Dynamic maternal and fetal Notch activity and expression in placentation

IntroductionMurine placentation requires trophoblast Notch2, while the Notch ligand, JAGGED1, is reduced in invasive trophoblasts from women with preeclampsia. However, the placental cells with active Notch signaling and expression of other Notch proteins and ligands in placentation have yet to be defined. We sought to identify endothelial cell and trophoblast subtypes with canonical Notch signaling in the decidua and placenta and correlate this to expression of Notch proteins and ligands.MethodsNotch reporter transgenic mice were used to define canonical Notch activity and immunofluorescence staining performed to characterize expression of Notch1, 2, 3, 4 and ligands, Delta-like 4 (Dll4) and Jagged1 (Jag1) during early placentation and in the mature placenta.ResultsNotch signaling is active in maternal and fetal endothelial cells and trophoblasts during early placentation and in the mature placenta. Dll4, Jag1, Notch1, and Notch4 are expressed in maternal vasculature in the decidua. Dll4, Jag1 and Notch1 are expressed in fetal vasculature in the labyrinth. Dll4, Notch2 and Notch4 are co-expressed in the ectoplacental cone. Notch2 and Notch4 are expressed in parietal-trophoblast giant cells and junctional zone trophoblasts with active canonical Notch signaling and in labyrinthine syncytiotrophoblasts and sinusoidal-trophoblast giant cells.DiscussionCanonical Notch activity and distinct expression patterns for Notch proteins and ligands was evident in endothelium and trophoblasts, suggesting Notch1, Notch2, Notch4, Dll4, and Jag1 have distinct and overlapping functions in placentation. Characterization of Notch signaling defects in existing mouse models of preeclampsia may shed light on the role of Notch in developing the preeclampsia phenotype.     

Eph and ephrin expression in normal placental development and preeclampsia

Eph receptors and their ephrin ligands play a fundamental role in embryogenesis. Their functions include cell targeting and angiogenesis. In placental development, trophoblasts migrate and invade maternal tissue and spiral arteries, where they play a role in both anchoring the placenta to the uterus and increasing blood flow to the developing fetus (interstitial and endovascular invasions). We investigated the cellular distribution and expression patterns of representative Eph and ephrin RNA and protein in an effort to identify the molecules involved in trophoblast migration during normal placental development and placental pathologies. We found ephrin-A1 expressed exclusively in the invasive extravillous trophoblast (EVT) cell lineage. We therefore proceeded to investigate ephrin-A1 in placental pathologies with defects in EVT invasion. In preeclampsia, where trophoblast invasion is shallow, we observed ephrin-A1 expression similar to normal placenta. Furthermore, in initial experiments on the deeply invading trophoblasts of placenta accreta, which lacks decidua, ephrin-A1 is found to be expressed highly in extravillous trophoblasts that have invaded the myometrium. In addition, we found the prototype ephrin-A1 receptor, EphA2, localized in several placental cell types. EphB4 and ephrin-B2 molecules, which have specific expression patterns during artery and vein development, respectively, were also expressed in the placenta. The cell specific distribution of ephrin-A1 suggests that it may play a role in targeting and migration of trophoblasts, and in the vascular remodeling induced by the invading extravillous trophoblasts. Failure of ephrin-A1 expression is unlikely to be the primary cause in defective migration of trophoblasts observed in preeclampsia. Specific roles for other Eph and ephrin proteins remain to be investigated.     

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.     

sFlt-1及PLGF与子痫前期发病的关系

可溶性血管内皮生长因子受体1 (sFlt-1)与Flt-1均属于血管内皮生长因子受体(VEGFR),其与Flt-1竞争结合VEGF,完全阻断VEGF的生物学活性,引起血管生成障碍并影响血管壁的完整性和通透性.胎盘生长因子(PLGF)是VEGF家族成员之一,PLGF与Flt-1受体结合时,发挥促血管生成和促绒毛滋养细胞增...     

Difference in the expression of alpha 7 nicotinic receptors in the placenta in normal versus severe preeclampsia pregnancies

OBJECTIVE: The prevalence of preeclampsia is low in smokers, suggesting a possible role of nicotinic receptor in the pathophysiology of the disease. Alpha 7 nicotinic acetylcholine receptor (alpha7 nAChR) was recently found in non-neuronal tissue with various mediating functions. Therefore, we investigated the difference in the placental expression of the alpha7 nAChR in normal versus severe preeclampsia placentas. STUDY DESIGN: Central portions of placenta were obtained from 9 severe preeclampsia women and 11 gestational-age-matched normal pregnant women delivered between the gestational ages of 33 and 40 weeks following elective or emergency cesarean section. RT-PCR, western blotting, and immunohistochemical staining were performed to evaluate the alpha7 nAChR expression difference. RESULTS: In all the placentas, the alpha7 nAChR was expressed in endothelial cells, vascular smooth muscle cells, and stromal cells, but not in trophoblasts. The vascular staining was more intense in the severe preeclampsia placenta (p=0.02). Although the gene expression did not differ between the two groups, protein expression was greater in 7 of 9 placenta samples from the severe preeclampsia group. CONCLUSION: Placental expression of alpha7 nAChR differs between normal and severe preeclampsia placentas, suggesting that it may be involved in the pathogenesis of preeclampsia.     

Angiogenesis and intrauterine growth restriction

Human placental development involves co-ordinated angiogenesis and trophoblast outgrowth that are compromised in intrauterine growth restriction. Adaptive angiogenesis in IUGR placental villi is a result of an imbalance in the orderly progression of the expression profile of vascular endothelial growth factor, placenta growth factor and angiopoietin during placental development. VEGF receptors and the angiopoietin receptor Tie-2 are expressed on trophoblast, and their activation leads to trophoblast proliferation, migration and production of nitric oxide. Thus, these vascular factors act as autocrine regulators of trophoblast behaviour in the development of the utero-/feto-placental circulation, an action independent of their well-established roles in vascular endothelium.     

Spatial and temporal patterns of expression of messenger RNA for insulin-like growth factors and their binding proteins in the placenta of man and laboratory animals

To better understand the role of the insulin-like growth factors (IGF-I and -II) and their binding proteins (IGFBPs 1-6) in placental development and function, it is important to review similarities and differences between species in expression of the respective mRNAs. In human placenta, IGF-II mRNA is expressed in chorionic mesoderm and first trimester villous cytotrophoblast, but not in syncytiotrophoblast. In contrast, in rhesus monkey placenta, IGF-II mRNA is expressed in syncytiotrophoblast but not in chorionic mesoderm. IGFBP-3 mRNA is present in the chorionic mesoderm of placental villi from both these species and may modulate IGF-II action through a paracrine mechanism. In rodent placentae, IGF-II mRNA is expressed both in fetal mesoderm and in the trophoblast of the placental labyrinth. In guinea pig, where IGFBP-5 mRNA is expressed in the marginal and interlobular syncytium and IGF-II mRNA in the labyrinth, interaction between IGF-II and IGFBP-5 mRNA may be involved in vascularization of the placenta by fetal vessels. In sheep placenta, IGF-II mRNA is expressed, not in the trophoblast layer, but in the fetal mesoderm immediately adjacent to it. In the basal plate of human, rhesus monkey and baboon placentae, extravillous trophoblasts express IGF-II mRNA and uterine decidual cells IGFBP 1-6 mRNAs. The inference is that there is interaction between IGF-II and IGFBPs at the maternal-fetal interface of the primate placenta during trophoblast invasion and decidualization. IGFBP-1 expressed by the decidua may also interact with alpha(5)beta(1)integrin expressed by the extravillous trophoblast. The placentae of rodents are also of the invasive type. Glycogen cells of the mouse placenta are analogous with human extravillous trophoblast and express IGF-II mRNA. However, expression of IGFBP mRNAs in the mouse, as in the guinea pig, is confined to non-decidualized endometrium and myometrium. IGF-II mRNA is strongly expressed by trophoblasts invading uterine vessels in human and guinea pig placentae. Interactions probably occur between IGF-II expressed by these trophoblasts and IGFBPs expressed in the vessel walls. However, it is possible that IGFBPs expressed by maternal vessels are associated with processes that are independent of trophoblast invasion. Thus, IGFBP-3 mRNA is highly expressed in the maternal blood vessels of the non-deciduate sheep placenta. Findings to date highlight the diversity in the expression of the IGF system among placentae of man and different laboratory animals, and even between closely related species. Comparative studies will continue to be required to understand the functional role of IGFs and IGFBPs in each species.     

Notch信号通路在胎盘血管中的作用及与子痫前期的关系

胎盘血管的发育和重建是胎盘功能、胎儿功能发育的关键,其异常将直接导致妊娠期并发症如子痫前期,并最终影响妊娠结局。对人类及其他哺乳动物胎盘的研究表明,Notch信号通路与胎盘血管形成、滋养细胞侵袭等多个过程相关。     

芳香烃受体(AHR)在胎盘生成中的作用

胎盘是连接母体与胎儿的重要器官,在维持正常的妊娠过程中发挥着重要的作用。胎盘的结构和功能异常不仅易引发妊娠期高血压和糖尿病等妊娠并发症,还易导致早产、胎儿宫内生长受限(intrauterine growth retardation,IUGR)、流产等不良妊娠结局。芳香烃受体(aryl hydrocarbon receptor,AHR)作为一种配体激活性转录蛋白,参与了生殖调控、免疫功能调节、血管重塑等一系列重要的生理活动。AHR与滋养细胞的增殖和凋亡密切相关,并且具有调节滋养细胞细胞周期的作用。AHR在胎盘血管的生成及血流量的调节中也发挥着重要的作用,它通过调节促血管生成因子与血管生成抑制因子的平衡,参与胎盘血管的正常发育生长;同时AHR还很可能在胎盘的生长发育中介导了胎盘血管的生成以及滋养细胞的侵袭能力;AHR表达异常直接导致了相关妊娠期疾病的发生。     

Nitric oxide generation affects pro- and anti-angiogenic growth factor expression in primary human trophoblast

Objectives

Preeclampsia is associated with reduced trophoblast placenta growth factor (PGF) expression, elevated soluble fms-like tyrosine kinase-1 (sFlt-1) and decreased bioactivity of nitric oxide (NO). Elevated sFlt-1 reduces bio-availability of PGF and vascular endothelial growth factor (VEGF) leading to maternal endothelial dysfunction. Although NO can regulate gene expression, its ability to regulate trophoblast expression of angiogenic growth factors is not known.

Study design

Human primary term trophoblast and JEG-3 choriocarcinoma cells were cultured under 21%O₂ or 1%O₂ conditions in the presence or absence of NO donor (SNP) or inhibitor (L-NAME). Effects on PGF, VEGF and Flt-1 isoform mRNA expression were determined by quantitative real-time PCR. Changes in expression of soluble protein isoforms of FLT-1 was monitored by ELISA.

Results

Hypoxia decreased PGF mRNA but increased VEGF, sFlt-1 and Flt-1 mRNA expression in trophoblast. Generation of NO in trophoblast under 1%O₂ culture conditions significantly reversed sFlt-1 mRNA and protein expression, independent of mFlt-1. Conversely NO generation in hypoxic trophoblast increased VEGF and PGF mRNA expression.

Conclusions

NO production in primary human trophoblast cultures had divergent effects on pro-angiogenic (PGF, VEGF) versus anti-angiogenic (sFlt-1) mRNA expression, resulting in an enhanced pro-angiogenic gene expression environment in vitro.     

IFPA Gabor Than Award lecture: Molecular control of placental growth: The emerging role of microRNAs

Fetal growth is dependent on appropriate growth and function of the placenta. This is modulated by a variety of factors, including maternal growth factors that exert their actions by binding to specific receptors on trophoblast to promote activation of signaling events. Kinases and phosphatases within trophoblast act in concert to regulate growth factor actions and recent studies have begun to elucidate a role for microRNAs (miRs) in regulating the levels of these proteins in the placenta. This review will discuss growth factor signaling in the placenta and describe the emerging role of miRs in regulating placental development.     

Immunological aspects of preeclampsia

Pre-eclampsia is a common obstetric syndrome affecting about 7-10% of pregnant women. Symptoms of this syndrome: hypertension and impaired renal function appear during the second or third trimester of pregnancy. Despite intensive efforts to find mechanisms and markers induced pre-eclampsia, no specific etiological factor has been identified until now. It is known that pre-eclampsia is a placental disorder developing in two stages. The first lies in the poor placentation with acute atheroma. It seems that abnormal cell adhesion molecule (integrin) expression can contribute to inappropriate invasion of trophoblasts. Furthermore, T helper 1 type cytokines which are present in decidua of patients with pre-eclampsia can alter the trophoblast invasion. Lower expression level of HLA-G molecule in pre-eclamptic placenta can influence on the profile of cytokines which are produced in pre-eclampsia. The second stage of the disease development comprises the consequences of placental ischemia. It has been suggested that TNF-alpha is produced by ischemic placenta and causes endothelial activation. It seems that some types of pre-eclampsia can be autoimmune origin, with the autoantibodies directed against phospholipids, laminin and endothelium. The events leading to pre-eclampsia are not known, but it seems that abnormal activation of the immune system may play a role in the etiology of this disorder.     

Interleukin-11 up-regulates endoplasmic reticulum stress induced target,PDIA4 in human first trimester placenta and in vivo in mice

Interleukin (IL)11 is a crucial factor for human trophoblast function and placentation. Elevated levels are associated with pregnancy complications including preeclampsia, intrauterine growth restriction (IUGR) and preterm birth. However, the regulation of IL11 in the placenta has not been investigated. We examined the effect of pro-inflammatory cytokines IL1β and TNFα, as well as low oxygen tension (2%) on IL11 levels in first trimester placental villous explants. IL1β upregulated IL11 mRNA and protein, while TNFα and low oxygen had no effect. Using mass spectrometry, we identified protein disulfide isomerase 4 (PDIA4) in IL11-treated first trimester human placental explants (100 ng/ml, 24 h, n = 3), but not PBS control tissues. PDIA4 is a member of the PDI family, also known as endoplasmic reticulum (ER) stress protein (ERP)72. We previously identified GRP78 (a master regulator for ER stress) in human placenta for the first time and demonstrated that IL11 up-regulates GRP78 in the placenta. In this report, we demonstrated that IL11 upregulates PDIA4 protein in human placental villous tissue, HTR8-SVneo trophoblasts (cell line) and in vivo in IL11-treated mouse placenta. We aimed to determine whether IL11 upregulates other ER stress proteins in human first trimester placental villous. IL11 stimulated ERP44, but not GRP94, or PDI. Placental endoplasmic reticulum stress has been postulated in the pathophysiology of preeclampsia and IUGR, but its activation remains elusive. Together, these data suggest that IL11 could trigger an ER stress response in the placenta, which may contribute to obstetric complications such as preeclampsia.     

Human Placental Adenosine Receptor Expression is Elevated in Preeclampsia and Hypoxia Increases Expression of the A2A Receptor

Placental hypoxia as a result of impaired trophoblast invasion is suggested to be involved in the pathophysiology of preeclampsia. Hypoxia is a potent stimulus for the release of adenosine, and the actions of adenosine are mediated through four adenosine receptors, A₁, A_2A, A_2B and A₃. We investigated the presence, distribution and expression of adenosine receptor subtypes in the human placenta, the expression of the adenosine receptors in placentas from pregnancies complicated by preeclampsia, small for gestational age (SGA) infants and uncomplicated pregnancies, and the effect of hypoxia on placental adenosine receptor expression. Immunofluorescent microscopy localized A₁, A_2A, A_2B and A₃ adenosine receptors to the syncytiotrophoblast, endothelial cells and myofibroblasts within the human placenta. Adenosine receptor protein and message expression levels were significantly higher in placentas from preeclamptic pregnancies with or without SGA infants, but not different in pregnancies with SGA infants alone. In vitro exposure of placental villous explants to hypoxia (2% oxygen) increased the expression of A_2A adenosine receptor 50%. These data indicate that all four known adenosine receptors are expressed in the human placenta and adenosine receptor expression is significantly higher in pregnancies complicated by preeclampsia. These data are consistent with the hypothesis that differences in placental adenosine receptors may contribute to alterations in placental function in preeclampsia.     

The role of the placenta in the initiation of spiral artery remodelling in an early pregnant chimpanzee uterus

IntroductionIn this study we evaluated the full extent of placental bed changes (centre to periphery) in a pregnant chimpanzee uterus, kept at the Museum for Central Africa in Tervuren, Belgium. According to placental size the specimen was equivalent to an 8 weeks pregnant human uterus.MethodsHistological sections from central to peripheral tissue blocks of the placental bed were stained to reveal the presence of trophoblast, endothelium, vascular smooth muscle and elastic laminae. As an indicator for early arterial remodelling, we evaluated endothelial nuclear rounding and subendothelial vascular changes within the maternal vasculature in decidua and adjacent inner myometrium.ResultsWhile interstitially invading trophoblasts were present, endovascular trophoblast invasion seemed about to start into one spiral artery outlet at the centre of the placental bed, confirming our previous impression of a later onset of endovascular trophoblast invasion as compared to the human. An early sign of spiral artery remodelling was rounding of the endothelial nuclei. This phenomenon was not related to the local presence of interstitial trophoblast.DiscussionEndothelial nuclear rounding turned out to be a feature of the placental bed as a whole, being significantly less prominent in the adjacent non-placental bed part of the uterus, indicating an effect of the presence of the placenta. The different time-course of early spiral artery remodelling in the chimpanzee as compared to the human may have had a significant impact upon our evolution.     

A hypothesis for preeclampsia: adenosine and inducible nitric oxide synthase in human placental microvascular endothelium

Preeclampsia is a human syndrome characterized by elevated maternal blood pressure and proteinuria. It is the main cause of maternal morbidity and mortality, and fetal metabolic disturbances in developed and developing countries. Fetal complications in preeclampsia have been related with lower placental blood flow. The placenta lacks of innervation, thus vascular tone regulation depends on endothelial release of vasoactive molecules such as adenosine and nitric oxide (NO). Information about NO synthesis and its action in the feto-placental vasculature in preeclamptic pregnancies is controversial mainly due to the use of different methodological approaches, severity of the disease and cell type that had been analyzed. A high plasma level of adenosine has been reported in umbilical vein from preeclampsia compared with normal pregnancies. Since this nucleoside is mainly involved in the regulation of vascular tone and angiogenesis, perhaps through the modulation of potassium channels, it is suggested that it would be involved in the maintenance of normal feto-placental function. In this review we hypothesize a potential adenosine-mediated, NO-dependent mechanism accounting for the feto-placental reduced blood flow exhibited in preeclampsia. We summarize the potential mechanisms involved in the modulation of inducible NO synthase expression and activity in preeclampsia, emphasizing metabolic alterations in the placenta microvascular endothelial function. The involvement of adenosine, nucleoside membrane transporters and adenosine receptors, nuclear factor kappa B (NF-kappaB), potassium channels and angiogenesis, are also discussed regarding abnormal endothelial function in preeclampsia.     

Preeclampsia is associated with reduced serum levels of placenta growth factor

Objectives: Adequate vascular development of the placental bed is essential for normal pregnancy. We assessed serum levels of placenta growth factor, an angiogenic factor, throughout normal pregnancy and determined its association with preeclampsia. Study Design: Serum samples were collected from (1) 308 healthy pregnant women throughout normal gestation, (2) at delivery from 30 each gestational age–matched patients with normal pregnancy and preeclampsia, and (3) maternal and cord blood samples from normal deliveries with and without labor (n = 37 each). Placenta growth factor levels were determined with an antigen-capture enzyme-linked immunosorbent assay. Results: Maternal placenta growth factor levels during normal pregnancy increased from the first trimester to the late second trimester; they subsequently declined from 30 weeks’ gestation to delivery. Significantly less maternal placenta growth factor (P < .0001) was found in pregnancies complicated by preeclampsia, and labor significantly lowered placenta growth factor levels in both maternal (P = .0189) and cord serum samples (P < .0001). Conclusion: Decreased levels of placenta growth factor during preeclampsia could influence endothelial cell and trophoblast function, thereby contributing to the pathogenesis of the disease. (Am J Obstet Gynecol 1998;179:1539-44.)     

Edaravone inhibits hypoxia-induced trophoblast-soluble Fms-like tyrosine kinase 1 expression: A possible therapeutic approach to preeclampsia

Objective

To investigate the effects of edaravone, a potent free radical scavenger used clinically, on hypoxia-induced trophoblast-soluble Fms-like tyrosine kinase 1 (sFlt-1) expression.

Methods

A trophoblast cell line (HRT-8/SVneo) impaired by cobalt chloride (CoCl₂) was used as the cell model under hypoxic conditions. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) was used to measure the viability of cells exposed to CoCl₂ and edaravone. The levels of intracellular reactive oxygen species (ROS) were analyzed by flow cytometry. mRNA expression of sFlt-1, vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) in trophoblasts was measured by real-time polymerase chain reaction, and the secretion of sFlt-1, VEGF, and PlGF proteins was analyzed by enzyme-linked immunosorbent assays (ELISAs). A human umbilical vein endothelial cell (HUVEC) tube-formation assay was performed to identify the effects of CoCl₂ and edaravone on vascular development.

Results

CoCl₂ treatment caused the loss of trophoblast viability, the formation of ROS, and sFlt-1 mRNA and protein expression in a dose-dependent manner. Pretreatment with edaravone significantly inhibited hypoxia-induced oxidative stress formation and sFlt-1 expression in trophoblasts. Neither PlGF nor VEGF mRNA or protein expression was increased by CoCl₂. In the in vitro tube formation assay, edaravone showed a protective role in vascular development under hypoxic conditions.

Conclusion

This study demonstrated that hypoxia leading to increased sFlt-1 release in trophoblasts may contribute to the placental vascular formation abnormalities observed in preeclampsia and suggested that the free radical scavenger edaravone could be a candidate for the effective treatment of preeclampsia.