Qinyin Deng

Wnt5a inhibited human trophoblast cell line HTR8/SVneo invasion: implications for early placentation and preeclampsia

Abstract Objective: Wnt5a and Wnt signaling play potential roles in human placental and fetal development. The objective of this study is to explore the role of Wnt5a in the invasion of the human trophoblast cell line HTR8/SVneo and the probable mechanism of early placentation and preeclampsia in which Wnt5a is involved. Methods: Human first trimester villous tissues from normal pregnancies and third trimester placentas from pregnancies with or without preeclampsia (PE) were used in the detection of the expression and subcellular location of Wnt5a. The human trophoblast cell line HTR8/SVneo was treated with 0–400 ng/ml recombinant Wnt5a to investigate the role of Wnt5a in human trophoblast invasion. Results: Human first trimester villous is accompanied by the decreased expression of Wnt5a compared with term placenta. Upregulated Wnt5a was detected in PE placenta compared with the normal control. Wnt5a inhibited the migration and invasion of HTR8/SVneo cells with decreased integrin β1, α5 and N-cadherin. Moreover, Wnt5a downregulated β-catenin in HTR8/SVneo cells. Conclusions: These findings strongly suggest that Wnt5a inhibits the invasion of HTR8/SVneo cells. Decreased Wnt5a facilitates early placentation, whereas increased Wnt5a contributes to the pathogenesis of PE with insufficient trophoblast invasion. Aberrant Wnt5a may function by impairing Wnt non-canonical/β-catenin signaling pathway in trophoblasts.

Laminin α4 (LAMA4) expression promotes trophoblast cell invasion, migration, and angiogenesis, and is lowered in preeclamptic placentas.

INTRODUCTION The laminin α4 subunit (LAMA4) has been shown to promote migration, proliferation, and survival of various cell types. This study investigated LAMA4s role in trophoblast cells during placental development. METHODS LAMA4 expression was immunohistochemically assessed in the first trimester and term human placentas. LAMA4 siRNA was applied to silence LAMA4 expression in extravillous explants and HTR8/SVneo cells. Hypoxia-reoxygenation (H/R) conditions were applied to mimic preeclampsia. LAMA4 expression and trophoblast cell invasion, migration, and tube formation (a measure of angiogenesis) were assessed in HTR8/SVneo cells. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 was used to study the mechanism underlying LAMA4 activity. LAMA4 promoter methylation was assessed by bisulfite-sequencing polymerase chain reaction (PCR) or methylation-specific PCR. RESULTS LAMA4 levels in preeclamptic placentas were significantly lower than those in controls. LAMA4 silencing significantly inhibited extravillous explant outgrowth as well as HTR8/SVneo cell invasion and migration. H/R conditions significantly lowered LAMA4 expression. Application of either H/R conditions or LAMA4 silencing both significantly decreased HTR8/SVneo cell invasion, migration, and tube formation, decreased MMP2 and MMP9 expression, and increased TIMP2 expression. SB203580 significantly reduced LAMA4 expression. LAMA4 silencing significantly decreased p-p38, p-c-Jun N-terminal kinase (JNK), and p-extracellular signal-regulated kinase (ERK) expressions; by contrast, H/R conditions induced significant upregulation of p-p38 and p-ERK but decreased p-JNK. LAMA4 promoter methylation was not significantly altered in preeclamptic placentas compared to controls. CONCLUSIONS LAMA4 expression is lowered in preeclamptic placentas and promotes trophoblast cell invasion, migration, and angiogenesis. H/R conditions decrease LAMA4 expression and appear to decouple the positive relationship between LAMA4 expression and p38 and ERK activation.

Expression of Gadd45α in human early placenta and its role in trophoblast invasion

OBJECTIVES Well-controlled trophoblast migration and invasion at the maternal-foetal interface are crucial events for normal placentation and successful pregnancy. Growing evidence has revealed that growth arrest and DNA damage-inducible 45 alpha (Gadd45α) participates in tumour migration and invasion as a tumour suppressor. However, the expression and function of Gadd45α in trophoblasts is unknown. This study aimed to determine the Gadd45α expression and function in the human first trimester placenta and identify the underlying mechanisms. METHODS The expression of Gadd45α in human first trimester placenta was determined using immunohistochemistry. HTR8/SVneo cell line was used to investigate the effects of Gadd45α on proliferation, apoptosis, migration/invasion, matrix metalloproteinase (MMP)2/9 activities, and tissue inhibitor of metalloproteinase (TIMP)1/2 expression using cell proliferation assays, flow cytometric analysis, transwell migration/invasion assays, gelatin gel zymography, and western blotting, respectively. Moreover, a placental villous explant model was employed to verify its functions in placentation. RESULTS Gadd45α was strongly expressed in syncytiotrophoblasts and trophoblast columns of human placental villi, extravillous trophoblast cells and glandular epithelium within the maternal decidua. Gadd45α knockdown significantly promoted migration and invasion of HTR8/SVneo cells, whereas it did not affect cell proliferation or apoptosis. Silencing Gadd45α also enhanced trophoblast outgrowth and migration in placental explants. These effects were related to increased activities of MMP2/9 and the decreased expression of TIMP1/2. DISCUSSION AND CONCLUSION Gadd45α may be involved in human trophoblast migration and invasion and may function as an important negative regulator at the foetal-maternal interface during early pregnancy by directly or indirectly regulating MMP2/9 activities.

N-acetylglucosaminyltransferase V inhibits the invasion of trophoblast cells by attenuating MMP2/9 activity in early human pregnancy

INTRODUCTION The invasion and migration of trophoblast cells are essential steps of normal placentation and successful pregnancy. The process is well-regulated by many factors at the fetal-maternal surface. Inadequate invasion by trophoblast cells may lead to poor perfusion of the placenta or complications such as preeclampsia (PE). Accumulating evidence suggests that N-acetylglucosaminyltransferase V (MGAT5) is correlated with tumor invasion and metastasis. Our objective was to characterize MGAT5 expression and function during placental development. METHODS The expression of MGAT5 in humans in placental tissue from the first trimester was determined by immunohistochemistry. To investigate whether MGAT5 regulates trophoblast invasion and migration, we investigated invasion/migration of the HTR8/SVneo trophoblast cells and used human villous explants. Cell proliferation and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. The activity of matrix metalloproteinase (MMP) 2/9, and the expression of tissue inhibitors of metalloproteinases (TIMPs) 1/2 were determined by gelatin zymography and Western blot, respectively. RESULTS MGAT5 was specifically localized within the cytotrophoblast, the syncytiotrophoblast and the trophoblast columns of human placental villi, decidual cells and some extravillous cells in the maternal decidua. MGAT5 shRNA significantly enhanced the invasion and migration capability of HTR8/SVneo cells, and increased villous explant outgrowth but did not affect proliferation and apoptosis of the trophoblast. The enhanced effect of MGAT5 shRNA on trophoblast cell invasion was associated with increased gelatinolytic activity of MMP2/9 and decreased expression of TIMP1/2. DISCUSSION AND CONCLUSION Our data support a role for MGAT5 in the inhibition of human trophoblast cell invasion and migration during early pregnancy by direct or indirect regulation of MMP2/9 activity.

IL-27 Activates Human Trophoblasts to Express IP-10 and IL-6: Implications in the Immunopathophysiology of Preeclampsia

Purpose. To investigate the effects of IL-27 on human trophoblasts and the underlying regulatory signaling mechanisms in preeclampsia. Methods. The expression of IL-27 and IL-27 receptor (WSX-1) was studied in the placenta or sera from patients with preeclampsia. In vitro, we investigated the effects of IL-27 alone or in combination with inflammatory cytokine tumor necrosis factor (TNF-α) on the proinflammatory activation of human trophoblast cells (HTR-8/SVneo) and the underlying intracellular signaling molecules. Results. The expression of IL-27 and IL-27 receptor α (WSX-1) was significantly elevated in the trophoblastic cells from the placenta of patients with preeclampsia compared with control specimens. In vitro, IL-27 could induce the expression of inflammatory factors IFN-γ-inducible protein 10 (CXCL10/IP-10) and IL-6 in trophoblasts, and a synergistic effect was observed in the combined treatment of IL-27 and TNF-α on the release of IP-10 and IL-6. Furthermore, the production of IP-10 and IL-6 stimulated by IL-27 was differentially regulated by intracellular activation of phosphatidylinositol 3-OH kinase-AKT, p38MAPK, and JAK/STAT pathways. Conclusions. These results provide a new insight into the IL-27-activated immunopathological effects mediated by distinct intracellular signal transduction molecules in preeclampsia.

Oxidative stress-induced Gadd45α inhibits trophoblast invasion and increases sFlt1/sEng secretions via p38 MAPK involving in the pathology of pre-eclampsia

Abstract Background: Pre-eclampsia (PE) is one of the most common pregnancy-related complications. We have previously reported that growth arrest and DNA damage-inducible 45 alpha (Gadd45α) is over-expressed in trophoblasts in pre-eclamptic placentas, with an excessive activation of p38 mitogen-activated protein kinase (MAPK) and increased levels of soluble Fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng) in maternal sera. Now we further investigate how Gadd45α regulates trophoblast functions and anti-angiogenesis factors secretions during placental development in patients with PE. Methods: Human placental villous explants were used to verify the effects of Gadd45α and p38 MAPK in placentation. Then HRT8/SVneo cells exposed to hypoxia/reoxygenation (H/R) were employed as an oxidative stress model to investigate the effects of Gadd45α on invasion and sFlt-1/sEng secretions. Through silencing Gadd45α with lentiviral vector-based short-hairpin RNA and inhibiting p38 MAPK with SB203580, we demonstrated that Gadd45α and its downstream p38 protein played roles in the pathology of pre-eclampsia. Results: Gadd45α was found to have increased expression in H/R-treated villous explants and HTR8/SVneo cells. Gadd45α knockdown or p38 blockage could promote trophoblast outgrowth and migration in H/R-exposed villous explants, and enhance the potentials of trophoblast migration/invasion and network formation in H/R-exposed HTR8/SVneo cells. These functional changes might be related to the increased activities of MMP2/9. Meanwhile, Gadd45α knockdown or p38 inhibition also decreases sFlt-1/sEng secretions via suppressing oxidative stress. Conclusions: Oxidative stress-induced overexpression of Gadd45α might influence the activity of MMPs through activation of p38 MAPK signaling to affect the invasion of trophoblast cells, and increase the secretions of sFlt-1/sEng, which then participate in the pathogenesis of pre-eclampsia.

Inhibition of Wnt Inhibitory Factor 1 Under Hypoxic Condition in Human Umbilical Vein Endothelial Cells Promoted Angiogenesis in Vitro.

Placentation is a complicated process critical for maternal–fetal exchange of nutrients and gases that includes stepwise vasculogenesis and angiogenesis. Wnt inhibitory factor 1 (WIF1) is a secreted Wnt antagonist that acts as a tumor-suppressor gene by antagonizing angiogenesis and proliferation and inducing apoptosis. The purpose of this study was to investigate the function of WIF1 on placental angiogenesis in human umbilical vein endothelial cells (HUVECs) under hypoxic conditions. We found that WIF1 was diversely expressed in placental vascular endothelial cells at different points during gestation and was weaker in the early placenta than in the term placenta. We validated the antiangiogenesis role of WIF1 by inhibiting proliferation, tube formation and migration, and inducing apoptosis of endothelial cells through antagonizing Wnt/β-catenin signaling pathway. We also identified that hypoxic conditions similar to the early placenta inhibited the expression of WIF1 and reversed the antiangiogenesis of WIF1 in HUVECs. In conclusion, our present study supported the hypothesis that WIF1 is crucial as a negative regulator of the functions of endothelial cells in angiogenesis and that hypoxia plays an important role in controlling WIF1 expression and angiogenesis. We also demonstrated that Wnt/β-catenin signaling pathway was activated in correspondence with the suppression of WIF1 in the angiogenesis of endothelial cells under hypoxic conditions.

Expression of DAB2IP in human trophoblast and its role in trophoblast invasion.

Abstract Objective: DAB2IP is a growth inhibitor present in many types of cancer cells and is associated with epigenetic regulations controlling tumor development. The primary objective of this study is to determine whether DAB2IP participates in the invasion and migration of trophoblasts during placental development. Methods: The expressions of DAB2IP in human placentas (10 villi, 18 term placentas and 20 pre-eclampsia placentas) were determined by immunohistochemistry, Western blotting and quantitative RT-PCR. HTR8/SVneo cells were treated with hypoxia–reoxygenation (H/R) to test how DAB2IP expression would affect the invasion and migration of trophoblasts. JEG-3 andHTR8/SVneo cells were treated with 5-aza-2-deoxycytidine (5-aza-dC) to study the role of DAB2IP promoter methylation in trophoblasts. Results: DAB2IP was strongly expressed in human villi and extravillous trophoblasts as well as in HTR8/SVneo cells, but not in pre-eclampsia placentas. DAB2IP expression increased after H/R treatment, but the invasive and migratory abilities of trophoblasts were reduced. DAB2IP expression in JEG-3 cells also increased after treatment with 5-aza-dC. Conclusions: These findings strongly suggest that DAB2IP is an important negative regulator at the maternal–fetal interface during early pregnancy. Excessive oxidative stress can increase DAB2IP expression in trophoblasts. The mechanism of DNA methylation may involve in its function during the development of pathologic pregnancy.

SRC-3 Plays a Critical Role in Human Umbilical Vein Endothelial Cells by Regulating the PI3K/Akt/mTOR Pathway in Preeclampsia:

Preeclampsia (PE) is currently thought to be characterized by oxidative stress which may lead to endothelial dysfunction. The normal function of vascular endothelium is essential to vascular homeostasis. Previous studies have shown that steroid receptor coactivator 3 (SRC-3) interacts with estrogen receptors (ERs) which are involved in the vasoprotective effects of estrogen and is also associated with cell migration, invasion, and inflammation; however, its role in PE remains unclear. The main purpose of this study is to identify the role of SRC-3 in the function of human umbilical vein endothelial cells (HUVECs) during the development of PE. Our study demonstrated that the expression of SRC-3 was significantly decreased in PE placentas compared to normal placentas. Additionally, lentivirus short hairpin RNA against SRC-3 and hypoxia/reoxygenation treatments attenuated migration and tube formation abilities and enhanced HUVEC apoptosis. Furthermore, we detected possible downstream in the PI3K/Akt/mammalian target of rapamycin (mTOR) signal pathway activity, which is involved in SRC-3-mediated HUVEC function. Our data suggest that oxidative stress plays a crucial role in controlling SRC-3 expression, which influences the migration and tube formation abilities of endothelial cells through the PI3K/Akt/mTOR signaling pathways. This action may then result in PE pathogenesis.

The Role of MGAT5 in Human Umbilical Vein Endothelial Cells Possible Relevance to the Pathological Mechanism of Preeclampsia

Preeclampsia (PE) is associated with shallow invasion of the trophoblast and insufficient remodeling of the uterine spiral artery. Glycosylation reactions are catalyzed by glycosyltransferases including N-acetylglucosaminyltransferase V (MGAT5) and accumulating evidence suggests that MGAT5 is correlated with the migration, proliferation, and survival of various cell types. Our previous study confirmed that MGAT5 is a negative regulator of trophoblast migration and invasion via the direct or indirect inhibition of matrix metalloproteinase 2/9 activity. The primary purpose of this study is to investigate the role of MGAT5 in the function of human umbilical vein endothelial cells (HUVECs) during the development of PE. We observed that MGAT5 was specifically localized within the decidual cells and endothelial cells in maternal decidual tissues. The expression of MGAT5 was elevated in PE placentas compared with the normal control placentas. Moreover, the expression of MGAT5 was increased in hypoxia–reoxygenation (H/R)-treated HUVECs. The knockdown of MGAT5 and PD98059 treatment significantly enhanced cell migration in vitro, promoted tube formation capacity, and inhibited apoptosis in H/R-exposed HUVECs. Our data suggest that oxidative stress induces the overexpression of MGAT5 via the regulation of the focal adhesion kinase–extracellular signal-regulated kinase signaling pathway, which, in turn, affects the function of endothelial cells, which then participates in the pathogenesis of PE.Preeclampsia (PE) is associated with shallow invasion of the trophoblast and insufficient remodeling of the uterine spiral artery. Glycosylation reactions are catalyzed by glycosyltransferases including N-acetylglucosaminyltransferase V (MGAT5) and accumulating evidence suggests that MGAT5 is correlated with the migration, proliferation, and survival of various cell types. Our previous study confirmed that MGAT5 is a negative regulator of trophoblast migration and invasion via the direct or indirect inhibition of matrix metalloproteinase 2/9 activity. The primary purpose of this study is to investigate the role of MGAT5 in the function of human umbilical vein endothelial cells (HUVECs) during the development of PE. We observed that MGAT5 was specifically localized within the decidual cells and endothelial cells in maternal decidual tissues. The expression of MGAT5 was elevated in PE placentas compared with the normal control placentas. Moreover, the expression of MGAT5 was increased in hypoxia-reoxygenation (H/R)-treated HUVECs. The knockdown of MGAT5 and PD98059 treatment significantly enhanced cell migration in vitro, promoted tube formation capacity, and inhibited apoptosis in H/R-exposed HUVECs. Our data suggest that oxidative stress induces the overexpression of MGAT5 via the regulation of the focal adhesion kinase-extracellular signalregulated kinase signaling pathway, which, in turn, affects the function of endothelial cells, which then participates in the pathogenesis of PE.