Apoptosis of Extravillous Trophoblast Cells Limits the Trophoblast Invasion in Uterine but not in Tubal Pregnancy During FirstTrimester

Abstract The adequate invasion of extravillous trophoblast cells (EVTs) is indispensable for the implantation of embryos and subsequent remodeling of uterine spiral arteries in early human gestation. Bone morphogenetic protein 2 (BMP2), which is abundantly expressed at the maternal-fetal interface, has been shown to promote the human EVT invasion process (1). Integrin switching (i.e., a switch from α6β4 to αvβ3) plays essential roles in cell-extracellular matrix adhesion and has been reported to influence EVT migration and invasion (2). Moreover, integrin β3 has been found to promote the adhesion, invasion, and migration abilities of embryonic trophoblasts (3). However, whether integrin β3 participates in BMP2 signaling and mediates BMP2-increased-human trophoblast invasion remains unknown. The purpose of our study was to explore the effects of BMP2 on integrin αvβ3 expression and the possible mediation role of integrin β3 in BMP2-regulated human trophoblast invasion. We used immortalized human trophoblast cell line (HTR8/SVneo) and primary human extravillous trophoblast cells (EVTs) isolated from first-trimester villi as study models. RT-qPCR and Western blot assay were respectively utilized to detect the messenger RNA and protein levels of intergrin αv and β3. The function of the target protein was studied by siRNA knockdown, and the trophoblast invasion ability was checked by Matrigel-coated transwell invasion assays. Our results demonstrated that the mRNA and protein levels of integrin β3, rather than integrin αv, were up-regulated after BMP2 treatment in HTR8/SVneo and primary EVT cells. Importantly, siRNA-mediated down-regulation of integrin β3 significantly inhibited basal and BMP2-induced HTR8/SVneo cell invasionas measured by transwell invasion assay. In conclusion, we findings support that BMP2 promotes human trophoblast cell invasion by up-regulating integrin β3 expression, benefiting the in-depth understanding of the pro-invasive effect of BMP2 on human trophoblasts during early pregnancy. Reference: (1) Hong-Jin Zhao et al., Cell Death Dis 2018;9:174. (2) Damsky, C.H. et al, Development 1994; 120, 3657-3666. (3) Dong-Mei He et al., Reproduction 2019;157:423-430.

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MON-025 Activin a Increases Human Trophoblast Invasion by Up-Regulating Integrinb1 Through ALK4

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.398 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Shiqin Zhu ◽

Peter C Leung ◽

Jinlong Ma ◽

Yan Li

Keyword(s):

Cell Invasion ◽

Activin A ◽

Extravillous Trophoblast ◽

Trophoblast Invasion ◽

Type I ◽

Trophoblast Cells ◽

Integrin Β1 ◽

Down Regulation ◽

Human Trophoblast

Abstract Activin A Increases Human Trophoblast Invasion by Up-regulating Integrin β1 Through ALK4 Following implantation, extravillous trophoblast cells (EVTs) derived from trophectoderm invade into the maternal decidua to a certain extent, which is tightly regulated by a variety of factors. Activin A, a member of the TGF-β superfamily, has been shown to stimulate the invasion of human trophoblasts (1). Integrin β1 has been implicated in cancer cell invasion and is consistently expressed in human preimplantation embryos (2). However, whether integrin β1 is integrated in activin A signaling and mediates activin A increased-human trophoblast invasion remain unknown. The objective of our study was to investigate the possible mediation role of integrin β1 in the pro-invasive effect of activin A on trophoblasts and illustrate the underlying molecular mechanisms. Primary and immortalized (HTR8/SVneo) cultures of human trophoblast cells were employed as study models. Real-time qPCR, Western blot, and small interfering RNA (siRNA)-mediated knockdown approaches were used to investigate the molecular determinants of activin A-mediated functions. The integrin β1 protein levels in poorly invasive BeWo,JAR and JEG-3 human choriocarcinoma cells were lower than that in highly invasive HTR8/SVneo cells and primary human EVTs, suggesting the possible essential role of integrin β1 in mediating human trophoblast invasion. The expression levels of integrin β1 were up-regulated in a time-dependent manner after activin A treatment in HTR8/SVneo cells. Importantly, siRNA-mediated down-regulation of integrin β1 significantly attenuated both basal and activin A-induced cell invasion in HTR8/SVneo cells as measured by transwell invasion assay. Interestingly, the TGF-β type I receptors (ALK4/5/7) inhibitor SB431542 abolished activin A-induced activation of SMAD2/SMAD3 as well as activin A-up-regulated integrin 1 expression. Moreover, siRNA-mediated down-regulation of ALK4 or SMAD4 attenuated activin A-up-regulated integrin β1 in both HTR8/SVneo cells and human primary EVT cells. These results reveal that activin A promotes human trophoblast cell invasion by up-regulating integrin β1 expression through ALK4-activated SMAD2/3-SMAD4 signaling pathway. Reference: (1) Bearfield et al., Eur J Endocrinol 2005;152:909–16. (2) Campbell et al., Hum Reprod 1995;10:1571–8.

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Regulation of Epigenetic Modifications in the Placenta During Preeclampsia: PPARγ Influences H3K4me3 and H3K9ac in Extravillous Trophoblast Cells

International Journal of Molecular Sciences ◽

10.3390/ijms222212469 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12469

Author(s):

Sarah Meister ◽

Laura Hahn ◽

Susanne Beyer ◽

Corinna Paul ◽

Sophie Mitter ◽

...

Keyword(s):

Histone Modifications ◽

Histone H3 ◽

Extravillous Trophoblast ◽

Trophoblast Invasion ◽

Peroxisome Proliferator ◽

Trophoblast Cells ◽

Peroxisome Proliferator Activated Receptor ◽

Pparγ Agonist ◽

Villous Trophoblast

The aim of this study was to analyze the expression of peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RxRα), a binding heterodimer playing a pivotal role in the successful trophoblast invasion, in the placental tissue of preeclamptic patients. Furthermore, we aimed to characterize a possible interaction between PPARγ and H3K4me3 (trimethylated lysine 4 of the histone H3), respectively H3K9ac (acetylated lysine 9 of the histone H3), to illuminate the role of histone modifications in a defective trophoblast invasion in preeclampsia (PE). Therefore, the expression of PPARγ and RxRα was analyzed in 26 PE and 25 control placentas by immunohistochemical peroxidase staining, as well as the co-expression with H3K4me3 and H3K9ac by double immunofluorescence staining. Further, the effect of a specific PPARγ-agonist (Ciglitazone) and PPARγ-antagonist (T0070907) on the histone modifications H3K9ac and H3K4me3 was analyzed in vitro. In PE placentas, we found a reduced expression of PPARγ and RxRα and a reduced co-expression with H3K4me3 and H3K9ac in the extravillous trophoblast (EVT). Furthermore, with the PPARγ-antagonist treated human villous trophoblast (HVT) cells and primary isolated EVT cells showed higher levels of the histone modification proteins whereas treatment with the PPARγ-agonist reduced respective histone modifications. Our results show that the stimulation of PPARγ-activity leads to a reduction of H3K4me3 and H3K9ac in trophoblast cells, but paradoxically decreases the nuclear PPARγ expression. As the importance of PPARγ, being involved in a successful trophoblast invasion has already been investigated, our results reveal a pathophysiologic connection between PPARγ and the epigenetic modulation via H3K4me3 and H3K9ac in PE.

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MBL Interferes with Endovascular Trophoblast Invasion in Pre-Eclampsia

Clinical and Developmental Immunology ◽

10.1155/2012/484321 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 14

Author(s):

Chiara Agostinis ◽

Fleur Bossi ◽

Elisa Masat ◽

Oriano Radillo ◽

Maddalena Tonon ◽

...

Keyword(s):

Endothelial Cells ◽

Mannose Binding Lectin ◽

Extravillous Trophoblast ◽

Trophoblast Invasion ◽

Vascular Remodelling ◽

Trophoblast Cells ◽

Mannose Binding ◽

Endovascular Trophoblast ◽

And Migration ◽

Binding Lectin

The spiral arteries undergo physiologic changes during pregnancy, and the failure of this process may lead to a spectrum of pregnancy disorders, including pre-eclampsia. Our recent data indicate that decidual endothelial cells (DECs), covering the inner side of the spiral arteries, acquire the ability to synthesize C1q, which acts as a link between endovascular trophoblast and DECs favouring the process of vascular remodelling. In this study, we have shown that sera obtained from pre-eclamptic patients strongly inhibit the interaction between extravillous trophoblast (EVT) and DECs, preventing endovascular invasion of trophoblast cells. We further demonstrated that mannose-binding lectin (MBL), one of the factor increased in pre-eclamptic patient sera, strongly inhibits the interaction of EVT with C1q interfering with the process of EVT adhesion to and migration through DECs. These data suggest that the increased level of MBL in pre-eclampsia may contribute to the failure of the endovascular invasion of trophoblast cells.

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Angiopoietin 2 stimulates trophoblast invasion via a mechanism associated with JNK signaling

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaab014 ◽

2021 ◽

Author(s):

Huomei Hou ◽

Fen Ning ◽

Joy Yue Zhang ◽

Qinsheng Lu ◽

Min Zhang ◽

...

Keyword(s):

Trophoblast Cell ◽

Extravillous Trophoblast ◽

Trophoblast Invasion ◽

Integrin Signaling ◽

Spiral Artery ◽

Trophoblast Cells ◽

Jnk Signaling ◽

Tie2 Receptor ◽

Artery Remodeling ◽

Angiopoietin 2

Abstract Extravillous trophoblast cell (EVT) invasion is tightly controlled, and its dysregulation can lead to altered spiral artery remodeling and contribute to a number of different pregnancy complications. Angiopoietin-2 (Ang-2) is expressed by trophoblast cells and various cells in the decidua, and trophoblast cells express its receptor, Tie2. Ang-2 has been shown to play roles in tumor progression and metastasis but it is not known if it also regulates EVT invasion. Here we show that both the HTR-8/SVneo cell line and primary isolates of human EVT expressed various integrins and the Tie2 receptor, and Ang-2 stimulated their migration and/or invasion. Ang-2 increased expression of matrix metalloproteinase (MMP)2 and MMP9, altered the cytoskeleton of HTR-8/SVneo cells and also induced phosphorylation of Tie2, JNK and c-Jun. Inhibition of p-JNK (using SP600125) blocked the Ang-2 induced invasion of HTR-8/SVneo cells. In addition, inhibition of Tie2 (pexmetinib) and integrin signaling (RGDS and ATN-161) also blocked Ang-2 induced invasion. In conclusion, we demonstrate that Ang-2 can stimulate EVT invasion via a mechanism associated with activation of both the Tie2 receptor and integrins, which appear to work through different pathways; Tie2 through the JNK/c-JUN pathway and integrins through an as yet unidentified pathway(s). We therefore propose that any alterations in Ang-2 expression in the decidua would lead to an imbalance in pro- and anti-invasive factors, disrupting regulation of EVT invasion and spiral artery remodeling and thereby contribute to the aetiology of several complications of pregnancy.

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The BMP2 Signaling Axis Promotes Invasive Differentiation of Human Trophoblasts

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.607332 ◽

2021 ◽

Vol 9 ◽

Author(s):

Jiali You ◽

Wei Wang ◽

Hsun-Ming Chang ◽

Yuyin Yi ◽

Hongjin Zhao ◽

...

Keyword(s):

Embryo Implantation ◽

First Trimester ◽

Limiting Factors ◽

Extravillous Trophoblast ◽

Trophoblast Invasion ◽

Cell Marker ◽

Trophoblast Cells ◽

Trophoblast Stem Cells ◽

Signaling Axis ◽

First Trimester Of Pregnancy

Embryo implantation and trophoblast invasion are principal limiting factors of pregnancy establishment. Aberrant embryo development or improper trophoblast differentiation and invasion may lead to various unfavorable pregnancy-related outcomes, including early pregnancy loss (EPL). Our clinical data show that the serum BMP2 levels were significantly increased during the first trimester of pregnancy and that the serum and BMP2 expression levels were lower in women with EPL than in women with normal early pregnancies. Moreover, we observed that BMP2 was expressed in oocytes and trophoblast cells of cleaved embryos and blastocysts prior to implantation in both humans and mice. Exogenous BMP2 promoted embryonic development by enhancing blastocyst formation and hatching in mice. LncRNA NR026833.1 was upregulated by BMP2 and promoted SNAIL expression by competitively binding to miR-502-5p. SNAIL induced MMP2 expression and promoted cell invasion in primary extravillous trophoblast cells. BMP2 promotes the invasive differentiation of mouse trophoblast stem cells by downregulating the expression of TS cell marker and upregulating the expression of trophoblast giant cell marker and labyrinthine/spongiotrophoblast marker. Our findings provide significant insights into the regulatory roles of BMP2 in the development of the placenta, which may give us a framework to explore new therapeutic strategies to pregnancy-related complications.

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ANXA4 promotes trophoblast invasion via the PI3K/Akt/eNOS pathway in preeclampsia

AJP Cell Physiology ◽

10.1152/ajpcell.00404.2018 ◽

2019 ◽

Vol 316 (4) ◽

pp. C481-C491 ◽

Cited By ~ 4

Author(s):

Yalan Xu ◽

Lili Sui ◽

Bintao Qiu ◽

Xiuju Yin ◽

Juntao Liu ◽

...

Keyword(s):

Cell Proliferation ◽

Western Blotting ◽

Underlying Mechanism ◽

Trophoblast Invasion ◽

Pathological Examination ◽

Trophoblast Cells ◽

Human Trophoblast ◽

Annexin A4 ◽

Extravillous Cytotrophoblasts ◽

Phosphoinositide 3 Kinase

The inadequate trophoblast invasion is associated with the development of preeclampsia (PE). Considering that annexin A4 (ANXA4) enhances tumor invasion, we aimed to explore the functional role of ANXA4 in trophoblast cells and to examine the underlying mechanism. ANXA4 expression in PE placentas was analyzed using immunohistochemistry and Western blotting. Cell proliferation, invasion, and apoptosis were determined using a MTT assay, Transwell assay, and flow cytometry, respectively. The expression levels of matrix metalloproteinase (MMP)-2, MMP-9, phosphoinositide 3-kinase (PI3K), Akt, phosphorylated (p)-Akt, and phosphorylated endothelial nitric oxide synthase (p-eNOS) were detected by Western blotting. Placentas were prepared for pathological examination using hematoxylin and eosin staining and apoptosis determination using the TUNEL method. Expression of ANXA4, PI3K, p-Akt and p-eNOS was downregulated in human PE placentas and PE placenta-derived extravillous cytotrophoblasts (EVCTs). Furthermore, ANXA4 overexpression promoted cell proliferation and invasion, inhibited cell apoptosis, and upregulated protein expression of PI3K, p-Akt, and p-eNOS in human trophoblast cells HTR-8/SVneo and JEG-3. By contrast, ANXA4 knockdown exerted the opposite effects. Furthermore, inhibition of the PI3K/Akt pathway by LY294002 abrogated the ANXA4 overexpression-mediated effects on trophoblast behavior. Furthermore, eNOS knockdown abrogated the ANXA4 overexpression-induced promotion of cell invasion and MMP2/9 expression. Additionally, in N-nitro-l-arginine methyl ester (l-NAME)-induced PE rats, ANXA4 overexpression alleviated PE progression, accompanied by an increase in expression of PI3K, p-Akt, and p-eNOS in rat placentas. Our findings demonstrate that ANXA4 expression is downregulated in PE. ANXA4 may promote trophoblast invasion via the PI3K/Akt/eNOS pathway.

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Low chorionic villous succinate accumulation associates with recurrent spontaneous abortion risk

Nature Communications ◽

10.1038/s41467-021-23827-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xiao-Hui Wang ◽

Sha Xu ◽

Xiang-Yu Zhou ◽

Rui Zhao ◽

Yan Lin ◽

...

Keyword(s):

Spontaneous Abortion ◽

Embryo Implantation ◽

Underlying Mechanism ◽

Recurrent Spontaneous Abortion ◽

Extravillous Trophoblast ◽

Trophoblast Invasion ◽

Iron Sulfur ◽

Succinate Dehydrogenase Complex ◽

Extravillous Trophoblasts ◽

Control Study

AbstractDysregulated extravillous trophoblast invasion and proliferation are known to increase the risk of recurrent spontaneous abortion (RSA); however, the underlying mechanism remains unclear. Herein, in our retrospective observational case-control study we show that villous samples from RSA patients, compared to healthy controls, display reduced succinate dehydrogenase complex iron sulfur subunit (SDHB) DNA methylation, elevated SDHB expression, and reduced succinate levels, indicating that low succinate levels correlate with RSA. Moreover, we find high succinate levels in early pregnant women are correlated with successful embryo implantation. SDHB promoter methylation recruited MBD1 and excluded c-Fos, inactivating SDHB expression and causing intracellular succinate accumulation which mimicked hypoxia in extravillous trophoblasts cell lines JEG3 and HTR8 via the PHD2-VHL-HIF-1α pathway; however, low succinate levels reversed this effect and increased the risk of abortion in mouse model. This study reveals that abnormal metabolite levels inhibit extravillous trophoblast function and highlights an approach for RSA intervention.

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