced Smad2 phosphorylation can be mediated via the ALK-5 receptor with subsequent transactivation of the Activin response element. Our studies identify an important regulatory signaling pathway in SM10 progenitor cells that is involved in labyrinthine trophoblast differentiation.

Elevated Adenosine Induces Placental DNA Hypomethylation Independent of A2B Receptor Signaling in Preeclampsia.

Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear. Using 2 independent experimental models of preeclampsia, adenosine deaminase-deficient mice and a pathogenic autoantibody-induced mouse model of preeclampsia, we demonstrate that elevated placental adenosine not only induces hallmark features of preeclampsia but also causes placental DNA hypomethylation. The use of genetic approaches to express an adenosine deaminase minigene specifically in placentas, or adenosine deaminase enzyme replacement therapy, restored placental adenosine to normal levels, attenuated preeclampsia features, and abolished placental DNA hypomethylation in adenosine deaminase-deficient mice. Genetic deletion of CD73 (an ectonucleotidase that converts AMP to adenosine) prevented the elevation of placental adenosine in the autoantibody-induced preeclampsia mouse model and ameliorated preeclampsia features and placental DNA hypomethylation. Immunohistochemical studies revealed that elevated placental adenosine-mediated DNA hypomethylation predominantly occurs in spongiotrophoblasts and labyrinthine trophoblasts and that this effect is independent of A2B adenosine receptor activation in both preeclampsia models. Extending our mouse findings to humans, we used cultured human trophoblasts to demonstrate that adenosine functions intracellularly and induces DNA hypomethylation without A2B adenosine receptor activation. Altogether, both mouse and human studies reveal novel mechanisms underlying placental DNA hypomethylation and potential therapeutic approaches for preeclampsia.

Id2 Mediates Differentiation of Labyrinthine Placental Progenitor Cell Line, SM10.

The placenta is an organ that is formed transiently during pregnancy, and appropriate placental development is necessary for fetal survival and growth. Proper differentiation of the labyrinthine layer of the placenta is especially crucial, as it establishes the fetal-maternal interface that is involved in physiological exchange processes. Although previous studies have indicated the importance of inhibitor of differentiation/inhibitor of DNA binding-2 (Id2) helix-loop-helix transcriptional regulator in mediating cell differentiation, the ability of Id2 to regulate differentiation toward the labyrinthine (transport) lineage of the placenta has yet to be determined. In the current study, we have generated labyrinthine trophoblast progenitor cells with increased (SM10-Id2) or decreased (SM10-Id2-shRNA) Id2 expression and determined the effect on TGF-β-induced differentiation. Our Id2 overexpression and knockdown analyses indicate that Id2 mediates TGF-β-induced morphological differentiation of labyrinthine trophoblast cells, as Id2 overexpression prevents differentiation and Id2 knockdown results in differentiation. Thus, our data indicate that Id2 is an important molecular mediator of labyrinthine trophoblast differentiation. An understanding of the regulators of trophoblast progenitor differentiation toward the labyrinthine lineage may offer insights into events governing pregnancy-associated disorders, such as placental insufficiency, fetal growth restriction, and preeclampsia.

Smoking Out Fetal Growth Restriction

Researchers have uncovered a mechanism behind one of the most damaging effects of maternal smoking—fetal growth restriction [1]. It seems that smoking boosts expression of a signaling molecule—secreted frizzled-related protein 1 (sFRP1)—that suppresses placental growth and development. The findings may help explain why infants of mothers who smoke weigh, on average, 200–400 g less than the infants of nonsmokers. Alice Wang et al. first found that sFRP1 mRNA levels were highly elevated in the placentas from smoking mothers who gave birth to babies with severe growth restriction. An extracellular antagonist of WNT signaling, sFRP1 is known to be involved in placental development. The researchers next overexpressed sFRP1 in pregnant mice, and observed defects in the placenta and decreased proliferation of labyrinthine trophoblasts. Exposing a trophoblast cell line to a carbon monoxide analog reduced WNT signaling, increasing sFRP1 mRNA expression and decreasing cellular proliferation. The researchers also exposed pregnant mice to the carbon monoxide analog, and found that these mice gave birth to underweight pups—though the effects on WNT and sFRP1 signaling remain to be explored. Other studies have hinted at other mechanisms behind fetal growth restriction in smoking mothers. Carbon monoxide has a great affinity for hemoglobin and can cause fetal hypoxia, and uterine and umbilical arteries constrict in response to tobacco smoke. Perhaps some of these factors may also affect sFRP1 expression. Smoking during pregnancy is a growing public health threat, and can also cause lung problems, cognitive effects, and other health issues in children. While smoking rates in the United States are on the decline, tobacco companies are tapping into the vast potential market in undeveloped countries—some of which, like Indonesia, currently have high rates of smoking in males but very low rates in females. The United States has yet to ratify the World Health Organization Framework Convention on Tobacco Control, which provides some counterweight to the growing number of lawsuits by tobacco companies claiming that country-wide efforts at tobacco control violate trade laws (http://www.nytimes.com/ 2013/12/13/health/tobacco-industry-tactics-limit-poorernations-smoking-laws.html).

Abnormal labyrinthine zone in the Hectd1-null placenta

IntroductionThe labyrinthine zone of the placenta is where exchange of nutrients and waste occurs between maternal and fetal circulations. Proper development of the placental labyrinth is essential for successful growth of the developing fetus and abnormalities in placental development are associated with intrauterine growth restriction (IUGR), preeclampsia and fetal demise. Our previous studies demonstrate that Hectd1 is essential for development of the junctional and labyrinthine zones of the placenta. Here we further characterize labyrinthine zone defects in the Hectd1 mutant placenta. MethodsThe structure of the mutant placenta was compared to wildtype littermates using histological methods. The expression of cell type specific markers was examined by immunohistochemistry and in situ hybridization. ResultsHectd1 is expressed in the labyrinthine zone throughout development and the protein is enriched in syncytiotrophoblast layer type I cells (SynT-I) and Sinusoidal Trophoblast Giant cells (S-TGCs) in the mature placenta. Mutation of Hectd1 results in pale placentas with frequent hemorrhages along with gross abnormalities in the structure of the labyrinthine zone including a smaller overall volume and a poorly elaborated fetal vasculature that contain fewer fetal blood cells. Examination of molecular markers of labyrinthine trophoblast cell types reveals increased Dlx3 positive cells and Syna positive SynT-I cells, along with decreased Hand1 and Ctsq positive sinusoidal trophoblast giant cells (S-TGCs). DiscussionTogether these defects indicate that Hectd1 is required for development of the labyrinthine zonethe mouse placenta.

Excess placental secreted frizzled-related protein 1 in maternal smokers impairs fetal growth

Maternal cigarette smoking during pregnancy remains one of the most common and preventable causes of fetal growth restriction (FGR), a condition in which a fetus is unable to achieve its genetically determined potential size. Even though epidemiologic evidence clearly links maternal cigarette smoking with FGR, insight into the molecular mechanisms of cigarette smoke-induced FGR is lacking. Here, we performed transcriptional profiling of placentas obtained from smoking mothers who delivered growth-restricted infants and identified secreted frizzled-related protein 1 (sFRP1), an extracellular antagonist of endogenous WNT signaling, as a candidate molecule. sFRP1 mRNA and protein levels were markedly upregulated (~10-fold) in placentas from smoking mothers compared with those from nonsmokers. In pregnant mice, adenovirus-mediated overexpression of sFRP1 led to FGR, increased karyorrhexis in the junctional zone, and decreased proliferation of labyrinthine trophoblasts. Consistent with our hypothesis that placental WNT signaling is suppressed in maternal smokers, we found that exposure to carbon monoxide analogs led to reduced WNT signaling, increased SFRP1 mRNA expression, and decreased cellular proliferation in a trophoblast cell line. Moreover, administration of carbon monoxide analogs to pregnant mice in late gestation led to FGR. In summary, our results indicate that the increased placental expression of sFRP1 seen in smokers impairs fetal growth by inhibiting WNT signaling and trophoblast proliferation.

Maternal serum omentin-1 profile is similar in humans and in the rat animal model

Omentin-1 is an adipocytokine with anti-inflammatory activity that has been associated with different metabolic disorders. The aim of this study is to investigate the serum profiles of omentin-1 throughout human and rat pregnancy.Serum omentin-1 levels were determined by ELISA in a prospective cohort study of healthy pregnant women (n=40) during the three trimesters of pregnancy and in twenty healthy non-pregnant women during the follicular and luteal phase of the menstrual cycle. In addition, serum omentin-1 levels were measured in rats during different periods of pregnancy (gestational days 8, 12, 16, 19, and 21) and in an age-matched control (virgin) group of rats (n=12rats/group). Finally, immunohistochemistry was used to demonstrate the presence of omentin-1 protein in human and rat placenta.Omentin-1 immunoreactivity was detected in cytotrophoblasts, syncytiotrophoblasts, sparse Hofbauer cells, and endothelial cells of the stem villi of human placenta. Additionally, it was detected in the labyrinthine trophoblast and yolk sac layer of the rat placenta. Human and rat serum omentin-1 levels were significantly lower in the late gestational period when compared with the non-pregnant women and virgin rats (p<0.05). Serum omentin-1 changes were not significant throughout the gestation in both species (p>0.05). Human serum omentin-1 levels have an inverse relationship with triglyceride levels during pregnancy.Our findings have not determined the exact role of omentin-1 during pregnancy, concerning the metabolic control of triglycerides and other energy sources. Whether omentin-1 decrease implies a regulatory function is still not clear. Further studies are needed to address this issue and determine the role of omentin-1 in metabolic adaptations during normal human and rat pregnancy.

Establishment of trophoblast stem cells under defined culture conditions in mice.

The inner cell mass (ICM) and trophoblast cell lineages duet early embryonic development in mammals. After implantation, the ICM forms the embryo proper as well as some extraembryonic tissues, whereas the trophoectoderm (TE) exclusively forms the fetal portion of the placenta and the trophoblast giant cells. Although embryonic stem (ES) cells can be derived from ICM in cultures of mouse blastocysts in the presence of LIF and/or combinations of small-molecule chemical compounds, and the undifferentiated pluripotent state can be stably maintained without use of serum and feeder cells, defined culture conditions for derivation and maintenance of undifferentiated trophoblast stem (TS) cells have not been established. Here, we report that addition of FGF2, activin A, XAV939, and Y27632 are necessary and sufficient for derivation of TS cells from both of E3.5 blastocysts and E6.5 early postimplantation extraembryonic ectoderm. Moreover, the undifferentiated TS cell state can be stably maintained in chemically defined culture conditions. Cells derived in this manner expressed TS cell marker genes, including Eomes, Elf5, Cdx2, Klf5, Cdh1, Esrrb, Sox2, and Tcfap2c; differentiated into all trophoblast subtypes (trophoblast giant cells, spongiotrophoblast, and labyrinthine trophoblast) in vitro; and exclusively contributed to trophoblast lineages in chimeric animals. This delineation of minimal requirements for derivation and self-renewal provides a defined platform for precise description and dissection of the molecular state of TS cells.

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.

Hypoxia and Trophoblast Differentiation: A Key Role for PPARγ

Tissue oxygen tension regulates differentiation of multiple types of stem cells. In the placenta, hypoxia has been associated with abnormal trophoblast differentiation and placental insufficiency syndromes of preeclampsia (PE) and intrauterine growth restriction (IUGR). Peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand-activated transcription factor involved in many cellular processes, including differentiation. We have previously shown that PPARγ-null trophoblast stem (TS) cells show a defect in differentiation to labyrinthine trophoblast, instead differentiating preferentially to trophoblast giant cells (TGC). Since PPARγ is known to be regulated by hypoxia in adipose tissue, we hypothesized that there may be a link between oxygen tension, PPARγ expression, and trophoblast differentiation. We found that hypoxia reduced PPARγ expression by a mechanism independent of both hypoxia-inducible factor (HIF) and histone deacetylases (HDACs). In addition, PPARγ partially rescued hypoxia-induced inhibition of labyrinthine differentiation in wild-type TS cells but was not required for hypoxia-induced inhibition of TGC differentiation. Finally, we show that induction of labyrinthine trophoblast differentiation by HDAC inhibitor treatment is independent of both PPARγ and Gcm1. We propose a model with two pathways for labyrinthine trophoblast differentiation of TS cells, one of which is dependent on PPARγ and inhibited by hypoxia. Since hypoxia is associated with PE and IUGR, we propose that PPARγ may at least partially mediate hypoxia-induced placental insufficiency and as such may be a promising therapeutic target for these disorders.

The c-Myc-Regulated MicroRNA-17∼92 (miR-17∼92) and miR-106a∼363 Clusters Target hCYP19A1 and hGCM1 To Inhibit Human Trophoblast Differentiation

Mononuclear cytotrophoblasts of the human placenta proliferate rapidly, subsequently fuse, and differentiate to form multinucleated syncytiotrophoblast with induction of aromatase (hCYP19A1) and chorionic gonadotropin (hCGβ) expression. Using microarray analysis, we identified members of the miR-17~92 cluster and its paralogs, miR-106a~363 and miR-106b~25, that are significantly downregulated upon syncytiotrophoblast differentiation. Interestingly, miR-19b and miR-106a directly targeted hCYP19A1 expression, while miR-19b also targeted human GCM1 (hGCM1), a transcription factor critical for mouse labyrinthine trophoblast development. Overexpression of these microRNAs (miRNAs) impaired syncytiotrophoblast differentiation. hGCM1 knockdown decreased hCYP19A1 and hCGβ expression, substantiating its important role in human trophoblast differentiation. Expression of the c-Myc proto-oncogene was increased in proliferating cytotrophoblasts compared to that in differentiated syncytiotrophoblast. Moreover, c-Myc overexpression upregulated miR-17~92 and inhibited hCYP19A1 and hCGβ expression. Binding of endogenous c-Myc to genomic regions upstream of the miR-17~92 and miR-106a~363 clusters in cytotrophoblasts dramatically decreased upon syncytiotrophoblast differentiation. Intriguingly, we observed higher levels of miR-106a and -19b and lower aromatase and hGCM1 expression in placentas from preeclamptic women than in placentas from gestation-matched normotensive women. Our findings reveal that c-Myc-regulated members of the miR-17~92 and miR-106a~363 clusters inhibit trophoblast differentiation by repressing hGCM1 and hCYP19A1 and suggest that aberrant regulation of these miRNAs may contribute to the pathogenesis of preeclampsia.

Morphometric analysis of the placenta in the New World mouse Necromys lasiurus (Rodentia, Cricetidae): a comparison of placental development in cricetids and murids

BackgroundStereology is an established method to extrapolate three-dimensional quantities from two-dimensional images. It was applied to placentation in the mouse, but not yet for other rodents. Herein, we provide the first study on quantitative placental development in a sigmodontine rodent species with relatively similar gestational time. Placental structure was also compared to the mouse, in order to evaluate similarities and differences in developmental patterns at the end of gestation.MethodsFetal and placental tissues of Necromys lasiurus were collected and weighed at 3 different stages of gestation (early, mid and late gestation) for placental stereology. The total and relative volumes of placenta and of its main layers were investigated. Volume fractions of labyrinth components were quantified by the One Stop method in 31 placentae collected from different individuals, using the Mercator® software. Data generated at the end of gestation from N. lasiurus placentae were compared to those of Mus musculus domesticus obtained at the same stage.ResultsA significant increase in the total absolute volumes of the placenta and its main layers occurred from early to mid-gestation, followed by a reduction near term, with the labyrinth layer becoming the most prominent area. Moreover, at the end of gestation, the total volume of the mouse placenta was significantly increased compared to that of N. lasiurus although the proportions of the labyrinth layer and junctional zones were similar. Analysis of the volume fractions of the components in the labyrinth indicated a significant increase in fetal vessels and sinusoidal giant cells, a decrease in labyrinthine trophoblast whereas the proportion of maternal blood space remained stable in the course of gestation. On the other hand, in the mouse, volume fractions of fetal vessels and sinusoidal giant cells decreased whereas the volume fraction of labyrinthine trophoblast increased compared to N. lasiurus placenta.ConclusionsPlacental development differed between N. lasiurus and M. musculus domesticus. In particular, the low placental efficiency in N. lasiurus seemed to induce morphological optimization of fetomaternal exchanges. In conclusion, despite similar structural aspects of placentation in these species, the quantitative dynamics showed important differences.

Protease-Activated Receptor 4 (PAR4) Plays a Critical Role in Fetal Loss and Peripheral Thrombosis in a Mouse Model of Thrombophilia

Abstract 373We generated a mouse model of thrombophilia by combining two common risk factors for thrombosis in humans; transgenic for high levels of FIX (TgFIX+) and activated protein C resistance due to factor V Leiden mutation (FVLQ). Breedings were used in which both parents were heterozygous for the FVL (FVLR/Q) mutation, but only one carried the TgFIX+(resulting in ∼5-fold higher of normal levels). More than 200 newborns were obtained but surprisingly no animal of FVLQ/Q/ TgFIX+ genotype was identified. When pregnancies were interrupted at embryonic age of E9.5 to E16.5, no deviation from the expected/observed embryo genotypes ratios was observed. However, the number of reabsorbed embryos increased significantly from 13% (E9.5) to 33% (E.16.5), p<0.02. All FVLQ/Q/ TgFIX+ embryos exhibited findings consistent with disseminated intravascular coagulation. Placental analysis (n=18) of these embryos harvested at E12.5 or later demonstrated prothrombotic abnormalities such as excessive fibrin deposition and innumerous apoptotic cells, determined by TUNEL assay, localized mainly in the labyrinthine trophoblast zone. The maternal genotype clearly influenced the pregnancy outcome since the number of newborns per litter was 2-fold lower when the mother carried the TgFIX+versus the paternal (average 4.3 vs. 8.3, respectively; p<0.02). No other genotypes analyzed (n=112) presented similar abnormalities in either embryos or in placental tissue. Maternal anticoagulant therapy with heparin or specific thrombin or factor Xa inhibitors reverted the prothrombotic placental phenotype, although a few newborns reached to term, none survived beyond day 1. Although FVLR/Q/ TgFIX+mice developed normally when reaching 8–10 months of age we observed a high rate of spontaneous peripheral thrombotic episodes (3/40; 7.5%) and histological analysis confirmed venous thrombosis. Thus, these mice represent a novel murine model of venous thrombosis. Moreover, this complication was also present in 5.2% of the FVLR/R/ TgFIX+ whereas no other age-matched mice (n=74) developed such complications. There was an age-dependent increase in TAT and D-Dimer levels in both prothrombotic strains by comparing samples collected at 2 and 10 months time points. In the FVLR/Q/ TgFIX+ or FVLR/R/ TgFIX+, levels of TAT (70ng/ml, n=28/group) and D-dimers (230ng/ml, n=17/group) were ∼ 2-fold higher (P<0.04), when compared to mice (TAT: 30ng/ml and D-Dimer: 100ng/ml) of other genotypes. Similarly, the highest fibrin deposition determined by Western blot analysis of tissues harvested was found in the FVLR/Q/ TgFIX+ followed by FVLR/R/ TgFIX+ mice when compared to the other animals (P<0.05). Thus, this model provides a unique opportunity to test the effect of modulators of thrombosis in a chronic thrombophilia status rather than simply upon chemical- or laser-induced acute vascular injury. Since thrombin-mediated mouse platelet activation is PAR4 dependent in an acute vascular injury model, we sought to determine whether PAR4 could modulate the prothrombotic phenotype in our chronic thrombophilia model. Using triallelic crosses FVLQ/Q/TgFIX+/PAR4(−/−) and FVLQ/Q/TgFIX+/PAR4(+/−) animals were generated. Interestingly, we were able to rescue the fetal mortality of FVLQ/Q/ TgFIX+by introducing just one PAR4 null allele. Notably, PAR4 deficiency in heterozygous or homozygous mice of FVLQ/Q/ TgFIX+allowed them to reach term and develop normally. Kaplan-Meier analysis of both FVLQ/Q/TgFIX+/PAR4(−/−)and FVLQ/Q/TgFIX+/PAR4(+/−)mice showed survival rates of 87.5% and 65%, respectively, at the 10-month time point (ongoing observation). Notably, these rescued mice showed no evidence of peripheral thrombosis. In conclusion, we have characterized a mouse model of chronic hypercoagulability and have shown that in this model PAR4 plays a central role in modulating thrombophilic complications. Disclosures:No relevant conflicts of interest to declare.

Evidence Implicating Peroxisome Proliferator-Activated Receptor-γ in the Pathogenesis of Preeclampsia

Preeclampsia, a major cause of maternal and perinatal mortality and morbidity, is thought to be attributed, in part, to impaired trophoblast invasion. Peroxisome proliferator-activated receptors are ligand-activated transcription factors expressed in trophoblasts, which regulate the expression of a number of genes involved in cell differentiation and proliferation. We investigated the effect of the administration of a peroxisome proliferator-activated receptor-γ antagonist during uncomplicated pregnancy in rats. Using an intraperitoneal miniosmotic pump, healthy pregnant rats were administered either vehicle or the peroxisome proliferator-activated receptor-γ-specific antagonist, T0070907 (1 mg/kg per day from gestational days 11-15). Rats treated with T0070907 developed key features of preeclampsia, including elevated mean arterial blood pressure, proteinuria, endothelial dysfunction, reduced pup weight, and increased platelet aggregation. T0070907-treated rats had reduced plasma vascular endothelial growth factor and increased plasma soluble fms-like tyrosine kinase 1. Furthermore, increases in total placental soluble fms-like tyrosine kinase 1 mRNA and fms-like tyrosine kinase 1 protein were also demonstrated, suggesting the placenta as the main contributor to the increased circulating levels of soluble fms-like tyrosine kinase 1. The labyrinthine trophoblast in the placentas of T0070907-treated rats were less differentiated, had increased cellular proliferation, and were strongly immunopositive for CD-31 staining, indicating adaptive angiogenesis. The present study suggests that peroxisome proliferator-activated receptor-γ may play a pivotal role in the progression of a healthy pregnancy and may critically regulate the risk of preeclampsia. These findings have important implications regarding the underlying etiology of preeclampsia and potential therapeutic targets.

Mapping of CIP/KIP inhibitors, G1 cyclins D1, D3, E and p53 proteins in the rat term placenta

As cell cycle regulation is fundamental to the normal growth and development of the placenta, the aim of the present study was to determine the immunolocalizations of cell cycle related proteins, which have key roles in proliferation, differentiation and apoptosis during the development of the rat placenta. Here immunohistochemistry has been used to localize G1 cyclins (D1, D3, E), which are major determinants of proliferation, CIP/KIP inhibitors (p21, p27, p57), p53 as a master regulator and proliferating cell nuclear antigen in all cell types of the rat term placenta. The proportion of each cell type immunolabeled was counted. Cyclin D1 and cyclin D3 were present mostly in cells of the fetal aspect of the placenta, whereas the G1/S cyclin E was present only in the spongio- and labyrinthine trophoblast populations. Among the CIP/KIP inhibitors, p21 was present only in cells of the fetal aspect whereas p27 and p57 were found in all cell types studied. p53 was only found in a small proportion of cells with no co-localization of p53 and p21. The data suggest that the cells of the fetal side of the rat placenta still have some proliferation potential which is kept in check by expression of the CIP/KIP cell cycle inhibitors, whereas cells of the maternal aspect have lost this potential. Apoptosis is only marginal in the term rat placenta. In conclusion, proliferation and apoptosis in rat placental cells appears controlled mostly by the CIP/KIP inhibitors in late pregnancy.

P45NF-E2 represses Gcm1 in trophoblast cells to regulate syncytium formation, placental vascularization and embryonic growth

Absence of the leucine zipper transcription factor p45NF-E2 results in thrombocytopenia, impaired placental vascularization and intrauterine growth restriction (IUGR) in mice. The mechanism underlying the p45NF-E2-dependent placental defect and IUGR remains unknown. Here, we show that the placental defect and IUGR of p45NF-E2 (Nfe2) null mouse embryos is unrelated to thrombocytopenia, establishing that embryonic platelets and platelet-released mediators are dispensable for placentation. Rather, p45NF-E2, which was hitherto thought to be specific to hematopoietic cells, is expressed in trophoblast cells, where it is required for normal syncytiotrophoblast formation, placental vascularization and embryonic growth. Expression of p45NF-E2 in labyrinthine trophoblast cells colocalizes with that of Gcm1, a transcription factor crucial for syncytiotrophoblast formation. In the absence of p45NF-E2, the width of syncytiotrophoblast layer 2 and the expression of Gcm1 and Gcm1-dependent genes (Synb and Cebpa) are increased. In vitro, p45NF-E2 deficiency results in spontaneous syncytiotrophoblast formation, which can be reversed by Gcm1 knockdown. Increased Gcm1 expression in the absence of p45NF-E2 is dependent on enhanced protein acetylation, including post-translational modification of Gcm1. Increasing and inhibiting acetylation in the placenta of wild-type control embryos phenocopies and corrects, respectively, the changes observed in p45NF-E2-deficient embryos. These studies identify a novel function of p45NF-E2 during placental development: in trophoblast cells, p45NF-E2 represses Gcm1 and syncytiotrophoblast formation via acetylation.

Transcriptional regulation of VEGF-A by the unfolded protein response pathway.

BackgroundAngiogenesis is crucial to many physiological and pathological processes including development and cancer cell survival. Vascular endothelial growth factor-A (VEGFA) is the predominant mediator of angiogenesis in the VEGF family. During development, adverse environmental conditions like nutrient deprivation, hypoxia and increased protein secretion occur. IRE1α, PERK, and ATF6α, master regulators of the unfolded protein response (UPR), are activated under these conditions and are proposed to have a role in mediating angiogenesis.Principal FindingsHere we show that IRE1α, PERK, and ATF6α powerfully regulate VEGFA mRNA expression under various stress conditions. In Ire1α−/− and Perk−/− mouse embryonic fibroblasts and ATF6α-knockdown HepG2 cells, induction of VEGFA mRNA by endoplasmic reticulum stress is attenuated as compared to control cells. Embryonic lethality of Ire1α−/− mice is due to the lack of VEGFA induction in labyrinthine trophoblast cells of the developing placenta. Rescue of IRE1α and PERK in Ire1α−/− and Perk−/− cells respectively, prevents VEGFA mRNA attenuation. We further report that the induction of VEGFA by IRE1α, PERK and ATF6 involves activation of transcription factors, spliced-XBP-1, ATF4 and cleaved ATF6 respectively.Conclusions/SignificanceOur results reveal that the IRE1α-XBP-1, PERK-ATF4, and ATF6α pathways constitute novel upstream regulatory pathways of angiogenesis by modulating VEGF transcription. Activation of these pathways helps the rapidly growing cells to obtain sufficient nutrients and growth factors for their survival under the prevailing hostile environmental conditions. These results establish an important role of the UPR in angiogenesis.

PPARγ Regulates Trophoblast Proliferation and Promotes Labyrinthine Trilineage Differentiation

BackgroundAbnormal trophoblast differentiation and function is the basis of many placenta-based pregnancy disorders, including pre-eclampsia and fetal growth restriction. PPARγ, a ligand-activated nuclear receptor, plays essential roles in placental development; null murine embryos die at midgestation due to abnormalities in all placental layers, in particular, small labyrinth and expanded giant cell layer. Previous studies have focused mostly on the role of PPARγ in trophoblast invasion. Based on the previously reported role of PPARγ in preadipocyte differentiation, we hypothesized that PPARγ also plays a pivotal role in trophoblast differentiation. To test this hypothesis, we report derivation of wild-type and PPARγ-null trophoblast stem (TS) cells.Methodology/Principal FindingsPPARγ-null TS cells showed defects in both proliferation and differentiation, specifically into labyrinthine trophoblast. Detailed marker analysis and functional studies revealed reduced differentiation of all three labyrinthine lineages, and enhanced giant cell differentiation, particularly the invasive subtypes. In addition, rosiglitazone, a specific PPARγ agonist, reduced giant cell differentiation, while inducing Gcm1, a key regulator in labyrinth. Finally, reintroducing PPARγ into null TS cells, using an adenovirus, normalized invasion and partially reversed defective labyrinthine differentiation, as assessed both by morphology and marker analysis.Conclusions/SignificanceIn addition to regulating trophoblast invasion, PPARγ plays a predominant role in differentiation of labyrinthine trophoblast lineages, which, along with fetal endothelium, form the vascular exchange interface with maternal blood. Elucidating cellular and molecular mechanisms mediating PPARγ action will help determine if modulating PPARγ activity, for which clinical pharmacologic agonists already exist, might modify the course of pregnancy disorders associated with placental dysfunction.