Human Trophoblast Cell Line Research Articles

Assembly of Cytoplasmic Stress Granules in Placentas in Women with Preeclampsia.

Inflammation is a well-recognized factor associated with preeclampsia (PE). Stress granules (SGs) have been shown to play an important role in regulating inflammation and immune responses. However, whether SGs are involved in the pathogenesis of PE has not been studied. Here, we evaluated the expression of SG components in placenta of pregnancies with PE. Placental samples or serum were collected from PE patients (n = 31) or healthy age-matched pregnancy (n = 17). mRNA expressions of SG-associated genes in placenta from PE or normal pregnancies were detected by real-time quantitative PCR, and protein expressions of HuR and G3BP were detected using western blot. Immunofluorescence staining was performed to evaluate SG components expression in placentas or 10% serum treated HTR-8/Svneo cells using antibodies against HuR and G3BP. Our study showed higher levels of elavl1, lsm2, lsm4, and ago1 mRNA expression and SG marker proteins expression in placental homogenates of PE patients. HuR/G3BP-positive SG structure was further observed in placental villi of PE by immunofluorescence assay. Besides, serum from PE patients could induce SG aggregation in human trophoblast cell line HTR-8/Svneo cells, suggesting the involvement of SGs in the development of PE.

Palmitoleate Protects against Zika Virus-Induced Placental Trophoblast Apoptosis.

Zika virus (ZIKV) infection in pregnancy is associated with the development of microcephaly, intrauterine growth restriction, and ocular damage in the fetus. ZIKV infection of the placenta plays a crucial role in the vertical transmission from the maternal circulation to the fetus. Our previous study suggested that ZIKV induces endoplasmic reticulum (ER) stress and apoptosis of placental trophoblasts. Here, we showed that palmitoleate, an omega-7 monounsaturated fatty acid, prevents ZIKV-induced ER stress and apoptosis in placental trophoblasts. Human trophoblast cell lines (JEG-3 and JAR) and normal immortalized trophoblasts (HTR-8) were used. We observed that ZIKV infection of the trophoblasts resulted in apoptosis and treatment of palmitoleate to ZIKV-infected cells significantly prevented apoptosis. However, palmitate (saturated fatty acid) did not offer protection from ZIKV-induced ER stress and apoptosis. We also observed that the Zika viral RNA copies were decreased, and the cell viability improved in ZIKV-infected cells treated with palmitoleate as compared to the infected cells without palmitoleate treatment. Further, palmitoleate was shown to protect against ZIKV-induced upregulation of ER stress markers, C/EBP homologous protein and X-box binding protein-1 splicing in placental trophoblasts. In conclusion, our studies suggest that palmitoleate protects placental trophoblasts against ZIKV-induced ER stress and apoptosis.

Different regulation of IRE1α and eIF2α pathways by oxygen and insulin in ACH-3P trophoblast model.

Endoplasmic reticulum (ER)-stress activates the unfolded protein response (UPR), which plays a (patho)physiological role in the placenta. Oxygen and hyperinsulinemia are major regulators of placental development. Thus, we hypothesized that oxygen, insulin and their interplay modulate ER-stress in early pregnancy. Using the human first-trimester trophoblast cell line ACH-3P, we quantified mRNA and protein of several members of UPR by RT-qPCR and Western blotting, respectively. ER-stress induction using tunicamycin and brefeldin A resulted in increased CHOP (4.6-fold change; P ≤ 0.001), XBP1 expression (1.7- and 1.3-fold change, respectively; P ≤ 0.001 and P < 0.05) and XBP1 splicing (7.9- and 12.8-fold change, respectively; P ≤ 0.001). We subsequently analyzed the effect of oxygen (6.5%, 2.5%), insulin (0.1-10 nM) and their interaction using ANCOVA adjusted for cell passage as co-variate. Although GRP78 protein remained unaffected, low oxygen (2.5% O2) increased IRE1α phosphorylation (+52%; P < 0.05) and XBP1 splicing (1.8-fold change; P ≤ 0.001) after 24 h, while eIF2α protein and CHOP expression were downregulated (-28%; P < 0.05 and -24%; P ≤ 0.001; respectively). eIF2α phosphorylation was also reduced after 48 h by low oxygen (-61%; P < 0.05) but increased in the presence of insulin (+46%; P ≤ 0.01). These changes were not PERK-mediated, since PERK phosphorylation and total protein were not altered. Overall, our results suggest that IRE1α and eIF2α UPR-pathways are differentially regulated by oxygen and insulin in early pregnancy.

Maternal Plasma Lipids During Pregnancy, Insulin-like Growth Factor-1, and Excess Fetal Growth.

Maternal lipids during pregnancy and placental growth factors are associated with excess fetal growth. However, how these factors interact to increase the risk of delivering large-for-gestational-age (LGA) neonates remains unclear. In this study, we investigated the relationship between maternal plasma triglycerides (TGs) and free fatty acids (FFAs) during pregnancy, cord blood insulin-like growth factors (IGF), and LGA. In a cell model, we studied the effect of different FAs on placental IGF-1 secretion. This cohort study included pregnant women with term pregnancy and without diabetes or hypertensive disorders in pregnancy. Maternal fasting plasma TGs and FFAs were measured in the second trimester. Cord blood IGF-1, IGF-2, and IGF binding protein-1 and protein-3 were measured at the time of delivery. A human trophoblast cell line, 3A-sub-E, was used to evaluate the effect of different FFAs on placental IGF-1 secretion. We recruited 598 pregnant women-neonate pairs. Maternal plasma TG (180 mg/dL [152.5-185.5 mg/dL] vs 166 mg/dL [133-206 mg/dL], P = .04) and cord blood IGF-1 concentrations (72.7 ± 23.0 vs 54.1 ± 22.8 ng/mL, P < .001) were higher in the LGA group and were significantly associated with birth weight z score. Maternal plasma free palmitic acid (PA) and stearic acid (SA), but not oleic acid (OA) or linoleic acid (LA), were significantly associated with cord blood IGF-1 concentrations. In 3A-sub-E cells, treatment with PA, SA, and LA, but not OA, induced IGF-1 expression and secretion. Certain FFAs can induce placental IGF-1 secretion, which suggests a potential pathophysiology linking maternal plasma lipids and LGA.

Bone Morphogenetic Protein 2 Increases Human Trophoblast Invasion by Up-Regulating Integrin Beta3

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.

Transcriptional profiling of the response to the trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine revealed activation of the eIF2α/ATF4 integrated stress response in two in vitro placental models.

Trichloroethylene (TCE) is an industrial solvent and widespread environmental contaminant. Although TCE exposure is prevalent, epidemiological studies of TCE exposure associations with adverse birth outcomes are inconclusive. Prior studies show that the TCE metabolite S-(1,2-dichlorovinyl)-l-cysteine (DCVC) exhibits toxicity in a placental cell line. In the current study, genome-wide gene expression and gene set enrichment analyses were used to identify novel genes and pathway alterations in the HTR-8/SVneo human trophoblast cell line and human placental villous explants treated with DCVC at concentrations relevant to human exposures. In the cells, concentration- and time-dependent effects were observed, as evidenced by the magnitude of altered gene expression after treatment with 20 µM DCVC versus 10 µM, and 12-h versus 6-h of treatment. Comparing the two models for the transcriptional response to 12-h 20 µM DCVC treatment, no differentially expressed genes reached significance in villous explants, whereas 301 differentially expressed genes were detected in HTR-8/SVneo cells compared with non-treated controls (FDR < 0.05 + LogFC > 0.35 [FC > 1.3]). GSEA revealed five upregulated enriched pathways in common between explants and cells (FDR < 0.05). Moreover, all 12-h DCVC treatment groups from both models contained upregulated pathways enriched for genes regulated by the ATF4 transcription factor. The overrepresentation of ATF4 regulation of differentially expressed genes indicated activation of the integrated stress response (ISR), a condition triggered by multiple stress stimuli, including the unfolded protein response. DCVC-induced ISR activation was confirmed by elevated eIF2α phosphorylation, ATF4 protein concentrations, and decreased global protein synthesis in HTR-8/SVneo cells. This study identifies a mechanism of DCVC-induced cytotoxicity by revealing the involvement of a specific stress signaling pathway.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00204-021-03011-5.

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.

A crossroad between placental and tumor biology: What have we learnt?

Placenta in certain species including the human has evolved as a highly invasive tumor-like organ invading the uterus aned its vasculature to derive oxygen and nutrients for the fetus and exchange waste products. While several excellent reviews have been written comparing hemochorial placentation with tumors, no comprehensive review is available dealing with mechanistic insights into what makes them different, and what tumor biologists can learn from placental biologists, and vice versa. In this review, we analyze the structure-function relationship of the human placenta, emphasizing the functional need of the spatio-temporally orchestrated trophoblast invasiveness for fetal development and growth, and pathological consequences of aberrant invasiveness for fetal and maternal health. We then analyze similarities and differences between the placenta and invasive tumors in terms of hallmarks of cancer, some key molecules regulating their invasive functions, and how placental cancers (choriocarcinomas) or other cancers become refractory or even addicted to these invasion-restraining molecules. We cite in vitro models of human trophoblast and choriocarcinoma cell lines utilized to study mechanisms in normal placental development as well as those responsible for tumor progression. We discuss the pathobiology of hyper-invasive placentas and show thattrophoblastic neoplasias are a unique and heterogeneous class of tumors. We delve into the questions as to why metastasis from other organs rarely occurs at the placental site and whether pregnancy makes the mother more or less vulnerable to cancer-related morbidity/mortality. We attempt to compare trophoblast stem cells and cancer stem cells. Finally, we leave the readers with some thoughts as foods of future investigations.

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.

Depletion of CTCF disrupts PSG gene expression in the human trophoblast cell line Swan 71.

Pregnancy‐specific glycoproteins (PSGs) are fetal proteins secreted by the placenta during pregnancy. The PSG level in maternal serum is an indicator of risk for pregnancy complications. However, little is known about the molecular mechanisms underlying PSG gene expression. Recently, the importance of epigenetic regulation of placental genes has been emphasized in the study of developmental defects and placental disease. In this study, the role of the CCCTC‐binding factor (CTCF) in regulation of PSG expression was investigated to better understand the epigenetic regulatory mechanisms of the PSG genes. Inhibition of CTCF expression disturbed transcription of several PSG genes: PSG1, PSG2, PSG4, PSG5, PSG8, and PSG9 were upregulated and PSG6 and PSG11 were downregulated. These transcriptional changes were correlated with decreased CTCF binding and changes in histone modification at the PSG promoters. Our data demonstrate that CTCF is a potential mediator in the regulation of PSG gene expression.

Delta-9-tetrahydrocannabinol inhibits invasion of HTR8/SVneo human extravillous trophoblast cells and negatively impacts mitochondrial function

Prenatal cannabis use is a significant problem and poses important health risks for the developing fetus. The molecular mechanisms underlying these changes are not fully elucidated but are thought to be attributed to delta-9-tetrahydrocannabinol (THC), the main bioactive constituent of cannabis. It has been reported that THC may target the mitochondria in several tissue types, including placental tissue and trophoblast cell lines, and alter their function. In the present study, in response to 48-h THC treatment of the human extravillous trophoblast cell line HTR8/SVneo, we demonstrate that cell proliferation and invasion are significantly reduced. We further demonstrate THC-treatment elevated levels of cellular reactive oxygen species and markers of lipid damage. This was accompanied by evidence of increased mitochondrial fission. We also observed increased expression of cellular stress markers, HSP70 and HSP60, following exposure to THC. These effects were coincident with reduced mitochondrial respiratory function and a decrease in mitochondrial membrane potential. Taken together, our results suggest that THC can induce mitochondrial dysfunction and reduce trophoblast invasion; outcomes that have been previously linked to poor placentation. We also demonstrate that these changes in HTR8/SVneo biology may be variably mediated by cannabinoid receptors CB1 and CB2.

Discovery of Zika virus host dependency factors in trophoblasts using CRISPR/Cas9 screening

Emerging mosquito-borne RNA viruses cause massive health complications worldwide. The Zika virus (ZIKV), in particular, has spread dramatically since 2007 and has provoked epidemics in the Americas and the South Pacific. The lack of antiviral therapy and vaccination has focused research on the investigation of ZIKV–host interactions, in order to understand underlying molecular infection mechanisms. We have established an approach for the analysis of ZIKV host dependency factors in a human trophoblast cell line and applied genome-wide CRISPR/Cas9 knockout mutagenesis. The presented method is especially of value for the identification of factors that are essential for placental infection with the potential to serve as targets for antiviral treatment.

30 Viral and bacterial signatures impair trophoblast migration which is reversed by aspirin, aspirin-triggered-lipoxin and hydroxychloroquine

While the etiology of preeclampsia remains elusive, an inflammatory response at the maternal-fetal interface is thought to play a role. Growing evidence suggests a relationship between maternal infection and risk of preeclampsia. Thus, infection may contribute to the pathogenesis by affecting trophoblast function. These cells express Toll-like receptors (TLRs) and Nod-like receptors (NLRs), which mediate infection-induced inflammation. We hypothesize that in addition to inducing inflammation via TLRs and NLRs, infections may limit trophoblast migration leading to a preeclampsia-like phenotype, and this may be reversed by potential therapeutics. A human first trimester trophoblast cell line (Sw.71) was treated with or without bacterial or viral signatures known to activate TLRs and NLRs (Table 1). For some experiments, cells were also treated with the therapeutics aspirin (ASA; 10ug/ml), aspirin-triggered lipoxin (ATL; 100nM), or hydroxychloroquine (HCQ; 1ug/ml). Cell viability was measured using the CellTiter assay. Cell migration was quantified using a two-chamber colorimetric assay. Cell viability was not altered by any infectious triggers with the exception of viral dsRNA which reduced viability by 50.4±4.7% (p<0.05). This was not tested further. Compared to media, viral ssRNA and bacterial CpG ODN significantly reduced trophoblast migration by 48.7±7.2% and 52.5±4.1%, respectively (p<0.05). Bacterial LPS, PGN, flagellin, iE-DAP and MDP did not alter cell migration. Compared to viral ssRNA alone, ASA, ATL and HCQ independently increased trophoblast migration by 1.6±0.3, 2.0±0.3 and 2.0±0.4-fold, respectively (p<0.05). Similarly, compared to CpG ODN alone, ASA, ATL and HCQ independently increased trophoblast migration by 1.6±0.1, 1.5±0.1 and 1.6±0.2-fold, respectively (p<0.05). Our findings suggest that viral ssRNA and bacterial CpG ODN may impair trophoblast migration leading to a preeclampsia-like phenotype. Further research is needed to understand the mechanisms by which ASA, ATL, and HCQ may reverse these effects and improve perinatal outcomes.

Genotoxic stress-activated DNA-PK-p53 cascade and autophagy cooperatively induce ciliogenesis to maintain the DNA damage response

The DNA-PK maintains cell survival when DNA damage occurs. In addition, aberrant activation of the DNA-PK induces centrosome amplification, suggesting additional roles for this kinase. Here, we showed that the DNA-PK-p53 cascade induced primary cilia formation (ciliogenesis), thus maintaining the DNA damage response under genotoxic stress. Treatment with genotoxic drugs (etoposide, neocarzinostatin, hydroxyurea, or cisplatin) led to ciliogenesis in human retina (RPE1), trophoblast (HTR8), lung (A459), and mouse Leydig progenitor (TM3) cell lines. Upon genotoxic stress, several DNA damage signaling were activated, but only the DNA-PK-p53 cascade contributed to ciliogenesis, as pharmacological inhibition or genetic depletion of this pathway decreased genotoxic stress-induced ciliogenesis. Interestingly, in addition to localizing to the nucleus, activated DNA-PK localized to the base of the primary cilium (mother centriole) and daughter centriole. Genotoxic stress also induced autophagy. Inhibition of autophagy initiation or lysosomal degradation or depletion of ATG7 decreased genotoxic stress-induced ciliogenesis. Besides, inhibition of ciliogenesis by depletion of IFT88 or CEP164 attenuated the genotoxic stress-induced DNA damage response. Thus, our study uncovered the interplay among genotoxic stress, the primary cilium, and the DNA damage response.

Effect of Inflammatory Signals on Progesterone Production at the Maternal-Fetal Interface

Introduction: In explant cultures of human fetal membranes (FM) granulocyte-macrophage-colony-stimulating-factor (GM-CSF) mediates the inflammation-induced FM weakening seen in preterm premature rupture of the membranes (pPROM) and exogenous progesterone (P4) inhibits GM-CSF and inflammation-induced FM weakening. Here we report that GM-CSF induces P4 production within the FM which then acts in a paracrine manner to counteract GM-CSF-induced weakening. Methods: FM explants mounted in Transwell inserts were cultured with control media and increasing GM-CSF, RU486 (blocks P4 action), or trilostane (blocks P4 production). P4 production, matrix metalloproteinase-2 (MMP-2) and FM rupture strength were determined. Effects of GM-CSF on P4 production and abundance of the 3ß-hydroxysteroid dehydrogenase (3ßHSD) enzyme in the BeWo human trophoblast cell line were also determined. Results: GM-CSF induced P4 production in both FM explants and BeWo cells in a concentration-dependent manner. GM-CSF also increased 3βHSD protein in BeWo cells. Incubation of FMs with RU486, or trilostane, each caused increased FM weakening. Trilostane also increased MMP-2. Exogenous P4 with trilostane repressed MMP-2 and restored FM strength. Conclusion: GM-CSF induced P4 production by FM and trophoblastic cells suggesting that locally produced P4 is increased by factors that weaken FM. Inhibition of local P4 production or action resulted in FM weakening with concomitant MMP-2 induction suggesting local P4 maintains FM structural integrity. This weakening is reversed by exogenous P4. These data are consistent with a negative-feedback system whereby P4 induced by GM-CSF, the mediator of inflammation-induced FM weakening, counteracts GM-CSF, inhibiting both its production and downstream action with resultant preservation of FM structural integrity.

Role of IL-36 Cytokines in the Regulation of Angiogenesis Potential of Trophoblast Cells.

IL-36 cytokines (the agonists IL-36α, IL-36β, IL-36γ, and the antagonist IL-36Ra) are expressed in the mouse uterus and associated with maternal immune response during pregnancy. Here, we characterize the expression of IL-36 members in human primary trophoblast cells (PTC) and trophoblastic cell lines (HTR-8/SVneo and JEG-3) and upon treatment with bacterial and viral components. Effects of recombinant IL-36 on the migration capacity of trophoblastic cells, their ability to interact with endothelial cells and the induction of angiogenic factors and miRNAs (angiomiRNAs) were examined. Constitutive protein expression of IL-36 (α, β, and γ) and their receptor (IL-36R) was found in all cell types. In PTC, transcripts for all IL-36 subtypes were found, whereas in trophoblastic cell lines only for IL36G and IL36RN. A synthetic analog of double-stranded RNA (poly I:C) and lipopolysaccharide (LPS) induced the expression of IL-36 members in a cell-specific and time-dependent manner. In HTR-8/SVneo cells, IL-36 cytokines increased cell migration and their capacity to interact with endothelial cells. VEGFA and PGF mRNA and protein, as well as the angiomiRNAs miR-146a-3p and miR-141-5p were upregulated as IL-36 response in PTC and HTR-8/SVneo cells. In conclusion, IL-36 cytokines are modulated by microbial components and regulate trophoblast migration and interaction with endothelial cells. Therefore, a fundamental role of these cytokines in the placentation process and in response to infections may be expected.

Pregnancy-Induced High Plasma Levels of Soluble Endoglin in Mice Lead to Preeclampsia Symptoms and Placental Abnormalities

Preeclampsia is a pregnancy-specific disease of high prevalence characterized by the onset of hypertension, among other maternal or fetal signs. Its etiopathogenesis remains elusive, but it is widely accepted that abnormal placentation results in the release of soluble factors that cause the clinical manifestations of the disease. An increased level of soluble endoglin (sEng) in plasma has been proposed to be an early diagnostic and prognostic biomarker of this disease. A pathogenic function of sEng involving hypertension has also been reported in several animal models with high levels of plasma sEng not directly dependent on pregnancy. The aim of this work was to study the functional effect of high plasma levels of sEng in the pathophysiology of preeclampsia in a model of pregnant mice, in which the levels of sEng in the maternal blood during pregnancy replicate the conditions of human preeclampsia. Our results show that wild type pregnant mice carrying human sEng-expressing transgenic fetuses (fWT(hsEng+)) present high plasma levels of sEng with a timing profile similar to that of human preeclampsia. High plasma levels of human sEng (hsEng) are associated with hypertension, proteinuria, fetal growth restriction, and the release of soluble factors to maternal plasma. In addition, fWT(hsEng+) mice also present placental alterations comparable to those caused by the poor remodeling of the spiral arteries characteristic of preeclampsia. In vitro and ex vivo experiments, performed in a human trophoblast cell line and human placental explants, show that sEng interferes with trophoblast invasion and the associated pseudovasculogenesis, a process by which cytotrophoblasts switch from an epithelial to an endothelial phenotype, both events being related to remodeling of the spiral arteries. Our findings provide a novel and useful animal model for future research in preeclampsia and reveal a much more relevant role of sEng in preeclampsia than initially proposed.

Modelling preeclampsia: a comparative analysis of the common human trophoblast cell lines.

ABSTRACTPreeclampsia remains a challenge without an effective therapy. Evidence supports targetability of soluble fms‐like tyrosine kinase‐1 (sFlt‐1) and soluble endoglin (sEng), which are released excessively from the placenta under ischemic and hypoxic stresses. We compared four trophoblast cell lines, BeWo, Jar, Jeg‐3, and HTR‐8/SVneo, in order to identify a suitable model for drug screening. Cultured trophoblasts were exposed to 1% oxygen vs. normoxia for 24‐48 hr; human umbilical vein and aortic endothelial cells were included for comparison. Supernatant sFlt‐1 and sEng concentrations were measured by ELISA, and sFlt‐1 mRNA expression determined by RT‐PCR. Cellular responses to experimental therapeutics were explored. All four trophoblast lines secreted sEng, which did not increase by hypoxia. BeWo, Jar, and Jeg‐3 exhibited significantly enhanced expression of sFlt‐1 i13 and e15a mRNA in response to hypoxia; however, only BeWo released a detectable level of sFlt‐1 protein, which was doubled by hypoxia. In contrast, hypoxia decreased sFlt‐1 mRNA expression and protein release in HTR‐8/SVneo, similarly to endothelial cells. The cellular mechanism involved HIFα. BeWo responded to representative agents similarly to human primary placental tissues in the literature. These data support that the BeWo‐hypoxia model mimics a key pathogenic mechanism of preeclampsia and has potential value for translational drug discovery.

Cyclin G2 upregulation impairs migration, invasion, and network formation through RNF123/Dvl2/JNK signaling in the trophoblast cell line HTR8/SVneo, a possible role in preeclampsia

Disruption of extravillous trophoblast (EVT) migration and invasion is considered to be responsible for pathological placentation in preeclampsia (PE). Cyclin G2 (CCNG2) is an atypical cyclin that inhibits cell cycle progression. However, its biological function and underlying molecular mechanism in PE are poorly understood. In this study, clinical data demonstrated that CCNG2 was significantly upregulated in PE placenta and associated with invasive EVT dysfunction. Additionally, Ccng2 knockout led to an attenuation of PE-like symptoms in the PE mouse model produced via treatment with NG-nitro-L-arginine methyl ester (L-NAME). In vitro, CCNG2 inhibited the migration, invasion, and endothelial-like network formation of human trophoblast cell line HTR8/SVneo. Mechanically, CCNG2 suppressed JNK-dependent Wnt/PCP signaling and its downstream indicators including epithelial-to-mesenchymal transition (EMT) markers and matrix metalloproteinases (MMPs) via promoting the polyubiquitination degradation of dishevelled 2 (Dvl2) protein in HTR8/SVneo cells. We also discovered that the E3 ligase Ring finger protein 123 (RNF123), as a novel CCNG2 target among HTR8/SVneo cells, interacted with Dvl2 and participated in CCNG2-induced polyubiquitination degradation of Dvl2. Moreover, we verified that the treatment of HTR8/SVneo cells with RNF123-specific siRNA improved polyubiquitination-induced degradation of Dvl2 and the activity of Wnt/PCP-JNK signaling mediated by CCNG2. Taken together, our results reveal that the CCNG2/RNF123/Dvl2/JNK axis may be involved in the pathogenesis and progression of PE through trophoblastic cell function modulation, thus probably providing us with new therapeutic strategies for PE treatment.

CCN3 Signaling Is Differently Regulated in Placental Diseases Preeclampsia and Abnormally Invasive Placenta.

ObjectivesAn adequate development of the placenta includes trophoblast differentiation with the processes of trophoblast migration, invasion, cellular senescence and apoptosis which are all crucial to establishing a successful pregnancy. Altered placental development and function lead to placental diseases such as preeclampsia (PE) which is mainly characterized by insufficient trophoblast invasion and abnormally invasive placenta (AIP) disorders (Placenta accreta, increta, or percreta) which are characterized by excessive trophoblast invasion. Both of them will cause maternal and fetal morbidity/mortality. However, the etiology of these diseases is still unclear. Our previous study has shown that the matricellular protein nephroblastoma overexpressed (NOV, CCN3) induces G0/G1 cell cycle arrest, drives trophoblast cells into senescence and activates FAK and Akt kinases resulting in reduced cell proliferation and enhanced migration capability of the human trophoblast cell line SGHPL-5. The present study focuses on whether CCN3 can alter cell cycle-regulated pathways associated with trophoblast senescence and invasion activity in pathological versus gestational age-matched control placentas.MethodsCell cycle regulator proteins were investigated by immunoblotting and qPCR. For localization of CCN3, p16, p21, and Cyclin D1 proteins, co-immunohistochemistry was performed.ResultsIn early-onset PE placentas, CCN3 was expressed at a significantly lower level compared to gestational age-matched controls. The decrease of CCN3 level is associated with an increase in p53, Cyclin E1 and pRb protein expression, whereas the level of cleaved Notch-1, p21, Cyclin D1, pFAK, pAKT, and pmTOR protein decreased. In term AIP placentas, the expression of CCN3 was significantly increased compared to matched term controls. This increase was correlated to an increase in p53, p16, p21, Cyclin D1, cleaved Notch-1, pFAK, pAkt, and pmTOR whereas pRb was significantly decreased. However, in late PE and early AIP placentas, no significant differences in CCN3, p16, p21, Cyclin D1, p53, and cleaved Notch-1 expression were found when matched to appropriate controls.ConclusionsCCN3 expression levels are correlated to markers of cell cycle arrest oppositely in PE and AIP by activating the FAK/AKT pathway in AIP or down-regulating in PE. This may be one mechanism to explain the different pathological features of placental diseases, PE and AIP.