Extravillous Cytotrophoblast Cells Research Articles

Abstract 066: Decoy Peptide Attenuates The Syndrome Of preeclampsia In A Rodent Model

Background: Preeclampsia (preE) is a serious complication of pregnancy manifested by high blood pressure, proteinuria, and edema. In a previous study, we demonstrated that circulating levels of soluble (pro)renin receptor ((P)RR) were elevated at delivery in patients with preE, and that both plasma and placental (pro)renin were elevated in preE patients and in a rat model of preE. Decapeptide based on this handle-region sequence (handle-region peptides or HRP) can block binding of (pro)renin to (P)RR. Objective: The goal of this study is to develop and characterize HRP as an innovative treatment for preE. Methods and Materials: The human extravillous cytotrophoblast (CTB) cell line Sw-71 used in these studies was derived from first trimester chorionic villous tissue. (P)RR (2 nM) was incubated in 1.0 mL medium with the receptor-expressing CTB cells for 0, 6, 12, 18, or 24 hours, followed by renin activity assay to determine the percent binding and activation of (P)RR. Female Sprague-Dawley rats (200-250 g, first-time pregnant with confirmation by vaginal plug, Harlan) are randomly assigned to three groups (n=8 per group): 1) NP: normal pregnant rats; 2) PDS: pregnant animals injected initially with 12.5 mg of DOCA in a depot form intraperitoneally, followed by a weekly injection of 6.5 mg, and whose drinking water was replaced with 0.9% saline; 3) PDS-HRP: rats administered DOCA and saline as for Group 2, and also given HRP (10 mg/kg, i.p.) daily from day 10 through day 20 of pregnancy. Results: (Pro)renin was non-proteolytically activated by binding to (P)RR on the cell membrane of CTB cells, while HRP inhibited binding and attenuated activation of (P)RR. The HRP decoy peptide normalizes blood pressure, proteinuria, and birth numbers in DOCA model of preE. When HRP was administered following the onset of hypertension, the BP, proteinuria, and number of pups were normalized to control normal pregnancy. Conclusions: HRP inhibits the binding of (pro)renin to (P)RR. This study in animal model of preE suggests HRP blockade of (P)RR can attenuate the disease. (P)RR and (pro)renin levels are elevated in preE patients and in their placenta, as well as in a rat model of preE. This data suggests HRP as a potential therapeutic for intervention of RAS signaling in preE.

The Molecular Mechanisms of HLA-G Regulatory Function on Immune Cells during Early Pregnancy.

Human leukocyte antigen-G (HLA-G) is a non-classical human major histocompatibility complex (MHC-I) molecule with the membrane-bound and soluble types. HLA-G is primarily expressed by extravillous cytotrophoblast cells located at the maternal-fetal interface during pregnancy and is essential in establishing immune tolerance. This review provides a comprehensive understanding of the multiple molecular mechanisms by which HLA-G regulates the immune function of NK cells. It highlights that HLA-G binds to microRNA to suppress NK cell cytotoxicity and stimulate the secretion of growth factors to support fetal growth. The interactions between HLA-G and NK cells also activate senescence signaling, promoting spiral artery remodeling and maintaining the balance of maternal-fetal immune responses. In addition, HLA-G can inhibit the function of decidual T cells, dendritic cells, and macrophages. Overall, the interaction between trophoblast cells and immune cells mediated by HLA-G plays a crucial role in understanding immune regulation at the maternal-fetal interface and offers insights into potential treatments for pregnancy-related diseases.

Hyperglycosylated-hCG: Its Role in Trophoblast Invasion and Intrauterine Growth Restriction

Human chorionic gonadotropin (hCG) is produced by the placenta and its roles have been studied for over a century, being the first known pregnancy-related protein. Although its main role is to stimulate the production of progesterone by corpus luteal cells, hCG does not represent just one biologically active molecule, but a group of at least five variants, produced by different cells and each with different functions. The hyperglycosylated variant of hCG (H-hCG) plays a key role in trophoblast invasion, placental development and fetal growth. During trophoblast invasion, H-hCG promotes extravillous cytotrophoblast cells to infiltrate the decidua, and also to colonize and remodel the spiral arteries in to low resistance, larger-diameter vessels. As fetal growth is heavily reliant on nutrient availability, impaired trophoblast invasion and remodeling of the uterine arteries, leads to a defective perfusion of the placenta and fetal growth restriction. Understanding the function of H-hCG in the evolution of the placenta might unveil new ways to manage and treat fetal growth restriction.

TGF-\u03b21 inhibits human trophoblast cell invasion by upregulating kisspeptin expression through ERK1/2 but not SMAD signaling pathway

BackgroundTightly regulation of extravillous cytotrophoblast (EVT) cell invasion is critical for the placentation and establishment of a successful pregnancy. Insufficient EVT cell invasion leads to the development of preeclampsia (PE) which is a leading cause of maternal and perinatal mortality and morbidity. Transforming growth factor-beta1 (TGF-β1) and kisspeptin are expressed in the human placenta and have been shown to inhibit EVT cell invasion. Kisspeptin is a downstream target of TGF-β1 in human breast cancer cells. However, whether kisspeptin is regulated by TGF-β1 and mediates TGF-β1-suppressed human EVT cell invasion remains unclear.MethodsThe effect of TGF-β1 on kisspeptin expression and the underlying mechanisms were explored by a series of in vitro experiments in a human EVT cell line, HTR-8/SVneo, and primary cultures of human EVT cells. Serum levels of TGF-β1 and kisspeptin in patients with or without PE were measured by ELISA.ResultsTGF-β1 upregulates kisspeptin expression in HTR-8/SVneo cells and primary cultures of human EVT cells. Using pharmacological inhibitor and siRNA, we demonstrate that the stimulatory effect of TGF-β1 on kisspeptin expression is mediated via the ALK5 receptor. Treatment with TGF-β1 activates SMAD2/3 canonical pathways as well as ERK1/2 and PI3K/AKT non-canonical pathways. However, only inhibition of ERK1/2 activation attenuates the stimulatory effect of TGF-β1 on kisspeptin expression. In addition, siRNA-mediated knockdown of kisspeptin attenuated TGF-β1-suppressed EVT cell invasion. Moreover, we report that serum levels of TGF-β1 and kisspeptin are significantly upregulated in patients with PE.ConclusionsBy illustrating the potential physiological role of TGF-β1 in the regulation of kisspeptin expression, our results may serve to improve current strategies used to treat placental diseases.

GDF-8 stimulates trophoblast cell invasion by inducing ALK5-SMAD2/3-mediated MMP2 expression.

Matrix metalloproteinases (MMPs) play a pivotal role in the regulation of cell invasion. Placental trophoblast cell invasion is a precisely regulated event. Dysregulation of MMPs has been linked to various placental diseases. Growth differentiation factor-8 (GDF-8), also known as myostatin, is a member of the transforming growth factor-beta (TGF-β) superfamily. GDF-8 and its putative receptors are expressed in human extravillous cytotrophoblast cells (EVTs). Although the pro-invasive effect of GDF-8 in human EVT cells has been recently reported, the underlying molecular mechanism remains largely unknown. In this study, we investigate the effects of GDF-8 on the expression of the two most important MMPs, MMP2 and MMP9, in the HTR-8/SVneo human EVT cell line. Our results show that GDF-8 significantly upregulates the expression of MMP2. The expression of MMP9 is not affected by GDF-8. Using a siRNA-mediated knockdown approach, we reveal that the stimulatory effect of GDF-8 on MMP2 expression is mediated by the ALK5-SMAD2/3 signaling pathway. Additionally, the knockdown of MMP2 attenuates the GDF-8-induced cell invasiveness. These findings deepen our understanding of the biological roles of GDF-8 in the regulation of human trophoblast cell invasion.

CD93 a potential player in cytotrophoblast and endothelial cell migration.

CD93, also known as complement component C1q receptor, is expressed on the surface of different cellular types such as monocytes, neutrophils, platelets, microglia, and endothelial cells, and it plays a pivotal role in cell proliferation, cell migration, and formation of capillary-like structures. These processes are strictly regulated, and many fetal and maternal players are involved during placental development. At present, there are no studies in literature regarding CD93 in placental development, so we investigated CD93 expression in first and third trimester and PE placentas by immunohistochemistry and western blotting analysis. In addition, we performed in vitro experiments under oxidative stress conditions to demonstrate how oxidative stress acts on CD93 protein expression. Our data showed that CD93 was expressed in villous cytotrophoblast cells, in some fetal vessels of first and third trimester and PE placentas and in the extravillous cytotrophoblast of cell columns in the first trimester placentas. Moreover, we detected a significant decrease of CD93 expression in third trimester and PE placentas compared to first trimester placentas, while no differences were detected between third and PE placentas. No differences of CD93 expression were detected in oxidative stress conditions. We suggest that CD93 can guide extravillous cytotrophoblast migration through β1-integrin in uterine spiral arteries during placentation in the first trimester of pregnancy and that the decrease of CD93 expression in third trimester and PE placentas could be linked to the poor extravillous cytotrophoblast cells migration. So, it might be interesting to understand the role of CD93 in the first phases of PE onset.

A re-examination of the origins of placental bed giant cells.

In view of controversy about the source of placental multinuclear giant cells, we have re-examined the literature which clearly shows they are derived from trophoblastic elements that have populated the decidua. Archival material for electron microscopy from 17 to 18 week placentae demonstrates they can be found connected via desmosomes to the outer extravillous cytotrophoblast cells of anchoring columns, thus identifying a primary source. We suggest their formation is a terminal differentiation step occurring at all stages of invasion from the cell column to the myometrium, progressively reducing the invasive population.

Term extravillous cytotrophoblast cells differential interferon response to Zika virus infection

Term extravillous cytotrophoblast cells differential interferon response to Zika virus infection

Kisspeptins and Glucose Homeostasis in Pregnancy: Implications for Gestational Diabetes Mellitus-a Review Article.

Gestational diabetes mellitus (GDM) is becoming an increasingly common complication of pregnancy with the global rise of obesity. The precise pathophysiological mechanisms underpinning GDM are yet to be fully elucidated. Kisspeptin, a peptide encoded by the KISS1 gene, is mainly expressed by placental syncytiotrophoblasts during pregnancy. It is an essential ligand for kisspeptin 1 receptor (KISS1R), which is expressed by both the villous and invasive extravillous cytotrophoblast cells. Circulatory kisspeptins rise dramatically in the second and third trimester of pregnancy coinciding with the period of peak insulin resistance. Kisspeptins stimulate glucose-dependent insulin secretion and decreased plasma levels inversely correlate with markers of insulin resistance. Additionally, kisspeptins play a critical role in the regulation of appetite, energy utilisation and glucose homeostasis. GDM pregnancies have been associated with low circulatory kisspeptins, despite higher placental kisspeptin and KISS1R expression. This review evaluates the role of kisspeptin in insulin secretion, resistance and regulation of appetite as well as its implications in GDM.

Development of a Microfluidic Device for Modeling Human Placenta

The placenta is a multifunctional organ essential for fetal development and survival and plays an important role in hormone production and the exchange of substances such as nutrients, gases, and waste products between the mother and fetus. Research on the mechanism of human placental development is important for elucidating the cause of infertility and intrauterine growth restriction and developing new medicines for them, but little is known about the mechanism. Studying the human placenta has been limited because of large species differences, the limited resources of the human placenta, and also the lack of an in vitro cell culture system that can be a good model of the human placenta. To accelerate the study of the human placenta, we recently established a human trophoblast stem (TS) cell lime from the human placenta [1]. This TS cell line could differentiate into the major placental trophoblast subpopulations, i.e. extravillous cytotrophoblast (EVT), and syncytiotrophoblast (ST). In vivo, EVT cells can invade the endometrium and remodel the maternal blood vessels, and ST cells can produce large quantities of placental hormones and mediate the maternal-fetal gas, nutrient, and waste exchange. The purpose of this study is to develop an in vitro human placental model that resembles the human placenta in vivo, using the TS cell line and microfabrication technology. In particular, we have attempted to fabricate a microfluidic device that recapitulates the function of fetal EVT cells that invade the endometrium and remodel the maternal blood vessels. This device could be an excellent model for studying the development and the function of the human placenta (Fig.1a). A two-layer microfluidic device was designed to imitate the fetal and maternal compartments in the placenta and to recapitulate the function of in vivo EVT cells regarding the invasion and remodeling. The upper layer has a small well(Φ4mm) for culturing EVT cells. The lower layer has a microchannel for the formation of perfusable vascular networks of human umbilical vein endothelial cells (HUVEC), as a cell culture space mimicking the maternal environment. A porous membrane was set between the upper layer and the lower layer, and this co-culture system was fabricated to aim to evaluate the migration and interaction of each cell in both layers. This two-layer microfluidic device was made using Polydimethylsiloxane (PDMS). To make each layer, PDMS was poured into silicon substrate molds that were made by photolithography and then cured. The polyester (PET) porous membrane underwent the silane-coupling-treatment and was inserted between the upper and lower layers. To bond all three components thoroughly, they were treated with oxygen plasma before assembly (Fig.1b). In the upper layer, for culturing EVT cells, TS cells were seeded to the small well and were differentiated into EVT cells by culturing cells in a differentiation medium containing Matrigel. In the lower layer, to form vascular networks of HUVEC, HUVEC suspension and a mixed solution of fibrin gel and thrombin were introduced into a channel. Two channels with multi micro-posts were also made in the lower layer to perfuse the fresh medium into the vascular system. The cells in the device were subjected to immunostaining and analyzed by fluorescence microscopy. Results of fluorescence microscopy showed that most of the TS cells in the upper layer were differentiated into EVT cells, which expressed an EVT cell marker HLA-G (Fig1.c). Furthermore, in the lower layer, HUVEC formed vascular networks with branched luminal structures. The fresh medium was successfully introduced into the networks through channels with micro-posts, and the vascular networks of HUVEC were able to be maintained for a long period of the time in this novel EVT-HUVEC co-cultured system (Fig.1c). In this device, these two types of cells are separated by a porous membrane and we would like to analyze in detail how these cells interact through the membrane in the future. Through the analysis, we hope to reveal the mechanism by which fetal EVT cells remodel the uterine vessels. We believe that this device would be an excellent model to examine the role of EVT cells in the human placenta, and we hope it will be a critical step in constructing a human placenta model.[1] Okae. H, Toh. H, Sato. T, Hiura. H, Takahashi. S, Shirane. K, Kabayama. Y, Suyama. M, Sasaki. H, and Arima. T, "Derivation of Human Trophoblast Stem Cells.", Cell Stem Cell 22, 50, 2018. Figure 1

GDF8 Promotes the Cell Invasiveness in Human Trophoblasts by Upregulating the Expression of Follistatin-Like 3 Through the ALK5-SMAD2/3 Signaling Pathway.

Growth differentiation factor 8 (GDF8) and its antagonist follistatin-like 3 (FSTL3) are expressed in the placenta during early pregnancy. These two factors may have a role to play in the regulation of normal placentation. However, whether GDF8 can regulate the expression of FSTL3 in human trophoblasts remains to be elucidated. In this study, we aimed to investigate the effects of GDF8 on the expression of FSTL3 and the underlying molecular mechanisms using human trophoblasts as a study model. Our results showed that GDF8 significantly upregulates the expression and production of FSTL3, which further promotes cell invasiveness in immortalized extravillous cytotrophoblast cells and primary extravillous cytotrophoblast cells obtained from human first-trimester placentae. Additionally, using an siRNA-mediated knockdown approach, we found that this regulatory effect is most likely mediated by the ALK5-Sma- and Mad-related protein (SMAD)2/3-induced signaling pathway. These findings deepen our understanding of the functional roles of GDF8 and FSTL3 in the regulation of cell invasiveness of trophoblasts.

AT-rich interactive domain 1A protein expression in normal and pathological pregnancies complicated by preeclampsia.

AT-rich interactive domain 1A (ARID1A, as known as BAF250a) is a subunit of human switch/sucrose nonfermentable chromatin remodeling complex with tumour suppressor function. Mutations of Arid1a have been reported in many human cancers and low expression of this protein has been correlated to a poor prognosis outcome in patients affected by some types of cancer. Although there are many studies regarding ARID1A functions in cancer, little is known about its role in regulating cell differentiation and normal tissues homeostasis. Here, we investigate ARID1A expression in normal placental tissues of first and third trimester of gestation and in pathological placental tissues of pregnancy complicated by preeclampsia (PE) and intrauterine growth restriction (IUGR) to evaluate a possible role of this protein in trophoblast differentiation. We found that ARID1A was specifically expressed in villous and extravillous cytotrophoblastic cells in normal placentas whereas syncytiotrophoblast was negative. Interestingly, ARID1A was expressed in both cytotrophoblastic cells and syncytiotrophoblast in placentas affected by PE and PE-IUGR. Moreover, ARID1A was also present in syncitial knots of pathological placentas. The present results indicate that ARID1A is a good marker of poor trophoblast differentiation in these pathologies, because the significant high positive staining in syncytiotrophoblast nuclei may suggest a poor differentiation of this trophoblast layer due to the cytotrophoblast cells fusion with the syncytiotrophoblast overlaying before arresting their cell cycle.

Pre-eclampsia onset and SPARC: A possible involvement in placenta development.

Pre-eclampsia (PE) is a multisystem disorder commonly diagnosed in the latter half of pregnancy and it is a leading cause of intrauterine fetal growth retardation (IUGR). The aim of this study was to investigate the localization and the role of SPARC, secreted protein acidic, and rich in cysteine, in PE and PE-IUGR placentas in comparison with normal placentas. SPARC was mainly expressed in the villous and extravillous cytotrophoblastic cells in first trimester, whereas in PE, PE-IUGR and at term placentas, SPARC immunostaining was visible in both cytotrophoblastic cells and syncytiotrophoblast. SPARC expression significantly decreased in normal placenta from first to third trimester and a further significant reduction was demonstrated in PE and PE-IUGR. The latter downregulation of SPARC depends on hypoxic condition as shown by in vitro models. In conclusion, SPARC can play a pivotal role in PE and PE-IUGR onset and it should be considered as a key molecule for future investigations in such pathologies.

The Carbohydrate Structure of the Hormone hCG, the Autocrine Hyper Glycosylated hCG, and the Extravillous Cytotrophoblast Hyperglycosylated hCG

Objective: Carbohydrate structure has been determined for pregnancy hCG, hydatidiform mole hCG and choriocarcinoma hCG by multiple authors. It has not, however, been discriminated for the three hCG components, the hormone hCG, the autocrine hyperglycosylated hCG or for invasive cell hyperglycosylated hCG. This is determined here. Methods: Three methodologies were used to discriminate the three components of hCG. The B152 immunoassay specifically determines the concentration of hyperglycosylated hCG. Matrigel invasion chambers determine invasiveness of first trimester trophoblast Results: B152 immunoassay binds only hyperglycosylated hCG, and identified all hyperglycosylated hCG in pregnancy hCG, hydatidiform mole hCG and choriocarcinoma hCG preparations. Distribution of triantennary sugar structures same as percent invasion of Matrigel membrane, indicating distribution of invasive extravillous cytotrophoblast component. Recombinant hCG is only hormone hCG, contains 100% Type 1 O-linked oligosaccharides. Identifies Type 1 O-linked oligosaccharide hCG as hormone hCG. Discussion: As determined, first trimester trophoblast cells produced 86.3% hormone hCG (biantennary N-linked and Type 1 O-linked oligosaccharides), 3.5% hyperglycosylated hCG (biantennary N-linked and Type 2 O-linked oligosaccharides) and 13.7% invasive extravillous cytotrophoblast hCG (triantennary N-linked and Type 2 O-linked oligosaccharides). Invasive extravillous cytotrophoblast cells produce 50.4% hormone hCG, 6.0% hyperglycosylated hCG and 42% invasive extravillous cytotrophoblast hCG. Hydatidiform mole cells produce 73.2% hormone hCG, 5.4% hyperglycosylated hCG and 23.4% invasive extravillous cytotrophoblast hCG. Choriocarcinoma cells produce 38.6% hormone hCG, 17.6% hyperglycosylated hCG and 42.8% invasive extravillous cytotrophoblast hCG.

Placental bed apoptosis is increased in pregnant women with pre-eclampsia versus normotensive pregnant women

abstractWe hypothesised that apoptosis in the placenta is increased in pregnant women whose pregnancies were complicated by pre-eclampsia as compared to normal pregnant women. Biopsy samples were obtained by punch biopsy from placental beds in 15 pre-eclamptic and 15 normotensive pregnant women during cesarean section. Apoptosis in syncytiotrophoblasts, syncytial cluster, extravillous cytotrophoblast, and decidual and stromal cells were evaluated by caspase-3, bax and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL) immunohistochemical methods. A significant involvement was observed via caspase-3 and TUNEL methods in the syncytiotrophoblasts, syncytial cluster and extravillous cytotrophoblast cells of the pre-eclamptic group versus normotensive group (p < 0.001). Caspase-3 method found significantly increased involvement in the pre-eclamptic group versus normotensive group (p < 0.001). Although bax method found significantly increased involvement in syncytiotrophoblasts in the pre-eclamptic group versus normotensive group (p < 0.001), no significant difference was found between the groups in terms of involvement of other cell groups (p > 0.05). Apoptosis in the placental bed is increased in pre-eclamptic woman.

Low oxygen tension induces Krüppel-Like Factor 6 expression in trophoblast cells

The transcription factor Krüppel-Like Factor 6 (KLF6) has important roles in cell differentiation, angiogenesis, apoptosis, and proliferation. Furthermore, there is evidence that KLF6 is required for proper placental development. While oxygen is a critical mediator of trophoblast differentiation and function, the involvement of oxygen in the regulation of KLF6 expression remains unexplored. In the present study we examined the expression of KLF6 in placental tissue from uncomplicated and preeclamptic pregnancies, the latter often characterized by an inadequately perfused placenta. We also determined the effect of hypoxia and the involvement of Hypoxia-Inducible Factor 1α (HIF-1α) on the expression of KLF6 in cultured trophoblast cells and placental tissues. Results revealed that villous, interstitial and endovascular extravillous cytotrophoblasts from placentas from normal and preeclamptic pregnancies express KLF6. In addition, KLF6 immunoreactivity was higher in the placental bed of preeclamptic pregnancies than in those of uncomplicated pregnancies. We demonstrated that hypoxia induced an early and transient increase in KLF6 protein levels in HTR8/SVneo extravillous cytotrophoblast cells and in placental explants. Reoxygenation returned KLF6 protein to basal levels. Moreover, hypoxia-induced up-regulation of KLF6 expression was dependent on HIF-1α as revealed by siRNA knockdown in HTR8/SVneo cells. These results indicate that KLF6 may mediate some of the effects of hypoxia in placental development. The regulation of KLF6 protein levels by oxygen has significant implications for understanding its putative role in diseases affected by tissue hypoxia.

Frequency of null allele of Human Leukocyte Antigen-G (HLA-G) locus in subjects to recurrent miscarriage

Human leukocyte antigen-G (HLA-G) is a non-classical class I molecule highly expressed by extravillous cytotrophoblast cells. Due to a single base pair deletion, its function can be compensated by other isoforms. Investigating the frequency of null allele in Recurrent Miscarriage (RM) subjects could be useful in understanding the relationship between frequency of this allele and RM in a given population. This study aimed to determine the frequency of HLA-G*0105N null allele and its potential association with down-regulation of HLA-G in subjects with RM. Western blotting was used to assess the level of HLA-G protein expression. For investigating the frequency of HLA-G*0105N null allele in RM subjects, PCR-RFLP method was used. Exon 3 of HLA-G gene was amplified by polymerase chain reaction (PCR). Subsequently, PpuM-1 enzyme was employed to digest the PCR products and fragments were analyzed using gel electrophoresis. Digestion using restriction enzyme showed the presence of heterozygous HLA-G*0105N null allele in 10% of the test population. Western blotting results confirmed the decrease in expression of HLA-G in the placental tissue of subjects with RM compared to subjects who could give normal birth. The frequency of heterozygous HLA-G*0105N null allele was high to some extent in subjects with RM. The mutation rate in subjects suggested that there is a significant association between RM and frequency of mutations in this allele.

MSX2 Induces Trophoblast Invasion in Human Placenta.

Normal implantation depends on appropriate trophoblast growth and invasion. Inadequate trophoblast invasion results in pregnancy-related disorders, such as early miscarriage and pre-eclampsia, which are dangerous to both the mother and fetus. Msh Homeobox 2 (MSX2), a member of the MSX family of homeobox proteins, plays a significant role in the proliferation and differentiation of various cells and tissues, including ectodermal organs, teeth, and chondrocytes. Recently, MSX2 was found to play important roles in the invasion of cancer cells into adjacent tissues via the epithelial-mesenchymal transition (EMT). However, the role of MSX2 in trophoblastic invasion during placental development has yet to be explored. In the present study, we detected MSX2 expression in cytotrophoblast, syncytiotrophoblast, and extravillous cytotrophoblast cells of first or third trimester human placentas via immunohistochemistry analysis. Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2), vimentin, and β-catenin. Conversely, treatment of HTR8/SVneo cells with MSX2-specific siRNAs resulted in decreased protein expression of MMP-2, vimentin, and β-catenin, and reduced invasion levels in a Matrigel invasion test. Notably, however, treatment with the MSX2 overexpression plasmid and the MSX2 siRNAs had no effect on the mRNA expression levels of β-catenin. Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells. Lastly, the protein expression levels of MSX2 were significantly lower in human pre-eclamptic placental villi than in the matched control placentas. Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may be associated with pre-eclampsia.

Activin A Increases Human Trophoblast Invasion by Inducing SNAIL-Mediated MMP2 Up-Regulation Through ALK4

Activin A increases matrix metalloproteinase (MMP) 2 expression and cell invasion in human trophoblasts, but whether the expression of MMP2 is essential for the proinvasive effect of activin A has yet to be determined. Moreover, the identity of the activin receptor-like kinase (ALK; TGF-β type I receptors) and downstream transcription factors (eg, SNAIL and SLUG) mediating the effects of activin on MMP2 expression and trophoblast cell invasion remains unknown. To elucidate the role of MMP2 in activin A-induced human trophoblast cell invasion as well as the involvement of ALK4 and SNAIL. HTR8/SVneo immortalized human extravillous cytotrophoblast (EVT) cells and primary cultures of human first-trimester EVT cells were used as study models. Small interfering RNA (siRNA)-mediated knockdown approaches were used to investigate the molecular determinants of activin A-mediated functions. Levels of mRNA and protein were examined by reverse transcription-quantitative real-time PCR and Western blot, respectively. Cell invasiveness was measured by Matrigel-coated transwell assays. Treatment of HTR8/SVneo cells with activin A increased the production of SNAIL, SLUG, and MMP2 without altering that of MMP9, TIMP1, TIMP2, TWIST, RUNX2, ZEB1, or ZEB2. Similarly, activin A up-regulated the mRNA and protein levels of SNAIL and MMP2 in primary EVT cells. Knockdown of SNAIL attenuated activin A-induced MMP2 up-regulation in HTR8/SVneo and primary EVT cells. In HTR8/SVneo cells, activin A-induced production of SNAIL and MMP2 was abolished by pretreatment with the TGF-β type I receptor (ALK4/5/7) inhibitor SB431542 or siRNA targeting ALK4, SMAD2/3, or common SMAD4. Likewise, knockdown of ALK4 or SMAD4 abolished the stimulatory effects of activin A on SNAIL and MMP2 expression in primary EVT cells. Importantly, activin A-induced HTR8/SVneo and primary EVT cell invasion were attenuated by siRNA-mediated depletion of ALK4 or MMP2. Activin A induces human trophoblast cell invasion by inducing SNAIL-mediated MMP2 expression through ALK4 in a SMAD2/3-SMAD4-dependent manner.

Decreased expression and activation of Stat3 in severe preeclampsia

Severe preeclampsia (PE) is a major cause of maternal mortality and morbidity worldwide. Signal transducer and activator of transcription 3 (Stat3) signal pathway can modulate various fundamental cellular processes. However, whether Stat3 plays a role in the pathogenesis of severe PE is unknown. Therefore, in this study, the expression levels of Stat3 pathway-related genes and proteins, Stat3, pStat3, IL-6, Mcl-1L, Bcl-xL, survivin, MMP-2, and MMP-9, were evaluated by immunohistochemistry (IHC), Western blot analysis and real-time PCR in the severe preeclamptic placentas. Our results showed that Stat3 and pStat3 immunoreactivity were localized in both extravillous cytotrophoblast cells and villous trophoblast cells in the placentas. As compared with normotensive pregnancies, significantly decreased expressions of Stat3 and pStat3 proteins were observed in extravillous cytotrophoblast cells, villous trophoblast cells and entire placentas in patients with severe PE. The expression levels of Stat3, IL-6, survivin and MMP-2 mRNA were significantly decreased in severe preeclamptic placentas, while Mcl-1L, Bcl-xL and MMP-9 mRNA levels were unchanged. IHC results further confirmed that there was a significant decrease of IL-6, survivin and MMP-2 proteins expression in the severe preeclamptic placentas compared with the normal specimens. These findings suggested that decreased expression and activation of the Stat3 may be caused by decreased expression of a Stat3 upstream gene, such as IL-6. Decreased Stat3 expression and activation may play an important role in the pathogenesis of PE through regulation of the transcription of the Stat3 targeted genes survivin and MMP-2 to modulate apoptosis and invasion of placental trophoblastic cells.