Human umbilical cord mesenchymal stem cell-derived extracellular vesicles loaded with TFCP2 activate Wnt/ß-catenin signaling to alleviate preeclampsia.

BACKGROUND: Failures in invasive extravillous trophoblasts (EVTs) migration into the maternal uterus have been noticed in preeclampsia (PE). Human umbilical cord mesenchymal stem cell (hUCMSC)-derived extracellular vesicles (EVs) have been highlighted for the role as a potential therapeutic method in PE. This study intends to investigate the mechanistic basis of hUCMSCs-derived EVs loaded with bioinformatically identified TFCP2 in the activities of EVTs of PE. METHODS: Primary human EVTs were exposed to hypoxic/reoxygenation (H/R) to mimic the environment encountered in PE. The in vivo PE-like phenotypes were induced in mice by reduced uterine perfusion pressure (RUPP) surgery. CCK-8, Transwell and flow cytometry assays were performed to detect proliferation, migration, invasion and apoptosis of H/R-exposed EVTs. More importantly, EVs were extracted from hUCMSCs and transduced with ectopically expressed TFCP2, followed by co-culture with EVTs. RESULTS: TFCP2 was found to be down-regulated in the preeclamptic placental tissues and in H/R-exposed EVTs. hUCMSCs-EVs loaded with TFCP2 activated the Wnt/ß-catenin pathway, thereby promoting the proliferative, migratory, and invasive potential of EVTs. Furthermore, overexpression of TFCP2 alleviated PE-like phenotypes in mice, which was associated with activated Wnt/ß-catenin pathway. CONCLUSION: From our data we conclude that hUCMSCs-EVs overexpressing TFCP2 may be instrumental for the therapeutic targeting and clinical management of PE.

Extracellular vesicle-derived microRNA-18b ameliorates preeclampsia by enhancing trophoblast proliferation and migration via Notch2/TIM3/mTORC1 axis.

Preeclampsia (PE), a common disorder of pregnancy, is characterized by insufficient trophoblast migration and inadequate vascular remodelling, such that promotion of trophoblast proliferation might ameliorate PE. In the current study, we sought to study the underlying mechanism of extracellular vesicle (EV)-derived microRNA-18 (miR-18b) in PE. Human umbilical cord mesenchymal stem cells (HUCMSCs) isolated from placental tissues were verified through osteogenic, adipogenic and chondrogenic differentiation assays. Bioinformatics analyses and dual-luciferase reporter gene assay were adopted to confirm the targeting relationship between miR-18b and Notch2. The functional roles of EV-derived miR-18b and Notch2 in trophoblasts were determined using loss- and gain-of-function experiments, and trophoblast proliferation and migration were assayed using CCK-8 and Transwell tests. In vivo experiments were conducted to determine the effect of EV-derived miR-18b, Notch2 and TIM3/mTORC1 in a rat model of PE, with monitoring of blood pressure and urine proteinuria. TUNEL staining was conducted to observe the cell apoptosis of placental tissues of PE rats. We found down-regulated miR-18b expression, and elevated Notch2, TIM3 and mTORC1 levels in the placental tissues of PE patients compared with normal placenta. miR-18b was delivered to trophoblasts and targeted Notch2 and negatively its expression, whereas Notch2 positively mediated the expression of TIM3/mTORC1. EV-derived miR-18b or Notch2 down-regulation enhanced trophoblast proliferation and migration in vitro and decreased blood pressure and 24 hours urinary protein in PE rats by deactivating the TIM3/mTORC1 axis in vivo. In summary, EV-derived miR-18b promoted trophoblast proliferation and migration via down-regulation of Notch2-dependent TIM3/mTORC1.

Downregulated N-acetylglucosaminyltransferase III is involved in attenuating trophoblast migration and invasion under hypoxia-reoxygenation condition.

OBJECTIVE: Many studies have confirmed that N-acetylglucosaminyltransferase III (GnT-III) is correlated with tumor invasion and metastasis. However, the expression of GnT-III and its role in normal pregnancy and preeclampsia (PE) has not been reached. So the primary objective of this study is to determine GnT-III expression in normal pregnancy and whether its expression is vulnerable to oxidative stress in the trophoblast cells. METHODS: Human first trimester villous tissues from normal pregnancies and third trimester placentas from pregnancies with or without preeclampsia (PE) were used for the detection of GnT-III expression. Human first trimester extravillous trophoblast cell line (HTR8/SVneo) exposed to hypoxia/reoxygenation (H/R) condition was employed as an oxidative stress model in vitro to investigate the expression of GnT-III. RESULTS: GnT-III was strongly expressed in cytotrophoblast (CTBs), syncytiotrophoblast (STBs) and the trophoblast columns (TCs) of human placental villi, and decidual cells in the maternal decidua. The expression of GnT-III was decreased in PE placentas compared with the normal control placentas. In addition, GnT-III was found to have decreased expression in H/R-exposed HTR8/SVneo cells, and the invasive and migratory abilities of HTR8/SVneo cells were attenuated, too. CONCLUSIONS: These findings suggest that GnT-III is an important regulator at the maternal-fetal interface during early pregnancy. Excessive oxidative stress can decrease GnT-III expression in trophoblast and the decreased expression of GnT-III may be involved in the development of preeclampsia.

Expression of N-Acetylglucosaminyltransferase III Promotes Trophoblast Invasion and Migration in Early Human Placenta.

INTRODUCTION: Trophoblast migration and invasion at the maternal-fetal interface are crucial events for normal placentation and successful pregnancy. This progress is well controlled by many placenta-specific factors. Inadequate trophoblast invasion results in poor placenta plantation or even complications such as preeclampsia. It has been shown that N-acetylglucosaminyltransferase III (GnT-III) participates in tumor invasion and metastasis as a suppressor; however, the expression of GnT-III and its role in normal pregnancy is unclear. Our objective was to characterize GnT-III expression and function during placental development and identify the underlying mechanisms. METHODS: The expression of GnT-III in human placental tissue from the first trimester was determined by immunohistochemistry. The HTR8/SVneo cell line was used to investigate the effects of GnT-III on proliferation, apoptosis, migration/invasion, matrix metalloproteinase (MMP) 2/9 activity, and the expression of the tissue inhibitor of metalloproteinase (TIMP) 1/2 using cell 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays, flow cytometric analysis, transwell migration/invasion assays, gelatin zymography, and Western blot, respectively. Moreover, a placental villous explant model was employed to determine its functions in placentation. RESULTS: In the first-trimester placental tissue, GnT-III was localized within the cytotrophoblast, the syncytiotrophoblast and the trophoblast columns of human placental villi, decidual cells, and some extravillous cells in the maternal decidua. GnT-III silencing significantly inhibited HTR8/SVneo cell invasion and migration as well as extravillous explant outgrowth. The application of GnT-III siRNA significantly attenuated MMP2/9 activity and increased TIMP1/2 expression. DISCUSSION AND CONCLUSION: GnT-III is expressed in trophoblasts during normal human pregnancy and is involved in regulating trophoblast function.

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.

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.

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/ß-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/ß-catenin signaling pathway was activated in correspondence with the suppression of WIF1 in the angiogenesis of endothelial cells under hypoxic conditions.

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

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 ß1, α5 and N-cadherin. Moreover, Wnt5a downregulated ß-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/ß-catenin signaling pathway in trophoblasts.

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

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.

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

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.

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.

Protective effects of curcumin against human immunodeficiency virus 1 gp120 V3 loop-induced neuronal injury in rats.

Curcumin improves the learning and memory deficits in rats induced by the gp120 V3 loop. The present study cultured rat hippocampal neurons with 1 nM gp120 V3 loop and 1 µM curcumin for 24 hours. The results showed that curcumin inhibited the gp120 V3 loop-induced mitochondrial membrane potential decrease, reduced the mRNA expression of the pro-apoptotic gene caspase-3, and attenuated hippocampal neuronal injury.