Efficacy of Low Molecular Heparin on Preeclampsia by Inhibiting Apoptosis of Trophoblasts via the p38MAPK Signaling Pathway.

OBJECTIVE: To explore the efficacy of low molecular heparin on preeclampsia by inhibiting apoptosis of trophoblasts via the p38MAPK signaling pathway. METHODS: A preeclampsia rat model was established, and the effects of low molecular heparin on preeclampsia via the p38MAPK signaling pathway were analyzed based on intervention of the rats with different combinations of low molecular heparin and p38MAPK signaling pathway activator. Furthermore, a hypoxia/reoxygenation model of trophoblasts in vitro was established to explore the effects of low molecular heparin on trophoblasts via the p38MAPK signaling pathway. RESULTS: After treatment with low molecular heparin, pregnant rats in the heparin group showed significantly decreased blood pressure, 24 h proteinuria, and p38MAPK protein levels in placenta tissues and decreased apoptosis rate of placenta tissue cells (all P < 0.05) and showed more fetal rats and lowered weight of them (both P < 0.05) but showed no significant change in the weight of placenta (all P > 0.05). Pregnant rats treated with low molecular heparin and p38MAPK activator showed significantly higher blood pressure, 24 h proteinuria, and p38MAPK protein levels in placenta tissues and apoptosis rate of placenta tissue cells than those of pregnant rats in the heparin group (all P < 0.05) and also showed less fetal rats and lighter fetal rats than those in the heparin group (both P < 0.05) but showed no difference with them in the weight of placenta (P > 0.05). Further analysis revealed that low molecular heparin could protect the survival and migration of trophoblasts under hypoxia/reoxygenation conditions and reduce apoptosis of them (all P < 0.05). CONCLUSION: Low molecular heparin can alleviate preeclampsia by inhibiting the p38MAPK signaling pathway and can inhibit apoptosis of trophoblasts and promote proliferation and migration of them.

N-acetylcysteine, xCT and suppression of Maxi-chloride channel activity in human placenta.

INTRODUCTION: Placental oxidative stress features in pregnancy pathologies but in clinical trials antioxidant supplementation has not improved outcomes. N-acetylcysteine (NAC) stimulates glutathione production and is proposed as a therapeutic agent in pregnancy. However, key elements of N-acetylcysteine biology, including its cellular uptake mechanism, remains unclear. This study explores how the cystine/glutamate transporter xCT may mediate N-acetylcysteine uptake and how N-acetylcysteine alters placental redox status. METHODS: The involvement of xCT in NAC uptake by the human placenta was studied in perfused placenta and Xenopus oocytes. The effect of short-term N-acetylcysteine exposure on the placental villous proteome was determined using LC-MS. The effect of N-acetylcysteine on Maxi-chloride channel activity was investigated in perfused placenta, villous fragments and cell culture. RESULTS: Maternoplacental N-acetylcysteine administration stimulated intracellular glutamate efflux suggesting a role of the exchange transporter xCT, which was localised to the microvillous membrane of the placental syncytiotrophoblast. Placental exposure to a bolus of N-acetylcysteine inhibited subsequent activation of the redox sensitive Maxi-chloride channel independently of glutathione synthesis. Stable isotope quantitative proteomics of placental villi treated with N-acetylcysteine demonstrated changes in pathways associated with oxidative stress, apoptosis and the acute phase response. DISCUSSION: This study suggests that xCT mediates N-acetylcysteine uptake into the placenta and that N-acetylcysteine treatment of placental tissue alters the placental proteome while regulating the redox sensitive Maxi-chloride channel. Interestingly N-acetylcysteine had antioxidant effects independent of the glutathione pathway. Effective placental antioxidant therapy in pregnancy may require maintaining the balance between normalising redox status without inhibiting physiological redox signalling.

miR-520h Inhibits cell survival by targeting mTOR in gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is a type of diabetes that occurs during pregnancy due to abnormal maternal glucose metabolism. This study aimed to investigate the effect of miR-520h and its potential target gene on the progression of GDM. The blood samples were taken from healthy pregnant women and GDM patients. Human villous trophoblasts HTR-8/SVNEO cells were treated with 25 mM glucose and were considered as the GDM cell model. The miR-520h level was detected using qRT-PCR in the serum and GDM cell model. The correlation analysis between fasting blood-glucose (FBG) level and miR-520h expression was analyzed. The target relationship between miR-520h and mTOR was verified using dual luciferase reporter assay. HG-induced cells were transfected with miR-520h mimic or miR-520h inhibitor and pCDNA3-mTOR vector or their NCs. Cell viability, apoptosis and mTOR expression level were detected using CCK-8, flow cytometry and western blotting, respectively. The results showed that the miR-520h serum level was up-regulated in the GDM patients' serum and GDM cell model, and was positively correlated with FBG of GDM patients. High glucose (HG) inhibited HTR-8/SVNEO cell viability and decreased mTOR expression, while it promoted apoptosis. Then, the effects of HG on HTR-8/SVNEO cells were reversed by miR-520h inhibitor. Moreover, mTOR was identified as a target gene downstream of miR-520h. The overexpression of mTOR alleviated miR-520h mimic-induced reduction in cell viability and enhancement in cell apoptosis in the GDM cell model. In conclusion, miR-520h could inhibit cell viability and promote cell apoptosis by regulating mTOR expression in the GDM cell model. Hence, miR-520h might be a potential and important marker for the diagnosis and treatment of GDM.

Protease Inhibitor Anti-HIV, Lopinavir, Impairs Placental Endocrine Function.

Protease Inhibitors (PI e.g., ritonavir (RTV) and lopinavir (LPV)) used to treat pregnant mothers infected by HIV induce prematurity and endocrine dysfunctions. The maintenance of pregnancy relies on placental hormone production (human Chorionic Gonadotrophin (hCG) and progesterone (P4)). Those functions are ensured by the villous trophoblast and are mainly regulated by the Unfolded Protein Response (UPR) pathway and mitochondria. We investigated, in vitro, if PI impair hCG and P4 production and the potential intracellular mechanisms involved. Term villous cytotrophoblast (VCT) were cultured with or without RTV or LPV from 6 to 48 h. VCT differentiation into syncytiotrophoblast (ST) was followed measuring hCG and P4 secretion. We evaluated the expression of P4 synthesis partners (Metastatic Lymph Node 64 (MLN64), cholesterol side-chain cleavage (P450SCC), Hydroxy-delta-5-Steroid Dehydrogenase and 3 Beta-and steroid delta-isomerase 1 (HSD3B1)), of mitochondrial pro-fusion factors (Mitofusin 2 (Mfn2), Optic Atrophy 1 (OPA1)) and of UPR factors (Glucose-Regulated Protein 78 (GRP78), Activating Transcription Factor 4 (ATF4), Activating Transcription Factor 6 (ATF6), spliced X-box Binding Protein 1 (sXBP1)). RTV had no significant effect on hCG and P4 secretion, whereas lopinavir significantly decreased both secretions. LPV also decreased P450SCC and HSD3B1 expression, whereas it increased Mfn2, GRP78 and sXBP1 expression in ST. RTV has no effect on the endocrine placenta. LPV impairs both villous trophoblast differentiation and P4 production. It is likely to act via mitochondrial fusion and UPR pathway activation. These trophoblastic alterations may end in decreased P4 levels in maternal circulation, inducing prematurity.

Immune Cells in the Placental Villi Contribute to Intra-amniotic Inflammation.

Intra-amniotic (IA) inflammation is associated with significant morbidities for both the mother and the fetus. Prior studies have illustrated many of the effects of IA inflammation on the uterine lining (decidua) and membranous layers of the placenta at the fetal-maternal interface. However, much less is known about the immunological response occurring within the villous placenta. Using a rhesus macaque model of lipopolysaccharide (LPS)-induced IA inflammation, we showed that pregnancy-matched choriodecidua and villi have distinct immunological profiles in rhesus pregnancies. In the choriodecidua, we show that the abundance of neutrophils, multiple populations of antigen-presenting cells, and two populations of natural killer (NK) cells changes with prenatal IA LPS exposure. In contrast, in immune cells within the villous placenta we observed alterations in the abundance of B cells, monocytes, and CD8 T cells. Prior work has illustrated that IA inflammation leads to an increase in tumor necrosis factor alpha (TNFα) at the fetal-maternal interface. In this study, pretreatment with a TNFα blockade partially reversed inflammation in the placental villi. Furthermore, we report that immune cells in the villous placenta sensed LPS during our experimental window, and subsequently activated T cells to produce proinflammatory cytokines. Moreover, this study is the first report of memory T cells in third-trimester non-human primate placental villi and provides evidence that manipulation of immune cells in the villi at the fetal-maternal interface should be considered as a potential therapeutic target for IA inflammation.

Estrogen induces IDO expression via TGF­ß in chorionic villi and decidua during early stages of pregnancy.

Indoleamine 2,3­dioxygenase (IDO) is one of the most important proteins protecting the embryos from the mother's immune system during pregnancy; however, little is known about the regulation of expression of this protein at the maternal­fetal interface. In the current study, chorionic villi and decidua were collected from women at early stages of pregnancy. Samples of chorionic villi and decidua were cultured in medium containing different concentrations of 17ß­estradiol and estriol respectively, with or without fulvestrant. Western blot analysis and/or immunofluorescent staining were used to detect the expression of transforming growth factor ß (TGF­ß) and IDO in chorionic villi and decidua tissues. Both TGF­ß and IDO were expressed in chorionic villi and decidua. The expression levels of these two proteins increased the most in samples of chorionic villi and decidua cultured in medium containing 17ß­estradiol at the concentration of 10 ng/ml, or estriol at the concentration of 1 µg/ml. This increase could be reversed when fulvestrant was added in the medium at the concentration of 10 µg/ml. IDO expression increased in a dose­dependent manner in tissue samples cultured in medium containing TGF­ß. The results of the current study revealed that administration of estrogen at doses similar to those observed in healthy pregnant women may upregulate the expression of IDO by TGF­ß, suggesting that estrogen may prevent allogeneic fetal rejection and may be used as an immunomodulator.

Immunomodulatory effects of Salvianolic acid B in a spontaneous abortion mouse model.

Pregnancy is a kind of natural immune tolerance. Immune factors play an important role in recurrent spontaneous abortion and repeated implantation failure. Salvianolic acid B (SalB) has anti-tumor, anti-inflammatory, anti-oxidation and immunomodulatory functions. However, there are few reports on the relationship between SalB and maternal-fetal immune tolerance. In this study, CBA/J × DBA/2 J mice as a spontaneous abortion mouse model were given SalB. The results showed that the abortion rate was significantly decreased after SalB treatment. The populations of Nkp46 and cytotoxic CD8+ T cells in the placenta of female mice treated with SalB were significantly decreased. The qRT-PCR showed that SalB was able to significantly reduce the expression of pro-inflammatory factors and Toll-like Receptor in the placenta. In addition, SalB was able to increase the area of the labyrinth in the placenta. In conclusion, these findings suggest that SalB is beneficial for the immune-modulation at the maternal-fetal interface in a spontaneous abortion mouse model, resulting in a decrease in the abortion rate. This may encourage new ideas for the treatment of patients with repeated implantation failure.

Estrogen induces indoleamine 2,3-dioxygenase expression via suppressors of cytokine signaling 3 in the chorionic villi and decidua of women in early pregnancy.

PROBLEM: Indoleamine 2,3-dioxygenase (IDO) is a key protein that participates in the protection of embryos against the mother's immune system during pregnancy. How the expression of this protein is regulated at the maternal-fetal interface remains largely unknown. METHOD OF STUDY: The chorionic villi and decidua of women in early pregnancy were collected. Tissue explants of the chorionic villi and decidua were cultured in media containing varying concentrations of 17ß-estradiol and estriol with or without fulvestrant. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to detect the expression of IDO and the suppressors of cytokine signaling 3 (SOCS3) in the cultured tissues from chorionic villi and decidua. RESULTS: Both IDO and SOCS3 were expressed in chorionic villi and decidua. The expression of IDO was increased in tissue explants from chorionic villi and decidua cultured in medium containing different concentrations of 17ß-estradiol or estriol, and this increase was reversed when fulvestrant was added to the medium. The expression of IDO was upregulated, and SOCS3 expression was downregulated the most in tissue explants from chorionic villi and decidua that were cultured in medium containing 17ß-estradiol at a concentration of 10 ng/mL or estriol at a concentration of 1 µg/mL. This increase in IDO and decrease in SOCS3 were reversed when fulvestrant was added to the medium at a concentration of 10 µg/mL. CONCLUSION: At a concentration similar to that present during pregnancy, estrogen may upregulate the expression of IDO via downregulating SOCS3, which implies that estrogen may contribute to the prevention of allogeneic fetal rejection, and further studies may strengthen the possibility of using estrogen as an immune modulator.

No evidence of attenuation of placental insulin-stimulated Akt phosphorylation and amino acid transport in maternal obesity and gestational diabetes mellitus.

Pregnancies complicated by obesity and/or gestational diabetes (GDM) are associated with peripheral insulin resistance; however, the insulin responsiveness of the placenta in these pregnancy complications remains largely unknown. We tested the hypothesis that primary human trophoblast cells and placental villous explants will be insulin responsive, characterized by amino acid transport, Akt and Erk activity with maternal obesity, and/or GDM. We evaluated term placentas from women with normal body mass index (BMI) (normal; n = 15), obesity (OB; n = 11), normal BMI with GDM (N-GDM; n = 11), and obesity with GDM (OB-GDM; n = 11). In a subgroup, primary human trophoblast cells (PHT) were isolated, and in an independent subgroup placental villous explants were exposed to varying concentrations of insulin. Amino acid transport capacity and insulin signaling activity were determined. Insulin significantly increased amino acid transport activity to a similar degree in PHT cells isolated from normal (+21%), N-GDM (+38%), OB (+37%), and OB-GDM (+35%) pregnancies. Insulin increased Akt and Erk phosphorylation in PHT cells (3-fold) and in villous explants (2-fold) in all groups to a similar degree. In contrast to the peripheral maternal insulin resistance commonly associated with obesity and/or GDM, we found that the placenta is insulin sensitive in these pregnancy complications. We suggest that elevated maternal insulin levels in pregnancies complicated by obesity and/or GDM promote critical placental functions, including amino acid transport. Insulin-stimulated placental nutrient delivery may contribute to the increased risk of fetal overgrowth and adiposity in these pregnancies. Moreover, our findings may inform efforts to optimize insulin regimens for women with GDM.

Increased Incidence of Villitis in Placentas Exposed to Chemotherapy During Pregnancy: Is There a Correlation?

We have identified 9 pregnant patients who were diagnosed with malignancy and initiated chemotherapy during their second trimester (cervical cancer [n = 3], leukemia [n = 3], breast cancer [n = 2], and Hodgkin's lymphoma [n = 1]). Five of the patients' placentas were small for gestational age (SGA). Pathologic examination revealed inflammatory changes in 4 of the placentas: 2 from the SGA placentas and 2 from non-SGA placentas. Examination revealed 3 placentas with villitis of unknown etiology (VUE) and 1 with intervillositis; all were negative for bacterial and viral cultures and by immunohistochemical (IHC) stains. In the VUE cases, IHC stains showed positivity of CD25+/FOXP3+ with focal positivity and CD3 and CD4 IHC were focally to strongly positive. Literature suggests that the use of chemotherapy during pregnancy can be detrimental to both the mother and the fetus; however, there has been limited focus on the effects of chemotherapy on the placenta. We suggest that the inflammatory process noted in the placentas is due to chemotherapy-induced toxic effects.

Polysaccharide from Spirulina platensis ameliorates diphenoxylate-induced constipation symptoms in mice.

The aim of this study is to probe new functions of a polysaccharide from Spirulina platensis (PSP) on constipation and intestinal microbiota in mice. Diphenoxylate-induced constipation in mice was treated with different doses of PSP, followed by examining the defecation patterns, levels of acetyl cholinesterase (AchE), nitric oxide (NO), and tissue section histopathology. The composition of intestinal microbiota was determined by genome sequencing analysis of the 16S rDNA. This study found that the average molecular weight of PSP was 29, 600 Da, and mainly monosaccharides of PSP were rhamnose (24.7%), glucose (16.15%) and galactose (13.32%). The beneficial effects of PSP treatment include defecation improvement, increase of AchE activity, reduction of NO concentration, renovation of the damaged intestinal villus and affection on the expression of some related genes in the constipated mice. In addition, PSP had significant effects on the gut microbiota, showing the enhancement in abundance of beneficial bacteria including Akkermansia, Lactobacillus, Butyricimonas, Candidatus Arthromitus and Prevotella, and the reduction in abundance of harmful bacteria such as Clostridium and Dorea. The present s uncovered a new function of PSP, indicating that PSP could be used in constipation therapies.

The effects of valproic acid on early pregnancy human placentas: Pilot ex vivo analysis in cultured placental villi.

Valproic acid is an established structural and neurodevelopmental teratogen. Recently, we demonstrated that valproate alters the barrier function of perfused term human placentas. Here, we conducted a pilot study to evaluate the effects of subchronic valproate exposure on carrier expression in cultured placental villous explants from early human pregnancies. Placental tissue of gestational age 6-13 weeks was collected from elective pregnancy terminations in women without known epilepsy. The effects of valproate (42, 83, or 166 µg/mL) on the mRNA expression of 37 major placental carriers and related genes were evaluated by a customized gene expression array (n = 5, 5 days). Five-day exposure to valproate was associated with high variability in gene expression. However, two main gene clusters were identified, including a cluster of three major folate carriers. Exposure to low therapeutic levels of valproate (42 µg/mL) was associated with a tendency toward reduced mRNA expression of genes encoding folate and amino acid and fatty acid carriers (P = 0.065, paired analysis). Our initial findings suggest that valproate can affect the function of the human placenta during early pregnancy.

The Impact of Immunosuppressive Drugs on Human Placental Explants.

The use of immunosuppressive drugs guarantees the vitality of the graft and allows gestation in spite of intercurrences such as prematurity and intrauterine growth restriction. However, little is known about the direct effects of immunosuppressive drugs on placental cells. We investigated the effects of immunosuppressive drugs in the chorionic villous explants from human term placentas of healthy gestations. Human placental explants from term gestations (37-39 week gestational age, n = 12) were exposed to cyclosporine A (CSA, 0, 62.5, 125, 1250 ng/mL) or azathioprine (AZA, 0, 5, 10, 100 ng/mL) separately or, in combination for up to 48 hours. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed a significant decrease in the explant metabolic activity between AZA and the control group (24 hours, 100 ng/mL, 48 hours, all concentrations, P < .005). Cyclosporin A (CsA) reduced cell activity when associated with AZA (48 hours, P < .005). Fibrinoid deposits increased in AZA-treated explants alone (5 ng/mL, 48 hours; 10 ng/mL, 24-48 hours; P < .005) or when associated with CsA (10 AZA/125 CsA, P < .05), whereas in CsA treatment alone, there was an augment in syncytial knots (24-48 hours, P < .005). The sFLT1 gene (24 hours, P < .05) and protein (P < .005) expression increased in AZA and CsA-treatments separately or in combination (P < .05). Placental growth factor increased in AZA (24 hours, 10 ng/mL) and CsA (125 ng/mL; P < .05). In conclusion, our data indicate that AZA primarily acts on the villous metabolism, perturbing placental homeostasis. Since these drugs may alter the balance of angiogenic factors in its selection for clinical application, their impact on the behavior of placental villous should be considered.

The role of various transporters in the placental uptake of ofloxacin in an in vitro model of human villous trophoblasts.

INTRODUCTION: Six years after the US Food and Drug Administration approval of the broad-spectrum antibiotic ofloxacin (OFLX), the chiral switching of this racemic mixture resulted in a drug composed of the L-optical isomer levofloxacin (LVFX). Since both fluoroquinolones (FQs) were introduced to the pharmaceutical market, they have been widely prescribed by physicians, with careful administration during pregnancy and breastfeeding. Therefore, the role of the influx and efflux placental transporters in the concentrations of these drugs that permeate through human placental barrier model was investigated in this study. METHODS: The contribution of major carriers on the transplacental flux of OFLX and LVFX uptake into choriocarcinoma BeWo cells was evaluated in the presence vs absence of well-known inhibitors. RESULTS: Our results reveal that neither the influx transporters such as organic cation transporters, organic anion transporters, and monocarboxylate transporters nor the efflux transporters such as P-glycoprotein or breast cancer resistance protein significantly affected the transport of OFLX. In contrast, multiple transporters revealed pronounced involvement in the transfer of the levorotatory enantiomer in and out of the in vitro placental barrier. These data suggest a non-carrier-mediated mechanism of transport of the racemic mixture, while LVFX is subjected to major influx and efflux passage through the placental brush border membranes. CONCLUSION: This study provides underlying insights to elucidate the governing factors that influence the flux of these FQs through organ barriers, in view of the controversial safety profile of these drugs in pregnant population.

MXRA5 is decreased in preeclampsia and affects trophoblast cell invasion through the MAPK pathway.

Preeclampsia causes gestational failure in a significant number of women annually. Insufficient trophoblast cell invasion plays an essential role in preeclampsia pathogenesis. Matrix-remodeling associated 5 (MXRA5) is a proteoglycan involved in adhesion and matrix remodeling. This study sought to explore the role of MXRA5 in trophoblast cell invasion. Preeclamptic villi were obtained for the delineation of MXRA5 expression. Specific MXRA5 siRNA and pcDNA3.1/MXRA5 were used to manipulate MXRA5 expression in HTR-8/SVneo. Cell viability was determined by MTT and apoptosis by flow cytometry. Cell invasion was evaluated using Matrigel invasion assay. MXRA5 expression was lower in preeclamptic villi and cytotrophoblasts. Silencing MXRA5 expression in HTR-8/SVneo decreased cell viability and invasion, which were augmented by MXRA5 overexpression. Furthermore, MXRA5 modulated N-cadherin, E-cadherin, MMP-2, and MMP-9 expression through p38 MAPK and ERK1/2 signaling transduction. In addition, the expression of MXRA5 was influenced by exogenous TNF-α but not by IFN-γ. Overexpression of MXRA5 attenuated HTR-8/SVneo apoptosis induced by TNF-α. MXRA5 is downregulated in preeclamptic cytotrophoblasts and can regulate trophoblast cell invasion via the MAPK pathway.

Antenatal corticosteroid treatment and placental pathology, with a focus on villous maturation.

INTRODUCTION: Mothers at risk of preterm birth are treated with antenatal corticosteroids, which have advantageous effects for prematurely born infants. Accelerated villous maturation in the placenta is also associated with improved perinatal outcome. The primary aim of this study was to examine the association between antenatal corticosteroids and accelerated villous maturation. The secondary aim was to study associations with other placental pathologies. MATERIAL AND METHODS: A retrospective cohort study including 105 women who had (n = 75) or had not (n = 30) been treated with antenatal corticosteroids. The women gave birth between 22+0 and 26+6  weeks of gestation in Stockholm County between 1 April 2004 and 31 March 2007. A pathologist blinded to all clinical data except gestational age examined the placental slides to identify pathology parameters. The outcomes were correlated with antenatal corticosteroid treatment, and confounding factors were adjusted using logistic regression. RESULTS: Accelerated villous maturation was significantly higher in the group treated with corticosteroids (odds ratio 16, 95% CI 2.4-690, p = 0.0005). After adjustment for gestational age and preeclampsia, the difference remained significant (odds ratio 8.9, 95% CI 1.2-389, p = 0.021). No significant associations were found regarding the secondary outcome variables, after adjusting for possible confounders. CONCLUSIONS: Antenatal corticosteroid treatment before preterm birth is associated with accelerated villous maturation. This could be one of the pathways by which corticosteroids are beneficial for preterm infants.

The HMGB1/RAGE Pro-Inflammatory Axis in the Human Placenta: Modulating Effect of Low Molecular Weight Heparin.

We evaluated whether physiological and pre-eclamptic (PE) placentae, characterized by exacerbated inflammation, presented alterations in pro-inflammatory High Mobility Group Box 1 (HMGB1) and its Receptor of Advanced Glycation End products (RAGE) expression. Moreover, we investigated, in physiological placental tissue, the ability of Low Molecular Weight Heparin (LMWH) to modify HMGB1 structural conformation thus inhibiting RAGE binding and HMGB1/RAGE axis inflammatory activity. HMGB1, RAGE, IL-6 and TNFα (HMGB1/RAGE targets) mRNA expression were assessed by Real Time PCR. HMGB1, RAGE protein levels were assessed by western blot assay. Physiological term placental explants were treated by 0.5 U LMWH for 24 or 48 h. HMGB1 and RAGE expression and association were evaluated in LMWH explants by RAGE immunoprecipitation followed by HMGB1 immunoblot. HMGB1 spatial localization was evaluated by immuofluorescent staining (IF). HMGB1 expression was increased in PE relative to physiological placentae while RAGE was unvaried. 24 h LMWH treatment significantly up-regulated HMGB1 expression but inhibited HMGB1/RAGE complex formation in physiological explants. RAGE expression decreased in treated relative to untreated explants at 48 h. IF showed HMGB1 localization in both cytoplasm and nucleus of mesenchymal and endothelial cells but not in the trophoblast. IL-6 and TNFα gene expression were significantly increased at 24 h relative to controls, while they were significantly down-regulated in 48 h vs. 24 h LMWH explants. Our data depicted a new molecular mechanism through which LMWH exerts its anti-inflammatory effect on PE placentae, underlying the importance of HMGB1/RAGE axis in PE inflammatory response.

Impact of chlorpyrifos on human villous trophoblasts and chorionic villi.

Placental barrier regulates maternal-fetal interchange protecting the baby from damage caused by substances found in the uterine environment or circulating in the vascular system. Organophosphate (OP) pesticides are a paramount group of environmental pollutants used in intensive agriculture for protection against diseases and pests. While many studies have reported an increased risk of pregnancy alterations in pregnant women exposed to OPs, few have analyzed the effects caused by these pesticides in the placenta. Herein, we evaluated the effects of chlorpyrifos (CPF), one of the most widely used OP insecticides, on human placenta using in vitro and ex vivo exposure models. Villous cytotrophoblast cells isolated from normal human term placentas maintained their cell viability, differentiated into syncytiotrophoblast-like structures, and increased the expression of ß-hCG, ABCG2, and P-gp in the presence of CPF at concentrations of 10 to 100µM. The same doses of CPF induced marked changes in chorionic villi samples. Indeed, CPF exposure increased stroma cell apoptosis, altered villi matrix composition, basement membrane thickness, and trophoblastic layer integrity. Histomorphological and ultrastructural alterations are compatible with those found in placentas where maternal-placenta injury is chronic and able to impair the placental barrier function and nutrient transport from mother to the fetus. Our study shows that placental ex vivo exposure to CPF produces tissue alterations and suggest that human placenta is a potential target of CPF toxicity. In addition, it highlights the importance of using different models to assess the effects of a toxic on human placenta.

Leptin reduces apoptosis triggered by high temperature in human placental villous explants: The role of the p53 pathway.

Maternal fever is common during pregnancy and has for many years been suspected to harm the developing fetus. Whether increased maternal temperature produces exaggerated apoptosis in trophoblast cells remains unclear. Since p53 is a critical regulator of apoptosis we hypothesized that increased temperature in placenta produces abnormal expression of proteins in the p53 pathway and finally caspase-3 activation. Moreover, leptin, produced by placenta, is known to promote the proliferation and survival of trophoblastic cells. Thus, we aimed to study the possible role of leptin preventing apoptosis triggered by high temperature, as well as the molecular mechanisms underlying this effect. Fresh placental tissue was collected from normal pregnancies. Explants of placental villi were exposed to 37 °C, 40 °C and 42 °C during 3 h in the presence or absence of 10 nM leptin in DMEM-F12 medium. Western blotting and qRT-PCR was performed to analyze the expression of p53 and downstream effector, P53AIP1, Mdm2, p21, BAX and BCL-2 as well as the activated cleaved form of caspase-3 and the fragment of cytokeratin-18 (CK-18) cleaved at Asp396 (neoepitope M30). Phosphorylation of the Ser 46 residue on p53, the expression of P53AIP1, Mdm2, p21, as well as caspase-3 and CK-18 were significantly increased in explants at 40 °C and 42 °C. Conversely, these effects were significantly attenuated by leptin 10 nM at both 40 °C and 42 °C. The BCL2/BAX ratio was also significantly decreased in explants at 40 °C and 42 °C compared with explants incubated at 37 °C, which was prevented by leptin stimulation. These data illustrate the potential role of leptin for reducing apoptosis in trophoblast explants, including trophoblastic cells, triggered by high temperature, by preventing the activation of p53 signaling.

Uterine Expression of NDRG4 Is Induced by Estrogen and Up-Regulated during Embryo Implantation Process in Mice.

Embryo implantation is an essential step for the establishment of pregnancy and dynamically regulated by estrogen and progesterone. NDRG4 (N-myc down-regulated gene 4) is a tumor suppressor that participates in cell survival, tumor invasion and angiogenesis. The objective of this study was to preliminarily explore the role of NDRG4 in embryo implantation. By immunohistochemistry (IHC) and quantitive RT-PCR (qRT-PCR), we found that uterine expression of NDRG4 was increased along with puberal development, and its expression in adult females reached the peak at the estrus stage during the estrus cycle. Furthermore, uterine NDRG4 expression was significantly induced by the treatment of estradiol (E2) both in pre-puberty females and ovariectomized adult females. Uterine expression pattern of NDRG4 during the peri-implantation period in mice was determined by IHC, qRT-PCR and Western blot. It was observed that NDRG4 expression was up-regulated during the implantation process, and its expression level at the implantation sites was significantly higher than that at the inter-implantation sites. Meanwhile, an increased expression in NDRG4 was associated with artificial decidualization as well as the activation of delayed implantation. By qRT-PCR and Western blot, we found that the in vitro decidualization of endometrial stromal cells (ESCs) was accompanied by up-regulation of NDRG4 expression, whereas knockdown of its expression in these cells by siRNA inhibited the decidualization process. In addition, Western blot analysis showed that NDRG4 protein expression was decreased in human villus tissues of recurrent miscarriage (RM) patients compared to normal pregnant women. Collectively, these data suggested that uterine NDRG4 expression could be induced by estrogen, and NDRG4 might play an important role during early pregnancy.