ABSTRACT
The fate of pluripotent cells in early mouse embryos is controlled by graded Nodal signals that are activated by the endoproteases Furin and Pace4. Soluble forms of Furin and Pace4 cleave proNodal in vitro and after secretion in transfected cells, but direct evidence for paracrine activity in vivo is elusive. Here, we show that Furin and Pace4 are released by the extraembryonic microenvironment, and that they cleave a membrane-bound reporter substrate in adjacent epiblast cells and activate Nodal to maintain pluripotency. Secreted Pace4 and Furin also stimulated mesoderm formation, whereas endoderm was only induced by Pace4, correlating with a difference in the spatiotemporal distribution of these proteolytic activities. Our analysis of paracrine Furin and Pace4 activities and their in vivo functions significantly advances our understanding of how the epiblast is patterned by its microenvironment. Adding cell-cell communication to the pleiotropic portfolio of these proteases provides a new framework to study proprotein processing also in other relevant contexts.
Subject(s)
Furin/metabolism , Germ Layers/enzymology , Paracrine Communication , Pluripotent Stem Cells/metabolism , Proprotein Convertases/metabolism , Animals , Ectoderm/embryology , Endoderm/drug effects , Endoderm/embryology , Extraembryonic Membranes/enzymology , Furin/pharmacology , Gene Expression Regulation, Developmental/drug effects , Mesoderm/drug effects , Mesoderm/embryology , Mice , Nodal Protein/metabolism , Proprotein Convertases/pharmacology , Signal Transduction/physiologyABSTRACT
OBJECTIVE: Lysyl oxidase (LOX) and LOX like enzymes (LOXL1-4) physiologically remodel extracellular matrix and pathologically contribute to cellular senescence under oxidative stress (OS). We characterized LOX and LOXL expressions and activity in human fetal membranes. METHODS: Human fetal membranes from women with uncomplicated pregnancies at term, preterm birth with intact membranes (PTB) or preterm prelabor rupture of membranes (pPROM), and in vitro fetal membranes stimulated with water-soluble cigarette smoke extract (CSE), an OS inducer, were analyzed by real-time PCR and immunohistochemistry for LOX and LOXL (1-4) expression and localization. LOX activity was measured by fluorometric assay. RESULTS: LOX gene expression was â¼2.5-fold higher in fetal membranes from pPROM compared to PTB and term (P=0.02). LOX and LOXL1, 2 and 4 were localized to both amniotic and chorionic cells, whereas LOXL3 was limited to chorion. LOX and LOXL isoform expressions were not different between CSE treated and untreated groups, while LOX activity was increased in the presence of an antioxidant (P=0.02). CONCLUSIONS: Increase of LOX expression in pPROM, an OS-related disease, and the apparent inhibition of LOX activity by CSE restored by antioxidant treatment suggest that reactive oxygen species might influence LOX-mediated tissue remodeling in fetal membranes. Balanced antioxidant supplementation during pregnancy may reduce the risk of pPROM by increasing LOX activity.
Subject(s)
Amino Acid Oxidoreductases/metabolism , Extraembryonic Membranes/enzymology , Fetal Membranes, Premature Rupture/enzymology , Protein-Lysine 6-Oxidase/metabolism , Adult , Amino Acid Oxidoreductases/genetics , Case-Control Studies , Extraembryonic Membranes/pathology , Female , Fetal Membranes, Premature Rupture/genetics , Fetal Membranes, Premature Rupture/pathology , Gene Expression , Humans , Immunohistochemistry , Infant, Newborn , Pregnancy , Premature Birth , Protein-Lysine 6-Oxidase/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Young AdultABSTRACT
Preterm birth remains the largest single cause of neonatal death and morbidity. Infection and/or inflammation are strongly associated with preterm delivery. Glycogen synthase kinase 3 (GSK3) is known to be a crucial mediator of inflammation homeostasis. The aims of this study were to determine the effect of spontaneous human labour in foetal membranes and myometrium on GSK3α/ß expression, and the effect of inhibition of GSK3α/ß on pro-labour mediators in foetal membranes and myometrium stimulated with Toll-like receptor (TLR) ligands and pro-inflammatory cytokines. Term and preterm labour in foetal membranes was associated with significantly decreased serine phosphorylated GSK3α and ß expression, and thus increased GSK3 activity. There was no effect of term labour on serine phosphorylated GSK3ß expression in myometrium. The specific GSK3α/ß inhibitor CHIR99021 significantly decreased lipopolysaccharide (ligand to TLR4)-stimulated pro-inflammatory cytokine gene expression and release; COX2 gene expression and prostaglandin release; and MMP9 gene expression and pro MMP9 release in foetal membranes and/or myometrium. CHIR99021 also decreased FSL1 (TLR2 ligand) and flagellin (TLR5 ligand)-induced pro-inflammatory cytokine gene expression and release and COX2 mRNA expression and prostaglandin release. GSK3ß siRNA knockdown in primary myometrial cells was associated with a significant decrease in IL1ß and TNFα-induced pro-inflammatory cytokine and prostaglandin release. In conclusion, GSK3α/ß activity is increased in foetal membranes after term and preterm labour. Pharmacological blockade of the kinase GSK3 markedly reduced pro-inflammatory and pro-labour mediators in human foetal membranes and myometrium, providing a possible therapeutics for the management of preterm labour.
Subject(s)
Glycogen Synthase Kinase 3/physiology , Labor, Obstetric/physiology , Extraembryonic Membranes/enzymology , Extraembryonic Membranes/physiology , Female , Gene Expression , Gene Silencing , Glycogen Synthase Kinase 3/analysis , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3 beta , Humans , Inflammation/prevention & control , Labor, Obstetric/drug effects , Lipopolysaccharides/pharmacology , Myometrium/enzymology , Myometrium/physiology , NF-kappa B/physiology , Pregnancy , Premature Birth/enzymology , Premature Birth/prevention & control , RNA, Small Interfering/genetics , Tissue Culture Techniques , Toll-Like Receptors/physiologyABSTRACT
PURPOSE: The present study was designed to examine apoptotic cell death via the caspase-dependent pathway in human fetal membranes. METHODS: Amniotic membrane samples were collected from three groups of women: group 1, women with preterm premature rupture of fetal membranes (PPROM) after cesarean delivery (n = 10), group 2, women with preterm labor (PTL) with intact membranes after cesarean delivery (n = 9) and group 3, women with term labor and vaginal delivery after an uncomplicated pregnancy (controls) (n = 11). RESULTS: Active caspase-3 immunopositivity (ACPI) of the PPROM group was significantly higher than that of the control group (p < 0.05). ACPI was higher in the PTL with intact membranes group as compared to the control group; however, it did not reach statistical significance (p > 0.05). CONCLUSION: Active caspase-3 positivity is increased in the fetal membranes of those women with PPROM.
Subject(s)
Apoptosis , Caspase 3/metabolism , Fetal Membranes, Premature Rupture/etiology , Adolescent , Adult , Case-Control Studies , Extraembryonic Membranes/enzymology , Female , Fetal Membranes, Premature Rupture/enzymology , Humans , Pregnancy , Young AdultABSTRACT
The maintenance of antioxidative/oxidative balance is crucial for cellular and extracellular environment. That is why antioxidative enzymes express their activity in different isoforms in different cell compartments and extracellular space. The aim of study was to verify the results of previous experiment on activities of antioxidative enzymes by the determination of their enzymatic proteins in bovine placental tissues by Western blotting technique. Moreover, the presence of particular isoenzymes was detected and differentiated. Homogenates of maternal and foetal part of both properly released and retained bovine placenta were subjected to PAGE electrophoresis in non-reducing and reducing conditions and Western blotting with appropriate antibodies against superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Electrophoresis allowed for the detection of protein bands of molecular weight related to CuZn-SOD as well as cGSH-Px isoenzymes. The reaction with appropriate antibodies confirmed this. Densitometric analysis, although semi-quantitative, allowed for the observation of trends in differences in antioxidative enzyme proteins, which may partly confirm previously described results in cases of retained and released placenta. Local antioxidative enzymatic mechanisms in bovine placental tissues are represented by CuZn-SOD and cGSH-Px, which show the changes in their expression during improper placental release.
Subject(s)
Cattle Diseases/enzymology , Extraembryonic Membranes/enzymology , Placenta, Retained/veterinary , Placenta/enzymology , Superoxide Dismutase/analysis , Animals , Antioxidants , Blotting, Western/veterinary , Cattle , Electrophoresis, Polyacrylamide Gel/veterinary , Female , Glutathione Peroxidase/analysis , Isoenzymes/analysis , Placenta, Retained/enzymology , Pregnancy , Superoxide Dismutase-1ABSTRACT
The present study investigated the expression of genes and proteins associated with PGF2alpha biosynthesis, catabolism, and transport in matched amnion and choriodecidua of human term placenta. The concentration of PGF2alpha within fetal membranes depends on the balance between complex enzymatic systems responsible for, respectively, its synthesis-by prostaglandin-endoperoxide synthase 2 (PTGS2) and members of the aldo-keto reductase (AKR) family, AKR1C3 and AKR1B1-and its catabolic inactivation-through hydroxy-prostaglandin-dehydrogenase (HPGD). We observed that AKR1C3 shows equal basal expression (mRNA and protein) in choriodecidua and amnion but that AKR1B1 exhibits preferential expression in the choriodecidua. Expression of HPGD and solute carrier organic anion transporter family member 2A1 (SLCO2A1) was found primarily in the choriodecidua. We also evaluated whether an inflammatory environment induced by the gram-negative bacterial endotoxin lipopolysaccharide (LPS) affects expression of each candidate enzymes. The amnion responded to LPS with a small but significant decrease of AKR1B1 mRNA expression. In contrast, we found a significant increase in PTGS2 and AKR1C3 mRNA expression in choriodecidua after LPS challenge, but such regulation was confirmed only at protein levels for PTGS2 and not for AKR1C3. Our results suggest that the choriodecidua appears to be the main tissue, which expresses maximally all the components (synthesis, degradation, and transport) controlling PGF2alpha levels.
Subject(s)
3-Hydroxysteroid Dehydrogenases/metabolism , Cyclooxygenase 2/metabolism , Dinoprost/metabolism , Extraembryonic Membranes/enzymology , Hydroxyprostaglandin Dehydrogenases/metabolism , Organic Anion Transporters/metabolism , Aldo-Keto Reductase Family 1 Member C3 , Female , Humans , Immunohistochemistry , Lipopolysaccharides , Placenta/enzymology , Pregnancy , RNA, Messenger/metabolismABSTRACT
Expression levels of cytochrome P450 (CYP) 3A4, CYP3A5 and CYP3A7 mRNAs in placentas and fetal membranes, which were split into amnion and chorion leave attached decidua (chorion/decidua), obtained from pregnant women with normal delivery (5 subjects) and Caesarean section (15 subjects) were determined. These CYP3A mRNAs were also expressed in amnion and chorion/decidua together with placenta, although the expression level of these mRNAs was strikingly different between subjects. The expression level of the CYP3A4 mRNA in the placenta was about 2-fold higher than those in amnion and chorion/decidua. On the other hand, the expression levels of CYP3A5 and CYP3A7 mRNAs were highest in chorion/decidua. The immunologically related protein(s) with CYP3A7 was detected in all tissues examined. Testosterone 6beta-hydroxylase activity in homogenate of human placenta, amnion and chorion/decidua were 26.6, 3.7 and 4.6 pmol/h/mg protein, respectively. These results suggest that CYP3As in fetal membranes have the metabolic function to protect the fetus from exposure to drugs.
Subject(s)
Aryl Hydrocarbon Hydroxylases/biosynthesis , Cytochrome P-450 CYP3A/biosynthesis , Extraembryonic Membranes/enzymology , Gene Expression Regulation, Enzymologic , Placenta/enzymology , Steroid Hydroxylases/metabolism , Term Birth/genetics , Aryl Hydrocarbon Hydroxylases/genetics , Cytochrome P-450 CYP3A/genetics , Enzyme Activation/genetics , Female , Humans , Infant, Newborn , Isoenzymes/biosynthesis , Isoenzymes/genetics , Pregnancy , Pregnancy Proteins/biosynthesis , Pregnancy Proteins/geneticsABSTRACT
A mechanism of mammalian teratogenesis involving inhibition of embryotrophic nutrition is suggested and exemplified by the action of trypan blue on pregnant rats. There is evidence for the localization of trypan blue in heterolysosomes of the epithelium of the visceral yolk sac, and our experiments indicate that the dye is an inhibitor of a selection of hydrolytic enzymes present in lysosomal fractions from homogenates of rat visceral yolk sac. It seems likely that trypan blue inhibits the intracellular digestion of embryotroph by the visceral yolk-sac epithelium; the conceptus may therefore be deprived of essential nutrients at critical stages of development.
Subject(s)
Abnormalities, Drug-Induced , Acid Phosphatase/metabolism , Deoxyribonucleases/metabolism , Extraembryonic Membranes/enzymology , Glucuronidase/metabolism , Lysosomes/enzymology , Pregnancy, Animal/drug effects , Ribonucleases/metabolism , Trypan Blue/pharmacology , Animals , Enzymes/pharmacology , Epithelial Cells , Female , Histocytochemistry , Lysosomes/drug effects , Microscopy, Electron , Pregnancy , RatsABSTRACT
OBJECTIVE: The purpose of this study was to evaluate the expression and function of catechol-O-methyltransferase in human fetal membranes at term. STUDY DESIGN: Fetal membranes obtained from women between 38-42 weeks of gestation, after (1) vaginal delivery with spontaneous labor and (2) prelabor elective cesarean section (no labor), were assayed for catechol-O-methyltransferase expression using quantitative real-time polymerase chain reaction analysis, immunohistochemistry, and Western blot analysis. Prostaglandin E(2) secretion from amnion and choriodecidua explants treated with or without catechol-O-methyltransferase inhibitor was assayed by enzyme-linked immunosorbent analysis. RESULTS: Amnion layer of fetal membranes from laboring women expressed significantly higher levels of catechol-O-methyltransferase, compared with those from women with no labor. Catechol-O-methyltransferase was higher in the amnion layer than in choriodecidua. Selective catechol-O-methyltransferase inhibition significantly decreased prostaglandin E(2) production from fetal membranes. CONCLUSION: Labor increases catechol-O-methyltransferase expression in the amnion of human fetal membranes. Selective catechol-O-methyltransferase inhibition decreased prostaglandin E(2) secretion in fetal explant cultures, suggesting a role for catechol-O-methyltransferase in human labor and delivery.
Subject(s)
Catechol O-Methyltransferase/biosynthesis , Dinoprostone/biosynthesis , Extraembryonic Membranes/enzymology , Labor, Obstetric/metabolism , Female , Humans , PregnancyABSTRACT
OBJECTIVES: To study the expression and the function of the 11beta-hydroxysteroid dehydrogenase enzyme 1 (11beta-HSD1) and 2 (11beta-HSD2) in placenta and the fetal membranes from pregnancies with intrauterine growth restriction (IUGR) and from controls. METHODS: Amnion, chorion, decidua and cotyledon were separated from placenta; mRNA was analyzed by TaqMan real-time technology and proteins by Western blot; enzyme activities were measured by the conversion of 3H-cortisol to 3H-cortisone and vice versa. RESULTS: Predominant mRNA expression (p < 0.001) was found for 11beta-HSD1 in chorion and for 11beta-HSD2 in decidua and cotyledon. In pregnancies with IUGR, 11beta-HSD1 was upregulated in chorion (mean DeltaCt 11beta-HSD:18S mRNA 193.5 vs. 103.0 in controls respectively, p < 0.05) and 11beta-HSD2 was downregulated in decidua (mean DeltaCt 11beta-HSD2:18S mRNA 0.18 vs. 15.88 in controls respectively, p < 0.05). 11beta-HSD1 protein levels were reduced in amnion and 11beta-HSD1 and 11beta-HSD2 oxidase activity in decidua and cotyledon were reduced from pregnancies with IUGR. CONCLUSION: Reduced synthesis or activity of 11beta-HSD1 or 2 in cases of IUGR is shown in some but not in all tissues. The local mRNA expression of 11beta-HSD1 in chorion may reflect a mechanism on the post-transcriptional gene regulation to stimulate the formation of cortisone in IUGR. To provoke increasing activity with oxidase stimulators could be a future therapy in cases of IUGR.
Subject(s)
11-beta-Hydroxysteroid Dehydrogenases/metabolism , Extraembryonic Membranes/enzymology , Fetal Growth Retardation/enzymology , Placenta/enzymology , 11-beta-Hydroxysteroid Dehydrogenases/genetics , Adult , Case-Control Studies , Down-Regulation , Female , Gestational Age , Humans , Pregnancy , RNA, Messenger/metabolism , Up-RegulationABSTRACT
Age-related cellular changes and limited replicative capacity of adult mesenchymal stem cells (MSCs) are few of the challenges confronting stem cell research. MSCs from human fetal membranes (hFM-MSCs), including placental, umbilical cord, and amniotic membrane, are considered an alternative to adult MSCs. However, the effect of mothers' age on hFM-MSC cellular properties is still not clearly established. This study aimed to evaluate the effect of mothers' age on hFM-MSC telomere length, telomerase activity, and proliferation ability in three different age groups: GI (20-29 years), GII (30-39 years), and GIII (≥40 years). hFM samples were collected from pregnant women ≤37 weeks after obtaining consent. hFM-MSCs were isolated and cultured to characterize them by flow cytometry and assess proliferation by MTT assay and doubling time. Telomere length and expression levels of human telomerase reverse transcriptase were assessed by quantitative real-time polymerase chain reaction (qRT-RCR). hFM-MSCs in the three age groups were spindle-shaped, plastic-adherent, and exhibited high proliferation rates and strong expression of hMSC markers. GI showed the longest telomere length in hMSCs in various FM regions, whereas GIII showed the highest level of telomerase expression. There was no difference in telomere length between GII and GIII, and both groups showed the same hMSC characteristics. In conclusion, although the hFM-MSCs derived from different fetal membranes maintained the MSC characteristics in all study groups, the hFM-MSCs of older mothers had shorter telomeres and higher telomerase activity and proliferation rate than did those derived from younger mothers. Thus, the hFM-MSCs of older mothers could be unsuitable for expansion in vitro or stem cell therapy. Determination of telomere length and telomerase expression level of hFM might help characterizing and understanding the biological differences of hFM-MSCs in different age groups.
Subject(s)
Adult Stem Cells/enzymology , Mesenchymal Stem Cells/enzymology , Telomerase/genetics , Telomere Homeostasis/genetics , Adult , Adult Stem Cells/metabolism , Age Factors , Cell Differentiation/genetics , Cell Proliferation/genetics , Extraembryonic Membranes/enzymology , Extraembryonic Membranes/growth & development , Female , Flow Cytometry , Humans , Mesenchymal Stem Cells/metabolism , Mothers , Placenta/cytology , Pregnancy , Telomere/genetics , Umbilical Cord/growth & development , Umbilical Cord/metabolismABSTRACT
Fetal membranes development is a complex process. The amniotic and exo-celomic cavities are appearing first. The rapid growth of the amniotic cavity is leading to the disappearance of the exo-celomic cavity and the chorion is merging with the decidua. Fetal membranes consist of three layers: the amnion and the chorion, issued from fetal tissues and the decidua issued from maternal tissue. A balance between the synthesis and the degradation of membranes components is physiologic throughout the gestation. Two main mechanisms are involved in the degradation process: apoptosis in the cellular compartment and matrix metalloproteinase (MMP) in the extracellular matrix. Regulation of MMP is depending on factors increasing their expression (cytokines) and factors decreasing their activity tissue inhibitor of metalloproteinases (TIMPS). Particular conditions can induce an unbalance between synthesis and degradation leading to the weakening of the membranes. Different factors can be associated to induce this unbalance: infection, hormonal factors, default in membranes fusion, oxidative stress and mechanic factors. In fine, the spontaneous rupture of the membranes is always occurring in regard of the uterine cervix after a process started several weeks before.
Subject(s)
Extraembryonic Membranes , Fetal Membranes, Premature Rupture/physiopathology , Amnion/enzymology , Chorion/enzymology , Decidua/enzymology , Extraembryonic Membranes/embryology , Extraembryonic Membranes/enzymology , Female , Fetal Membranes, Premature Rupture/enzymology , Humans , Matrix Metalloproteinases/metabolism , PregnancyABSTRACT
INTRODUCTION: The way in which tobacco smoking increases the risk of preterm labor remains uncertain. Altered prostaglandin metabolism is one potential mechanism. METHODS: Proteins in fetal membrane samples (amniochoriodecidua) from 20 women were relatively quantified using Tandem Mass Tagging nano-liquid chromatography mass spectrometry. RESULTS: Prostaglandin synthases and two enzymes involved in prostaglandin degradation, hydroxyprostaglandin dehydrogenase (HPGD) and CBR1, were detected by the mass spectrometer. The expression of HPGD was significantly lower in smokers relative to non-smokers (0.43 fold, p = 0.016). There was no effect of labor, inflammatory status or gestational age on the HPGD levels. DISCUSSION: We describe for the first time an association between maternal smoking and HPGD expression. We propose that reduced expression of HPGD is one mechanism through which smoking may contribute to preterm labor. Lower levels of this enzyme, key to metabolising prostaglandins, may result in higher levels of prostaglandins and therefore precipitate labor prematurely.
Subject(s)
Extraembryonic Membranes/enzymology , Hydroxyprostaglandin Dehydrogenases/metabolism , Obstetric Labor, Premature/etiology , Tobacco Smoking/adverse effects , Female , Humans , Infant, Newborn , Male , Pregnancy , ProteomicsABSTRACT
Cancer procoagulant (CP), a direct activator of coagulation factor X, is among one of the tumour cell products or activities which may promote fibrin formation and has been suggested to be selectively associated with the malignant phenotype. At present, the most reliable assay for the quantification of CP activity is the three-stage chromogenic assay which utilises the ability of CP to activate factor X. In this assay, the activation of factor X leads to the formation of activated thrombin from prothrombin and the eventual hydrolyses of a thrombin chromogenic substrate which contains a p-nitroaniline leaving group. The complexity of the three-stage chromogenic assay suggests a need for a direct method of assaying CP activity. This study focuses on the design of a fluorogenic substrate that would enable the direct quantification of CP activity. The results of the study show two promising substrates for the determination of CP activity: Boc-PQVR-AMC and PQVR-AMC. Further analysis showed that Boc-PQVR-AMC could be excluded as a potential substrate for CP since it was also cleaved by collagenase.
Subject(s)
Cysteine Endopeptidases , Early Detection of Cancer/methods , Fluorescent Dyes/metabolism , Neoplasm Proteins , Cysteine Endopeptidases/analysis , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/isolation & purification , Cysteine Endopeptidases/metabolism , Enzyme Stability , Extraembryonic Membranes/enzymology , Factor X/metabolism , Fibronectins/metabolism , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Humans , Hydrogen-Ion Concentration , Kinetics , Neoplasm Proteins/analysis , Neoplasm Proteins/chemistry , Neoplasm Proteins/isolation & purification , Neoplasm Proteins/metabolism , Oligopeptides/metabolism , Thrombin/metabolismABSTRACT
An assay procedure was developed in which phosphatidyl[2-(3)H]inositol was employed as substrate for the measurement of phosphatidylinositol-specific phospholipase C activity. Employing this assay, phosphatidylinositol-specific phospholipase C activity in human fetal membranes and uterine decidua was identified and characterized. The specific activity of this enzyme in amnion (4.4 mumol x mg(-1) protein x h(-1)) was three times that in uterine decidua and more than five times that in chorion laeve. No difference was found between the specific activity of phosphatidylinositol-specific phospholipase C in placental amnion and that in reflected amnion. The products of phosphatidylinositol hydrolysis in short-term incubations were stoichiometric amounts of diacylglycerol and inositol-1,2-cyclic-phosphate plus inositol-1-phosphate. After longer periods of incubation, monoacylglycerol also was detected. Diacylglycerol lipase activity also was demonstrated in these tissues. More than 90% of phosphatidylinositol-specific phospholipase C activity of amnion tissue was recovered in the 105,000-g supernatant fraction, and optimal enzymatic activity in vitro was observed at pH 6.5-7.5 in the presence of Ca(2+) (8 mM) and mercaptoethanol (4 mM). Phosphatidylinositol-specific phospholipase C activity was stimulated by fatty acids in low concentrations, but was inhibited by lysophosphatidylcholine and a variety of detergents. No effect of labor on the specific activity of phosphatidylinositol-specific phospholipase C in either fetal membranes or uterine decidua could be detected. The finding of an active phosphatidylinositol-specific phospholipase C activity in human fetal membranes and uterine decidua is complementary to our previous finding of a selective loss of arachidonic acid from phosphatidylinositol of human fetal membranes during labor. The action of phosphatidylinositol-specific phospholipase C, coupled to diacylglycerol lipase action, could provide a mechanism for the release of arachidonic acid for prostaglandin biosynthesis during parturition.
Subject(s)
Decidua/enzymology , Extraembryonic Membranes/enzymology , Phosphatidylinositols/metabolism , Phospholipases/metabolism , Type C Phospholipases/metabolism , Arachidonic Acids/biosynthesis , Calcium/pharmacology , Detergents/pharmacology , Female , Humans , Hydrogen-Ion Concentration , Labor, Obstetric , Lipase/metabolism , Pregnancy , Subcellular Fractions/enzymologyABSTRACT
The role of the matrix metalloproteinases (MMPs) in the decidua, fetal membranes and amniotic fluid (AF) has been receiving more and more attention. The MMPs are not only important intermediaries in pathological processes leading to preterm labor but it seems that they also play a crucial role in the activation of labor at term. During normal gestation MMP-1, -2, -3, -7 and -9 are found in the amniotic fluid and fetal membranes. MMP-2 and MMP-3 are expressed constitutively while MMP-9 is barely detectable until labor. At labor, while MMP-9 is the major MMP responsible for gelatinolytic activity in the membranes, MMP-2 is dominant in the decidua. MMP-7 (AF) increases with gestation but does not appear to play a major role in labor. The expression of MMPs is attenuated through the expression of relaxins, integrins and extracellular matrix metalloproteinase inducer (EMMPRIN). Spontaneous preterm delivery (PTD) may be a product of preterm labor (PTL), preterm premature rupture of membranes (P-PROM) or placental abruption. Each of these processes may have differing pathways but the presence of an intrinsic inflammatory response with or without infection seems to involve all etiologies. The inflammatory response is mediated with cytokines such as interleukins -1, -6 and -8 and tumor necrosis factor alpha. MMP-3, MMP-7 and MMP-8 appear to be important in these processes. MMP-9, which is the major MMP involved in normal labor, plays an important role in pathological labor as well. Finally, apoptosis seems to play a role in pathological labor, particularly deliveries involving P-PROM. African-American are at greater risk of PTD than white or Hispanic Americans. Environmental differences may not suffice to explain this phenomenon. Genetic polymorphisms of the MMP genes may help explain the greater risk among this population. Finally, manipulating MMPs may have a role in the prevention of PTD. Agents suggested include indomethacin, N-acetylcysteine, progesterone and specific inhibitors of phosphodiesterase 4.
Subject(s)
Decidua/enzymology , Extraembryonic Membranes/enzymology , Labor, Obstetric/metabolism , Matrix Metalloproteinases/physiology , Female , Humans , Matrix Metalloproteinase Inhibitors , Matrix Metalloproteinases/metabolism , Obstetric Labor, Premature/enzymology , PregnancyABSTRACT
Processes of human labour include increased oxidative stress, formation of inflammatory mediators (e.g. cytokines) and uterotonic phospholipid metabolites (e.g. prostaglandins). In non-gestational tissues, advanced glycation endproducts (AGE) induce the expression of pro-inflammatory molecules through mitogen-activated protein kinase and nuclear factor kappaB (NF-kappaB)-dependent pathways. Thus, the aim of this study was to investigate the effects of AGE on 8-isoprostane (a marker of oxidative stress), pro-inflammatory cytokine and prostaglandin release in human gestational tissues, and to define the signalling pathways involved. Human placenta and gestational membranes (amnion and choriodecidua combined; n=5) were incubated in the absence or presence of AGE-BSA (0.25, 0.5, 1 and 2 mg/ml) for 18 h. AGE significantly increased in vitro release of tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-8, prostaglandin (PG)E(2), PGF(2alpha) and 8-isoprostane from human placenta and gestational membranes. This was associated with a concomitant increase in NF-kappaB p65 activation and ERK 1/2 phosphorylation. AGE-stimulated 8-isoprostane, cytokine and prostaglandin production was significantly suppressed by the ERK 1/2 inhibitor U0126 and the NF-kappaB inhibitor BAY 11-7082. In conclusion, AGE mediates inflammatory actions in human gestational tissues. Protein kinases and the NF-kappaB pathway play an essential role in AGE signalling in human gestational tissues.
Subject(s)
Extraembryonic Membranes/metabolism , Glycation End Products, Advanced/pharmacology , Mitogen-Activated Protein Kinase 3/metabolism , NF-kappa B/metabolism , Placenta/metabolism , Pregnancy/metabolism , Analysis of Variance , Biomarkers/analysis , Butadienes/pharmacology , Dinoprost/analogs & derivatives , Dinoprost/analysis , Dinoprost/metabolism , Dinoprostone/analysis , Dinoprostone/metabolism , Extraembryonic Membranes/enzymology , Extraembryonic Membranes/immunology , Female , Humans , Interleukin-1/analysis , Interleukin-1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , NF-kappa B/antagonists & inhibitors , Nitriles/pharmacology , Phosphorylation , Placenta/enzymology , Placenta/immunology , Stimulation, Chemical , Sulfones/pharmacology , Tissue Culture Techniques , Tumor Necrosis Factor-alpha/analysis , Tumor Necrosis Factor-alpha/metabolismABSTRACT
OBJECTIVE: This study was aimed to explore the effect of progesterone on gelatinase expression in the decidua and fetal membranes before and after contractions. STUDY DESIGN: Zymography was conducted for matrix metalloproteinase (MMP) secretion. Semiquantitative reverse transcriptase-polymerase chain reaction was performed to examine MMP2 transcripts, and the effect of progesterone on MMP2 promoter activity was determined with the use of luciferase activity. RESULTS: Progesterone decreased pro-MMP2 secretion, expression, and promoter activity in decidua before contractions began. The effect of progesterone was reversed completely by mifepristone (RU486). Progesterone failed to inhibit MMP2 expression in the amnion and chorion before contractions began. After contractions, progesterone failed to inhibit MMP2 expression in both the decidua and fetal membranes. CONCLUSION: MMP2 expression is inhibited by progesterone only in the decidua and only before contractions begin.
Subject(s)
Decidua/enzymology , Extraembryonic Membranes/enzymology , Gelatinases/metabolism , Progesterone/pharmacology , Progestins/pharmacology , Uterine Contraction/drug effects , Uterine Contraction/physiology , Amnion/enzymology , Cells, Cultured , Chorion/enzymology , Dose-Response Relationship, Drug , Female , Humans , Luciferases/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinases/metabolism , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
Chorioamnionitis increases the risk of preterm labour and is associated with adverse neonatal outcomes including cerebral palsy. Tumour necrosis factor-alpha (TNF-alpha) derived from the gestational tissues (placenta, fetal membranes and maternal decidua) is thought to play a pivotal role in the induction of cytokine response in chorioamnionitis. Tumour necrosis factor-alpha converting enzyme (TACE) is essential for the release of TNF-alpha. Our aim was to determine whether the expression of TACE is increased in human gestational tissues from pregnancies complicated by chorioamnionitis, and whether lipopolysaccharide (LPS) causes increased expression of TACE in the human gestational tissues in vitro. The immunostaining of TACE was generally more intense, in particular in the syncytiotrophoblast and stromal cells, in villous samples from pregnancies complicated by chorioamnionitis than those from normal pregnancies. Increased immunoreactivity of TACE was also noted in the amnion and choriodecidua. In parallel, there was an increased infiltration of monocytes/macrophages within the villous stroma and choriodecidua. As a complement to our in vivo findings, LPS significantly increased the levels of mRNA and protein of TACE in a dose-dependent response in villous and fetal membrane explant cultures. Together, our results imply a potential role of TACE in the pathogenesis of chorioamnionitis.
Subject(s)
ADAM Proteins/metabolism , Chorioamnionitis/enzymology , Extraembryonic Membranes/enzymology , Placenta/enzymology , ADAM17 Protein , Chorioamnionitis/immunology , Female , Humans , Immunoenzyme Techniques , Lipopolysaccharides , Macrophages/physiology , Placenta/immunology , Pregnancy , RNA, Messenger/metabolismABSTRACT
Nitric oxide (NO) synthesized by fetal membranes may act either directly inhibiting myometrium contractility or indirectly interacting with tocolytic agents as prostaglandins (PGs). Here we examined if NO could modulate prostaglandin E(2) 9-ketoreductase (9-KPR) activity in human fetal membranes (HFM). 9-KPR is the enzyme that converts PGE(2) into PGF(2alpha), the main PGs known to induce uterine contractility at term. Chorioamnion explants obtained from elective caesareans were incubated with aminoguanidine (AG), an iNOS inhibitor, or NOC-18, a NO donor. NOC-18 (2mM) increased PGE(2) production and diminished PGF(2alpha) synthesis in HFM. AG presented the opposite effect. When we evaluated the activity of 9-KPR by the conversion of [(3)H]-PGE(2) into [(3)H]-PGF(2alpha) and 13,14-dihidro-15-keto prostaglandin F(2alpha) (the PGF(2alpha) metabolite), we found that NOC-18 inhibited 9-KPR activity. Interestingly, AG did not elicit any effect on 9-KPR but l-NAME, a non-selective NOS inhibitor, significantly increased its activity. Our data suggests that exogenous NO inhibits 9-KPR activity in HFM, thus modulating the synthesis of important labor mediators as PGF(2alpha).