Cardiotonic steroids induce anti-angiogenic and anti-proliferative profiles in first trimester extravillous cytotrophoblast cells

ObjectivePreeclampsia (preE), is characterized by abnormal placental invasion and function. Marinobufagenin (MBG), a cardiotonic steroid (CTS), inhibits cytotrophoblast (CTB) cell functions that are critical for normal placental development. This study tests the hypothesis that CTSs induce anti-angiogenic and anti-proliferative effects in CTB cells.MethodsHuman extravillous CTB cells of the line Sw-71, derived from first trimester chorionic villus tissue, were incubated with 0, 0.1, 1, 10, and 100 nM of each of three CTSs (MBG, cinobufatalin (CINO) and ouabain (OUB)) for 48 h. Thereafter, levels of pro-angiogenic (vascular endothelial growth factor (VEGF165), placental growth factor (PlGF)) and anti-angiogenic (soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng)) factors were measured in culture media using ELISA kits. Expression of three receptors (VEGF receptor 1 (VEGFR1), angiogenic angiotensin type 1 receptor (AT₁) and anti-angiogenic angiotensin type 2 receptor (AT₂)) were assayed using immunoblotting (western blots) in cell lysates.ResultssFlt-1 and sEng secretion were increased while VEGF165 and PIGF were decreased in the culture media of CTB cells treated with 1 nM or more of each CTSs (p < 0.01 for each). The AT₂ receptor expression was up-regulated (p < 0.05) in CTB cells treated with 1 nM or more of MBG and CINO and with 100 nM OUB, while AT₁ and VEGFR1 expressions decreased (p < 0.05) with 1 nM or more of MBG and 10 nM or more of CINO and OUB.ConclusionsCTSs influence extravillous CTB cells to induce an anti-angiogenic and anti-proliferative profile.     

Secretion of cytokines by villous cytotrophoblast and extravillous trophoblast in the first trimester of human pregnancy

Cytokines are proposed to play roles in regulation of trophoblast invasion, spiral artery remodeling and immunoregulation during early pregnancy. Secretion of 12 cytokines (interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-13, IFNγ, GM-CSF, MCP-1 and RANTES) by first trimester extravillous trophoblast and villous cytotrophoblast cells was examined using multiplex cytokine array technology. Seven (IL-1β, IL-8, IL-12p70, IL-13, GM-CSF, MCP-1 and RANTES) of the 12 cytokines examined were detectable in the samples studied (n=10 each group). Villous cytotrophoblast production of IL-1β and IL-8 increased with gestational age. Extravillous trophoblast production of IL-8, IL-13 and RANTES increased with gestational age. At 12-14 weeks gestation extravillous trophoblast cells secreted higher levels of IL-8, IL-13 and RANTES than villous cytotrophoblast cells.     

Marinobufagenin impairs first trimester cytotrophoblast differentiation

Preeclampsia is a pregnancy-specific syndrome that is the leading cause of maternal death during pregnancy in the developed world. In preeclampsia, a combination of immunological, genetic and environmental factors can lead to altered cytotrophoblast (CTB) invasion of the uterine wall, a process that is critical for normal placental development and pregnancy maintenance. Marinobufagenin (MBG) is an endogenous inhibitor of the sodium pump Na(+)/K(+) ATPase, and increased plasma MBG is associated with hypertension, chronic renal failure and preeclampsia. In the present study, the effects of MBG on CTB differentiation and invasion were investigated utilizing the first trimester extravillous CTB cell line SGHPL-4. MBG significantly inhibited SGHPL-4 proliferation in a dose-dependent manner. In addition, growth factor-induced migration and invasion were significantly inhibited by MBG treatment. These findings demonstrate that MBG impairs CTB differentiation along the invasive pathway. Elucidating the mechanisms by which MBG impairs placental development may increase our understanding of fetal and maternal pathologies associated with preeclampsia.     

过氧化物酶体增殖物激活受体γ及其配体对早孕期绒毛组织及细胞滋养细胞浸润能力的影响

目的探讨过氧化物酶体增殖物激活受体γ（PPARγ）及其配体对早孕期绒毛组织及细胞滋养细胞浸润能力的影响。方法采用免疫组化方法、免疫荧光细胞化学染色法、蛋白印迹法和RT-PCR技术检测20例孕6～8周（早孕早期组）及20例孕11～12周（早孕晚期组）绒毛组织及细胞滋养细胞中的PPARγ蛋白及其mRNA的表达;并检测不同浓度PPARγ激动配体——15-脱氧-前列腺素J2（15-d-PGJ2）和曲格列酮,以及不同浓度拮抗配体——双酚丙烷二环氧甘油醚（BADGE）对原代无血清培养的细胞滋养细胞浸润能力的影响。结果（1）PPARγ／蛋白在早孕早期组和早孕晚期组绒毛组织中均有表达,主要定位在细胞滋养细胞核中,合体滋养细胞及绒毛间质细胞中无表达。（2）早孕早期组绒毛组织和培养的细胞滋养细胞中,PPARγ／蛋白表达水平分别为1．35±0．08、1．13±0．11,PPARγ／mRNA表达水平分别为36．0±5．1、13．4±3．1;早孕晚期组绒毛组织和培养的细胞滋养细胞中,PPARγ／蛋白表达水平分别为1．17±0．03、0．86±0．05,PPARγ mRNA表达水平分别为23．3±5．5、6．1±1．3,早孕晚期组PPARγ蛋白及其mRNA表达水平明显低于早孕早期组,两组分别比较,差异均有统计学意义（P〈0．05）。（3）PPARγ／激动配体15-d-PGJ2和曲格列酮均有抑制细胞滋养细胞的浸润的作用。15-d-PGJ2浓度为1、10μmol／L,曲格列酮浓度为10μmol／L时,早孕早期组细胞滋养细胞浸润指数分别为0．57±0．03、0．43±0．02、0．50±0．06,早孕晚期组分别为0．69±0．02、0．59±0．03、0．66±0．05,两组分别比较,差异均有统计学意义（P〈0．05）。（4）PPARγ拮抗配体BADGE浓度为20、50μmol／L时,早孕早期组细胞滋养细胞浸润指数分别为1．23±0．07和1．58±0．04;早孕晚期组分别为1．05±0．03和1．38±0．08,两组分别比较,差异均有统计学意义（P〈0．05）。结论PPARγ／在调节滋养细胞浸润过程中起重要作用;在早孕期胎盘绒毛组织,PPARγ／激动配体可抑制滋养细胞浸润;PPARγ／拮抗配体可促进滋养细胞浸润,且能部分逆转激动配体的作用。     

Apoptosis of extravillous trophoblast cells limits the trophoblast invasion in uterine but not in tubal pregnancy during first trimester

During the first trimester of pregnancy extravillous trophoblast cells (EVT) invade the maternal decidua. Invasion normally is reduced from the second trimester onwards and stops in the inner third of the myometrium. By contrast, in extrauterine tubal pregnancy, trophoblast invasion may even penetrate the tubal wall, which ultimately leads to the rupture of the fallopian tube. Induction of apoptosis of EVT cells, by maternal immune competent cells, may be an important mechanism to limit EVT invasion in uterine pregnancy. Tissue specimens from first and second trimester uterine pregnancy and first trimester tubal pregnancy were analyzed for apoptosis by TUNEL- and M30-staining. By immunohistochemical double labelling, maternal leukocyte subtypes were co-localized to apoptotic cells and in this context, the number of CD56(+)NK cells was analyzed. Our data show that apoptosis is confined to the decidua basalis. Most apoptotic cells are single cytokeratin-positive epithelial cells residing in the stromal compartment. Consequently these cells can only be EVT cells. Maternal leukocytes are not apoptotic. They are located in close contact to apoptotic cells. The number of apoptotic cells in the second trimester (1.8+/-0.7 per cent) is reduced compared to first trimester (5.6+/-0.7 per cent) of uterine pregnancy. In parallel, the number of NK cells declines from first (24.4+/-2.9) to second (12.4+/-1.8) trimester. Furthermore, apoptosis is significantly reduced in ectopic (0.9+/-0.3 per cent) compared to eutopic first trimester pregnancies. Consequently, we suggest that in first trimester uterine pregnancy, induction of EVT cell apoptosis by the maternal immune system is one mechanism to limit EVT invasion. During the second trimester, in parallel to declining numbers of NK cells, the mechanism changes. However, in tubal pregnancy due to differing immunological microenvironments at the ectopic implantation site, apoptosis induction fails, which deleteriously may result in uncontrolled invasion and penetration of the tubal wall.     

Expression and regulation of vascular endothelial growth factor in a first trimester trophoblast cell line

Embryo implantation and development are critically dependent upon the spatial and temporal regulation of angiogenesis and localized vascular permeability. A key mediator of these effects is the endothelial cell mitogen vascular endothelial growth factor (VEGF). VEGF has been shown to promote endometrial vascular permeability, fetal vasculogenesis and placental, fetal and maternal angiogenesis. However, the mechanism through which this regulation occurs in the placenta is poorly understood. This study was conducted to determine if the pro-angiogenic cytokines, TNF-alpha and TGF-beta1, affect VEGF expression in human first trimester trophoblasts. Culture of a first trimester trophoblast cell line (HTR-8/SVneo), in the presence of either TNF-alpha or TGF-beta1, resulted in the expression of significant levels of VEGF in culture. The trophoblast cell line also showed a time-dependent and a dose-dependent increase in VEGF mRNA levels when cultured in the presence of either TNF-alpha or TGF-beta1. These results suggest that both TNF-alpha and TGF-beta1 may regulate the production of VEGF in early gestational trophoblasts and may therefore serve to modulate placental vascular permeability and angiogenesis that are necessary for embryo implantation and placentation.     

Stimulation of hCG protein and mRNA in first trimester villous cytotrophoblast cells in vitro by glycodelin A

AIM: Human chorionic gonadotropin (hCG) is produced by fetal trophoblast cells and secreted into maternal circulation mainly in the first trimester of pregnancy. Another glycoprotein, glycodelin A, is one of the main products of the maternal decidua during this period. The purpose of this study was to investigate the effect of glycodelin A on hCG release by isolated cytotrophoblast cells in vitro. METHODS: Cytotrophoblast cells were prepared from human first trimester placenta and incubated with varying concentrations of glycodelin A. Supernatants were assayed for hCG protein concentrations, and quantification of beta hCG mRNA was carried out by RT-PCR. Expression of hCG was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry. RESULTS: Glycodelin A induces a dose-dependent increase of hCG production. An increase of hCG expression was measured at 100 and 200 microg/mL glycodelin-A treatment in trophoblast cell culture by TaqMan assay on mRNA level. We found a moderate staining of hCG in control trophoblast cells, whereas a strong hCG staining was seen in glycodelin A-treated trophoblast cells. CONCLUSIONS: HCG is a marker for the differentiation process of trophoblast cells. Our results suggest that glycodelin A secreted by the decidualized endometrium is involved in the regulation of hormones produced by the trophoblast.     

Human endogenous retrovirus K (HERV-K) is expressed in villous and extravillous cytotrophoblast cells of the human placenta

Human endogenous retroviruses (HERVs) have been shown to be important in physiological and pathophysiological processes in humans. Several HERVs have been found to be expressed in the placenta-a tissue with special immunomodulatory functions that is responsible for nutrition of the embryo and the ability of the semiallogenic trophoblast to invade. The envelope proteins of HERV-W (also known as syncytin 1) and HERV-FRD (syncytin 2) were shown to be involved in cell fusion leading to the generation of the syncytiotrophoblast. Syncytin 2 was further shown to have immunosuppressive properties. Herein we analyse the expression of another HERV, HERV-K, which is characterised by open reading frames for all viral genes. Using immunohistochemistry and Western blot analysis, expression of the transmembrane envelope (TM) protein of HERV-K was studied in normal placental and decidual tissues obtained at different gestational ages. The TM protein was expressed exclusively in villous (VT) and extravillous cytotrophoblast (EVT) cells, but not in the syncytiotrophoblast or other cells. The expression of the TM protein of HERV-K in EVT cells was confirmed by Western blot analysis of isolated c-erbB2-expressing cytotrophoblast cells. Thus, this is the first report showing expression of the TM protein of HERV-K in normal human placental tissue with an exclusive expression in cytotrophoblast cells, suggesting a potential involvement of HERV-K in placentogenesis and pregnancy. Since retroviral TM proteins including the TM protein of HERV-K have immunosuppressive properties, expression of the TM protein of HERV-K may contribute to immune protection of the fetus.     

Detection of class I MHC mRNA in subpopulations of first trimester cytotrophoblast cells by in situ hybridization

Class I MHC mRNA has been identified in both villous and extravillous cytotrophoblast cells in first trimester placentas by in situ hybridization. In this report, we expand those observations to additional morphologically and anatomically distinct subpopulations of trophoblast cells in early placentas using the same experimental approach. In the transition zone of first trimester placental villi, where cytotrophoblast cells are proliferating to form new villi or to migrate into adjacent tissue, both cytotrophoblast cells beneath the uninterrupted syncytium and the proliferating cytotrophoblast cells contained class I mRNA whereas a layer of cytotrophoblast cells proximal to the villus core did not contain class I mRNA. In the placental bed, migrating cytotrophoblast cells contained high levels of class I mRNA as determined by the intensity of staining. In contrast, multinucleated giant trophoblast cells contained little specific message. Alterations in levels of class I mRNA seem therefore to be associated with trophoblast proliferation, migration and differentiation.     

Villous culture of first trimester human placenta--model to study extravillous trophoblast (EVT) differentiation.

During implantation and subsequent placentation the human extravillous trophoblast (EVT) cells invade the endometrium and maternal vasculature within the uterus. The origin of the EVT and signals triggering its differentiation, migration and invasion are poorly understood. First and second trimester human chorionic villi explants were used as a source of EVT and a variety of substrates which resemble extracellular matrix (ECM) in vivo have been tested to induce EVT differentiation and migration. The obtained results demonstrate that villous explants from both 5-7 and 8-10 weeks of gestation give rise to EVT cells in vitro if maintained on the surface of Matrigel or decidual extract supplemented collagen gel. Fetal calf serum (FCS) supplemented media was essential for EVT differentiation and villous trophoblast viability. Immunostaining of both EVT cells and cells from the cytotrophoblastic column with monoclonal antibody Ki67 (cell proliferation marker) indicate that EVT cells differentiate in vitro by proliferation from the tip of anchoring villi. These mononucleated, round-shaped, migrating cells are HLA-A,B,C class I antigen (W6/32) antibody and low molecular weight cytokeratin positive, and do not immunostain with PAI-1 (plasminogen activator inhibitor) and HPL antibodies. Differentiation of EVT was restricted to first trimester villous tissue; explants from second trimester placentae did not give rise to EVT. Tissue viability as monitored by glucose utilization, lactate, progesterone and hCG production rates correlated with EVT differentiation. The production rates for hCG demonstrated significant variation among individual placentae and was maintained constant for 10 days consistently only in explants cultured on decidual extract supplemented collagen matrix. The described villous tissue culture system may be, therefore, a unique in vitro model to study proliferation and differentiation of EVT from cytotrophoblastic columns, the regulation of EVT proliferation and differentiation, the role of ECM in the induction of the migration and the interaction of extravillous and villous trophoblast at the level of the cytotrophoblastic column.     

Hypoxia inhibits invasion of extravillous trophoblast cells through reduction of matrix metalloproteinase (MMP)-2 activation in the early first trimester of human pregnancy

During early pregnancy, extravillous trophoblast (EVT) cells are exposed to very low pO₂ values. In this study, we investigated the proteolytic functions and invasiveness of human primary EVT cells under hypoxic conditions to show the early placental pathophysiology.Placental samples (from 5 to 10 weeks gestation) were obtained at termination of pregnancy. Cytotrophoblast cells were separated by Percoll^® gradient method and cultured on Matrigel^® to obtain an invasive phenotype (similar to EVT). The invasion capacity (Matrigel-coated invasion assay), migration of the cells (wound healing assay), activity and expression of matrix metalloproteinase (MMP)-2 and tissue inhibitor for MMP (TIMP)-2 (gelatin gel zymography, ELISA, and quantitative RT-PCR), and expression of membrane-type (MT)1-MMP (western blot) were investigated. All cultures (except for quantitative RT-PCR) were performed under 20% oxygen, 5% oxygen, and 5% oxygen with 3 repetitions of 0.1% oxygen hypoxic stimulation for 1 h.Invasion and MMP2 activity of the cells were significantly increased in 20% and decreased in 0.1% oxygen. There was no significant difference in cell migration among the oxygen environments. Concentrations of MMP2 in the supernatant and expression of MT1-MMP were increased in both the 0.1% and 20% oxygen environments. The MMP2 mRNA level was increased after 1-h stimulation with 0.1% oxygen. The TIMP2 concentration was increased only in 20% oxygen, but the mRNA level was decreased in 0.1% oxygen.These results suggested that hypoxia might inhibit the invasive capacity and MMP2 activation of EVT cells in the early first trimester of pregnancy. Decrease in TIMP2 production may reduce the MMP2/TIMP2/MT1-MMP complex and lead to this unique behavior of EVT cells under hypoxic conditions.     

人早孕绒毛组织的体外原代培养和人滋养细胞株的建立

目的:建立快速、有效的绒毛滋养细胞体外原代培养的方法以及建立人滋养细胞株和初步评价。方法:采用胰酶和胶原酶I混合消化法消化绒毛组织,用胰蛋白酶消化传代体外培养,通过光镜、透射电镜和免疫荧光激光共聚焦方法对培养出的细胞进行形态和骨架评价。结果:显微镜下可见细胞呈多边形,细胞平铺生长,有细胞融合后形成的合体滋养层细胞;透射电镜下可见细胞表面的微绒毛丰富;胞质丰富,内质网扩张明显,有丰富的糖原颗粒、髓样小体和明显的脂滴,以及细胞间紧密的桥粒样结构连接;直接免疫荧光双标记染色检测到角蛋白18和波形蛋白均为阳性。结论:采用胰酶和胶原酶I混合消化法可获得较纯的滋养细胞,通过光镜、透射电镜和免疫荧光激光共聚焦检测细胞外形和骨架,确定该体外传至56代的细胞株为滋养细胞株。