RESUMO
Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2015 there were twelve themed workshops, three of which are summarized in this report. These workshops covered areas of placental regulation and nutrient handling: 1) placental epigenetics; 2) placental mitochondrial function; 3) placental transport systems.
Assuntos
Epigênese Genética , Mitocôndrias/metabolismo , Placenta/metabolismo , Placentação/fisiologia , Animais , Transporte Biológico/fisiologia , Feminino , Humanos , GravidezRESUMO
BACKGROUND: Maternal obesity is a major problem in obstetrics, and the placenta is involved in obesity-related complications via its roles at the maternal-fetal interface. We have recently shown a causative role for micro(mi)RNA-210, a so called 'hypoxamir' regulated by HIF-1α, in mitochondrial dysfunction in placentas from women with preeclampsia. We also reported mitochondrial dysfunction in placentas with maternal obesity. Here we hypothesized that expression of miR-210 is dysregulated in the placentas with obesity. METHODS: Placentas from uncomplicated pregnancies were collected at term from healthy weight or control (CTRL, pre-pregnancy body mass index (BMI)<25), overweight (OW, BMI=25-24.9) and obese (OB, BMI>30) women following C-section with no labor. Expression of miRNA-210 and its target genes was measured by reverse transcription-PCR and Western Blot, respectively. Mitochondrial respiration was assessed by Seahorse Analyzer in syncytiotrophoblast (ST) 72 h after cytotrophoblast isolation. RESULTS: Expression of miR-210 was significantly increased in placentas of OB and OW women with female but not male fetuses compared with CTRL placentas of females. However, expression of HIF-1α in these placentas remained unchanged. Levels of tumor-necrosis factor-alpha (TNFα) were increased in OW and OB placentas of females but not males, and in silico analysis suggested that activation of miR-210 expression in these placentas might be activated by NFκB1 (p50) signaling. Indeed, chromatin Immunoprecipitation assay showed that NFkB1 binds to placental miR-210 promoter in a fetal sex-dependent manner. Female but not male STs treated with TNFα showed overexpression of miR-210, reduction of mitochondrial target genes and decreased mitochondrial respiration. Pre-treatment of these STs with small interfering RNA to NFkB1 or antagomiR-210 prevented the TNFα-mediated inhibition of mitochondrial respiration. CONCLUSIONS: Our data suggest that the inflammatory intrauterine environment associated with maternal obesity induces an NFκB1-mediated increase in miR-210 in a fetal sex-dependent manner, leading to inhibition of mitochondrial respiration and placental dysfunction in the placentas of female fetuses.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/metabolismo , MicroRNAs/metabolismo , Obesidade/metabolismo , Placenta/metabolismo , Complicações na Gravidez/metabolismo , Adulto , Western Blotting , Hipóxia Celular , Feminino , Desenvolvimento Fetal , Humanos , Imunidade Inata , Inflamação/imunologia , Mitocôndrias , NF-kappa B/metabolismo , Obesidade/complicações , Obesidade/fisiopatologia , Placenta/imunologia , Gravidez , Complicações na Gravidez/fisiopatologia , Gestantes , Transporte Proteico , Caracteres Sexuais , Nascimento a TermoRESUMO
OBJECTIVE: Smoking and pre-eclampsia (PE) are associated with increases in preterm birth, placental abruption and low birthweight. We evaluated the relationship between prenatal vitamin C and E (C/E) supplementation and perinatal outcomes by maternal self-reported smoking status focusing on outcomes known to be impacted by maternal smoking. DESIGN/SETTING/POPULATION: A secondary analysis of a multi-centre trial of vitamin C/E supplementation starting at 9-16 weeks in low-risk nulliparous women with singleton gestations. METHODS: We examined the effect of vitamin C/E by smoking status at randomisation using the Breslow-Day test for interaction. MAIN OUTCOME MEASURES: The trial's primary outcomes were PE and a composite outcome of pregnancy-associated hypertension (PAH) with serious adverse outcomes. Perinatal outcomes included preterm birth and abruption. RESULTS: There were no differences in baseline characteristics within subgroups (smokers versus nonsmokers) by vitamin supplementation status. The effect of prenatal vitamin C/E on the risk of PE (P = 0.66) or PAH composite outcome (P = 0.86) did not differ by smoking status. Vitamin C/E was protective for placental abruption in smokers (relative risk [RR] 0.09; 95% CI 0.00-0.87], but not in nonsmokers (RR 0.92; 95% CI 0.52-1.62) (P = 0.01), and for preterm birth in smokers (RR 0.76; 95% CI 0.58-0.99) but not in nonsmokers (RR 1.03; 95% CI 0.90-1.17) (P = 0.046). CONCLUSION: In this cohort of women, smoking was not associated with a reduction in PE or the composite outcome of PAH. Vitamin C/E supplementation appears to be associated with a reduction in placental abruption and preterm birth among smokers.
Assuntos
Descolamento Prematuro da Placenta/epidemiologia , Hipertensão Induzida pela Gravidez/epidemiologia , Pré-Eclâmpsia/epidemiologia , Nascimento Prematuro/epidemiologia , Fumar/epidemiologia , Vitaminas/administração & dosagem , Adolescente , Adulto , Ácido Ascórbico/administração & dosagem , Suplementos Nutricionais , Método Duplo-Cego , Feminino , Humanos , Gravidez , Vitamina E/administração & dosagem , Adulto JovemRESUMO
The pivotal role of glucocorticoids in the initiation of parturition has been very well documented in several domestic mammalian animal species. However the role of glucocorticoids in human parturition remains controversial mainly because of the absence of effect of synthetic glucocorticoids, given to promote fetal organ maturation in pregnant women with threatened preterm delivery, on the length of gestation. This article will review studies of glucocorticoids in human parturition and provide evidence for an important role of glucocorticoids in human parturition as well but a simultaneous high concentration of estrogen within the intrauterine tissues may be necessary for GCs to initiate parturition. The synthetic GCs dexamethasone and betamethasone pass through the placenta intact resulting in potent negative feedback on the fetal HPA axis and diminished production of DHEA from fetal adrenal glands for estrogen synthesis by the placenta. This may negate the effect of systemic administration of GCs on the induction of labor, especially in cases where the myometrium is not yet fully primed by estrogen. Endogenous glucocorticoids are inactivated by the placental 11ß-HSD2 thus limiting the negative feedback of maternal cortisol on the fetal HPA axis and allowing the simultaneous rise of cortisol and estrogen levels towards the end of gestation. Therefore, endogenous glucocorticoids, particularly glucocorticoids produced locally in the intrauterine tissues may play an important role in parturition in humans by enhancing prostaglandin production in the fetal membranes and stimulating estrogen and CRH production in the placenta.
Assuntos
Glucocorticoides/metabolismo , Parto/metabolismo , Placenta/metabolismo , Feminino , Humanos , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , GravidezRESUMO
Biobanks provide an important repository of samples for research purposes. However, for those samples to reflect the in vivo state, and for experimental reliability and reproducibility, careful attention to collection, processing and storage is essential. This is particularly true for the placenta, which is potentially subjected to stressful conditions during delivery, and sample collection may be delayed owing to routine postpartum inspection by clinical staff. In addition, standardisation of the collection procedure enables samples to be shared among research groups, allowing larger datasets to be established. Here, we provide an evidence-based and experts' review of the factors surrounding collection that may influence data obtained from the human placenta. We outline particular requirements for specific techniques, and propose a protocol for optimal sample collection. We recognise that the relevance of these factors, and of the sample types collected to a particular study will depend on the research questions being addressed. We therefore anticipate that researchers will select from the protocol to meet their needs and resources available. Wherever possible, we encourage researchers to extend their collection to include additional samples that can be shared on an international collaborative basis, with appropriate informed consent, to raise the quality, as well as quantity, of placental research.
Assuntos
Bancos de Espécimes Biológicos , Placenta , Manejo de Espécimes/métodos , Feminino , Humanos , Imuno-Histoquímica , Consentimento Livre e Esclarecido/ética , Metabolômica/métodos , Microscopia Eletrônica , Tamanho do Órgão , Estresse Oxidativo , Placenta/anatomia & histologia , Placenta/microbiologia , Gravidez , Manejo de Espécimes/normas , Cordão UmbilicalRESUMO
Preeclampsia (PE) affects 5-8% of pregnancies and is responsible for 18% of maternal deaths in the US, and for long-term complications in mother and child. PE is an inflammatory state and may influence placental function in a sex-specific manner. We determined if there is a sexual dimorphism in the placental inflammatory and apoptotic responses in preeclamptic pregnancies. Placentas were collected from normotensive and preeclamptic pregnancies with either male or female fetuses (MPE and FPE respectively) after c-section at term with no labor. Expression patterns of markers of inflammation measured by ELISA, as well as hypoxia, apoptosis and angiogenesis markers measured by Western blotting were determined in the placenta. Consistent with previous studies, an increase in inflammation, hypoxia, and apoptotic cell death was observed in PE compared to normotensive pregnancies. Levels of TNFα, IL-6 and IL-8, and HIF-1α were significantly greater, whereas the angiogenic marker VEGF was significantly reduced in MPE vs. FPE. Sexual dimorphism was also observed in the activation of cell death: the number of TUNEL-positive cells, and the expression pro-apoptotic markers PUMA and Bax being higher in MPE vs. FPE. We also found an increase in the levels of protein and DNA-binding activity of NFκB p65 in MPE vs. FPE. In summary, we show here that in preeclamptic pregnancies the placentas of males were associated with significantly higher expression of inflammatory, hypoxia and apoptotic molecules but reduced expression of a pro-angiogenic marker compared to placentas of female fetuses. We propose that the transcription factor NFκB p65 might, at least partially, be involved in sexual dimorphism during PE.
Assuntos
Núcleo Celular/metabolismo , Desenvolvimento Fetal , Placenta/fisiopatologia , Pré-Eclâmpsia/fisiopatologia , Fator de Transcrição RelA/metabolismo , Adulto , Proteínas Angiogênicas/metabolismo , Apoptose , Biomarcadores/metabolismo , Hipóxia Celular , Cesárea , Citocinas/metabolismo , Feminino , Humanos , Recém-Nascido , Masculino , Placenta/imunologia , Placenta/metabolismo , Placenta/patologia , Pré-Eclâmpsia/imunologia , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Transporte Proteico , Índice de Gravidade de Doença , Caracteres Sexuais , Nascimento a Termo , Adulto JovemRESUMO
OBJECTIVE: To determine if change in maternal angiogenic biomarkers between the first and second trimesters predicts pre-eclampsia in low-risk nulliparous women. DESIGN: A nested case-control study of change in maternal plasma soluble Flt-1 (sFlt-1), soluble endoglin (sEng) and placenta growth factor (PlGF). We studied 158 pregnancies complicated by pre-eclampsia and 468 normotensive nonproteinuric controls. SETTING: A multicentre study in 16 academic medical centres in the USA. POPULATION: Low-risk nulliparous women. METHODS: Luminex assays for PlGF, sFlt-1 and sEng performed on maternal EDTA plasma collected at 9-12, 15-18 and 23-26 weeks of gestation. Rate of change of analyte between first and either early or late second trimester was calculated with and without adjustment for baseline clinical characteristics. MAIN OUTCOME MEASURES: Change in PlGF, sFlt-1 and sEng. RESULTS: Rates of change of PlGF, sEng and sFlt-1 between first and either early or late second trimesters were significantly different in women who developed pre-eclampsia, severe pre-eclampsia or early-onset pre-eclampsia compared with women who remained normotensive. Inclusion of clinical characteristics (race, body mass index and blood pressure at entry) increased sensitivity for detecting severe and particularly early-onset pre-eclampsia but not pre-eclampsia overall. Receiver operating characteristics curves for change from first to early second trimester in sEng, PlGF and sFlt-1 with clinical characteristics had areas under the curve of 0.88, 0.84 and 0.86, respectively, and for early-onset pre-eclampsia with sensitivities of 88% (95% CI 64-99), 77% (95% CI 50-93) and 77% (95% CI 50-93) for 80% specificity, respectively. Similar results were seen in the change from first to late second trimester. CONCLUSION: Change in angiogenic biomarkers between first and early second trimester combined with clinical characteristics has strong utility for predicting early-onset pre-eclampsia.
Assuntos
Antígenos CD/sangue , Pré-Eclâmpsia/sangue , Proteínas da Gravidez/sangue , Primeiro Trimestre da Gravidez/sangue , Segundo Trimestre da Gravidez/sangue , Receptores de Superfície Celular/sangue , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/sangue , Adulto , Biomarcadores/sangue , Pressão Sanguínea , Índice de Massa Corporal , Diagnóstico Precoce , Endoglina , Feminino , Humanos , Estudos Longitudinais , Paridade , Fator de Crescimento Placentário , Pré-Eclâmpsia/diagnóstico , Pré-Eclâmpsia/etnologia , Gravidez , Fatores de Risco , Adulto JovemRESUMO
Compelling evidence indicates a crucial role of prostaglandin F2α (PGF2α) in parturition. Both the maternal and fetal sides of the fetal membranes synthesize PGF2α, which exerts effects via the prostaglandin F2α receptor (FP) that is coupled to the activation of protein kinase C (PKC). Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step of the inducible synthesis of prostaglandin. Although activation of PKC is known to induce COX-2 expression, it is not clear whether PGF2α can induce COX-2 via FP receptor-coupled PKC activation. COX-2 promoter carries a cAMP-response element (CRE) and phosphorylation of CRE binding protein 1 (CREB1) is associated with COX-2 expression in human amnion fibroblasts. We demonstrated that human amnion fibroblasts produced PGF2α and expressed FP receptor. PGF2α increased COX-2 expression and CREB1 phosphorylation, which could be blocked by either the FP receptor antagonist AL8810 or PKC inhibitor Ro31-7549. The PKC activator, phorbol-12-myristate-13-acetate (PMA), could mimic the induction of COX-2 and CREB1 phosphorylation. The induction of COX-2 by PGF2α and PMA could be attenuated by the small interfering RNA-mediated knockdown of CREB1 expression or overexpressing dominant-negative CREB1. A chromatin immunoprecipitation assay showed that the binding of CREB1 to the COX-2 promoter was increased by PGF2α and PMA in amnion fibroblasts. In conclusion, we provide evidence that PGF2α induces COX-2 expression via the FP receptor and phosphorylates CREB1 by PKC, thus increasing CREB1 binding to the COX-2 promoter and the expression of COX-2 in human amnion fibroblasts. This feed-forward loop may be crucial for the production of prostaglandins in the fetal membranes prior to the onset of labor.
Assuntos
Âmnio/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprosta/metabolismo , Fibroblastos/metabolismo , Proteína Quinase C/metabolismo , Âmnio/citologia , Âmnio/efeitos dos fármacos , Células Cultivadas , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Dinoprosta/biossíntese , Dinoprosta/farmacologia , Dinoprostona/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Regiões Promotoras Genéticas , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Preeclampsia (PE) affects 5-8% of all pregnancies and is associated with significant maternal and fetal morbidity and mortality. Placental mitochondrial dysfunction has been reported in PE. MicroRNAs (miRNA) are small non-coding RNAs that regulate gene expression through mRNA degradation and translational repression. MiR-210 has been previously shown to be upregulated in placentas from pregnancies complicated by PE. We hypothesized that placental mitochondrial dysfunction during PE can be mediated by miR-210. Placentas were collected at term from normotensive pregnancies (CTRL) and those complicated by severe PE (n = 6 each) following c-section (no labor). Villous tissue from PE showed significantly increased levels of HIF-1α compared to CTRL with no change in corresponding mRNA expression but with reduced DNA-binding activity. Mitochondrial complex III was significantly decreased in PE along with significantly reduced protein expression in complex I and IV during PE. Among the four miRNAs tested, miR-210 showed significant up regulation in PE and significant downregulation of its target, ISCU mRNA. To understand the role of miR-210 in PE, loss- and gain-of-function studies were performed using primary trophoblasts. Trophoblasts were transfected with miR-210 inhibitor or pre-miR-210 and mitochondrial function was measured using Seahorse Extracellular Flux Analyzer. Cells transfected with pre-miR-210 showed significant reduction in oxygen consumption. In contrast, transfection of trophoblast with AntagomiR-210 was sufficient to prevent the DFO-mediated respiratory deficiency. These data collectively suggest that miR-210 overexpression during PE could be responsible for placental mitochondria dysfunction.
Assuntos
MicroRNAs/fisiologia , Mitocôndrias/metabolismo , Placenta/fisiopatologia , Pré-Eclâmpsia/fisiopatologia , Adulto , Regulação para Baixo , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , GravidezRESUMO
Pregnancy is a state of oxidative stress, which becomes exaggerated under pathological conditions, such as preeclampsia, IUGR, diabetes and obesity, where placental mitochondrial dysfunction is observed. The majority of investigations utilize isolated mitochondria when measuring mitochondrial activity in placenta. However, this does not provide a complete physiological readout of mitochondrial function. This technical note describes a method to measure respiratory function in intact primary syncytiotrophoblast from human term placenta.
Assuntos
Técnicas de Cultura de Células , Mitocôndrias/metabolismo , Consumo de Oxigênio , Trofoblastos/metabolismo , Respiração Celular , Células Cultivadas , Feminino , Humanos , Hipóxia/metabolismo , GravidezRESUMO
CONTEXT: Overexposure of the fetus to glucocorticoids early in gestation is detrimental to fetal development. Glucocorticoid concentrations in the fetal circulation are kept low by 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2, encoded by HSD11B2) in the placental syncytiotrophoblasts. However, cytotrophoblasts, the progenitors of syncytiotrophoblasts, express low levels of 11ß-HSD2. Here we studied the molecular mechanisms underlying 11ß-HSD2 induction upon syncytialization. METHODS: Freshly isolated human term placental cytotrophoblasts and in vitro differentiated syncytiotrophoblasts were examined to determine the methylation status of HSD11B2 promoter. The transcription factor responsible for 11ß-HSD2 induction was identified by observing its expression upon syncytialization, the effect of its attenuation, and its binding to the HSD11B2 promoter. RESULTS: 11ß-HSD2 expression was markedly increased upon syncytialization in vitro. No methylation differences of HSD11B2 promoter were found between cytotrophoblasts and syncytiotrophoblasts. Expression of the transcription factor Sp1 was markedly induced during syncytialization and further increased by activation of the cAMP pathway, which correlated with 11ß-HSD2 expression. Importantly, small interfering RNA-mediated knockdown of Sp1 expression or inhibition of Sp1 activity with mithramycin A markedly attenuated not only basal but also cAMP pathway-stimulated expression of 11ß-HSD2 in the syncytiotrophoblasts. Stimulation of the cAMP pathway also increased the binding of Sp1 and RNA polymerase II to HSD11B promoter in syncytiotrophoblasts. Concomitantly, acetylation at histone H3K9 was increased whereas methylation at histone H3K9 was decreased. CONCLUSIONS: 11ß-HSD2 induction upon syncytialization is at least in part due to the increased expression of Sp1 upon activation of the cAMP pathway rather than the differential methylation of the HSD11B2 promoter.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , Placenta/metabolismo , Fator de Transcrição Sp1/genética , Trofoblastos/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Análise de Variância , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , AMP Cíclico/genética , AMP Cíclico/metabolismo , Metilação de DNA , Feminino , Humanos , Imuno-Histoquímica , Placenta/citologia , Gravidez , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Fator de Transcrição Sp1/metabolismoRESUMO
Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 there were twelve themed workshops, six of which are summarized in this report. 1. The immunology workshop focused on normal and pathological functions of the maternal immune system in pregnancy. 2. The transport workshop dealt with regulation of ion and water transport across the syncytiotrophoblast of human placenta. 3. The epigenetics workshop covered DNA methylation and its potential role in regulating gene expression in placental development and disease. 4. The vascular reactivity workshop concentrated on methodological approaches used to study placental vascular function. 5. The workshop on epitheliochorial placentation covered current advances from in vivo and in vitro studies of different domestic species. 6. The proteomics workshop focused on a variety of techniques and procedures necessary for proteomic analysis and how they may be implemented for placental research.
Assuntos
Feto/fisiologia , Placenta/fisiologia , Trofoblastos/fisiologia , Animais , Educação , Epigênese Genética/fisiologia , Feminino , Feto/irrigação sanguínea , Feto/citologia , Feto/imunologia , Humanos , Transporte de Íons/fisiologia , Troca Materno-Fetal/fisiologia , Placenta/irrigação sanguínea , Placenta/citologia , Placenta/imunologia , Placentação/fisiologia , Gravidez , Proteômica/métodos , Trofoblastos/citologia , Trofoblastos/imunologiaRESUMO
UNLABELLED: HYPOTHESIS AND STUDY OBJECTIVES: We hypothesized that maternal obesity is associated with increased placental amino acid transport and hyperleptinemia. Our objectives were to study placental amino acid transport and the effect of leptin on placental amino acid transport in vitro in the setting of maternal obesity. MATERIALS AND METHODS: Seven lean, BMI at entry 22.4, and seven obese, BMI at entry 31.5 (p < 0.001), pregnant women were studied at 39 weeks. We measured baseline and leptin-stimulated placental system A sodium-dependent neutral amino acid transporter (SNAT) activity, placental immunoreactive protein expression of SNAT, leptin and leptin receptor, and maternal and fetal plasma leptin concentrations, with significance set at p Assuntos
Sistema A de Transporte de Aminoácidos/metabolismo
, Leptina/sangue
, Obesidade/metabolismo
, Placenta/metabolismo
, Complicações na Gravidez/metabolismo
, Índice de Massa Corporal
, Feminino
, Humanos
, Imuno-Histoquímica
, Recém-Nascido
, Insulina/sangue
, Gravidez
RESUMO
The placenta regulates fetal growth and development via transport of nutrients and gases, and synthesis and secretion of steroid and peptide hormones. These functions are determined by vascular development and blood flow and by growth and differentiation of the trophoblast, which contains receptors, transporters and enzymes. The placenta generates reactive oxygen species which may contribute to the oxidative stress seen even in normal pregnancy but this is increased in pregnancies complicated by preeclampsia, IUGR and pregestational diabetes where oxidative and nitrative stress have been clearly documented. Nitrative stress is the covalent modification of proteins and DNA by peroxynitrite formed by the interaction of superoxide and nitric oxide. We have demonstrated nitrative stress by localizing nitrotyrosine residues in these placentas and found increased expression of NADPH oxidase (NOX) enzyme isoforms 1 and 5 as a potential source of superoxide generation. The presence of nitrative stress was associated with diminished vascular reactivity of the fetal placental circulation, a situation that could be reproduced by treatment with peroxynitrite in vitro. We find many nitrated proteins in the placenta, including p38 MAP kinase which has a role in development of the villous vasculature. Nitration of p38 MAPK was increased in the preeclamptic placenta and associated with loss of catalytic activity. We hypothesize that nitration of proteins in the placenta including receptors, transporters, enzymes and structural proteins can alter protein and placental function and this influences fetal growth and development. Increasing nitrative stress but a decrease in oxidative stress, measured as protein carbonylation, is found in the placenta with increasing BMI. Formation of peroxynitrite may then consume superoxide, decreasing nitrative stress. As protein carbonylation is a covalent modification at Lys, Arg, Pro and Thr residues the switch from carbonylation to nitration at tyrosine residues may alter protein function and hence placental function.
Assuntos
Placenta/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Feminino , Humanos , Troca Materno-Fetal , Oxirredução , Estresse Oxidativo , Pré-Eclâmpsia/etiologia , Pré-Eclâmpsia/metabolismo , GravidezRESUMO
CONTEXT: Human amnion fibroblasts produce abundant prostaglandins toward the end of gestation, which is believed to be one of the major events leading to parturition. Glucocorticoids have been shown to up-regulate cyclooxygenase-2 (COX-2) expression, the crucial enzyme catalyzing prostaglandin synthesis, in human amnion fibroblasts. Although a major propregnancy hormone, the effect of progesterone and the associated progesterone receptor subtypes in the regulation of both basal and glucocorticoid-induced COX-2 expression in human amnion fibroblasts have not been resolved. METHODS AND RESULTS: Cultured human amnion fibroblasts prepared from the fetal membranes at term pregnancy without labor mainly expressed the progesterone receptor A form (PRA). Inhibition of endogenous progesterone production with trilostane or knockdown of PRA expression with small interfering RNA significantly enhanced the glucocorticoid receptor (GR)-mediated COX-2 induction by cortisol, whereas overexpression of PRA attenuated the induction by cortisol. Co-immunoprecipitation assay revealed PRA in the GR protein complex. Although exogenous progesterone did not alter COX-2 expression under basal conditions, it attenuated cortisol-induced COX-2 expression at concentrations about 10- to 50-fold higher, which might be achieved by competition with cortisol for GR. CONCLUSIONS: We demonstrated in this study that endogenous progesterone might counteract the induction of prostaglandin synthesis by cortisol via PRA transdominant repression of GR function, whereas high levels of progesterone might further inhibit the induction by cortisol via competitive binding to GR in human amnion fibroblasts. These inhibitory actions of progesterone and PRA on glucocorticoids and GR may partly explain the inconsistent effects of glucocorticoids on parturition in humans.
Assuntos
Líquido Amniótico/enzimologia , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Fibroblastos/enzimologia , Hidrocortisona/fisiologia , Progesterona/fisiologia , Receptores de Progesterona/biossíntese , Receptores de Progesterona/genética , Adulto , Líquido Amniótico/citologia , Western Blotting , Células Cultivadas , Dinoprostona/biossíntese , Dinoprostona/genética , Feminino , Feto/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Imunoprecipitação , Plasmídeos/genética , Gravidez , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Human amnion fibroblasts produce abundant prostaglandins toward the end of gestation, which is one of the major events leading to parturition. In marked contrast to its well-described antiinflammatory effect, glucocorticoids have been shown to up-regulate cyclooxygenase-2 (COX-2) expression in human amnion fibroblasts. The mechanisms underlying this paradoxical induction of COX-2 by glucocorticoids have not been resolved. Using cultured human amnion fibroblasts, we found that the induction of COX-2 mRNA expression by cortisol was a glucocorticoid receptor (GR)-dependent process requiring ongoing transcription. Upon transfection of a COX-2 promoter-driven reporter gene into the amnion fibroblasts, cortisol stimulated the COX-2 promoter activity. This was abolished by mutagenesis of a cAMP response element (CRE) at -53 to approximately -59bp as well as by cotransfection of a plasmid expressing dominant-negative CRE-binding protein (CREB). The phosphorylation level of CREB-1 was significantly increased by cortisol treatment of the amnion fibroblasts, whereas the effect was attenuated either by the protein kinase A inhibitor H89 or the p38 -MAPK inhibitor SB203580. The induction of the COX-2 promoter activity and the phosphorylation of CREB-1 were also blocked by the GR antagonist RU486. Chromatin immunoprecipitation (ChIP) assay revealed that the binding of CREB-1 to the CRE of the COX-2 promoter was increased by cortisol treatment of the amnion fibroblasts. In conclusion, cortisol, via binding to GR, stimulated COX-2 expression by increasing phosphorylated CREB-1 binding to the CRE of the COX-2 gene. Cortisol may phosphorylate CREB-1 by activating either protein kinase A or p38-MAPK in the amnion fibroblasts.
Assuntos
Âmnio/citologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/biossíntese , Fibroblastos/metabolismo , Regulação Enzimológica da Expressão Gênica , Glucocorticoides/metabolismo , Elementos de Resposta , Sequência de Bases , Inibidores Enzimáticos/farmacologia , Antagonistas de Hormônios/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases , Mifepristona/farmacologia , Dados de Sequência Molecular , FosforilaçãoRESUMO
Maternal obesity is an increasing problem in obstetrics associated with adverse pregnancy outcomes and delivery complications. As an inflammatory state, where elevated levels of pro-inflammatory cytokines are found, obesity can lead to the increased incidence of oxidative and nitrative stress. These stresses may result in protein oxidation and protein nitration respectively, which are post- translational covalent modifications that can modify the structure and subsequently alter the function of a protein. The objective of this study was to examine whether placental oxidative and nitrative stress increase with increasing maternal body mass index. Placental tissue was collected from three groups of patients categorized as lean, overweight and obese. The presence of nitrotyrosine residues, a marker of nitrative stress, and antioxidant enzymes, as markers of oxidative stress, were assessed by immunohistochemistry, Western blot and ELISA. Protein carbonyl formation, a specific measure of protein oxidation, was measured by OxyBlot kit. Nitrotyrosine residues were increased in obese compared to lean and overweight groups although localization was unaltered across the three groups. Superoxide dismutase enzyme expression, localization and activity was unaltered between the groups. Protein carbonyl formation was greater in the lean compared to the overweight individuals. This study demonstrates that with increasing maternal body mass index there is an increase in placental nitrative stress. There does not appear to be a corresponding increase in oxidative stress and indeed we demonstrate some evidence of a decrease in oxidative effects in these placenta samples. Potentially the formation of peroxynitrite may be consuming reactive oxygen species and reducing oxidative stress. There may be a shift in the balance between nitrative and oxidative stress, which may be a protective mechanism for the placenta.
Assuntos
Índice de Massa Corporal , Obesidade/metabolismo , Estresse Oxidativo , Placenta/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Magreza/metabolismo , Adulto , Biomarcadores/metabolismo , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Gravidez , Carbonilação Proteica , Superóxido Dismutase/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Adulto JovemRESUMO
Preeclampsia, a pregnancy-specific syndrome characterized by hypertension, proteinuria and edema, resolves on delivery of the placenta. Normal pregnancy is itself characterized by systemic inflammation, oxidative stress and alterations in levels of angiogenic factors and vascular reactivity. This is exacerbated in preeclampsia with an associated breakdown of compensatory mechanisms, eventually leading to placental and vascular dysfunction. The underlying pathology of preeclampsia is thought to be a relatively hypoxic or ischemic placenta. Both the placenta and maternal vasculatures are major sources of reactive oxygen and nitrogen species which can interact to produce peroxynitrite a powerful prooxidant that covalently modifies proteins by nitration of tyrosine residues, to possibly alter vascular function in preeclampsia. The linkage between placental hypoxia and maternal vascular dysfunction has been proposed to be via placental syncytiotrophoblast basement membranes shed by the placenta or via angiogenic factors which include soluble flt1 and endoglin secreted by the placenta that bind vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) in the maternal circulation. There is also abundant evidence of altered reactivity of the maternal and placental vasculature and of the altered production of autocoids in preeclampsia. The occurrence of preeclampsia is increased in women with preexisting vascular disease and confers a long-term risk for development of cardiovascular disease. The vascular stress test of pregnancy thus identifies those women with a previously unrecognized at risk vascular system and promotes the development of preeclampsia. Preexisting maternal vascular dysfunction intensified by placental factors is possibly responsible for the individual pathologies of preeclampsia.
Assuntos
Vasos Sanguíneos/fisiopatologia , Pré-Eclâmpsia/fisiopatologia , Endotélio Vascular/patologia , Feminino , Humanos , Placenta/irrigação sanguínea , GravidezRESUMO
Crucial roles of the placenta are disrupted in early and mid-trimester pregnancy loss, preeclampsia, eclampsia and intrauterine growth restriction. The pathophysiology of these disorders includes a relative hypoxia of the placenta, ischemia/reperfusion injury, an inflammatory response and oxidative stress. Reactive oxygen species including nitric oxide (NO), carbon monoxide and superoxide have been shown to participate in trophoblast invasion, regulation of placental vascular reactivity and other events. Superoxide, which regulates expression of redox sensitive genes, has been implicated in up-regulation of transcription factors, antioxidant production, angiogenesis, proliferation and matrix remodeling. When superoxide and nitric oxide are present in abundance, their interaction yields peroxynitrite a potent pro-oxidant, but also alters levels of nitric oxide, which in turn affect physiological functions. The peroxynitrite anion is extremely unstable thus evidence of its formation in vivo has been indirect via the occurrence of nitrated moieties including nitrated lipids and nitrotyrosine residues in proteins. Formation of 3-nitrotyrosine (protein nitration) is a "molecular fingerprint" of peroxynitrite formation. Protein nitration has been widely reported in a number of pathological states associated with inflammation but is reported to occur in normal physiology and is thought of as a prevalent, functionally relevant post-translational modification of proteins. Nitration of proteins can give either no effect, a gain or a loss of function. Nitration of a range of placental proteins is found in normal pregnancy but increased in pathologic pregnancies. Evidence is presented for nitration of placental signal transduction enzymes and transporters. The targets and extent of nitration of enzymes, receptors, transporters and structural proteins may markedly influence placental cellular function in both physiologic and pathologic settings.