Impaired Trophoblast Invasion Research Articles

Impaired Sphingosine-1-Phosphate Synthesis Induces Preeclampsia by Deactivating Trophoblastic YAP (Yes-Associated Protein) Through S1PR2 (Sphingosine-1-Phosphate Receptor-2)-Induced Actin Polymerizations.

Incomplete spiral artery remodeling, caused by impaired extravillous trophoblast invasion, is a fundamental pathogenic process associated with malplacentation and the development of preeclampsia. Nevertheless, the mechanisms controlling this regulation of trophoblast invasion are largely unknown. We report that sphingosine-1-phosphate synthesis and expression is abundant in healthy trophoblast, whereas in pregnancies complicated by preeclampsia the placentae are associated with reduced sphingosine-1-phosphate and lower SPHK1 (sphingosine kinase 1) expression and activity. In vivo inhibition of sphingosine kinase 1 activity during placentation in pregnant mice led to decreased placental sphingosine-1-phosphate production and defective placentation, resulting in a preeclampsia phenotype. Moreover, sphingosine-1-phosphate increased HTR8/SVneo (immortalized human trophoblst cells) cell invasion in a Hippo-signaling-dependent transcriptional coactivator YAP (Yes-associated protein) dependent manner, which is activated by S1PR2 (sphingosine-1-phosphate receptor-2) and downstream RhoA (Ras homolog gene family, member A)/ROCK (Rho-associated protein kinase) induced actin polymerization. Mutation-based YAP-5SA (S61A, S109A, S127A, S164A, S381A) demonstrated that sphingosine-1-phosphate activation of YAP could be either dependent or independent of Hippo signaling. Together, these findings suggest a novel pathogenic pathway of preeclampsia via disrupted sphingosine-1-phosphate metabolism and signaling-induced, interrupted actin dynamics and YAP deactivation; this may lead to potential novel intervention targets for the prevention and management of preeclampsia.

Advanced Maternal Age-associated SIRT1 Deficiency Compromises Trophoblast Epithelial-Mesenchymal Transition through an Increase in Vimentin Acetylation.

Advanced maternal age (AMA) pregnancies are rapidly increasing and are associated with aberrant trophoblast cell function, poor placentation, and unfavorable pregnancy outcomes, presumably due to premature placental senescence. SIRT1 is an NAD+‐dependent deacetylase with well‐known antiaging effects, but its connection with placental senescence is unreported. In this study, human term placentas and first‐trimester villi were collected from AMA and normal pregnancies, and a mouse AMA model was established by cross breeding young and aged male and female C57 mice. SIRT1 expression and activity in HTR8/SVneo cells were genetically or pharmacologically manipulated. Trophoblast‐specific Sirt1‐knockout (KO) mouse placentas were generated by mating Elf5‐Cre and Sirt1 fl/fl mice. Trophoblast cell mobility was assessed with transwell invasion and wound‐healing assays. SIRT1‐binding proteins in HTR8/SVneo cells and human placental tissue were identified by mass spectrometry. We identified SIRT1 as the only differentially expressed sirtuin between AMA and normal placentas. It is downregulated in AMA placentas early in the placental life cycle and is barely impacted by paternal age. SIRT1 loss upregulates P53 acetylation and P21 expression and impairs trophoblast invasion and migration. Sirt1‐KO mouse placentas exhibit senescence markers and morphological disruption, along with decreased fetal weight. In trophoblasts, SIRT1 interacts with vimentin, regulating its acetylation. In conclusion, SIRT1 promotes trophoblast epithelial−mesenchymal transition (EMT) to enhance invasiveness by modulating vimentin acetylation. AMA placentas are associated with premature senescence during placentation due to SIRT1 loss. Therefore, SIRT1 may be an antiaging therapeutic target for improving placental development and perinatal outcomes in AMA pregnancies.

Abstract P136: First Steps In Uncovering Mechanisms In Late Onset Preeclampsia

Preeclampsia is a leading cause of maternal morbidity and mortality worldwide, complicating 2-8% of pregnancies. The etiology of PE is poorly understood, the role of placental extracellular matrix (pECM) in PE is understudied. Extravillous trophoblasts (HTR8/svneo) and pECM are crucial in placental blood vessel formation, including spiral arteries. The objective is to identify changes in signaling mechanisms in late onset PE and how HTR8/svneo respond. Additionally, we explored a novel therapeutic to restore HTR8/svneo phenotype by phototherapy using a 670nm LED light. Western blot analysis shows eNOS, phospho-eNOS and Hsp90 expressions are unchanged in late onset PE placental samples despite previous reports in early onset of PE. The association of Hsp90 to eNOS and eNOS to phospho-eNOS is reduced in the PE sample. Soluble Flt, a diagnostic marker for PE, levels are increased in media from HTR8/svneo trophoblasts cultured on PE pECM. Endothelial function is significantly reduced in an isolated vessel as determined by acetylcholine stimulation. 670nm light reverses this impairment. Migration of HTR8/SVneo on PE pECM is reduced (control 86.29±18.58, PE 30.16±15.49) but proliferation of HTR8/SVneo on PE pECM is unaffected in patients (control: 59.125±6262.9, PE:67.125±7706.5). Proliferation and migration are improved by 670nm light (proliferation control 101.375±2075.4; PE 106.875±4870.7, migration control 125±11.97, PE 144.84±16.01). An increase of apoptotic HTR8/SVneo cultured on PE pECM is observed compared to healthy pECM in patients control 2.4±0.24, PE 3±0.4 samples. 670nm light treatment decreases the number of apoptotic cells, control 1.2 ±0.58, PE 1.4±0.5. Conclusion: In late onset preeclampsia, endothelial function is decreased, sFlt release is increased, the eNOS/Hsp90/p-eNOS axis is slightly reduced pointing to O 2 - synthesis. Migration is decreased likely due to impaired trophoblast invasion, but proliferation is unaffected. 670nm light treatment can reverse some of the effects of the preeclamptic microenvironment. Extracellular matrix is altered in late onset PE leading to an abnormal HTR8/SVneo phenotype. 670nm light restores the phenotype of HTR8/SVneo on PE pECM suggesting 670nm light as a therapeutic treatment.

Tissue Transglutaminase Impairs HTR-8/SVneo Trophoblast Cell Invasion via the PI3K/AKT Signaling Pathway

Objectives: The pathogenesis of preeclampsia (PE) is associated with impaired trophoblast invasion, which results in placental insufficiency. Our earlier studies demonstrated that tissue transglutaminase (tTG) is highly expressed in human PE serum. However, whether tTG participates in trophoblast invasion remains unclear. The aim of the present study was to determine the role and mechanism of tTG in regulating matrix metalloproteinase (MMP)-2/MMP-9 expression to reduce trophoblast invasiveness in PE. Methods: HTR-8/SVneo cells were transfected with a lentivirus vector and small interfering RNA targeting tTG. The protein level was detected by Western blotting. Cell proliferation and apoptosis were assessed by MTS and flow cytometry assays, respectively. Cell invasion was investigated by Transwell assay. In addition, the influence of tTG on PI3K and AKT mRNA levels in HTR-8/SVneo cells was evaluated using reverse transcription-quantitative PCR. Results: tTG-overexpression inhibited HTR-8/SVneo cell proliferation and invasion and promoted apoptosis. In addition, upregulation of tTG induced an increase of PI3K and phosphorylated AKT and a decrease of MMP-2 and MMP-9 expression. tTG-knockdown significantly promoted the proliferation and invasion of HTR-8/SVneo cells and inhibited the apoptosis. Furthermore, the PI3K expression level was reduced, and the MMP-2/MMP-9 protein levels were increased. Conclusion: Taken together, the present study demonstrated that tTG-overexpression inhibited HTR-8/SVneo cell invasion via reducing the expression of MMP-2 and MMP-9 by activating PI3K/AKT signaling pathway, which may lead to the occurrence or development of PE. The present data provide new insights into modulation of tTG expression as a potential therapeutic target for PE.

Elevated Serum Reactive Oxygen Species Level Predicts Early Abortion

Background: Impaired trophoblast invasion is associated with early abortion. The calorie needed for the trophoblast cell (TC) invasion is mainly met by adenosine triphosphate (ATP) produced in the mitochondria. Reactive oxygen species (ROS), byproduct of ATP synthesis, plays an important role in cellular physiology, but a high level of ROS may result in deoxyribonucleic acid (DNA) damage or cell dysfunction, thereby impaired TC invasion leading to early abortion. The study aims to determine elevated serum ROS level to predicts early abortion.Materials and method: This was an observational study with a cross-sectional design. Fifty subjects with gestational age less than 12 weeks, consist of 25 early abortions and 25 normal pregnancies subjects, were included in this study. Clinical examination and diagnosis are carried out in 2 Hospitals and 5 Public Health Centers in Padang. Examination of ROS levels was carried out by enzyme-linked immunosorbent assay (ELISA) in the Biomedical Laboratory, Faculty of Medicine, Universitas Andalas. The Mann-Whitney test was used to analyze the difference of serum ROS levels, with a significance level of 0.05.Results: The subjects of the two study groups were equivalent in terms of age, gestational age, and gravidity (p=0.051, p=0.453, and p=1.000). The median ROS levels were found to be 1.36 (1.02-26.30) ng/mL in the early abortion and 1.20 (0.43-2.75) ng/mL in the normal pregnancy (p=0.003).Conclusion: There is a significant difference between ROS levels in early abortion and normal pregnancy.Keywords: ROS, early abortion, normal pregnancy

Acetaminophen Attenuates invasion and alters the expression of extracellular matrix enzymes and vascular factors in human first trimester trophoblast cells

Acetaminophen is one of the most common medications taken during pregnancy, considered safe for maternal health and fetal development. However, recent epidemiological studies have associated prenatal acetaminophen use with several developmental disorders in offspring. As acetaminophen can freely cross into and through the placenta, epidemiological associations with prenatal acetaminophen use may reflect direct actions on the fetus and/or the impact of altered placental functions. In the absence of rigorous mechanistic studies, our understanding of how prenatal acetaminophen exposure can cause long-term effects in offspring is limited. The objective of this study was to determine whether acetaminophen can alter key functions of a major placental cell type by utilizing immortalized human first trimester trophoblast cells. This study employed a comparative analysis with the nonsteroidal, anti-inflammatory drug aspirin, which has established effects in first trimester trophoblast cells. We report that immortalized trophoblast cells express the target proteins of acetaminophen and aspirin: cyclooxygenase (COX) −1 and −2. Unlike aspirin, acetaminophen significantly repressed the expression of angiogenesis and vascular remodeling genes in HTR-8/SVneo cells. Moreover, acetaminophen impaired trophoblast invasion by over 80%, while aspirin had no effect on invasion. Acetaminophen exposure reduced the expression of matrix metalloproteinase (MMP)-2 and −9 and increased the expression of tissue inhibitors of matrix metalloproteinases 2, leading to an imbalance in the ratio of proteolytic enzymes. Finally, a bioinformatic approach identified novel acetaminophen-responsive gene networks associated with key trophoblast functions and disease. Together these results suggest that prenatal acetaminophen use may interfere with critical trophoblast functions early in gestation, which may subsequently impact fetal development.

Galectin Family in Placental Insufficiency Syndromes

Galectin Family in Placental Insufficiency Syndromes

Downregulation of cAMP-Dependent Protein Kinase Inhibitor-b Promotes Preeclampsia by Decreasing Phosphorylated Akt

Preeclampsia is a multi-system disease that is unique to human pregnancy. Impaired extravillous trophoblast migration and invasion accompanied by poor spiral vascular remodeling is thought to be the initial reason. This study investigated cAMP-dependent protein kinase inhibitor-b(PKIB) expression in placentas and its involvement in the pathogenesis of PE. We used immunohistochemistry and western blotting to calculate PKIB levels in the placentas. Then we knocked down PKIB by siRNA and used real-time cell analysis to assess the invasion and migration ability of trophoblasts. Tube formation assay and spheroid sprouting assay were utilized to identify the ability to form vessels of trophoblasts. At last, western blotting was used to demonstrate the level of phosphorylated Akt, as well as downstream-related genes of Akt signaling pathway in trophoblasts. We first found that PKIB expression level was lower in the PE placentas than in the normal placentas. In addition, we found that downregulation of PKIB can inhibit the migration, invasion, and the ability to form vessels of HTR8/SVneo cells. Downregulation of PKIB leaded to a decrease in phosphorylated Akt, as well as downstream proteins such as matrix metalloproteinase 2, matrix metalloproteinase 9, and glycogen synthase kinase 3β, which are related to migration and invasion. Our study revealed that the downregulation of PKIB expression resulted in decreased migration, invasion, and vessel formation ability by regulating Akt signaling pathway in placental trophoblasts in PE.

The relationship among vitamin D, TLR4 pathway and preeclampsia.

Preeclampsia is a pregnancy-specific syndrome that has been the greatest cause of maternal and fetal morbidity and mortality. The impaired outcomes are related to maternal and the offspring healthy in the short and long-term. Although preeclampsia origins remain unclear, it is well known that there is impaired trophoblast invasion with culminant abnormal immune response. The early and late-onset preeclampsia have been studied, the subtypes have the same difference in the placentation and inflammatory features. Dietary compounds can stimulate or inhibit the activation of immune cells. Low vitamin D intake has been linked to impaired fetal development, intrauterine growth restriction, and preeclampsia. Vitamin D has been described as an anti-inflammatory effect. It can downregulate pro-inflammatory cytokines expression by the inhibition of the Nuclear Factor-ĸB pathway signaling cascade. High vitamin D levels could attenuate the immune response. On the other hand, vitamin D deficiency may contribute to increasing pro-inflammatory state. In preeclampsia, there is a reduced expression of vitamin D receptor and its metabolism is disrupted. In this review, we aimed to discuss the role of vitamin D as an anti-inflammatory agent in relation to the pro-inflammatory process of preeclampsia through the activation of the TLR4 pathway. Although there are limited studies showing the relation between vitamin D and lower risk of preeclampsia, the maternal status of vitamin D seems to influence the risk of PE development. Therefore, vitamin D supplementation in women may be a strategy to improve pregnancy outcomes.

Phosphorylation of Yes-associated protein impairs trophoblast invasion and migration: implications for the pathogenesis of fetal growth restriction†.

Fetal growth restriction (FGR) is a condition in which a newborn fails to achieve his or her prospective hereditary growth potential. This condition is associated with high newborn mortality, second only to that associated with premature birth. FGR is associated with maternal, fetal, and placental abnormalities. Although the placenta is considered to be an important organ for supplying nutrition for fetal growth, research on FGR is limited, and treatment through the placenta remains challenging, as neither proper uterine intervention nor its pathogenesis have been fully elucidated. Yes-associated protein (YAP), as the effector of the Hippo pathway, is widely known to regulate organ growth and cancer development. Therefore, the correlation of the placenta and YAP was investigated to elucidate the pathogenic mechanism of FGR. Placental samples from humans and mice were collected for histological and biomechanical analysis. After investigating the location and role of YAP in the placenta by immunohistochemistry, we observed that YAP and cytokeratin 7 have corresponding locations in human and mouse placentas. Moreover, phosphorylated YAP (p-YAP) was upregulated in FGR and gradually increased as gestational age increased during pregnancy. Cell function experiments and mRNA-Seq demonstrated impaired YAP activity mediated by extracellular signal-regulated kinase inhibition. Established FGR-like mice also recapitulated a number of the features of human FGR. The results of this study may help to elucidate the association of FGR development with YAP and provide an intrauterine target that may be helpful in alleviating placental dysfunction.

UL16-Binding Protein 1 Induced HTR-8/SVneo Autophagy via NF-κB Suppression Mediated by TNF-α Secreted through uNK Cells.

UL16-binding protein 1(ULBP1) has been reported to inhibit trophoblast invasion through the modification of secretion functions of uNK cells in the previous study, but its mechanisms remain unclear. In this study, we investigated the related mechanism by which upregulated ULBP1 expression impaired trophoblast invasion. We found that conditioned media with ULBP1 increased autophagy in HTR-8/SVneo, and anti-TNF-α-neutralizing antibody rescued the autophagy caused by the conditioned medium. We further found TNF-α induced autophagy in trophoblast cells in a dose-dependent way and accompanied by a decreased activity of nuclear factor-kappa B (NF-κB). Inhibition of NF-κB activation by chemical inhibitor augmented these autophagic responses to TNF-α in the cells. In addition, interruption NF-κB caused a significant decrease in HTR-8/SVneo invasion and enhanced the inhibition effect of TNF-α on HTR-8/SVneo invasion. Taken together, these findings suggest that TNF-α is able to regulate autophagic activity via suppressing NF-κB, which might be the mechanism related to ULBP1 in preeclampsia pathogenesis.

Long non-coding RNA lnc-DC in dendritic cells regulates trophoblast invasion via p-STAT3-mediated TIMP/MMP expression.

Dendritic cells are the primary antigen-presenting cells that contact trophoblasts at the beginning of pregnancy. Excessive DCs maturity is described in some pregnancy complications, such as pre-eclampsia and fetal growth restriction, which are characterized by impaired trophoblast invasion. However, the mechanism is unclear. The long non-coding RNA long non-coding RNA DC (lnc-DC) is expressed exclusively in conventional human DCs and induces DC differentiation and maturation by promoting signal transducer and activator of transcription 3 (STAT3) phosphorylation. Our previous investigation proved lnc-DC and p-STAT3 are elevated in pre-eclampsia. This research is to study the mechanism of lnc-DC and trophoblast invasion. We transfected DCs with lnc-DC shRNA or a lentivirus for lnc-DC overexpression and cocultured these treated DCs with trophoblast under different conditions. Transwell assay and wound healing assay were used to detect the trophoblast invasion ability. We also tested the matured DCs and Th1 cells as well as the p-STAT3. We found that lnc-DC promoted DC maturation and inhibited trophoblast invasion without the involvement of CD4+ T cells. And the p-STAT3 agonist could reverse the lnc-DC function. Mature DCs may be involved in altering trophoblast invasion through the overexpression of lnc-DC, which increases p-STAT3 levels and the tissue inhibitor of metalloproteinase-1 (TIMP-1)/matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-2 (TIMP-2)/matrix metalloproteinase-2 (MMP-2) ratios. Thus, lnc-DC is a promising novel target for regulating trophoblast invasion.

Brominated diphenyl ether-47 differentially regulates cellular migration and invasion in a human first trimester trophoblast cell line

Polybrominated diphenyl ethers (PBDEs) are flame retardant compounds detected in human placenta and linked to adverse pregnancy outcomes. Impaired trophoblast migration and invasion during early pregnancy have been implicated as potential mechanisms of pregnancy disorders. The present study investigated the effect of BDE-47, a prevalent PBDE congener, on cell migration, invasion, and matrix metalloproteinase (MMP) expression in a human first trimester extravillous trophoblast cell line, HTR-8/SVneo. BDE-47 stimulated cell migration in HTR-SV/neo cells while decreasing invasion of cells into Matrigel. In addition, BDE-47 led to differential expression of MMP-1, -2, -3, and -9 at protein and mRNA levels. In summary, BDE-47 differentially regulated cellular migration and invasion with divergent changes in MMP expression in trophoblasts. Because proper regulation of trophoblast migration and invasion is critical for placental development and function, further research is warranted to determine if exposure to PBDEs disrupts trophoblast functions with increased risk for adverse pregnancy outcomes.

TNF-related apoptosis-inducing ligand (TRAIL) promotes trophoblast cell invasion via miR-146a-EGFR/CXCR4 axis: A novel mechanism for preeclampsia?

IntroductionPreeclampsia (PE) is associated with failure of uterovascular transformation due to impaired trophoblast invasion. Previously, TNF-related apoptosis-inducing ligand (TRAIL) has been controversially reported to correlate with PE, but whether it regulates trophoblast invasion yet to be defined. MethodsWe treated HTR8/SVneo cells with sTRAIL at concentrations of 0.1 ng/mL, 1 ng/mL and 10 ng/mL for a 72-h time course and compared cell proliferation, apoptosis and invasion ability by CCK8 assay, flow cytometry analysis and Matrigel cell invasion assay, respectively. The expressions levels of miR-146a, CXCR4, EGFR and MMP2 were quantified by qRT-PCR and Western blot. Moreover, HTR8/SVneo cells were transfected with miR-146a mimics and miR-146a inhibitor, followed by analyzing invasion ability and gene expressions of CXCR4, EGFR and MMP2. Finally, both serum and placental samples from preeclamptic and gestational week-matched normotensive women were collected and assessed for the expression levels of TRAIL by ELISA assay and immunochemistry staining. ResultssTRAIL treatment of HTR8/SVneo cells resulted in no change in proliferation or apoptosis, but dose- and time-dependently enhanced invasion. TRAIL downregulated the expressions of miR-146a and upregulated CXCR4, EGFR and MMP2. Transfection of miR-146a resulted in the inhibition of invasion and downregulation of CXCR4, EGFR and MMP2. Lastly, the expression levels of TRAIL decreased in both serum and placenta of preeclamptic pregnancies and correlated with the disease severity. DiscussionTRAIL regulated miR-146a-CXCR/EGFR axis to promote the invasion of trophoblast like cells. Its deceased levels in preeclamptic sera and placenta, suggest that low levels of TRAIL might contribute to the pathogenesis of preeclampsia.

Sestrin2 alleviates palmitate-induced endoplasmic reticulum stress, apoptosis, and defective invasion of human trophoblast cells.

Maternal obesity induces elevated saturated fatty acid palmitate levels in the blood and causes pregnancy complications such as gestational diabetes, preeclampsia, fetal growth abnormalities, and stillbirth. Sestrin2, a highly conserved stress-inducible protein, is involved in the cellular responses of various stress conditions and homeostatic regulation. However, the effects of Sestrin2 on trophoblast cells have not yet been investigated. Here, we investigated the role of Sestrin2 in palmitate-induced lipotoxicity and its underlying mechanisms in human first-trimester trophoblast cells (Sw.71). Mouse placental tissues were obtained from low-fat diet-fed mice (n=14) and high-fat diet-fed mice (n=14) at gestation day 17.5. Sw.71 cells were treated with palmitate or bovine serum albumin as vehicle controls. The role of Sestrin2 in palmitate-induced lipotoxicity was examined by immunocytochemistry, immunoblot analysis, quantitative real-time PCR, and invasion assay. Expression of placental Sestrin2 was elevated in high-fat diet-fed dams compared to that of low-fat diet-fed dams. Prolonged treatment of Sw.71 cells with palmitate-induced endoplasmic reticulum (ER) stress-dependent expressions of Sestrin2 protein and mRNA, and the treatment also triggered apoptosis. Knockdown of Sestrin2 increased palmitate-mediated ER stress, inflammatory signaling, and apoptosis. Furthermore, Sestrin2 suppressed impaired trophoblast invasion caused by palmitate and attenuated palmitate-induced ER stress and inflammation via AMPK/mTORC1 pathways. Our study provides the relationship between Sestrin2, AMPK/mTORC1 pathway, and trophoblast function, suggesting that Sestrin2 may be a novel potential therapeutic target for the prevention of pregnancy complications.

Upregulation of PUM1 Expression in Preeclampsia Impairs Trophoblast Invasion by Negatively Regulating the Expression of the lncRNA HOTAIR.

Pumilio (PUM) proteins are members of a highly conserved RNA-binding protein family that posttranscriptionally regulate gene expression in many organisms. However, their roles in the placenta are unclear. In the present study, we report the requirement for the PUM homolog 1 (PUM1) gene in preeclampsia (PE). Immunofluorescence and immunohistochemical data showed that PUM1 was highly expressed in human placental villi from women with PE compared to healthy controls (HCs). Further, PUM1 overexpression repressed, and knockdown enhanced, the invasion and proliferation of trophoblasts. Interestingly, PUM1 knockdown promoted trophoblast invasion in a villous explant culture model, while PUM1 overexpression repressed these effects. Furthermore, lncRNA transcriptome sequencing coupled with RNA immunoprecipitation (RIP) revealed that PUM1 inhibits trophoblast invasion in PE by downregulating the expression of lncRNA HOTAIR. Moreover, PUM1 regulates HOTAIR expression via a posttranscriptional mechanism. Using RNA-protein pull-down and mRNA stability assays, we identified PUM1 as a specific binding partner that decreased the half-life of HOTAIR and lowered the steady-state level of HOTAIR expression, suggesting a novel posttranscriptional regulatory mechanism. Collectively, these findings identified a novel RNA regulatory mechanism, revealing a new pathway governing the regulation of PUM1/HOTAIR in trophoblast invasion in the pathogenesis of PE.

Low-Dose Aspirin May Prevent Trophoblast Dysfunction in Women With Chlamydia Pneumoniae Infection

Previously, we demonstrated that live Chlamydia pneumoniae (Cp) impaired extravillous trophoblast (EVT) viability and invasion and that Cp DNA was detected in placentas from cases with preeclampsia. We sought to elucidate whether (1) inactive forms of Cp also affect EVT function; (2) potential therapeutic interventions protect against the effects of Cp; and (3) anti-Cp antibodies are associated with preeclampsia. Human first-trimester EVTs were infected with ultraviolet light-inactivated Cp. Subgroups of EVTs were pretreated with low-dose acetyl-salicylic acid (ASA), dexamethasone, heparin, and indomethacin. We conducted functional assays after infection with inactivated Cp and measured interleukin 8 (IL8), C-reactive protein (CRP), heat shock protein 60 (HSP60), and tumor necrosis factor-α (TNFα) in culture media. We measured anti-Cp IgG serum levels from women who developed preeclampsia (N = 105) and controls (N = 121). Inactivated Cp reduced EVT invasion when compared to noninfected cells (P < .00001) without adversely affecting cell viability. Increased levels of IL8, CRP, HSP60, and TNFα were detected in EVTs infected with inactivated Cp compared to noninfected cells (P < .0001). Only pretreatment with low-dose ASA prevented reduced EVT invasion and decreased release of inflammatory mediators (P < .01). Elevated anti-Cp IgG antibodies were more prevalent in serum from cases with preeclampsia compared to controls (67/105 vs 53/121; adjusted P = .013); elevated IgG correlated significantly with elevated serum CRP and elevated soluble fms-like tyrosine kinase-1-placental growth factor ratio. Inactivated Cp induces decreased EVT invasion and a proinflammatory response; these effects were abrogated by pretreatment with low-dose ASA. Our results suggest an association between Cp infection, trophoblast dysfunction, and preeclampsia.

MiR-141-5p regulate ATF2 via effecting MAPK1/ERK2 signaling to promote preeclampsia

ObjectivePreeclampsia is a pregnancy-specific syndrome characterized by hypertension and proteinuria. Impaired trophoblast invasion partly modulated by abnormal MAPK1/ERK2 signaling played important roles in the pathological process of preeclampsia. The objective of this study is to investigate miR-141-5p regulate ATF2 via effecting MAPK1/ERK2 signaling to promote preeclampsia. Study designThe maternal placentae and clinical data of 30 patients with preeclampsia and 30 healthy pregnant women were collected in the Second Hospital of Shanxi Medical University from July 2015 to April 2016. Transcriptional levels of miR-141-5p in placentae were monitored using quantitative real-time reverse transcription-polymerase chain reaction. The target gene of miR-141-5p was analyzed with “TargetScanHuman Release 7.2″. To evaluate the pathways of this response, MAPK1 and ERK1/2 in placentae were detected using immunohistochemistry and Western Blot. Transfection experiment was used to verify the function of miR-141-5p regulating ATF2 to effect MAPK1/ERK2 signaling in JEG-3 cells. ResultsmiR-141-5p was significantly down-regulated in placentae of patients with preeclampsia, in comparison to the healthy pregnant women groups. There was no difference in MAPK1 expression between placentae of patients with preeclampsia and healthy pregnant women groups. While p-MAPK1 expression was lower in preeclampsia placentae, in comparison to the healthy pregnant women groups. Moreover, inhibition and activation experiments also validate the function of miR-141-5p in effecting p-MAPK1 level in JEG-3 cells. Bioinformatic analysis identified that ATF2 was a target gene of miR-141-5p, which was one DNA-binding protein to effect phosphatase DUSP1 transcription. DUSP1 effect MAPK1/ERK2 signaling in preeclampsia. ConclusionmiR-141-5p up-regulated transcription factor ATF2 to promote phosphatase DUSP1 expression. DUSP1 expression reduces p-MAPK1 and ERK1/2 expression to promote preeclampsia.

Hypoxia-induced microRNA-141 regulates trophoblast apoptosis, invasion, and vascularization by blocking CXCL12β/CXCR2/4 signal transduction

BackgroundAn impaired trophoblast invasion ability contributes to the development of pre-eclampsia (PE), and can be induced by the altered expression of various microRNAs (miRs). MiR-141 and CXCL12β (C-X-C motif chemokine ligand 12) signaling regulate trophoblast invasion and vascularization capabilities during PE pathogenesis; however, their interactions and underlying mechanisms of action remain unclear. We investigated how miR-141 modulates trophoblast invasion, with a focus on its interaction with CXCL12β signaling. MethodsA PE model was established by using HTR-8/SVneo cells, which were first cultured with 2% O2 for 48 h, and then with 5% O2. The expression of miR-141 in human villous trophoblast HTR-8/SVneo cells was modulated with mimics or an inhibitor, and analyzed by quantitative RT-PCR. CXCL12β levels were determined by ELISA. Cell apoptosis was determined by flow cytometry, and the invasion and vascularization capabilities of trophoblasts were evaluated by Transwell and tube formation assays, respectively. Binding of miR-141 with CXCL12β mRNA was verified by the dual luciferase assay. Protein levels were estimated by western blotting. ResultsMiR-141 expression was significantly induced by hypoxia in HTR-8/SVneo cells. MiR-141 was found to promote apoptosis and inhibit the invasion and vascularization abilities of HTR-8/SVneo cells under conditions of hypoxia. MiR-141 could directly bind with the 3′UTR region of CXCL12β mRNA and inhibit its translation. In addition, we proved that miR-141 could inhibit the invasion and vascularization abilities, and promote the apoptosis of HTR-8/SVneo cells by targeting CXCL12β under hypoxic conditions. Furthermore, we demonstrated that arachidonic acid could reverse the invasion and apoptosis abilities of HTR-8/SVneo cells mediated by CXCL12β during hypoxia. In terms of mechanism, MiR-141 could downregulate MMP2, p62, and LC3B expression, and upregulate ROCK1 and RhoA expression in HTR-8/SVneo cells by targeting the CXCL12β gene during hypoxia. The effects of CXCL12βon HTR-8/SVneo cells could be reversed by arachidonic acid (ARA). ConclusionInduction of miR-141 by hypoxia promotes apoptosis, and inhibits the invasion and vascularization capabilities of HTR-8/SVneo cells by suppressing the CXCL12β and CXCR2/4 signaling pathways.

Nutrient sensor signaling pathways and cellular stress in fetal growth restriction.

Fetal growth restriction is one of the most common obstetrical complications resulting in significant perinatal morbidity and mortality. The most frequent etiology of human singleton fetal growth restriction is placental insufficiency, which occurs secondary to reduced utero-placental perfusion, abnormal placentation, impaired trophoblast invasion and spiral artery remodeling, resulting in altered nutrient and oxygen transport. Two nutrient-sensing proteins involved in placental development and glucose and amino acid transport are mechanistic target of rapamycin (mTOR) and O-linked N-acetylglucosamine transferase (OGT), which are both regulated by availability of oxygen. Impairment in either of these pathways is associated with fetal growth restriction and accompanied by cellular stress in the forms of hypoxia, oxidative and endoplasmic reticulum (ER) stress, metabolic dysfunction and nutrient starvation in the placenta. Recent evidence has emerged regarding the potential impact of nutrient sensors on fetal stress response, which occurs in a sexual dysmorphic manner, indicating a potential element of genetic gender susceptibility to fetal growth restriction. In this mini review, we focus on the known role of mTOR and OGT in placental development, nutrient regulation and response to cellular stress in human fetal growth restriction with supporting evidence from rodent models.