Promotes Trophoblast Migration Research Articles

MiR-95-3p/EPM2A/MMP2 contributes to the pathogenesis of severe preeclampsia through the regulation of trophoblast biological behaviour

ObjectivePreeclampsia (PE) is a maternal multisystem disease with an unclear mechanism. Data showed that MiR-95-3p promoted cell migration, invasion and proliferation, leading to the occurrence and development of many cancers, and placental trophoblasts and tumor cells had similar migration, invasion and proliferation abilities. Meanwhile we found that MiR-95-3p was differentially expressed in PE and normal placenta. Therefore, this article aimed to explore the biological function and mechanism of miR-95-3p in PE. MethodsThe expression of miR-95-3p in PE and normal placental tissue was explored by high-throughput sequencing and qRT–PCR. The effects of miR-95-3p on trophoblast migration, invasion, proliferation, angiogenesis and apoptosis were investigated by Transwell migration and invasion assays, cell viability assay, tube formation assay and flow cytometry in two trophoblast cell lines (HTR-8/SVneo and JAR). The miR-95-3p target gene EPM2A was identified and verified by unique identifier mRNA next-generation sequencing and dual-luciferase reporter gene experiments. Rescue experiments were conducted to investigate whether miR-95-3p regulated EPM2A to participate in trophoblast migration and invasion. Finally, the effects of miR-95-3p and EPM2A on the expression of angiogenic factors and inflammation-related factors were investigated by ELISA. ResultsWe found that miR-95-3p was expressed at low levels in the placental tissue of patients with PE and was negatively correlated with EPM2A expression. In vitro upregulation of miR-95-3p and downregulation of EPM2A promote trophoblast migration, invasion and proliferation. Furthermore, EPM2A was confirmed as a target mRNA of miR-95-3p. Upregulation of EPM2A mitigated miR-95-3p-mediated promotion of trophoblast migration and invasion and vice versa. Finally, both miR-95-3p and EPM2A regulate the expression of trophoblast angiogenesis-related factors and inflammation-related factors. ConclusionOur findings demonstrated that miR-95-3p promoted the migration and invasion of trophoblast cells by targeting EPM2A to inhibit the occurrence and development of PE.

Placenta exosomal miRNA-30d-5p facilitates decidual macrophage polarization by targeting HDAC9.

Pregnancy involves a wide range of adaptations in the maternal body. Maternal immune tolerance toward the foreign fetus is critical for a successful pregnancy. Decidual macrophages are the primary antigen-presenting and phagocytic cells responsible for antigen presentation and apoptotic cell removal. Their phenotype changes dynamically during pregnancy. Placenta-derived exosomes are small vesicles carrying active biological molecules such as microRNAs, proteins, and lipids. The placenta-derived exosomes have been implicated in endothelial cell activation, smooth muscle cell migration, and T-cell apoptosis, but it is unknown whether placenta-derived exosomes would affect the development and functions of decidual macrophages. In this study, we reported that placenta-derived exosomes stimulated macrophage polarization into alternatively activated (M2) macrophages. Mechanistically, miRNA-30d-5p from the placenta-derived exosomes induced macrophage polarization to the M2 phenotype by targeting histone deacetylase 9. Furthermore, the conditioned medium of placenta-derived exosome-treated macrophages promoted trophoblast migration and invasion. By contrast, the conditioned medium impaired the ability of endothelial cell tube formation and migration. Placenta-derived exosome-treated macrophages had no impact on T-cell proliferation. Together, we demonstrated that placenta-derived exosomes polarize macrophages to acquire a decidua-like macrophage phenotype to modulate trophoblast and endothelial cell functions.

Dysfunction of CCR1+ decidual macrophages is a potential risk factor in the occurrence of unexplained recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) puzzles 1-3% of women of childbearing age worldwide. Immunological factors account for more than 60% of cases of unexplained RPL (URPL); however, the underlying mechanism remains unclear. Here, using single-cell sequencing data and functional experiments with clinical samples, we identified a distinct population of CCR1+ decidual macrophages (dMφ) that were preferentially enriched in the decidua from normal early pregnancies but were substantially decreased in patients with URPL. Specific gene signatures endowed CCR1+ dMφ with immunosuppressive and migration-regulatory properties, which were attenuated in URPL. Additionally, CCR1+ dMφ promoted epithelial-to-mesenchymal transition (EMT) to promote trophoblast migration and invasion by activating the ERK1/2 signaling pathway. Decidual stromal cell (DSC)-derived CCL8 was the key regulator of CCR1+ dMφ as CCL8 recruited peripheral CCR1+ monocytes, induced a CCR1+ dMφ-like phenotype, and reinforced the CCR1+ dMφ-exerted modulation of trophoblasts. In patients with URPL, CCL8 expression in DSCs was decreased and trophoblast EMT was defective. Our findings revealed that CCR1+ dMφ play an important role in immune tolerance and trophoblast functions at the maternal-fetal interface. Additionally, decreased quantity and dysregulated function of CCR1+ dMφ result in URPL. In conclusion, we provide insights into the crosstalk between CCR1+ dMφ, trophoblasts, and DSCs at the maternal-fetal interface and macrophage-targeted interventions of URPL.

Altered vitamin D metabolism is involved in the dysregulation of γδT cell function and their crosstalk with trophoblasts in recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is a common disease characterized by immune dysfunction and vitamin D deficiency. This study aimed to investigate vitamin D metabolism and γδT cell phenotypes at the maternal-fetal interface in women with early normal pregnancy (NP) and RPL and to determine the effects of vitamin D on the functions of γδT cells and their crosstalk with trophoblasts. The levels of 25-(OH)VD3 , the expression of vitamin D metabolic enzymes in the villi, and the proportion of γδT cells in the decidua were detected in women with NP and RPL. After treatment with different concentrations of vitamin D, the mRNA expression of the vitamin D receptor (VDR), cytokines, and transcription factors were detected in Vδ2+ γδT cells. In addition, the proliferation, migration, and invasion of HTR-8/SVneo trophoblasts were determined by coculturing them with vitamin D-treated Vδ2+ γδT cells and their supernatants. In women with RPL, the level of 25-(OH)VD3 in the villi was increased; however, that of CYP27B1 (enzyme converting 25-(OH)VD3 into 1,25-(OH)2 VD3 ) was decreased. In addition, the proportion of Vδ2+ γδT cells increased, whereas that of Foxp3+ Vδ2+ γδT cells decreased in the decidua of women with RPL. An in vitro study showed that vitamin D increased the expression of VDR mRNA and Foxp3, but decreased the expression of IFN-γ mRNA, in Vδ2+ γδT cells. Finally, vitamin D-treated Vδ2+ γδT cells promoted trophoblast migration and invasion. Abnormal vitamin D metabolism and γδT cell proportions were present at the maternal-fetal interface in women with RPL. Under normal pregnancy conditions, vitamin D can induce the differentiation of decidual Vδ2+ γδT cells toward an anti-inflammatory phenotype (Treg-like γδT cells) and modulate the crosstalk between Vδ2+ γδT cells and trophoblasts.

Chorionic villus-derived mesenchymal stem cells induce E3 ligase TRIM72 expression and regulate cell behaviors through ubiquitination of p53 in trophoblasts.

Preeclampsia is a significant contributor for maternal or fetal morbidity and mortality, which is characterized by reduced invasion capacity of trophoblasts and is regulated by extracellular matrix (ECM). It is still under investigation whether chorionic villus-derived mesenchymal stem cells (CVMSC) could affect the functionality of trophoblasts. In this study, CVMSC-derived exosomes were isolated; their effect on trophoblasts was investigated based on the CCK8 assay, migration assay, and apoptosis detection. And the underlying mechanism of this effect was investigated using mRNA sequencing, western blot, co-immunoprecipitation, luciferase report assay, and ubiquitination assay. The results show that CVMSC-derived exosomes promote migration and proliferation of trophoblasts, and also reduce cell apoptosis. mRNA sequencing confirmed that after treatment of CVMSC-derived exosomes, Tripartite Motif Containing 72 (TRIM72) expression was upregulated and Tumor Protein P53 (P53) expression was downregulated, both significantly in trophoblasts. Subsequent study confirms that TRM72 can directly interact with P53 and promote P53 ubiquitination and proteasomal degradation, reducing apoptosis rate and elevating proliferation and migration in trophoblasts. Our study confirms that CVMSC-derived exosomes promote trophoblast migration and proliferation by upregulating TRIM72 expression, and subsequently advance P53 ubiquitination and proteasomal degradation.

Exosomal microRNA‑302a promotes trophoblast migration and proliferation, and represses angiogenesis by regulating the expression levels of VEGFA in preeclampsia.

The global morbidity rate of preeclampsia (PE) is 3‑7,and10‑20% of maternal deaths are associated with PE. However, the mechanism of its pathogenesis remains unknown. The aim of the present study was to examine the relationship between microRNA‑302a (miR‑302a) and PE. Firstly, the relative expression levels of miR‑302a in placental tissues from patients with PE and normal controls were analyzed using reverse transcription‑quantitative PCR. miR‑302a expression was upregulated in PE tissues, particularly in severe PE. Subsequently, HTR‑8/SVneo cells were transfected with miR‑302a vectors to overexpress miR‑302a. The overexpression of miR‑302a markedly promoted cell proliferation, colony formation, migration and invasion invitro. Subsequently, the present study examined the function of exosomes secreted by HTR‑8/SVneo cells transfected with miR‑302a vectors. Compared with the negative control vector group, miR‑302a expression was markedly increased in exosomes in the miR‑302a overexpression group. Additionally, exosomes with miR‑302a overexpression had repressed HUVEC invasion and ring formation. The luciferase reporter assay indicated that VEGFA was a direct target of miR‑302a, and miR‑302a expression was negatively correlated with VEGFA expression. In conclusion, the present results demonstrated that upregulation of miR‑302a may promote HTR‑8/SVneo cell proliferation, migration and invasion, and repress angiogenesis by targeting VEGFA, indicating that miR‑302a may be a potential target for the development of PE therapies.

Galectin-14 Promotes Trophoblast Migration and Invasion by Upregulating the Expression of MMP-9 and N-Cadherin.

Galectin-14 is specifically expressed in placental trophoblasts, and its expression is reduced in trophoblasts retrieved from the cervix of women destined to develop early pregnancy loss. However, the roles of galectin-14 in regulating trophoblasts and in the pathogenesis of pregnancy complication have never been investigated. In the current research, we aimed to investigate the roles of galectin-14 in the regulation of trophoblasts. Tissues of the placenta and villi were collected. Primary trophoblasts and human trophoblast cell line HTR-8/SVneo were used. Western blotting and RT-PCR were used to quantify gene expression. The siRNA-mediated galectin-14 knockdown and lentivirus-mediated overexpression were performed to manipulate the gene expression in trophoblasts. Transwell migration and invasion assays were used to evaluate cell migration and invasion capacity. Gelatin zymography was used to determine the gelatinase activity. Galectin-14 was significantly decreased in the villi of early pregnancy loss and the placenta of preeclampsia. Knockdown of galectin-14 in primary trophoblasts inhibited cell migration and invasion, downregulated the expression of matrix metalloproteinase (MMP)-9 and N-cadherin, the activity of MMP-9, and decreased the phosphorylation of Akt. Meanwhile, the overexpression of galectin-14 in HTR-8/SVneo promoted cell migration and invasion, upregulated the expression of MMP-9 and N-cadherin, the activity of MMP-9, and increased the phosphorylation of Akt. Increased Akt phosphorylation promoted cell migration and invasion and upregulated the expression and activity of MMP-9, while decreased Akt phosphorylation inhibited cell migration and invasion and downregulated the expression and activity of MMP-9. Thus, galectin-14 promotes trophoblast migration and invasion by enhancing the expression of MMP-9 and N-cadherin through Akt phosphorylation. The dysregulation of galectin-14 is involved in the pathogenesis of early pregnancy loss and preeclampsia.

Implications for preeclampsia: hypoxia-induced Notch promotes trophoblast migration.

In the first trimester of human pregnancy, low oxygen tension or hypoxia is essential for proper placentation and placenta function. Low oxygen levels and activation of signaling pathways have been implicated as critical mediators in the promotion of trophoblast differentiation, migration, and invasion with inappropriate changes in oxygen tension and aberrant Notch signaling both individually reported as causative to abnormal placentation. Despite crosstalk between hypoxia and Notch signaling in multiple cell types, the relationship between hypoxia and Notch in first trimester trophoblast function is not understood. To determine how a low oxygen environment impacts Notch signaling and cellular motility, we utilized the human first trimester trophoblast cell line, HTR-8/SVneo. Gene set enrichment and ontology analyses identified pathways involved in angiogenesis, Notch and cellular migration as upregulated in HTR-8/SVneo cells exposed to hypoxic conditions. DAPT, a γ-secretase inhibitor that inhibits Notch activation, was used to interrogate the crosstalk between Notch and hypoxia pathways in HTR-8/SVneo cells. We found that hypoxia requires Notch activation to mediate HTR-8/SVneo cell migration, but not invasion. To determine if our in vitro findings were associated with preeclampsia, we analyzed the second trimester chorionic villous sampling (CVS) samples and third trimester placentas. We found a significant decrease in expression of migration and invasion genes in CVS from preeclamptic pregnancies and significantly lower levels of JAG1 in placentas from pregnancies with early-onset preeclampsia with severe features. Our data support a role for Notch in mediating hypoxia-induced trophoblast migration, which may contribute to preeclampsia development.

Chorionic Villus-Derived Mesenchymal Stem Cells Induce E3 Ligase TRIM72 Expression and Regulate Cell Behaviors Through Ubiquitination of p53 in Trophoblasts

Background: It was designed to investigate whether umbilical cord matrix stem cells (CVMSC) could affect the functionality of trophoblasts to prevent preeclampsia. Methods: In this study, CVMSC-derived exosomes were isolated; its effect on trophoblasts was investigated based on the CCK8 assay, migration assay, and apoptosis detection. And the underlying mechanism of this effect was investigated using mRNA sequencing, western blot, co-immunoprecipitation, luciferase report assay, and ubiquitination assay. Findings: The results showed that CVMSC-derived exosomes promoted migration and proliferation of trophoblasts, and also reduced cell apoptosis. mRNA sequencing confirmed that after treatment of CVMSC-derived exosomes, TRIM72 expression was upregulated and P53 expression was downregulated, both significantly in trophoblasts. Subsequent study confirmed that TRM72 could directly interact with P53 and promoted P53 ubiquitination and proteasomal degradation, reducing apoptosis rate and elevating proliferation and migration in trophoblasts. Interpretation: Our study confirmed that CVMSC-derived exosomes promoted trophoblast migration and proliferation by upregulating TRIM72 expression, and subsequently advanced P53 ubiquitination and proteasomal degradation. Funding Statement: This study received no specific grant from public funding agencies, commercial, or not-for-profit sectors. Declaration of Interests: All authors declare there is no competing interest. Ethics Approval Statement: This study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University.

LncRNA MALAT1 affects the migration and invasion of trophoblast cells by regulating FOS expression in early-onset preeclampsia.

Preeclampsia (PE), particularly early-onset PE (ePE), causes maternal and fetal complications and remains a major health problem in modern society. Aberrant uterine spiral artery remodeling leads to ePE through poor placentation. In this study, we investigated the role of the long non-coding RNA (lncRNA) MALAT1 in ePE pathogenesis. Total RNA was extracted from 40 paired placental ePE tissues and control groups. RNA levels were quantified by qRT-PCR and protein expression was determined by western blotand immunohistochemistry (IHC) analysis. The effects of MALAT1 on trophoblast migration and invasion were evaluated in HTR-8/SVneo and JAR cells. FOS was identified as a downstream functional gene of MALAT1 by RNA-seq. RNA binding protein immunoprecipitations (RIPs) were performed to reveal the cellular targets of MALAT1. MALAT1 was poorly expressed in ePE placentas and its silencing (-/-) inhibited trophoblast invasion and migration. MALAT1 -/- also decreased N-cadherin and vimentin expression, but increased E-cadherin expression. RNA-seq analysis and subsequent RIP assays showed that MALAT1 improved FOS through Hu-antigen R (HuR) binding. FOS overexpression similarly enhanced trophoblast migration and invasion. IHC staining showed that E-cadherin was upregulated in placenta tissue from ePE groups, whilst FOS, N-cadherin, and vimentin were downregulated. MALAT1 promotes trophoblast migration and invasion through FOS-induced epithelial mesenchymal transition (EMT). This highlights new roles for MALAT1 in the impairment of spiral artery remodeling in ePE pathogenesis.

4 Sperm, but not seminal plasma, elicit changes in the bovine endometrial transcriptome after natural mating

Studies in mice and pigs have shown that seminal plasma (SP) can modulate the uterine environment, leading to an increase in embryo survival and implantation. However, in cattle, conflicting evidence has resulted from studies in which SP was infused into the uterus. In contrast to mice and pigs, it is questionable whether any SP reaches the bovine uterus during natural mating. However, sperm could act as vehicle for SP proteins that bind to their plasma membrane at ejaculation. Thus, the aim of this study was to analyse the effect of exposure to SP, in the presence or absence of sperm, on the endometrial transcriptome. Beef heifers were oestrus synchronized and mated after being seen in standing oestrus with intact bulls (which ejaculate sperm and SP, n=7) or vasectomized bulls (VX, which only ejaculate SP, n=8) or left unmated (control, n=7). To reduce a potential bull effect, 3 vasectomized and 2 intact Holstein bulls were used. Each heifer was mounted once to one intact or VX bull. Heifers were slaughtered 24h (±6h) after mating, and their reproductive tracts were recovered. Endometrial tissue from the base of the uterine horn ipsilateral to the ovulatory follicle was processed for RNA sequencing. Surprisingly, mating with a VX bull resulted in no differentially expressed genes (DEG) in the endometrium compared with unmated controls. In contrast, mating with intact bulls resulted in 22 DEG (10 down regulated and 12 upregulated) compared with unmated controls and 24 DEG (6 downregulated and 18 upregulated) compared with VX samples. The upregulated genes in intact versus VX samples included several that may have a potential role in implantation: CX3CL1, C4BPA, VCAM1-like, and IL17F. Gene CX3CL1 promotes trophoblast migration in women and has been described as a potential bovine embryokine. In addition, decreased levels of C4BPA have been correlated with implantation failure and unexplained recurrent abortions in women. Further, VCAM1 is involved in bovine conceptus implantation and is downregulated in women with unexplained infertility. In mice, SP regulates inflammation of IL17F in the uterus through the γδT/IL-17 pathway. On the other hand, the immune-related genes PRSS2 and CEBPD were downregulated in intact versus control samples, and BOLA-DQB was downregulated in intact versus VX samples. Gene PRSS2, involved in tissue remodelling, is upregulated in the endometrium of repeated breeder cows. Gene CEBPD, is a transcription factor that enhances IL6 expression, whereas BOLA-DQB is involved in antigen presentation. Interestingly, the gene coding for the α-chain of this protein is also downregulated in high fertility cows. In conclusion, these results do not support a direct effect of SP in regulating pregnancy success in cattle. However, sperm can elicit modest endometrial transcriptomic changes related to implantation and immune system regulation. Further research is needed to elucidate the role of intrinsic sperm proteins, or SP proteins transported by these cells, in the modulation of the uterine environment. This work was supported by EU Horizon 2020 Marie Sklodowska-Curie (No. 792212).

Down-regulation of CCR7 via AKT pathway and GATA2 inactivation suppressed trophoblast migration and invasion in recurrent spontaneous abortion†.

The underlying mechanism of the chemokine-C receptor 7 (CCR7) that leads to aberrant trophoblast migration and invasion in recurrent spontaneous abortion (RSA) remains unknown. CCR7 is considered crucial for migration and invasion and has been associated with the risk of miscarriage. However, the functional role of CCR7 in RSA is not fully understood. Our study found that CCR7 mRNA and protein abundance were significantly decreased in the villous from RSA patients compared with healthy controls. Knockdown of CCR7 caused a significant reduction of migration and invasion in JAR and JEG-3 cells. Meanwhile, CCR7 functioned as a positive upstream factor of the AKT pathway contributing to the expression of GATA2, promoting trophoblast migration, and invasion via MMP2. Notably, a decreased abundance of CCR7 was positively correlated with the phosphorylation of AKT and with an abundance of GATA2 and MMP2 in human villous specimens of RSA compared with the control group. CCL19, a ligand of CCR7, could promote trophoblast migration and invasion by activating the deregulation of the CCR7-mediated pathway in RSA. We are convinced that CCR7 and its downstream factors may be possible mechanisms for the pathogenesis of RSA.

P57KIP2‑mediated inhibition of human trophoblast apoptosis and promotion of invasion in vitro.

Placental hypoxia serves a role in the early stages of normal pregnancy and is involved in the pathophysiology of preeclampsia. Previously, it was suggested that p57kinase inhibitory protein (KIP)2 regulates the cell cycle during embryogenesis and apoptosis. Recent evidence has indicated that p57KIP2 is increased in preeclamptic placentas and absence of p57KIP2 induces preeclampsia‑type symptoms in rats. However, effects of p57KIP2 on apoptosis under hypoxic conditions remain to be elucidated. In the present study, HTR‑8/SVneo trophoblasts were cultured under hypoxic conditions (2% O2). Knockdown using small interfering (si)RNA and overexpression of p57KIP2 were utilized to explore the biological function of p57KIP2 in apoptosis and cell function in vitro. Furthermore, expression of p57KIP2 and apoptosis were evaluated by western blotting, flow cytometry and TUNEL assays, and the response of trophoblasts to hypoxia and the role of p57KIP2 in trophoblast migration and invasion was assessed. The role of p57KIP2 in the JNK signaling pathway in HTR‑8/SVneo trophoblasts was further studies. In vitro, protein expression of p57KIP2 was increased in HTR‑8/SVneo cells exposed to 2% O2. Exogenous p57KIP2 overexpression significantly decreased the expression of pro‑apoptosis proteins, including p53, Bax and cleaved caspase3, under hypoxic conditions for 24 h. In addition, knockdown of p57KIP2 increased the response to apoptosis following hypoxia for 24 h. The present study revealed that overexpression of p57KIP2 decreased the levels of phosphorylated‑JNK. JNK inhibitor treatment combined with the overexpression of p57KIP2 significantly decreased the levels of apoptosis and increased cell invasion and migration. Taken together, p57KIP2 knockdown significantly increased apoptosis in HTR‑8/SVneo cells exposed to 2% O2, whereas overexpression of p57KIP2 had opposite effects, mediated by the JNK/stress activated protein kinase (SAPK) signaling pathway. The results indicated that hypoxia‑induced expression of p57KIP2 promoted trophoblast migration and invasion by mediating the JNK/SAPK signaling pathway, which is crucial during placentation. These results may provide a novel molecular mechanism to understand the involvement of p57KIP2 in the pathogenesis of preeclampsia.

EIF5A1 promotes trophoblast migration and invasion via ARAF-mediated activation of the integrin/ERK signaling pathway

Trophoblast dysfunction is one mechanism implicated in the etiology of recurrent miscarriage (RM). Regulation of trophoblast function, however, is complex and the mechanisms contributing to dysregulation remain to be elucidated. Herein, we found EIF5A1 expression levels to be significantly decreased in cytotrophoblasts in RM villous tissues compared with healthy controls. Using the HTR-8/SVneo cell line as a model system, we found that overexpression of EIF5A1 promotes trophoblast proliferation, migration and invasion in vitro. Knockdown of EIF5A1 or inhibiting its hypusination with N1-guanyl-1,7-diaminoheptane (GC7) suppresses these activities. Similarly, mutating EIF5A1 to EIF5A1K50A to prevent hypusination abolishes its effects on proliferation, migration and invasion. Furthermore, upregulation of EIF5A1 increases the outgrowth of trophoblasts in a villous explant culture model, whereas knockdown has the opposite effect. Suppression of EIF5A1 hypusination also inhibits the outgrowth of trophoblasts in explants. Mechanistically, ARAF mediates the regulation of trophoblast migration and invasion by EIF5A1. Hypusinated EIF5A1 regulates the integrin/ERK signaling pathway via controlling the translation of ARAF. ARAF level is also downregulated in trophoblasts of RM villous tissues and expression of ARAF is positively correlated with EIF5A1. Together, our results suggest that EIF5A1 may be a regulator of trophoblast function at the maternal–fetal interface and low levels of EIF5A1 and ARAF may be associated with RM.

ADAM8 localizes to extravillous trophoblasts within the maternal-fetal interface and potentiates trophoblast cell line migration through a β1 integrin-mediated mechanism.

Does A Disintegrin And Metalloproteinase 8 (ADAM8) control extravillous trophoblast (EVT) differentiation and migration in early human placental development? ADAM8 mRNA preferentially localizes to invasive HLA-G-positive trophoblasts, associates with the acquirement of an EVT phenotype and promotes trophoblast migration through a mechanism requiring β1-integrin. Placental establishment in the first trimester of pregnancy requires the differentiation of progenitor trophoblasts into invasive EVTs that produce a diverse repertoire of proteases that facilitate matrix remodeling and activation of signaling pathways important in controlling cell migration. While multiple ADAM proteases, including ADAM8, are highly expressed by invasive trophoblasts, the role of ADAM8 in controlling EVT-related processes is unknown. First trimester placental villi and decidua (6-12 weeks' gestation), primary trophoblasts and trophoblastic cell lines (JEG3, JAR, Bewo, HTR8/SVNeo) were used to examine ADAM8 expression, localization and function. All experiments were performed on at least three independent occasions (n = 3). Placental villi and primary trophoblasts derived from IRB approved first trimester placental (n = 24) and decidual (n = 4) were used to examine ADAM8 localization and expression by in situ RNAScope hybridization, flow cytometry, quantitative PCR and immunoblot analyses. Primary trophoblasts were differentiated into EVT-like cells by plating on fibronectin and were assessed by immunofluorescence microscopy and immunoblot analysis of keratin-7, vimentin, epidermal growth factor receptor (EGFR), HLA-G and ADAM8. ADAM8 function was examined in primary EVTs and trophoblastic cell lines utilizing siRNA-directed silencing and over-expression strategies. Trophoblast migration was assessed using Transwell chambers, cell-matrix binding was tested using fibronectin-adhesion assays, and ADAM8-β1-integrin interactions were determined by immunofluorescence microscopy, co-immunoprecipitation experiments and function-promoting/inhibiting antibodies. Within first trimester placental tissues, ADAM8 preferentially localized to HLA-G+ trophoblasts residing within anchoring columns and decidua. Functional experiments in primary trophoblasts and trophoblastic cell lines show that ADAM8 promotes trophoblast migration through a mechanism independent of intrinsic protease activity. We show that ADAM8 localizes to peri-nuclear and cell-membrane actin-rich structures during cell-matrix attachment and promotes trophoblast binding to fibronectin matrix. Moreover, ADAM8 potentiates β1-integrin activation and promotes cell migration through a mechanism dependent on β1-integrin function. The primary limitation of this study was the use of in vitro experiments in examining ADAM8 function, as well as the implementation of immortalized trophoblastic cell lines. Histological localization of ADAM8 within placental and decidual tissue sections was limited to mRNA level analysis. Further, patient information corresponding to tissues obtained by elective terminations was not available. The novel non-proteolytic pro-migratory role for ADAM8 in controlling trophoblast migration revealed by this study sheds insight into the importance of ADAM8 in EVT biology and placental development. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC-Discovery Grant) and the Canadian Institutes of Health Research (CIHR-Open Operating Grant). There are no conflicts or competing interests. NA.

P34: Inflammatory signals from the stroma promotes trophoblast migration and invasion

P34: Inflammatory signals from the stroma promotes trophoblast migration and invasion

Vaccinium angustifolium (lowbush blueberry) leaf extract increases extravillous trophoblast cell migration and invasion in vitro.

Perturbations to extravillous trophoblast (EVT) cell migration and invasion are associated with the development of placenta-mediated diseases. Phytochemicals found in the lowbush blueberry plant (Vaccinium angustifolium) have been shown to influence cell migration and invasion in models of tumorigenesis and noncancerous, healthy cells, however never in EVT cells. We hypothesized that the phenolic compounds present in V.angustifolium leaf extract promote trophoblast migration and invasion. Using the HTR-8/SVneo human EVT cell line and Boyden chamber assays, the influence of V.angustifolium leaf extract (0 to 2×104 ng/ml) on trophoblast cell migration (n=4) and invasion (n=4) was determined. Cellular proliferation and viability were assessed using immunoreactivity to Ki67 (n=3) and trypan blue exclusion assays (n=3), respectively. At 20ng/ml, V.angustifolium leaf extract increased HTR-8/SVneo cell migration and invasion (p<.01) and did not affect cell proliferation or viability. Chlorogenic acid was identified as a major phenolic compound of the leaf extract and the most active compound. Evidence from Western blot analysis (n=3) suggests that the effects of the leaf extract and chlorogenic acid on trophoblast migration and invasion are mediated through an adenosine monophosphate-activated protein (AMP) kinase-dependent mechanism. Further investigations examining the potential therapeutic applications of this natural health product extract and its major chemical compounds in the context of placenta-mediated diseases are warranted.

Low-dose aspirin plus low-molecular-weight heparin for the prevention of pre-eclampsia: yes, no or maybe.

Low-dose aspirin plus low-molecular-weight heparin for the prevention of pre-eclampsia: yes, no or maybe.

Modeling defects in trophoblast differentiation using Trisomy 21-affected human pluripotent stem cells

s / Placenta 35 (2014) A1eA112 A10 Methods: miR-218 levels were assessed with real time PCR in tissues collected from healthy and PE patients. Invasion andmigration of EVTs was studied in the HTR-8/SVneo trophoblast cell line and first trimester placenta. The switch to an endothelial adhesion molecule phenotype was measured with VE-Cadherin, CD31 as well as integrin a5b1, a1b1 and aVb3 mRNA levels in first trimester placentas. Finally, an in vitro model of spiral artery remodelling was used to further investigate the role of miR-218 in EVT differentiation, invasion and remodelling of the maternal vasculature. Results:miR-218 was expressed in placenta throughout pregnancy and its level was significantly down-regulated in placenta obtained from patients with PE as compared to gestational age-matched controls. miR-218 mimic promoted, while anti-miR-218 suppressed, the outgrowth of EVTs as well as migration and invasion in vitro. Dissected placental columns treated with miR-218 mimic showed an increase in all of these endothelial markers. Immunohistochemistry analysis revealed accelerated spiral artery remodelling and more invasive EVTs in the miR218 treated samples. Conclusion: Taken together, these novel findings suggest that miR-218 promotes trophoblast migration, invasion and differentiation into endovascular EVTs required for proper spiral artery remodelling. NI.8. MODELING DEFECTS IN TROPHOBLAST DIFFERENTIATION USING TRISOMY 21-AFFECTED HUMAN PLURIPOTENT STEM CELLS Mariko Horii , Younghan Kim , Yingchun Li , Mina Lee , Karen Sabatini , Louise C. Laurent , Ying Liu , Mana M. Parast a Department of Pathology, University of California San Diego, La Jolla, California, USA; Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, Severance Hospital, Institute of Women's Life Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea; Department of Obstetrics and Gynecology ,College of Medicine, Chungnam National University, Daejeon, Republic of Korea; Department of Reproductive Medicine, University of California San Diego, La Jolla, California, USA; Department of Neurosurgery, Medical School, Houston, Texas, USA; Center for Stem Cell and Regenerative Medicine Senator Lloyd and B.A. Bentsen Center for Stroke Research The Brown Foundation Institute of Molecular Medicine The University of Texas Health Science Ce,

MicroRNA-378a-5p targets cyclin G2 to inhibit fusion and differentiation in BeWo cells.

MicroRNAs are expressed abundantly in the placenta throughout pregnancy. We have previously reported that microRNA (miR)-378a-5p promoted trophoblast migration and invasion. To further understand the role of miR-378a-5p during placental development, we investigated whether it may regulate the differentiation of syncytiotrophoblast (STB). Using a choriocarcinoma cell line, BeWo, we found that miR-378a-5p was down-regulated during forskolin-induced STB differentiation. Transfection of a miR-378a-5p mimic into BeWo cells decreased the formation of multinucleated STB, increased E-cadherin, and decreased the expression level of STB marker genes. On the other hand, transfection of anti-miR-378a-5p resulted in an increase in formation of multinucleated STB and expression of STB marker genes, as well as the loss of E-cadherin. Bioinformatic analysis revealed that miR-378a-5p has four potential binding sites at the 3' untranslated region (UTR) of cyclin G2 (CCNG2). Using luciferase reporter assays, we showed that miR-378a-5p decreased the luciferase activity of reporter constructs that contain CCNG2 3' UTR. In addition, miR-378a-5p decreased, whereas anti-miR-378a-5p increased, CCNG2 mRNA levels. Overexpression of CCNG2 increased the expression of syncytin-1 and fusion index and reversed the inhibitory effects of miR-378a-5p. In contrast, silencing of CCNG2 using siRNA increased E-cadherin and decreased syncytin-1 levels. These findings provide initial evidence that CCNG2 promotes STB differentiation and suggest that miR-378a-5p exerts an inhibitory role in STB differentiation, in part, by down-regulating CCNG2 expression, in the BeWo cell model.