Differential expression of human placental PAPP-A2 over gestation and in preeclampsia

IntroductionPregnancy Associated Plasma Protein A2 (PAPP-A2) is a pregnancy related insulin-like growth factor binding protein-5 (IGFBP-5) protease, known to be elevated in preeclampsia. As the insulin-like growth factors are important in human implantation and placentation, we sought to determine the expression pattern of PAPP-A2 over human gestation in normal and preeclamptic pregnancies to evaluate its role in placental development and the pathogenesis of preeclampsia.MethodsPlacental basal plate and chorionic villi samples, maternal and fetal cord blood sera were obtained from preeclamptic and control pregnancies. Formalin-fixed tissue sections from across gestation were stained for cytokeratin-7, HLA-G, and PAPP-A2. PAPP-A2 immunoblot analysis was also performed on protein lysates and sera.ResultsPAPP-A2 expression is predominately expressed by differentiated trophoblasts and fetal endothelium. Chorionic villi show strong expression in the first trimester, followed by a progressive decrease in the second trimester, which returns in the third trimester. PAPP-A2 expression is not impacted by labor. PAPP-A2 is increased in the basal plate, chorionic villi and maternal sera in preeclampsia compared to controls, but is not detectable in cord blood.DiscussionPAPP-A2 is differentially expressed in different trophoblast populations and shows strong down regulation in the mid second trimester in chorionic villous samples. Both maternal sera and placental tissue from pregnancies complicated by preeclampsia show increased levels of PAPP-A2. PAPP-A2 levels are not altered by labor. Additionally, PAPP-A2 cannot be detected in cord blood demonstrating that the alterations in maternal and placental PAPP-A2 are not recapitulated in the fetal circulation.     

Evidence for insulin-mediated control of AQP9 expression in human placenta

The AQP9 gene contains a negative insulin response element, suggesting that it may be modulated by insulin. Previously, we reported AQP9 overexpression in preeclamptic placentas but a lack of functionality of AQP9 in water and mannitol transport. We also observed high serum levels of insulin and TNF-α in preeclamptic women.

Objective

To evaluate whether AQP9 expression is regulated by insulin in the human placenta, and whether the dysregulation of AQP9 observed in preeclamptic placentas may be related to the inability to respond to insulin stimuli.

Methods

Explants from normal and preeclamptic placentas were cultured at different concentrations of insulin. Treatment with TNF-α was used to induce phosphorylation of insulin receptor substrate (IRS), which may desensitize insulin action. AQP9 molecular expression and water uptake was determined.

Results

Insulin decreased the molecular expression of AQP9 exclusively in explants from normal placentas in a concentration-dependent manner. Treatment with TNF-α previous to insulin addition prevented these changes. Moreover, insulin treatment did not modify water uptake neither its sensitivity to HgCl_2.

Conclusion

AQP9 water permeability seems to be independent of its molecular expression, strongly suggesting that AQP9 might not have a key role in water transport in human placenta. We also propose another mechanism of down-regulation of AQP9 molecular expression mediated by insulin in a concentration-dependent manner in human placenta and provide new evidence that in preeclamptic placentas the mechanisms of insulin signaling may be altered, producing an overexpression of AQP9 that does not correlate with an increase in its functionality.     

14-3-3 tau (YWHAQ) gene promoter hypermethylation in human placenta of preeclampsia

IntroductionDisruption of the 14-3-3 tau (YWHAQ) gene has been shown to be involved in preeclampsia (PE). The YWHAQ promoter could be differentially regulated by methylation in severe PE patients.MethodsPlacental genomic DNA from patients with severe PE (n = 21) and controls who experienced a normal pregnancy (n = 16) was analyzed using dot-blot and immunohistochemistry. The placental methylation patterns of YWHAQ, expression of 14-3-3 tau and ten-eleven translocation (TET), were confirmed by bisulfite sequencing, immunohistochemistry, western blot and real-time PCR, respectively.ResultsGenomic 5 hmC (P < 0.001), expression of 14-3-3 tau (P < 0.01) and TET (P < 0.05) were down-regulated, whereas 5 mC was up-regulated (P < 0.001) in preeclamptic placentas. Significant hypermethylation of the YWHAQ promoter was detected in PE placentas compared with control samples (19.1% vs. 9.4%, P = 0.0095). PE-specific hypermethylation of CpG2 – 4, CpG9, CpG17, CpG19 was identified in PE patients compared with controls (CpG2: 13.3% vs. 2.5%, P < 0.0001; CpG3: 14.8% vs. 3.1%, P < 0.0001; CpG4: 19.5% vs. 5.0%, P < 0.0001; CpG9: 15.7% vs. 5.0%, P = 0.0018; CpG17: 16.2% vs. 6.3%, P = 0.0003; and CpG19: 78.1% vs. 59.4%, P < 0.0001).DiscussionThe observed participation of 14-3-3 tau in the regulation of the placental epigenome may participate in the molecular mechanisms that govern the pathological process of PE, although this requires further evaluation.     

miR-223-3p靶向NLRP3调控子痫前期胎盘中炎性反应的机制研究

目的:探讨miR-223-3 p能否通过靶向NLRP3参与调节子痫前期(PE)胎盘中的炎性反应.方法:选取2018年12月至2020年01月在江苏省苏北人民医院住院行剖宫产分娩的60例孕妇,其中PE组30例,正常对照组30例.采用实时定量聚合酶链式反应(RT-qPCR)、蛋白免疫印迹、免疫组化法对胎盘组织中NLRP3和...     

子痫前期孕妇血清与胎盘尿紧张素Ⅱ的表达及意义

目的:检测子痫前期孕妇外周血和新生儿脐血血清中尿紧张素Ⅱ(urotensinⅡ,UⅡ)水平和UⅡmRNA在胎盘组织的表达水平,探讨其在子痫前期发生发展中的作用。方法:(1)ELISA测定30例子痫前期患者及15例正常晚期妊娠孕妇(对照组)外周血和新生儿脐血血清UⅡ水平;(2)用RT-PCR法检测各组孕妇胎盘组织中UⅡmRNA的表达水平。结果:(1)重度子痫前期组孕妇外周血血清UⅡ水平明显高于轻度子痫前期组和对照组,差异均有显著性(P<0.05)。轻度和重度子痫前期组的脐静脉血清UⅡ浓度均明显高于对照组(P<0.05,P<0.001)。(2)胎盘组织UⅡmRNA表达水平在轻度和重度子痫前期组均明显高于对照组,差异有显著性(P<0.05)。结论:UⅡ可能参与子痫前期全身小血管痉挛的机制并在胎盘组织缺血缺氧和动脉粥样硬化的发生起重要作用。     

Placental mRNA expression of angiopoietins (Ang)-1, Ang-2 and their receptor Tie-2 is altered in pregnancies complicated by preeclampsia

Objective

To investigate the placental expression of angiopoietin (Ang)-1, Ang-2 and their receptor, Tie-2, in preeclampsia (PE) with or without intrauterine growth restriction (IUGR).

Methods

Case-control study including placentas from 28 PE pregnancies, 30 PE-IUGR pregnancies and 40 controls. The expression status of the genes was evaluated by quantitative real-time PCR.

Results

In both PE and PE-IUGR groups, compared to the control group, there was significantly higher expression of Ang-2 (p < 0.001) and Tie-2 (p = 0.008) and lower expression of Ang-1 (p = 0.001). The magnitude of the difference was similar for Ang-1 for both groups, whereas the magnitude of the differences was higher for Ang-2 and Tie-2 in PE-IUGR group compared to controls. Ang-2 and Tie-2 were correlated in both PE (r = 0.8602, p < 0.001) and PE-IUGR (r = 0.6342, p < 0.001) groups. In PE-IUGR group, Ang-1 was associated to Ang-2 (r = 0.3458, p = 0.0452) and Tie-2 (r = 0.4448, p = 0.0084). Log₁₀Ang-1 but not Ang-2 was gestational age dependent (R2 = 0.40, p < 0.001). After conversion in Multiples of the Median (MoM) log₁₀ MoM Ang-1 was reduced in the PE group (mean = −0.8181, p < 0.001) and the PE-IUGR group (mean = −1.2583, p < 0.001) compared to control group (mean = −0.0924).

Discussion

We have demonstrated increased placental expression of Ang-2 and Tie-2 along with lower expression levels of Ang-1 in pregnancies with PE and PE-IUGR.

Conclusion

The angiopoietin axis seems to be disrupted in PE pregnancies. Whether the results of this study represent the angiogenic imbalance observed in PE pregnancies or they are part of the pathophysiology of this condition has to be further investigated.     

Exocyst complex protein expression in the human placenta

Introduction

Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal–fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood.

Objective

While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens.

Methods

A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections.

Results

The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion.

Discussion/conclusion

Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst's regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion.     

Dysregulation of maternal and placental vitamin D metabolism in preeclampsia

IntroductionEpidemiology has linked preeclampsia (PET) to decreased maternal serum 25-hydroxyvitamin D3 (25(OH)D3). However, alterations in systemic and placental/decidual transport and metabolism of 25(OH)D3 during pregnancy suggest that other forms of vitamin D may also contribute to the pathophysiology of PET.MethodsIn a cross sectional analysis of normal pregnant women at 1st (n = 25) and 3rd trimester (n = 21), pregnant women with PET (n = 22), and non-pregnant female controls (n = 20) vitamin D metabolites were quantified in paired maternal serum, placental, and decidual tissue.ResultsSerum 25(OH)D3 was not significantly different in sera across all four groups. In normal 3rd trimester pregnant women serum active 1,25-dihydroxyvitamin D3 (1,25(OH)₂D3) was significantly higher than non-pregnant, normal 1st trimester pregnant, and PET women. Conversely, PET sera showed highest levels of the catabolites 3-epi-25(OH)D3 and 24,25-dihydroxyvitamin D3 (24,25(OH)₂D3). Serum albumin was significantly lower in normal 3rd trimester pregnant women and PET relative to normal 1st trimester pregnant women, but there was no change in free/bioavailable 25(OH)D3. In PET placental tissue, 25(OH)D3 and 3-epi-25(OH)D3 were lower than normal 3rd trimester tissue, whilst placental 24,25(OH)₂D3 was highest in PET. Tissue 1,25(OH)₂D3 was detectable in 1st trimester decidua, which also showed 10-fold higher 25(OH)D3 relative to paired placentae. 3-epi-25(OH)D3 and 24,25(OH)₂D3 were not different for decidua and placenta. In normal 3rd trimester pregnant women, total, free and bioavailable maternal 25(OH)D3 correlated with placental 25(OH)D3, but this was not conserved for PET.DiscussionThese data indicate that PET is associated with decreased activation, increased catabolism, and impaired placental uptake of 25(OH)D3.     

子(癎)前期患者胎盘组织中Toll样受体2和Toll样受体4的表达及意义

目的研究子痫前期（PE）患者胎盘组织中Toll样受体2（TLR2）和Toll样受体4（TLR4）的表达及其与胎盘中肿瘤坏死因子α（TNF-α）、白介素6（IL-6）表达的关系,探讨PE胎盘中Toll样受体变化的意义。方法2005年1月至2006年12月在哈尔滨医科大学第一临床医学院采用免疫组化和RT-PCR法检测PE患者和正常晚期妊娠妇女胎盘组织中TLR2和TLR4的定位及表达,同时用脂多糖（LPS）体外刺激胎盘组织中的TLR4,检测其上清液中TNF-α、IL-6的变化。其中PE患者44例（PE组）,同期正常晚期妊娠妇女22例（对照组）。结果PE组和对照组胎盘组织中均有TLR2和TLR4的表达,其中PE组TLR4表达明显比对照组增强,组间差异有显著性意义（P〈0.05）;LPS体外刺激PE组胎盘组织,导致TNF-α、IL-6分泌量增加,刺激后2hPE组TNF-α、IL-6分泌量与对照组比较差异无显著意义（P〉0.05）,刺激后6hPE组TNF-α、IL-6的分泌量升高明显,与对照组比较,差异有显著性意义（P〈0.01）。结论PE患者胎盘中TLR4表达增加与TNF-α、IL-6改变密切相关,是造成局部细胞因子微环境异常的主要因素,其增加可能是PE发病机制中的关键环节之一。     

Genes,epigenetics and miRNA regulation in the placenta

This text reviews briefly the context in which epigenetics regulate gene expression in trophoblast development and function. It is an attempt to focus on a limited number of recent papers that, according to the author, shed new light on placental development, and constitute possible trails for improving knowledge and women follow-up in pathological pregnancies.     

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.     

人类白细胞相关抗原-G及其各亚型mRNA在正常妊娠及重度子痫前期患者胎盘组织中的表达

目的 研究人类白细胞相关抗原-G（HLA-G）及其各个亚型mRNA在正常妊娠及重度子痫前期患者胎盘组织中的表达.方法 选取2005-01-2005-06第四军医大学唐都医院10例重度子痫前期患者（研究组）及10例正常妊娠胎盘组织（对照组）,应用荧光定量RT-PCR比较两组间HLA-G及其各个亚型mRNA（HLA-G1、G2、G3、G4、G5、G6）的表达.结果 与对照组相比,重度子痫前期患者胎盘组织中总HLA-G mRNA显著降低,以HLA-G1、HLA-G3降低为主,差异有统计学意义（P＜0.05）.结论 HLA-G1与HLA-G3 在胎盘组织中的低表达可能与妊娠期高血压疾病的发病和病理生理过程有关.     

Neither normal nor diseased placentas contain lymphatic vessels

Background

Scant data on placental lymphatic vessels have pointed to the absence of lymphatic circulation. A recent study on mesenchymal dysplasia (MD), however, has identified pathologic lymphangiogenesis using the D2-40 lymphatic marker. These conflicting data have prompted us to investigate whether lymphatic vessels are present in normal developing placentas and in placental disorders characterized by cistern formation.

Design

Seventeen human placentas without significant pathological abnormality ranging from 12 to 39 weeks of gestational age were studied. Cisternal placental disorders were represented by mesenchymal dysplasia (n = 1), partial hydatitiform mole (n = 2), spontaneous abortion (n = 3) and complete hydatiform mole (n = 2). To identify lymphatic vessels, we used lymphatic endothelial markers Prox-1 and D2-40. The pan-endothelial marker CD31 was used to highlight overall placental vasculature and to determine if the lining cells of cisterns were of endothelial origin. Lymphatic marker positivity was assessed in maternal (decidual) as well as in fetal (chorionic villous) vasculature.

Results

No staining with Prox-1 or D2-40 was identified in fetal vessels in developing or term placentas, or in selected cisternal placental disorders, although both markers highlighted a number of thin-walled decidual vessels. Cistern lining cells were negative for Prox-1, D2-40 and CD31. D2-40 consistently marked stromal cells in chorionic villi and highlighted perivascular/pericellular extracellular matrix.

Conclusion

We established that no lymphatic vasculature is present in the chorionic villi during development, at term or in selected edematous placental disorders. The cisternal lining cells are not endothelial cells; most likely they are of stromal cell origin. Lymphangiogenesis is a part of decidual vascular remodeling during gestation.     

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.     

Wnt2在植入胎盘组织中的表达及其对滋养细胞迁移和侵袭功能的影响

目的检测Wnt2在植入胎盘组织中的表达,分析Wnt2对滋养细胞凋亡、迁移和侵袭功能的影响。方法收集2020年7月至2021年1月于武汉大学中南医院妇产科行剖宫产的胎盘植入孕产妇胎盘组织15例(植入组)和正常孕产妇胎盘组织15例(对照组)。RT-PCR法和Western blot法检测胎盘组织中Wnt2表达水平,采用siRNA敲低HTR-8/Svneo细胞中Wnt2表达水平,流式细胞学法检测细胞凋亡,Transwell侵袭和迁移实验评估细胞的迁移和侵袭能力,RT-PCR法和Western blot法检测β-catenin、Bcl-2、Caspase-3、MMP-2和MMP-9表达水平。结果与对照组相比,植入组胎盘组织中Wnt2、MMP-2和MMP-9 mRNA和蛋白表达量均显著升高(均P<0.05)。转染敲低HTR-8/Svneo细胞Wnt2水平后,细胞凋亡率显著增加(P<0.05),细胞迁移和侵袭能力显著抑制(P<0.01),β-catenin、Bcl-2、MMP-2和MMP-9表达显著下降(均P<0.05),而Caspase-3表达显著升高(P<0.05)。结论植入胎盘组织中Wnt2蛋白表达显著升高,且Wnt2可能通过wnt/β-catenin信号通路参与了胎盘滋养细胞的凋亡、迁移和侵袭。     

Elevation of granulocyte-macrophage colony-stimulating factor in the placenta and blood in preeclampsia

OBJECTIVE: This study investigated whether granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in the placenta and blood in preeclampsia differed from those in normal pregnancies. Macrophage colony-stimulating factor (M-CSF) levels in the placenta were also measured. STUDY DESIGN: The subjects were 44 pregnant women carrying single fetuses, of whom 22 were women with normal pregnancies and 22 were women with preeclampsia. Their average gestational age at entry was 37 to 38 weeks of gestation. Peripheral blood was collected before the onset of labor. Separated serum was obtained after centrifugation and stored at -20 degrees C. A tissue segment of the placenta was cut immediately after delivery. The frozen placental tissue was placed into a plastic tube containing phosphate-buffered saline solution. The tissue was fully homogenized and then centrifuged. Separated supernatant was frozen at -80 degrees C for subsequent determination. GM-CSF levels in separated serum were measured, and GM-CSF, M-CSF, and total protein (TP) levels in separated supernatant were also measured. RESULTS: Not only GM-CSF levels in blood but also GM-CSF/TP levels in the placenta were significantly higher (P<.05) in preeclampsia than in normal pregnancies. Similar results were obtained for M-CSF/TP levels in the placenta. CONCLUSIONS: We demonstrated a significant increase in placenta levels of GM-CSF/TP in preeclampsia. Elevated GM-CSF in the placenta may be related to immunologic abnormalities contributing to the etiology of preeclampsia.     

Syncytin-2 mRNA及其受体MFSD2 mRNA在正常妊娠及子痫前期胎盘组织中的表达研究

目的检测syncytin-2 mRNA及其受体MFSD2 mRNA在正常妊娠及子痫前期胎盘组织中的表达及其与子痫前期发病的关系。 方法分析2010年3月至2011年12月就诊于广州医学院第三附属医院住院分娩46例孕妇资料,正常妊娠组(21例),子痫前期组(25例)。在剖宫产术中采集产妇胎盘组织,采用实时荧光定量PCR法检测胎盘组织中syncytin-2 mRNA及其受体MFSD2 mRNA的表达,两组表达水平的比较采用t检验。 结果Syncytin-2 mRNA及其受体MFSD2 mRNA在正常妊娠组及子痫前期组患者胎盘组织中均有表达。在子痫前期组,两种指标分别为5.59±0.29和0.79±0.08;在正常妊娠组,两种指标分别为6.99±0.62和0.83±0.10。两组中,syncytin-2 mRNA表达水平差异有统计学意义(t＝2.331,P＝0.029),MFSD2 mRNA表达水平差异无统计学意义(t＝0.258,P＝0.778)。 结论胎盘组织syncytin2 mRNA表达下调可能与子痫前期发病密切相关,其受体MFSD2 mRNA与子痫前期发生无相关性。