轴突导向因子netrin-1在子痫前期患者胎盘组织中的表达及其与胎盘新生血管形成的关系

目的探讨轴突导向因子 netrin-1在子痫前期孕妇胎盘组织中的表达及其对胎盘新生血管形成中的作用。方法采用 RT-PCR 和免疫印迹技术分别检测正常妊娠妇女20例(正常妊娠组)和子痫前期孕妇20例(子痫前期组,其中轻度12例,重度8例)的胎盘组织中轴突导向因子netrin-1 mRNA 和蛋白的表达;采用免疫组化并通过抗 F8因子抗体检测两组孕妇胎盘组织中的血管密度。结果 (1)正常妊娠组及子痫前期组孕妇胎盘组织中 netrin-1 mRNA 相对吸光度(A)值分别为0.51±0.08和0.41±0.06,netrin-1蛋白 A 值分别为26.4±1.8和20.5±1.3。两组分别比较,差异有统计学意义(P<0.01)。子痫前期组轻度和重度孕妇 netrin-1 mRNA A 值分别为0.48±0.08和0.34±0.07,netrin-1蛋白 A 值分别为22.8±1.3和18.2±1.0。两者分别比较,差异有统计学意义(P<0.01)。(2)子痫前期组孕妇胎盘血管密度为(54±8)个,正常妊娠组孕妇为(65±10)个,两组比较,差异有统计学意义(P<0.01);子痫前期组中重度孕妇血管密度为(48±7)个,轻度孕妇为(60±9)个,两者比较,差异有统计学意义(P<0.01)。(3)正常妊娠组孕妇胎盘组织中 netrin-1 mRNA 及其蛋白表达与血管密度呈正相关关系,相关系数(r)分别为0.67和0.71(P 均<0.01);子痫前期组孕妇胎盘组织中 netrin-1 mRNA 及其蛋白表达与血管密度呈正相关关系,r 分别为0.61和0.70(P 均<0.01);子痫前期组轻度孕妇胎盘组织中 netrin-1 mRNA 及其蛋白表达与血管密度呈正相关关系,r 分别为0.69和0.73(P 均<0.01);重度孕妇胎盘组织中 netrin-1 mRNA 及其蛋白表达与血管密度呈正相关关系,r 分别为0.71和0.75(P 均<0.01)。结论子痫前期孕妇胎盘组织中 netrin-1表达下降,可能是胎盘血管密度降低的原因之一。     

轴突导向因子netrin-1基因沉默对胎盘血管生成影响的体内外实验研究

目的 研究轴突导向因子netrin-1基因沉默对胎盘血管生成的影响.方法 应用RNA 干扰技术分别沉默netrin-1基因在人脐静脉内皮细胞(HUVEC)和孕鼠胎盘组织中的表达;用四甲基偶氮唑蓝(MTT)比色法检测细胞活性;克隆形成实验检测细胞增殖能力;细胞迁移实验检测细胞迁移能力;小管形成实验检测细胞管腔形成能力;观察胎鼠体质量;CD34因子的免疫组化链霉亲和素-生物素-过氧化物酶复合物(SABC)法检测孕鼠胎盘的微血管密度.结果 (1)HUVEC:沉默netrin-1基因后,HUVEC细胞活性受到抑制,克隆形成率由(69±6)%降至(46±5)%,迁移细胞数由(86±17)个降至(46±13)个,小管形成的分支点数由(37±9)个降至(17±5)个,HUVEC的netrin-1基因沉默前后比较,差异均有统计学意义(P＜0.05).(2)孕鼠胎盘组织:沉默孕鼠胎盘组织netrin-1基因后,胎鼠体质量由(2.39±0.17)g降至(2.12±0.10)g,胎盘微血管密度由(258±38)条/mm2降至(197±32)条/mm2,孕鼠胎盘组织netrin-1基因沉默前后比较,差异均有统计学意义(P＜0.05).结论 沉默netrin-1基因,可以抑制体外HUVEC的细胞活性及增殖、迁移和管腔形成能力,同时使体内胎盘组织的血管生成能力下降.netrin-1是重要的促胎盘血管生长基因.

Abstract：

Objective To study influence on angiogenesis of placenta by gene silencing of netrin-1.Methods Netrin-1 gene in human umbilical vein endothelial cells(HUVEC)and placenta of pregnant rats were silenced by RNA interference.The following methods were used in this study,including the phenytetrazoliumromide(MTT)for viability,clone formation for proliferation,transwell for migration,and tube formation for angiogenesis in vitro.The change of fetal growth was recorded.Placental microvessel density in pregnant rats was measured by immunohistochemical CD34 staining in vivo.Results (1)HUVEC:viability and proliferation of HUVEC were remarkably inhibited by gene silencing of netrin-1.which number of clone formation,migration cell,tube formation were from(69±6)%,86±17,37±9 decreased to(46±5)%,46±13 and 17±5(P＜0.05)respectively.(2)Placenta of pregnant rats:after netrin-1gene silenced,fetal weight were decreased from(2.39 ±0.17)g to(2.12±0.10)g(P＜0.05).Placental microvessel density was decreased from(258±38)/mm2 to(197±32)/mm2 in vivo(P＜0.05).Conclusions Gene silencing of netrin-1 could inhibit viability,proliferation,migration,tubal formation of HUVEC and angiogcnesis of placenta.Netrin-1 plays an important role in regulating angiogenesis in placenta.     

Cord blood netrin-1 and -4 concentrations in term pregnancies with normal,restricted and increased fetal growth

Abstract

Objective: To determine levels of the possible angioregulatory molecules netrin-1 and -4, in intrauterine-growth-restricted (IUGR), large for gestational age (LGA) (both groups characterized by altered angiogenic mechanisms) and appropriate-for-gestational-age (AGA) pregnancies.

Methods: Cord blood (UC) netrin-1 and -4 concentrations were measured in 30 IUGR, 30 LGA and 20 AGA infants and their mothers (MS).

Results: Netrin-1 and -4 concentrations did not differ in all groups. UC netrin-4 increased with gestational age (b?=?0.075, 95% CI 0.029–0.121, p?=?0.002). In the IUGR group, MS netrin-4 decreased as birth-weight centiles increased [b?=??0.058, 95% CI ?0.112 to ?0.004, p?=?0.036]. In the LGA group, MS netrin-1 decreased with advanced gestational age [b?=??0.063, 95% CI ?0.105 to ?0.022, p?=?0.004]. In all cases, MS netrin-1 positively correlated with MS netrin-4 (r?=?0.299, p?=?0.007), while UC netrin-1 negatively correlated with UC netrin-4 (r?=??0.239, p?=?0.033).

Conclusions: Increased UC netrin-4 levels with advancing gestational age may reflect its effect on fetal development. Decreased maternal netrin-1 levels in the LGA group possibly represent a negative feedback mechanism against increased angiogenesis. Increased maternal netrin-4 levels in IUGR neonates may reflect in utero hypoxia, while the negative correlations between fetal netrin-1 and -4 levels may exert the dynamic balance between their angio- and anti-angiogenic properties.     

The expression level of C19MC miRNAs in early pregnancy and in response to viral infection

IntroductionWe have previously shown that miRNAs produced from the Chromosome 19 MiRNA Cluster (C19MC), which are expressed almost exclusively in primate trophoblasts and are released into the maternal circulation, reduce viral replication in non-placental cells and can modulate migratory behavior of extravillous trophoblast. We sought to define the expression pattern of C19MC miRNA in early pregnancy and in response to viral infection in vitro and in vivo.MethodsWe prospectively followed women undergoing in vitro fertilization (IVF) and determined their blood level of C19MC miRNA using RT-qPCR. To examine the effect of viral exposure on C19MC miRNAs expression, we used three systems: (1) a transgenic mouse overexpressing the C19MC cluster and exposed to Togaviridae during pregnancy, (2) cultured primary human trophoblasts exposed to Vesicular Stomatitis Virus in vitro, and (3) amniotic fluid from women exposed to cytomegalovirus during pregnancy.ResultsIn 27 IVF pregnancies, C19MC miRNAs were detected as early as 2 weeks after implantation, and their levels increased thereafter. There was no change in C19MC miRNA expression levels in the mouse placenta in response to viral exposure. Similarly, Vesicular Stomatitis Virus infection of primary human trophoblast did not selectively increase C19MC miRNA expression. C19MC miRNA expression in the amniotic fluid was not affected by vertical transmission of cytomegalovirus.DiscussionThe expression of C19MC miRNAs in maternal circulation very early in pregnancy suggests a role in the establishment of the maternal-fetal interface. The levels of C19MC miRNA are not influenced by diverse types of viral infection.     

EFEMP1 expression promotes angiogenesis and accelerates the growth of cervical cancer in vivo

Objective

The study was to investigate the role of EFEMP1 in angiogenesis and growth of cervical carcinoma in vivo.

Methods

Effects of EFEMP1 on proliferation of Hela cells and HUVECs, invasion of Hela cells and migration of HUVECs, and adhesion of Hela cells to HUVECs were evaluated by MTT, Transwell chamber assay and adhesion assay, respectively. EFEMP1 overexpression in Hela cells was achieved by stable EFEMP1 gene transfection into Hela cells by Lipofectamin™ 2000 and the effectiveness of transfection was verified with western-blotting. The effect of EFEMP1 transfection upon the VEGF expression of Hela cells was detected with ELISA. The nude mouse models bearing cervical cancer were established with Hela cells transfected with EFEMP1 gene to observe the role of EFEMP1 in angiogenesis and growth of cervical cancer in vivo. VEGF expression and microvascular density of cervical cancer tissues were detected with immunohistochemistry and CD34 labeling respectively to elucidate the pathway by which EFEMP1 influences the growth of cervical cancer.

Results

Proliferation and invasion of Hela cells were promoted by the EFEMP1 protein. The EFEMP1 gene transfection into Hela cells was successful and EFEMP1 gene obtained stable high expression in Hela cells. Compared to the control, the tumors with EFEMP1 overexpression showed a faster growth rate and had a higher level of VEGF expression and microvascular density. EFEMP1 gene transfection elevated the VEGF protein level in Hela cells and EFEMP1 protein facilitated the adhesion of Hela cells to HUVECs. However, no direct effect of EFEMP1 was observed on proliferation, migration and tube formation of HUVECs.

Conclusions

EFEMP1 promoted the angiogenesis and accelerated the growth of cervical carcinoma in vivo through a VEGF up-regulation pathway.     

Trophoblast deportation part II: a review of the maternal consequences of trophoblast deportation

The deportation of trophoblast debris from the placenta was first documented over 100 years ago, and today we know that the deported material ranges from multinucleated syncytial knots/sprouts to trophoblast-derived nanoparticles. However little is known about the effect of trophoblast debris on maternal physiology since it is difficult to investigate these effects in vivo in women. Animal models have been reported but they have provided relatively little information. Most of our current knowledge regarding the effects of trophoblast debris on maternal systems is provided by studies using trophoblast debris obtained from in vitro models of the human placenta. Herein we review the animal models and the in vitro studies, which, between them, suggest that deported trophoblast material may play a role in tolerising the maternal immune system during normal pregnancy, and conversely that in pathological pregnancies aberrant maternal immune and/or endothelial/vascular responses may result from a change in either the quantity or quality of deported trophoblast debris.     

Mesenchymal stromal cells from the human placenta promote neovascularization in a mouse model in vivo

Cell transplantation is a promising strategy in regenerative medicine for revascularization of ischemic tissues. Based on our observation that placental mesenchymal stromal cells (PMSC) enhance endothelial cell viability in vitro via secretion of angiogenic factors, we asked whether PMSC support vascular growth in vivo. PMSC were isolated from amnion and placental endothelial cells (PLEC) from chorion and either separately or co-transplanted subcutaneously into immune-deficient mice. Co-transplantation resulted in a higher number of perfused human vessels (CD31+/vimentin+) containing mouse glycophorin A+ erythrocytes. Results indicate positive effects of PMSC on neovascularization in vivo, making them attractive candidates to create autologous PMSC/PLEC pairs for research and transplantation.     

A placental phenotype for intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy specific liver disease associated with significant risk of fetal complications. It is hypothesised that the risk of adverse fetal outcomes relates to the toxic effects of bile acids, the levels of which are increased in both maternal and fetal serum. Human and rodent studies have shown that transplacental transfer of bile acids is impaired in ICP. Furthermore, the morphology of placentas from the rodent model of ICP is markedly abnormal, and is associated with increased expression of apoptotic markers and oxidative stress. Using placental tissue from ICP cases and normal pregnancies and cultured placental explant fragments we investigated the histological and molecular effects of cholestasis. We also examined the influence of ursodeoxycholic acid (UDCA) administration on these parameters. Here we report that ICP is associated with several morphological abnormalities of the placenta, including an increase in the number of syncytial knots, and that these can be reproduced in an in vitro (explant) model exposed to the bile acids taurocholic acid and taurochenodoexycholic acid. Furthermore, we demonstrate that ursodeoxycholic acid, a drug commonly used in the management of ICP, has a protective effect on placental tissue both in vivo and in vitro.     

DNA methylation patterns of imprinting centers for H19, SNRPN,and KCNQ1OT1 in single-cell clones of human amniotic fluid mesenchymal stem cell

ObjectiveTo test the hypothesis that human amniotic fluid mesenchymal stem cells contain a unique epigenetic signature in imprinting centers of H19, SNRPN, and KCNQ1OT1 during in vitro cell culture.Materials and MethodsBy bisulfite genomic sequencing, we analyzed the imprinting centers of three imprinted genes (including H19, SNRPN, and KCNQ1OT/) in a total of six single-cell clones of human amniotic fluid mesenchymal stem cells at cell passages 7, 8, 9, and 10 during in vitro cell culture.ResultsThe imprinting centers of H19 and KCNQ1OT1 showed hypermethylation at passage 7 in all single-cell clones of human amniotic fluid mesenchymal stem cells, and there was no significant change in DNA methylation patterns during in vitro cell culture. The imprinting centers of SNRPN showed variable methylation patterns at passage 7 in six single-cell clones, and DNA methylation patterns varied during in vitro cell culture from passages 8 to 10.ConclusionIn conclusion, human amniotic fluid mesenchymal stem cells contain a unique epigenetic signature during in vitro cell culture. H19 and KCNQ1OT1 possessed a substantial degree of hypermethylation status, and variable DNA methylation patterns of SNRPN was observed during in vitro cell culture of human amniotic fluid mesenchymal stem cells. Our results urge further understanding of epigenetic status of human amniotic fluid mesenchymal stem cells before it is applied in cell replacement therapy.     

Ex vivo culture of pre-placental tissues reveals that the allantois is required for maintained expression of Gcm1 and Tpbpα

IntroductionChorioallantoic fusion is essential for development of the labyrinth layer of the mouse placenta. However, events that occur after chorioallantoic attachment remain poorly described, partly due to difficulties of conducting ex vivo analysis of the placenta. Herein, we report conditions for ex vivo culture of the developing murine placenta.MethodsMesometrial halves of decidua containing pre-attachment chorions were cultured alone or with explants of allantoides from stage-matched controls and analyzed by confocal and immunofluorescence microscopy. Expression and levels of marker genes associated with specific placental cell types were measured by in situ hybridization and qRT-PCR, respectively.ResultsAfter 24 h (hr) of co-culture, a mosaic pattern of eGFP⁺ and eGFP⁻ cells were found when explants of pre-attachment chorions from eGFP⁺ embryos were co-cultured with stage-matched allantoides from eGFP⁻ embryos or vice versa. In addition, proliferation increased in the allantoic region and folds formed on the chorionic plate. PECAM positive cells derived from the allantois were found in the chorionic region. Levels of the SynT-II marker, Gcm1, significantly increased at 24 h, although expression of Gcm1, was only found in explants co-cultured with an allantois at 12 h and 24 h. In addition, though levels of Tpbpα was not altered by co-culture with an allantois, Tpbpα was only detected in explants co-cultured with an allantois for 24 h.DiscussionOur data show that chorioallantoic fusion and events associated with initiation of labyrinth layer formation can be modeled ex vivo, and reveal a previously unsuspected requirement of chorioallantoic fusion for Tpbpα expression.     

Ex vivo endothelin dependent contraction of the remodeled rat spiral artery

Introduction

Similarities of the rat to the human placenta make rat pregnancy models relevant to the study of human gestational diseases. Understanding of species differences is necessary to extrapolate from animal models to humans. We observed alpha-smooth muscle actin (αSMA) expression in rat endovascular trophoblasts (EVasT) and investigated the spatial and temporal expression of smooth muscle (SM) proteins and their potential function in remodeled spiral artery.

Methods

Rat placentas were examined from gestational day 13 to term, and were immunostained for cytokeratin, αSMA, alpha heavy chain of SM myosin, non-muscle myosin, Rho proteins, regulators of SM gene expression, myocardin, an early marker of SM differentiation and endothelin receptors A and B (ETA, ETB). Transmission electron microscopy (TEM) was performed. Modified spiral artery rings were studied ex vivo for endothelin-1- induced contraction.

Results

EVasT expressed SM proteins co-localizing with cytokeratin confirming their trophoblastic origin from gestational day 13 to term. Thin fibers, consistent with actin fibers, were observed by TEM, in the cellular localization of αSMA in EVasT.Functional experiments revealed that addition of 10⁻⁷ M endothelin-1 ex vivo reduced vascular lumen area by 11.1% ± 1.8% compared with control. This effect was reduced to only 1.0 ± 1.7% with ETA antagonist, and to 5.4 ± 1.7% contraction by ETB antagonist, p < 0.002, for all.

Discussion

The expression of SM proteins in EVasT along with the contractibility of the rat remodeled spiral artery ex vivo, suggest that some vascular tone is potentially maintained by endothelin-1, and may play a role in situations of dysregulation of the vasoactive systems.     

HDAC2 was involved in placental P-glycoprotein regulation both in vitro and vivo

IntroductionPlacental P-glycoprotein (P-gp) plays a significant role in regulating drugs' transplacental transfer rates. Investigations on placental P-gp regulation could provide more therapeutic targets for individualized and safe pharmacotherapy during pregnancy. Currently, the epigenetic regulation of placental P-gp is rare. Our previous study has demonstrated that HDACs inhibition could up-regulate placental P-gp and HDAC1/2/3 might be involved in this process. The present study was carried out to further explore whether HDAC1/2/3 were indeed involved in the regulation of placental P-gp or not and screen out the subtype engaged in this process.MethodsBeWo and JAR cells were transfected with HDAC1/2/3 specific siRNA. After 48 h of transfection, cells were harvested for real-time quantitative PCR (qRT-PCR), Western blot, immunofluorescence and fluorescent dye efflux assay to evaluate P-gp expression, localization, and efflux activity, respectively. Hdac2 siRNA was intraperitoneally injected to pregnant mice every 48 h from E7.5 to E15.5 and digoxin was administered by gavages 1 h prior to euthanasia at E16.5. Placental Hdac1/2/3 and P-gp expression were determined by qRT-PCR and Western blot. Maternal plasma and fetal-unit digoxin concentrations were detected by enzyme-multiplied immunoassay.ResultsIn vitro, HDAC2 inhibition could significantly elevate P-gp expression and reduce intracellular accumulation of P-gp substrates (DiOC₂ (3) and Rh 123) both in BeWo and JAR, while knockdown of HDAC1/3 had no influence on P-gp expression and its efflux activity. Additionally, in vivo, Hdac2 silencing in pregnant mice also elevated placental P-gp expression and decreased digoxin transplacental transfer rate.ConclusionHDAC2 inhibition could result in induction of placental P-gp expression and functionality both in vitro and in vivo.