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.     

Corticotrophin-releasing factor and urocortin inhibit system A activity in term human placental villous explants

Plasma corticotrophin-releasing factor (CRF) and urocortin are elevated in preterm labour and/or fetal growth restriction (FGR). FGR is associated with reduced placental system A amino acid transporter activity and in vitro data suggest altered endocrine status could be responsible. Here we test the hypothesis that CRF and urocortin inhibit placental system A activity. Chronic (48 h) exposure of term placental villous explants to these hormones (10⁻⁷ M) significantly reduced system A activity (Na⁺-dependent ¹⁴C-methylaminoisobutyric acid uptake), whereas 1 h exposure had no effect. We propose elevated CRF and urocortin contribute to FGR through negative regulation of placental system A activity.     

Different expression of placental pyruvate kinase in normal,preeclamptic and intrauterine growth restriction pregnancies

IntroductionPreeclampsia (PE) and intrauterine growth restriction (IUGR) are two diseases that affect pregnant women and their unborn children. These diseases cause low birth weight, pre-term delivery, and neurological and cardiovascular disorders in babies. Combined they account for 20% of preterm deliveries. Pyruvate kinase M2 (PKM2) is a metabolism enzyme found in developing embryonic and cancer tissues. Our objective is to determine the expression of PKM2 in human PE and IUGR compared to normal pregnancies. Understanding expression of PKM2 in PE and IUGR could help us to better understand the mechanisms and find treatments for PE and IUGR.MethodsHuman placental tissues were obtained for PKM2 determination and analyzed by immunohistochemistry, Western blot, and a pyruvate assay. Placental samples were homogenized and cytoplasmic and nuclear proteins were extracted for Western blot analysis.ResultsPreeclampsia samples had elevated levels of p-PKM2, p-ERK, and ERK in the cytoplasm. Beta-catenin and lactose dehydrogenase (LDH) were also elevated in preeclampsia placenta samples.Discussion and conclusionWe conclude that PKM2 is expressed in normal, PE and IUGR pregnancies. Also, that this expression is increased in the PE placenta at delivery. These results suggest placental metabolism through PKM2 could play a role in human preeclampsia.     

Predominant basal directional release of thromboxane, but not prostacyclin, by placental trophoblasts from normal and preeclamptic pregnancies

OBJECTIVE: To investigate apical and basal releases of thromboxane (TX) and prostacyclin (PGI2) by trophoblasts (TCs) from normal and preeclamptic (PE) placentas. METHODS: TCs isolated from normal and PE placentas were incubated in cell culture inserts for 48h. Medium from the upper (apical) and the lower (basal) chambers were then collected separately and measured for TX and PGI2 by their stable metabolites of TXB2 and 6-keto PGF1alpha by ELISA. Apical and basal releases of TX and PGI were also examined with apical exposure of TCs to arachidonic acid (AA)+/-aspirin at different concentrations. Villous tissue expressions for PGI synthase, TX synthase and TX (TP) receptor were examined by immunohistochemistry. RESULTS: (1) TXB2, but not 6-keto PGF1alpha, concentrations were significantly higher in the lower than in the upper chambers with both normal and PE TCs (p<0.01); (2) apical exposure of TCs to AA resulted in a significant increase in TX release towards both the upper and the lower chambers in normal TCs (p<0.01), but only a significant increase in the upper chamber in PE TCs (p<0.01); (3) aspirin could attenuate AA-induced TX release both in the upper and the lower chambers in normal, but not in PE, TCs (p<0.01), respectively; (4) there were no differences in 6-keto PGF1alpha productions both in normal and PE TCs treated with AA+/-aspirin; (5) intense staining of TX synthase and TP receptor was seen in syncytiotrophoblast layer, villous core vessels and stromal cells in preeclamptic placental tissue sections. CONCLUSION: Predominant basal release of TX together with intense staining of TX synthase and TP receptor in trophoblasts, stromal cells and villous core vessels are found in placentas from PE. We speculate if predominant basal release of TX by TCs occurs in vivo as we found in our in vitro culture condition, basal released TX may play a significant role in increased placental vasoconstriction such as in PE.     

Progesterone and human placental lactogen inhibit leptin secretion on cultured trophoblast cells from human placentas at term

The placenta is an important source of leptin production that contributes to the state of hyperleptinemia observed in pregnant women. Moreover, the synthesis of leptin and its receptors by syncytiotrophoblast cells suggests a potential paracrine or autocrine action of leptin in the placenta. In the present study we examined the effect of gestational hormones, human chorionic gonadotropin (hCG), human placental lactogen (hPL), progesterone and estradiol, on in vitro leptin release by human term trophoblast cells in culture. Placentas at term were obtained immediately after delivery from mothers with uncomplicated pregnancies. Leptin levels were measured by enzyme-linked immunosorbent assay in culture media of trophoblasts maintained in monolayer culture for 24, 48 and 72?h with different hormonal treatments or placebo. Treatment with hPL and progesterone led to a time- and dose-dependent decrease in leptin release that was statistically significant after 24?h, with a maximal effect after 72?h of incubation. In contrast, incubation with estradiol and hCG did not have exhibit any effect on leptin secretion at any of the doses and times assayed in this work. The results obtained in this study support that leptin can be considered a gestational hormone implied in the endocrine function of the placenta and that its secretion is at least partially regulated by steroid and peptidic reproductive hormones in trophoblast cells in vitro.     

Evidence of endothelial cytotoxic compounds in placental extracts from preeclamptic women

OBJECTIVE: To determine whether placenta and plasma of preeclamptic women contain factors that cause endothelial cell damage. METHODS: Placental extracts and plasma from preeclamptic and normotensive women were added to cultures of normal human umbilical vein endothelial cells and their effect on their viability, was determined by MTT reduction and 51chromium release. RESULTS: Placental extracts from normotensive and preeclamptic women were cytotoxic to endothelial cells, but not the plasma from both groups. Mean +/- standard deviation values of cytotoxicity index in preeclamptic and normotensive placental extracts using the MTT reduction were 70.3 +/- 6.76% and 51.4 +/- 8.81%, respectively, showing a significant difference (P < .0001). Using the 51chromium-release assay, preeclamptic placental extracts showed cytotoxic effects of 87.6 +/- 13.47% compared with 17 +/- 20.60% in control patients. The cytotoxic activity decreased after trypsin digestion and heat treatment in both groups. CONCLUSIONS: A cytotoxic factor to endothelial cells in placental extracts of preeclamptic women was identified. This compound is thermolabile and sensitive to trypsin digestion.     

Progesterone and human placental lactogen inhibit leptin secretion on cultured trophoblast cells from human placentas at term.

The placenta is an important source of leptin production that contributes to the state of hyperleptinemia observed in pregnant women. Moreover, the synthesis of leptin and its receptors by syncytiotrophoblast cells suggests a potential paracrine or autocrine action of leptin in the placenta. In the present study we examined the effect of gestational hormones, human chorionic gonadotropin (hCG), human placental lactogen (hPL), progesterone and estradiol, on in vitro leptin release by human term trophoblast cells in culture. Placentas at term were obtained immediately after delivery from mothers with uncomplicated pregnancies. Leptin levels were measured by enzyme-linked immunosorbent assay in culture media of trophoblasts maintained in monolayer culture for 24, 48 and 72 h with different hormonal treatments or placebo. Treatment with hPL and progesterone led to a time- and dose-dependent decrease in leptin release that was statistically significant after 24 h, with a maximal effect after 72 h of incubation. In contrast, incubation with estradiol and hCG did not have exhibit any effect on leptin secretion at any of the doses and times assayed in this work. The results obtained in this study support that leptin can be considered a gestational hormone implied in the endocrine function of the placenta and that its secretion is at least partially regulated by steroid and peptidic reproductive hormones in trophoblast cells in vitro.     

Evaluation of VEGF in placental bed biopsies from preeclamptic women by immunohistochemistry

OBJECTIVE: The aim of the study was to determine VEGF protein with immunohistochemical staining in placental bed biopsies of preeclamptic pregnancies in comparison to normal controls. DESIGN: Prospective cohort study. METHODS: The placental bed biopsies were obtained from 12 patients with preeclapmsia and ten patients for a control group at the time of cesarean delivery. Tissue samples of the placental bed were examined for VEGF protein distribution with avidin-biotin-peroxidase immunohistochemistry. Two blinded histopathologists were asked to score each sample for the intensity of staining and the number of cells stained in a randomly selected HPF of each sample. The resulting "H-score" was computed as a product of intensity and percent of cells stained. RESULTS: VEGF expression was significantly lower in both the myometrium and stroma of the preeclamptic group compared to the control group (77.2 +/- 25.4 vs 134 +/- 44.3, p = 0.007; 194.1 +/- 20.7 vs 170.2 +/- 17, p = 0.017, respectively). CONCLUSION: VEGF expression is significantly lower in placental bed biopsies of preeclamptic pregnancies.     

正常妊娠和子痫前期患者外周血树突细胞亚群的变化

目的:探讨树突细胞(DCs)及其亚群在正常妊娠和子痫前期患者间的变化,及与Th1/Th2型反应的关系。方法:选取正常妊娠孕妇25例、子痫前期患者17例和正常未孕妇女15例,用流式细胞术检测3组外周血树突细胞及其髓样(MDC)和淋巴样(PDC)亚群,比较其数量和比值在妊娠前后及子痫前期患者的变化,并与Th1/Th2型细胞因子的含量比较。结果:与正常妊娠早期和晚期相比,妊娠中期MDC和PDC数量减少,MDC/PDC比值升高,妊娠早、晚期相比无显著差异。与正常晚期妊娠妇女比较,子痫前期患者PDC数量减少,MDC数量改变不明显,MDC/PDC比值升高,两组相比差异显著。与正常晚期妊娠妇女相比较,子痫前期患者Th1型细胞因子IL-2含量增加,IFN-γ无显著差异,Th2型细胞因子IL-10减少,IL-2/IL-10、IFN-γ/IL-10比值升高。结论:DCs在正常妊娠的不同阶段其数量和亚群发生变化,子痫前期患者出现PDC减少和MDC/PDC比值升高现象,并与Th1/Th2型细胞因子的变化趋势一致。     

Catalase activity in normal and preeclamptic placentas

OBJECTIVE: The decrease of placental catalase (CAT) activity may lead to an increase of placental amounts of reactive oxygen species and can contribute to preeclampsia pathogenesis. DESIGN: The aim of the study was to determine CAT activity in placentas from normal and preeclamptic pregnancies (with and without intrauterine growth restriction--IUGR). MATERIALS AND METHODS: The investigations comprised placentas obtained immediately after delivery from 22 normal pregnancies (group K), 26 pregnancies complicated by severe preeclampsia-PE without IUGR (group PE) and 23 pregnancies complicated by severe PE and IUGR (group PEI). The activity of CAT was determined using a spectrophotometric method and expressed as IU/mg protein. Comparative analysis was performed using U Mann-Whitney test. RESULTS: Mean activity of CAT (MCAT) in the PEI group--0.38 +/- 0.14 (M +/- SD), was significantly lower (p < 0.001) as compared to MCAT in the group K (0.55 +/- 0.16). MCAT in the PE group (0.48 +/- 0.14) was lower than MCAT in the group K, but this difference was not statistically significant (p > 0.05). MCAT in the group PEI was significantly lower (p = 0.026) as compared to MCAT in the PE group. CONCLUSIONS: The activity of CAT is decreased in placentas from pregnancies complicated by severe PE and IUGR. Obtained results may indicate that the decrease of placental CAT activity may be involved in pathogenesis of IUGR in preeclamptic pregnancies.     

The effects of placental extracts from normotensive and preeclamptic women on vasoconstriction and oxidative metabolism

OBJECTIVE: A circulating factor derived from the placenta has been implicated in the pathogenesis of preeclampsia. The aim of this study was to determine whether placental extracts from normotensive women and women with preeclampsia increase oxidative metabolism and histamine-induced vasoconstriction in porcine carotid artery. STUDY DESIGN: Placental extracts from normotensive women and women with preeclampsia were applied to porcine carotid artery, and oxidative metabolism was measured. Histamine-induced isometric force responses were also determined in the absence and presence of placental extracts. RESULTS: Application of placental extracts to porcine carotid artery caused a fall in oxygen tension, which reflects increased consumption. Extracts from placentas taken from women with preeclampsia caused a greater fall than those from normotensive women (0.117 +/- 0.026 vs 0.018 +/- 0.0024 micromol oxygen per milligram; P < or =.01). Histamine-induced contractions were potentiated by extracts from preeclampsia but not from those of women without hypertension. The maximal steady-state force values were 13,137 +/- 3647, 12,921 +/- 3684, and 21,673 +/- 7189 N/m(-2) for control, normotensive, and preeclamptic samples at 10-micromol/L histamine (P < or =.05, compared with control placental extracts). CONCLUSIONS: Placental extracts from women with preeclampsia cause a greater stimulation of porcine artery oxygen consumption and exacerbation of histamine-induced vasoconstriction than extracts from normotensive women.     

PAF levels and PAF-AH activities in placentas from normal and preeclamptic pregnancies

OBJECTIVE: The aim of this study was to determine: (1) platelet-activating factor (PAF) levels and PAF-acetylhydrolase (PAF-AH) activities in normal and preeclamptic placentas; (2) lipid peroxide production by placental tissues stimulated with PAF. METHODS: Placentas were obtained immediately after delivery from normal and preeclamptic pregnancies. Tissue pieces were snap frozen in liquid nitrogen and stored at -70 degrees C. One gram of tissue from each placenta was used for PAF extraction and for total RNA isolation. PAF was measured by PAF [3H] scintillation proximity assay (SPA) system. Trophoblast PAF-AH activity was determined by enzyme-linked immunosorbent assay (ELISA). mRNA expression for PAF receptor was assessed by RNase protection assay (RPA). Normal placental explants were incubated with PAF at concentrations of 1 and 10 microg/ml for 48 h. Lipid peroxide productions of 8-isoprostane and malondialdehyde (MDA) were measured by ELISA and thiobarbituric acid reaction, respectively. Data were presented as mean+/-SE and analyzed by nonparametric Mann-Whitney U test and ANOVA. A p level less than 0.05 was considered statistically significant. RESULTS: (1) The mean tissue level for PAF was elevated, but not statistically different, in preeclamptic (n=7) than in normal (n=8) placentas, 6.45+/-1.05 versus 4.47+/-0.60 ng/g, p=0.42. (2) PAF-AH activity was higher in trophoblasts from preeclamptic (n=7) placentas than that in trophoblasts from normal (n=8) placentas, 0.69+/-0.16 versus 0.38+/-0.03 micromol/min/microg protein, p<0.05. (3) The relative mRNA expression for PAF receptor was not different between normal (0.70+/-0.08) and preeclamptic (0.76+/-0.13) placental tissues, p=0.60. (4) Productions of 8-isoprostane and MDA were not increased in tissues with PAF in culture, 8-isoprostane: 0.37+/-0.09 ng/mg (control) versus 0.32+/-0.09 ng/mg (1 microg/ml) and 0.37+/-0.07 ng/mg (10 microg/ml), p>0.5; MDA: 0.62+/-0.05 nmol/mg (control) versus 0.68+/-0.04 nmol/mg (1 microg/ml) and 0.69+/-0.03 nmol/mg (10 microg/ml), p>0.5. CONCLUSIONS: Increased PAF-AH activity in trophoblasts may be a compensatory effect to control PAF level in the preeclamptic placenta. The co-existence of PAF-AH and PAF receptor in trophoblasts suggests an autocrine regulation of PAF in these cells to limit PAF and its metabolites within the placenta.     

Maternal plasma levels of cytokines in normal and preeclamptic pregnancies and their relationship with diastolic blood pressure and fibronectin levels

BACKGROUND: To determine the plasma concentrations of placental growth factor (PLGF), vascular endothelial growth factor (VEGF), transforming growth factor-beta1 (TGF-beta1), soluble tumor necrosis factor alpha receptor (sTNFp55), interleukin-2 receptor (IL-2R), and interleukins 6 and 10 (IL-6, IL-10) in normotensive and preeclamptic women, and to evaluate the correlations between these cytokines and the diastolic blood pressure and fibronectin levels. METHODS: A prospective case-control study. Thirty-five women with preeclampsia were compared with 34 healthy women with uncomplicated pregnancies. Peripheral venous blood samples were obtained and plasma levels of PLGF, VEGF, TGF-beta1, sTNFp55, IL-2R, IL-6 and IL-10 were measured by an enzyme-linked immunoassay and fibronectin by a radial immundiffusion technic. RESULTS: In preeclampsia PLGF and VEGF levels were significantly lower, and TGF-beta1, sTNFp55, IL-2R, IL-6 and IL-10 levels were significantly higher than in normotensive pregnancy (p < 0.001). The plasma levels of PLGF and VEGF significantly decreased, whereas TGF-beta1, sTNFp55, IL-2R, IL-6 and IL-10 levels significantly increased with the increments in diastolic blood pressure and fibronectin levels (p < 0.001). CONCLUSIONS: Altered concentrations of various cytokines might explain the shallow placentation and endothelial cell dysfunction described in preeclampsia. The clinical severity of preeclampsia seems to correlate with the severity of the cytokine abnormalities.     

Superoxide dismutase activity in normal and preeclamptic placentas

OBJECTIVES: A deficiency in superoxide dismutase (SOD) activity in preeclamptic placentas can lead to an excess of superoxide radicals and may be responsible for the development and the severity of preeclampsia (PE). DESIGN: Our studies were undertaken in order to determine placental SOD activity and to investigate their association with the development and the severity of PE. MATERIALS AND METHODS: The activity of SOD was determined using a spectrophotometric method in 22 placentas from normal term pregnancies (group K), 24 placentas from pregnancies complicated by severe PE (group PE), and 21 placentas from pregnancies complicated by severe PE and intrauterine growth retardation (IUGR) (group PEI). RESULTS: Mean activity of SOD (MSOD) in 45 preeclamptic placentas 3.89 +/- 1.32 (M +/- SD) was significantly lower (P = 0.008) as compared to MSOD in the group K (6.75 +/- 1.96). MSOD in the PEI group (3.5 +/- 1.29) was significantly lower (P = 0.03) as compared to MSOD in the group K. MSOD in the PE group (4.23 +/- 1.25) was lower than MSOD in the group K, but this difference was not statistically significant (P = 0.11). MSOD in the group PEI was lower as compared to MSOD in the PE group, however this difference was not statistically significant (P = 0.23). CONCLUSIONS: The studies revealed decreased SOD activity in preeclamptic placentas in comparison to normal placentas.     

Plasma visfatin levels in preeclamptic and normal pregnancies

Objective  

Increasing evidences support the participation of adipokines in the pathogenesis of preeclampsia (PE). Visfatin is a novel adipokine secreted by fat tissue and macrophages and is involved in the regulation of glucose homeostasis. Our aim is to investigate visfatin levels in women with PE, women with the third trimester of normal pregnancy and healthy non-pregnant women.     

Glutathione peroxidase activity in normal and preeclamptic placentas

OBJECTIVE: The decrease of placental glutathione peroxidase (GSH-Px) activity may lead to exacerbation of lipid peroxidation in preeclamptic placentas. DESIGN: The aim of the study was the determination of GSH-Px activity in placentas from normal and preeclamptic pregnancies (with and without intrauterine growth retardation-IUGR). MATERIALS AND METHODS: The investigations comprised placentas obtained immediately after delivery from 24 normal pregnancies (group K), 26 pregnancies complicated by severe preeclampsia-PE (group PE) and 23 pregnancies complicated by severe PE and IUGR (group PEI). The activity of GSH-Px was determined using a spectrophotometric method and expressed as IU/g protein. Comparative analysis was performed using u Mann-Whitney test. RESULTS: Mean activity of GSH-Px (MGSH-Px) in the PEI group--5.38 +/- 1.59 (M +/- SD), was significantly lower (p < 0.001) as compared to MGSH-Px in the group K (7.22 +/- 1.21). MGSH-Px in the PE group (6.47 +/- 1.31) was lower than MGSH-Px in the group K, but this difference was not statistically significant (p > 0.05). MGSH-Px in the group PEI was significantly lower (p = 0.023) as compared to MGSH-Px in the PE group. CONCLUSIONS: The activity of GSH-Px is decreased in placentas from pregnancies complicated by severe PE and IUGR. Received results may indicate that the decrease of placental GSH-Px activity may be involved in pathogenesis of IUGR in preeclamptic pregnancies.