Extra-placental expression of vascular endothelial growth factor receptor-1, (Flt-1) and soluble Flt-1 (sFlt-1), by peripheral blood mononuclear cells (PBMCs) in normotensive and preeclamptic pregnant women

The soluble VEGF receptor, sFlt-1 (otherwise referred to as sVEGFR-1), has been implicated in the pathogenesis of preeclampsia. The preeclamptic placenta has been previously demonstrated to produce high levels of the soluble VEGF receptor. Here we tested the hypothesis that peripheral blood mononuclear cells (PBMCs) may also represent an additional source for circulating sFlt-1 during normal and preeclamptic pregnancies. We first demonstrate that preeclamptic placentae show five-fold increased Flt-1 and sFlt-1 mRNA levels. We also show that the Flt-1 and sFlt-1 levels are eight-fold higher in preeclamptic placentae if we collect biopsies without rinsing them in saline to remove excess blood. Cultured villous explants from women with preeclampsia failed to show the increased amount of Flt-1 and sFlt-1 mRNA that was observed in the placental biopsies of normal pregnancy and preeclampsia. Under normoxic conditions the Flt-1 and sFlt-1 mRNA levels in the explants were 3.11+/-0.6 fold in normal pregnancy and 3.6+/-0.4 fold in women with preeclampsia (p = NS by ANOVA). However, the same villous explants showed hypoxic induction of Flt-1 mRNA (NP 3.96+/-0.4 fold, p = NS and PE 5.24+/-0.6 fold, p < 0.05 by ANOVA). We analyzed Flt-1 and sFlt-1 protein levels in the peripheral blood mononuclear cells (PBMCs) to analyze the possibility of an extra-placental sFlt-1 source. Our results indicate that PBMCs of pregnant women are capable of expressing variable amounts of Flt-1 proteins. PBMCs from pregnant women exposed to hypoxia show up-regulation of HIF-1alpha and Flt-1 proteins. PBMCs obtained from women with preeclampsia (n = 9) produced significantly higher amounts of sFlt-1 under normal tissue culture conditions (104.6+/-14.3 pg/ml vs. 46.23+/-5.03 pg/ml, p < 0.05 by ANOVA) and much higher concentrations under hypoxia (196.74+/-26.3pg/ml vs. 83.3+/-13.6pg/ml, p < 0.05 by ANOVA) than PBMCs from normal pregnant women (n = 11). Moreover, analysis of PBMCs from a different group of women with a history of preeclampsia showed persistent abnormality of Flt-1 women one year post-partum. The present study indicates that Flt-1 dysregulation in PBMCs of pregnant women resulting in over-expression of sFlt-1 could be an additional (extra-placental) source of sFlt-1 that contributes to the pathogenesis of preeclampsia.     

Circulating and placental endoglin concentrations in pregnancies complicated by intrauterine growth restriction and preeclampsia

Inadequate trophoblast invasion and spiral artery remodeling leading to poor placental perfusion and hypoxia are believed to underlie preeclampsia (PE) and intrauterine growth restriction (IUGR). Recent studies implicate increased circulating endoglin as a contributor to the pathogenesis of PE. The objective of this study was to determine whether placental and circulating endoglin concentrations are altered in pregnancies complicated by intrauterine growth restricted (IUGR) infants and to address the role of hypoxia on the regulation of placental endoglin. We analyzed 10 placentas each from normal pregnant (NP), PE, and IUGR subjects. Endoglin levels were 2.5-fold higher in preeclamptic placentas compared to NP (15.4+/-2.6 versus 5.7+/-1.0, p<0.01). In contrast, endoglin levels were similar in NP and IUGR placentas (5.7+/-1.0 vs 5.9+/-1.1, p=NS). Placentas from pregnancies with both PE and IUGR exhibited endoglin levels comparable to the PE group and significantly different from normotensive pregnancies with and without IUGR pregnancies (mean 14.9+/-4.0, n=9, p=0.013). Soluble endoglin concentrations in maternal plasma were comparable in NP and IUGR, but higher in women with PE (n=10 per group, p<0.05). Despite a 2-fold increase in hypoxia inducible factor, HIF-1alpha, we did not observe endoglin upregulation in NP, PE, or IUGR placental villous explants exposed to hypoxia (2% oxygen). In contrast to PE, placental or circulating endoglin is not increased in normotensive women delivering small, asymmetrically grown (IUGR) infants at term. The placentas of women with IUGR appear to be fundamentally different from PE women with respect to endoglin, despite the proposed common pathology of deficient trophoblast invasion/spiral artery remodeling and poor placental perfusion.     

Melatonin prevents preeclamptic sera and antiphospholipid antibodies inducing the production of reactive nitrogen species and extrusion of toxic trophoblastic debris from first trimester placentae

BackgroundThe exact cause of preeclampsia is unknown. However a “toxin” from the placenta triggers the condition via activation of the maternal endothelium. Extracellular vesicles (EVs) from the syncytiotrophoblast, may be an endothelial-activating toxin. Antiphospholipid antibodies (aPL) and preeclamptic sera both induce the production of endothelial cell-activating EVs by mechanisms which may produce excess free-radicals in the placenta. Melatonin is produced by the human placenta and has both direct and indirect anti-free-radical properties and may therefore counter the effects of aPL and preeclamptic sera.MethodsFirst trimester placental explants were exposed to preeclamptic sera or aPL in the presence or absence of melatonin. Nitrosylative damage was assessed in the explants by immunohistochemistry and the effect of EVs from these explants on endothelial cell activation determined by ICAM-1. Release of nitrosylated proteins from the explants was also measured.ResultsPlacental explants showed reduced secretion of melatonin after treatment with preeclamptic sera. Nitrosylated proteins were more abundant in placentae that had been treated with aPL or preeclamptic sera and EVs from such placentae induced endothelial cell activation. Adding melatonin to the aPL or preeclamptic sera reversed the protein nitrosylation and production of endothelial-activating EVs.DiscussionOur data are consistent with reports that the levels of circulating melatonin are reduced in preeclampsia and suggest that aPL and factors in preeclamptic sera induce free-radical-mediated damage in the placenta leading to the production of endothelial-activating EVs. Melatonin reversing production of endothelial-activating EVs indicates that melatonin may have therapeutic benefits in women with preeclampsia and/or aPL.     

Increased expression of interleukin-1 beta is associated with persistence of the disease and invasion in complete hydatidiform moles (CHM)

Complete hydatidiform moles (CHM), a post-conceptual pathologic condition of the placenta, have a high prevalence rate (12/1,000 deliveries) in Kerala, India. This study addresses the expression of IL-1 alpha and beta by immunohistochemistry in relation to persistence and invasion of the disease. Mild to moderate expression of IL-1 alpha in the villous cytotrophoblasts, syncytiotrophoblasts and decidua of the first trimester in the normal placenta and all gestational ages in the molar placenta were observed. IL-1 beta expression was observed in the extravillous trophoblasts, syncytiotrophoblasts and decidua in both the normal and molar placentae and also in the villous cytotrophoblasts and the stromal Haufbaur cells in molar placentae. Strong expression of IL-1 beta in the placenta suggests its involvement in placental physiology supporting earlier reports. Higher expression of IL-1 beta correlated well with the invasive and persistent nature of the tumour and holds potential as a marker of persistence and invasion in CHM.     

Reduced expression of survivin, the inhibitor of apoptosis protein correlates with severity of preeclampsia

Introduction

Preeclampsia is a major complication of pregnancy affecting maternal and fetal health. Although the pathogenesis of preeclampsia is unclear, it is believed that trophoblast apoptosis plays an important role in the pathogenesis of preeclampsia during pregnancy. Survivin is a member of the inhibitor of apoptosis protein family that uniquely promotes trophoblast proliferation. In this study we investigated the alteration of survivin levels during pregnancy and compared the survivin protein and mRNA between preeclampsia and normal pregnancy.

Methods

The mRNA level of survivin in first, second and third trimester placentae was measured by Real-time PCR. The expression of survivin in preeclamptic placentae (including severe and mild preeclampsia) and in age-matched normal placentae was measured by immunohistochemistry. The mRNA levels of survivin in preeclamptic or normal placentae were measured by Real-time PCR.

Results

The mRNA level of survivin was significantly reduced throughout gestation. The mRNA level of survivin in preeclamptic placentae was significantly reduced compared to that in normal placenta. The mRNA level of survivin in severe preeclamptic placentae was further significantly reduced compared to that in mild preeclamptic placentae. In addition, survivin was expressed on syncytiotrophoblasts and cytotrophoblasts and the expression of survivin was significantly decreased in preeclamptic placenta compared to that in normal placenta (p = 0.01). Furthermore the expression of survivin in severe preeclamptic placentae was significantly lower than that in mild preeclamptic placentae.

Conclusion

Our current data suggests lower placental expression of survivin may be associated with the severity of preeclampsia.     

CFTR May Modulate AQP9 Functionality in Preeclamptic Placentas

Preeclampsia (PE) is a hypertensive disorder unique to human pregnancy. Although its causes remain unclear, it is known that altered placental villous angiogenesis and a poorly developed fetoplacental vasculature can affect the transport functions of the syncytiotrophoblast (hST).We have previously observed that in preeclamptic placentas there is an increase in AQP9 protein expression, with a lack of functionality. Up to now, the mechanisms for AQP9 regulation and the role of AQP9 in the human placenta remain unknown. However, there is strong evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) regulates AQP9 functionality.ObjectiveHere, we studied CFTR expression and localization in hST from preeclamptic placentas in order to investigate if alterations in CFTR may be associated with the lack of activity of AQP9 observed in PE.MethodsThe expression of CFTR in normal and preeclamptic placentas was determined by Western Blot and immunohistochemistry, and CFTR-AQP9 co-localization was determined by immunoflurescence. Water uptake experiments were performed using explants from human normal term and preeclamptic placentas treated with CFTR inhibitors.ResultsWe found that CFTR expression significantly decreased in preeclamptic placentas, and that the hST apical labeling almost disappeared, losing its co-localization with AQP9. Functional experiments demonstrated that water uptake diminished in normal term explants incubated with CFTR inhibitors.ConclusionsThese results suggest that CFTR expression decreases in preeclampsia and may thus be implicated in the regulation of AQP9 activity.     

Over-expression of hypoxia-inducible factor 1 alpha in ovarian clear cell carcinoma

OBJECTIVE: Unlike other histological types of epithelial ovarian carcinoma, ovarian clear cell carcinoma is known to have very poor response to therapy even when discovered in its early stages. Since tumor hypoxia has been shown to be strongly associated with poor prognosis, deregulation of the representative factor of tissue hypoxia; hypoxia-inducible factor 1 alpha (HIF-1alpha) and related protein; Von Hippel-Lindau (VHL) may be associated with poor prognosis of ovarian clear cell carcinoma. METHODS: Immunolocalization of both HIF-1alpha and VHL was performed on 56 cases of paraffin-embedded tissue sections of four different histological types of epithelial ovarian carcinoma and 5 cases of benign ovarian tumors as a control. Quantitative RT-PCR analysis of both HIF1A and VHL was performed on RNA isolated from 61 microdissected frozen tissues of four different histological types of epithelial ovarian carcinoma and 6 cases of normal ovarian epithelial cells. Expression levels of HIF-1alpha and VHL in different histological types and correlation between HIF-1alpha and VHL were determined by nonparametric analysis by Kruskal-Wallis and Spearman's test. RESULTS: HIF-1alpha expression levels were significantly higher in ovarian clear cell carcinoma than in other histological types (P=0.001). We found no correlation between mRNA and protein expression level in any type of carcinoma specimens. Among endometrioid, serous, and mucinous carcinoma, there were no differences in HIF-1alpha expression (P=0.643). There was a negative correlation between HIF-1alpha and VHL in serous (r=-0.661, P=0.027) and in endometrioid carcinoma (r=-0.657 P=0.039), but no correlation was found between HIF-1alpha and VHL expression levels in ovarian clear cell carcinoma (P=0.60). CONCLUSIONS: The results suggest that the role of hypoxia may change according to the histological type of ovarian carcinoma. High expression of HIF-1alpha and its independence from VHL in ovarian clear cell carcinoma may confer chemoresistance in this histological type.     

血红素氧合酶在正常妊娠绒毛和胎盘组织中的表达及定位

目的　探讨血红素氧合酶 (HO)的两种同工酶基因及蛋白在正常早期妊娠绒毛和晚期妊娠胎盘组织中的表达。方法　选择正常妊娠 6～ 10周的孕妇 (早孕组 )和妊娠 3 7～ 41周的孕妇 (晚孕组 )各 2 0例 ,采用逆转录聚合酶链反应 (RT PCR)技术 ,检测绒毛和胎盘组织中诱导型HO(HO 1)mRNA及结构型HO(HO 2 )mRNA的表达 ;并采用免疫组织化学 (免疫组化 )方法 ,进行HO蛋白定位和半定量分析。结果　两组HO 1mRNA的表达均较弱 ,早孕组绒毛HO 1mRNA表达水平为 ( 0 3 1±0 19) ,晚孕组胎盘HO 1mRNA表达水平为 ( 0 2 8± 0 14 ) ,两组比较 ,差异无显著性 (P >0 0 5) ;而两组HO 2mRNA的表达均较强 ,晚孕组胎盘表达水平为 ( 1 12± 0 58) ,早孕组绒毛表达水平为 ( 0 70±0 48) ,两组比较 ,差异有显著性 (P <0 0 5)。免疫组化定位结果显示 ,HO 1蛋白主要定位于绒毛间质细胞和胎盘滋养细胞 ,HO 2蛋白主要定位于胎盘滋养细胞及血管内皮细胞 ,绒毛间质也有染色。蛋白半定量结果显示 ,胎盘细胞染色分数为非正态分布 ,HO 1在早孕组绒毛滋养细胞、间质细胞和血管内皮细胞中的染色分数中位数分别为 9 0、2 6和 2 8,晚孕组分别为 8 7、2 0和 1 4,两组比较 ,差异均无显著性 (P >0 0 5)。HO 2在早孕组绒毛血管内皮细胞中     

Differential expression of the receptor tyrosine kinase EphB4 and its ligand Ephrin-B2 during human placental development

Normal placentation involves the development of an utero-placental circulation following the migration of the extravillous cytotrophoblasts into the decidua and invasion of the spiral arteries, which are thereby transformed into large vessels of low resistance. Given the documented role of the receptor tyrosine kinase EphB4 and its ligand ephrin-B2 in the establishment of the embryonal vascular network, we hypothesized that these molecules are also instrumental in the development of the human placenta. Monitoring the expression during placental development revealed that in first trimester and term placentae both molecules are equally expressed at the RNA level. In contrast, the protein levels were significantly reduced during gestation. Immunohistochemistry revealed a distinct localization of the EphB4 and ephrin-B2 proteins. EphB4 was predominantly expressed in the villous syncytial trophoblast layer and in a subset of intravillous capillaries. Prominent expression was also observed in the extravillous cytotrophoblast giant cells. In contrast, ephrin-B2 expression was detected in the villous cytotrophoblast and syncytial trophoblast cell layers, as well as initially in all intravillous capillaries. Strong expression was also observed in extravillous anchoring cytotrophoblast cells. Hypoxia is a major inducer of placental development. In vitro studies employing trophoblast-derived cell lines revealed that predominantly ephrin-B2 expression is induced by hypoxia, however, in an Hif-1alpha independent manner. These experiments suggest that EphB4 and ephrin-B2 are instrumental in the establishment of a functional placental structure and of the utero-placental circulation.     

Hypoxia attenuates PGE(2)but increases prostacyclin and thromboxane production in human term villous trophoblast.

Prostanoids have been proposed to play a major role in the regulation of uteroplacental blood flow. We examined the effect of hypoxia on the production of prostaglandin E(2)(PGE(2)) thromboxane B(2)(TXB(2)), and prostacyclin (measured as 6-keto-PGF(1alpha)) by human term trophoblast cells and villous placental explants. Explants (n=8) and purified trophoblast cells (n=5) were incubated for 24-72 h under either normoxic (21 per cent O(2)) or hypoxic (2 per cent O(2)) conditions. In trophoblast monolayer cultures, hypoxia attentuated PGE(2)production rates to 52+/-9.4 per cent (mean+/-sem, P< 0.05) but recovered to control rates within 48 h. In villous explants, PGE(2)production was significantly decreased after 48 and 72 h of hypoxia versus the normoxic control, accompanied by increased production of 6-keto-PGF(1alpha)to 173.9+/-26.7 per cent after 48 h. TXB(2)production was increased to 172.3+/-25.9 per cent and 653.2+/-135.7 per cent (P< 0.05) control after 48 and 72 h of hypoxia, respectively. These results were confirmed in villous explants (n=3) cultured in the presence of exogenous 10 microm arachidonic acid. Hypoxia had no significant effect on TXB(2)and 6-keto-PGF(1alpha)in trophoblast cells. In summary, our findings suggest that hypoxia could be responsible for abnormal profiles of prostanoid production commonly observed in women with pre-eclampsia. These results indicate a putative link between hypoxia and compromised placental perfusion.     

Plasma fibronectin receptor levels during pregnancy complicated by preeclampsia and abruptio placentae.

The level of human fibronectin receptor (FNR) in plasma was measured by enzyme-linked immunosorbent assay in samples from normal pregnant women in the 1st trimester (n = 5), 2nd trimester (n = 7), 3rd trimester (n = 23), normal postpartum women day 1 (n = 4), day 2 (n = 5), day 3 (n = 8), nonpregnant women (n = 18), 20 preeclamptic patients in the 3rd trimester, and 8 patients with abruptio placentae in the 3rd trimester. In normal pregnancy, the mean value of FNR was 1.4 +/- 0.4 micrograms/ml in the 1st, 1.4 +/- 0.2 micrograms/ml in the 2nd, and 1.9 +/- 0.3 micrograms/ml (p less than 0.05) in the 3rd trimester. FNR values increased with pregnancy. During the puerperium, its level decreased with time, being 1.4 +/- 0.5 micrograms/ml (p less than 0.01) on day 1, 1.0 +/- 0.3 micrograms/ml on day 2, and 0.8 +/- 0.2 micrograms/ml on day 3. The level in preeclamptic patients was 2.0 +/- 0.4 micrograms/ml, and that in abruptio placentae was 2.7 +/- 0.4 micrograms/ml. There were significant differences between the levels in abruptio placentae versus preeclampsia (p less than 0.05) and 3rd-trimester normal pregnant women (p less than 0.01). In the immunohistochemical study, the surface of normal decidual cells stained weakly for FNR, and the decidual cell membranes of the cases of preeclampsia stained moderately or strongly. Decidual cells and their extracellular matrix close to hematomas of abruptio placentae stained very strongly for FNR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoxia inhibits activin A production by term villous trophoblast in vitro

Elevated activin A and inhibin A levels have been associated with pre-eclampsia, a pregnancy-related disorder associated with placental hypoxaemia. We investigated the effect of in vitro hypoxia on the production of inhibin A, activin A and its binding protein follistatin in term villous placental explants (n=4-7) and trophoblast monolayer cultures (n=4). Explants and trophoblasts were incubated for 24-72 h under either normoxic (21 per cent O(2)) or hypoxic (2 per cent O(2)) conditions. Production of activin A, inhibin A, and follistatin was determined by specific ELISA. After 48 h of hypoxia, villous explants exhibited a significant reduction in activin A production rates to 53.2 +/- 8.9 per cent (mean +/- SEM, P<0.05) of normoxic controls which was sustained after 72 h in culture (46.8 +/- 5.9 per cent), whereas production by trophoblast monolayers was not affected by hypoxia. Follistatin production was decreased to 53.7 +/- 9.2 per cent of control (P<0.05) after 48 h of hypoxia. Inhibin A production remained unaltered in both culture systems. Our data demonstrate for the first time that hypoxia lowers term placental activin A and follistatin production in vitro. These findings do not support the notion that elevated circulating activin A levels in pre-eclampsia originate from the placenta as a result of placental hypoxia. Other as yet unknown maternal/placental factors may contribute to elevated activin A production in women with severe pre-eclampsia.     

Antioxidant gene expression in preeclamptic placentae: a preliminary investigation

Oxidative stress has been implicated in the pathogenesis of preeclampsia. This study measured the relative mRNA expression of antioxidant proteins glutathione peroxidase 1 and 4, glutathione reductase, thioredoxin 1 and 2, thioredoxin reductase 1, thioredoxin peroxidase 3 and superoxide dismutase 1 and 2 in preeclamptic and non-preeclamptic placentae. Quantitative real-time PCR was conducted on placental mRNA isolated from preeclamptic and control patients. Cycle threshold numbers and fold differences were calculated as a measure of linear product amplification and used for comparison. The mRNA expression of glutathione reductase was significantly reduced (fold difference 0.41, p<0.05) in preeclamptic placenta when compared to controls while the expression of thioredoxin peroxidase 3 was significantly increased (fold difference 3.25, p<0.001) in the preeclamptic placentae. No significant difference in expression was observed for glutathione peroxidase 1 and 4, thioredoxin 1 and 2, thioredoxin reductase 1 and superoxide dismutase 1 and 2. These results suggest that it is the abnormal oxidative insult associated with preeclampsia not mRNA expression of antioxidant proteins that may be responsible for reduced antioxidant enzyme activity in preeclamptic placentae.     

Increased phospholipase A2 and thromboxane but not prostacyclin production by placental trophoblast cells from normal and preeclamptic pregnancies cultured under hypoxia condition

In this study we determined whether hypoxia could promote vasoactivator thromboxane (TX) and prostacyclin (PGI2) as well as phospholipase A2 (PLA2) production by placental trophoblast cells (TCs) from normal and preeclamptic (PE) pregnancies. Placentas were obtained immediately after delivery from normal (n=9) and preeclamptic (n=9) pregnancies. TCs were isolated by dispase digestion of villous tissue and purified by Percoll gradient centrifugation. TCs (5x10(6) cells/well) were cultured with Dulbecco's Modified Eagles Medium (DMEM) under hypoxia condition (2% O2/5% CO2/93% N2) for 48 h. TCs cultured under normoxia condition (5% CO2/air) were used as control. Culture medium was collected at the end of incubation. Productions for TX, PGI2 and PLA2 were measured by ACE competitive enzyme immunoassay (EIA). Comparisons were made using the Mann-Whitney U test or paired t-test and the data are expressed as mean+/-SE (pg/microg cellular protein). Significance was set at a p-value of <0.05. We found: (1) PE-TCs produced more TXB2 and PLA2 than normal-TCs under normoxia conditions, TXB2: 4.33+/-1.03 vs. 1.84+/-0.29 pg/microg protein, p<0.05; PLA2: 0.38+/-0.08 vs. 0.21+/-0.03 pg/microg protein, p<0.05, respectively. (2) Hypoxia promoted both PE- and normal-TCs to generate more TXB2 and PLA2, TXB2: 6.36+/-1.72 vs. 3.05+/-0.45 pg/microg; PLA2: 0.52+/-0.10 vs. 0.30+/-0.04 pg/microg, respectively. (3) No change in 6-keto PGF1alpha production was observed for normal-TCs or PE-TCs when compared under normoxia vs. hypoxia condition, normal-TCs: 0.20+/-0.05 vs. 0.21+/-0.05 pg/microg; PE-TCs: 0.38+/-0.05 vs. 0.36+/-0.04 pg/microg, respectively. We concluded that hypoxia promotes both PLA2 and TX, but not PGI2, production by placental trophoblast cells cultured under hypoxia condition. These results suggest that increased PLA2 release may alter the arachidonic acid cascade and promote TX synthesis. Relative hypoxia could contribute to the increase in TX production and result in vasoconstriction in placental vasculature in preeclampsia.