309 results on '"Amnion enzymology"'
Search Results
2. Drastic induction of MMP-7 by cortisol in the human amnion: implications for membrane rupture at parturition.
- Author
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Wang LY, Wang WS, Wang YW, Lu JW, Lu Y, Zhang CY, Li WJ, Sun K, and Ying H
- Subjects
- Amnion drug effects, Anti-Inflammatory Agents adverse effects, Cells, Cultured, Enzyme Activation, Female, Fetal Membranes, Premature Rupture chemically induced, Fetal Membranes, Premature Rupture enzymology, Fibroblasts drug effects, Humans, Pregnancy, Amnion enzymology, Fetal Membranes, Premature Rupture pathology, Fibroblasts enzymology, Gene Expression Regulation, Enzymologic drug effects, Hydrocortisone adverse effects, Matrix Metalloproteinase 7 metabolism, Parturition
- Abstract
Preterm premature rupture of fetal membranes precedes 30-40% of preterm births. Activation of matrix metalloproteases (MMPs) is the one of the major causes of extracellular matrix (ECM) degradation in membrane rupture. Increased cortisol, regenerated by 11β-hydroxysteroid dehydrogenase 1 in the amnion at parturition, is known to participate in a number of parturition-pertinent events. However, whether cortisol has a role in the regulation of MMPs in the membranes is not known. Here, we addressed this issue using human amnion tissue, the most tensile layer of the membranes. RNA-sequencing revealed that cortisol induced MMP7 expression dramatically in amnion fibroblasts, which was confirmed by real-time quantitative RT-PCR and Western blotting analysis in cortisol-treated amnion explants and fibroblasts. Measurement of collagen IV α5 chain (COL4A5), a substrate for MMP-7, showed that cortisol reduced its extracellular abundance, which was blocked by an antibody against MMP-7. Moreover, increased MMP-7 but decreased COL4A5 abundance was observed in the amnion tissue following labor-initiated spontaneous rupture of membranes. Mechanistic studies showed that cortisol increased the phosphorylation of c-Jun and the expression of c-Fos, the 2 major components of activated protein 1 (AP-1), respectively. The knocking down of c-Fos or c-Jun significantly attenuated the induction of MMP7 expression by cortisol. Chromatin immunoprecipitation assays showed that cortisol stimulated the enrichment of c-Fos and c-Jun at the AP-1 binding site in the MMP7 promoter. The data suggest that induction of MMP7 by cortisol via AP-1 may be a contributing factor to ECM degradation in membrane rupture at parturition.-Wang, L.-Y., Wang, W.-S., Wang, Y.-W., Lu, J.-W., Lu, Y., Zhang, C.-Y., Li, W.-J., Sun, K., Ying, H. Drastic induction of MMP-7 by cortisol in the human amnion: implications for membrane rupture at parturition.
- Published
- 2019
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3. Human amnion-derived mesenchymal stem cells induced osteogenesis and angiogenesis in human adipose-derived stem cells via ERK1/2 MAPK signaling pathway.
- Author
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Wang Y, Chen X, Yin Y, and Li S
- Subjects
- Adipocytes metabolism, Adipose Tissue cytology, Amniotic Fluid cytology, Amniotic Fluid enzymology, Cell Differentiation drug effects, Cell Proliferation drug effects, Cells, Cultured, Humans, MAP Kinase Kinase 1 metabolism, Mitogen-Activated Protein Kinase 1 metabolism, Neovascularization, Physiologic drug effects, Osteogenesis physiology, Stem Cells cytology, Stem Cells enzymology, Amnion cytology, Amnion enzymology, MAP Kinase Signaling System, Mesenchymal Stem Cells cytology, Mesenchymal Stem Cells enzymology, Osteogenesis drug effects
- Abstract
Mesenchymal stem cells (MSCs) have shown great potential in treating bone deficiency. Human adipose-derived stem cells (HASCs) are multipotent progenitor cells with multi-lineage differentiation potential. Human amnion-derived mesenchymal stem cells (HAMSCs) are capable of promoting osteogenic differentiation of MSCs. In this study, we investigated the effect of HAMSCs on HASCs by a transwell co-culture system. HAMSCs promoted proliferation, osteogenic differentiation, angiogenic potential and adiponectin (APN) secretion of HASCs. Moreover, the positive effect of HAMSCs was significantly inhibited by U0126, a highly selective inhibitor of extracellular signaling-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway. These observations suggested that HAMSCs induced bone regeneration in HASCs via ERK1/2 MAPK signaling pathway. [BMB Reports 2018; 51(4): 194-199].
- Published
- 2018
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4. Inhibition of Lysyl Oxidase by Cortisol Regeneration in Human Amnion: Implications for Rupture of Fetal Membranes.
- Author
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Liu C, Guo C, Wang W, Zhu P, Li W, Mi Y, Myatt L, and Sun K
- Subjects
- Cortisone, Dinoprostone metabolism, Down-Regulation, Epithelial Cells physiology, Female, Fibroblasts physiology, Humans, Pregnancy, Receptors, Glucocorticoid metabolism, 11-beta-Hydroxysteroid Dehydrogenase Type 1 metabolism, Amnion enzymology, Hydrocortisone metabolism, Parturition metabolism, Protein-Lysine 6-Oxidase metabolism
- Abstract
The mechanisms underlying human parturition are still not understood, yet we need this knowledge to combat preterm birth. Fetal membranes express abundant 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which converts inert cortisone to active cortisol. We examined whether cortisol regeneration in the amnion might play a role in human parturition through regulation of lysyl oxidase (LOX), a collagen cross-linking enzyme, thereby contributing to the rupture of fetal membranes. By using cultured human primary amnion fibroblasts, we demonstrated that, in addition to the induction of the key enzymes involved in prostaglandin E2 (PGE2) synthesis, cortisol stimulated 11β-HSD1 and inhibited LOX reciprocally. These results were reproduced in human amnion tissue explants after cortisol treatment. Cortisone also inhibited LOX expression, which was abolished by the inhibition of 11β-HSD1. Despite the inhibition of LOX by PGE2, inhibition of the PGE2 pathway failed to block the inhibition of LOX by cortisol. However, inhibition of glucocorticoid receptor and mutation of a negative glucocorticoid response element in LOX promoter abolished the inhibition of LOX by cortisol. Chromatin immunoprecipitation assay revealed that cortisol increased GR binding to the LOX promoter. Moreover, increased cortisol and 11β-HSD1 abundance and decreased LOX abundance were observed in human amnion tissue after the labor-initiated spontaneous rupture of membranes. These data highlight a crucial role for local cortisol regeneration by 11β-HSD1 in the down-regulation of LOX expression via glucocorticoid receptor binding to a negative glucocorticoid response element to its promoter in the amnion, which may contribute to rupture of fetal membranes at parturition.
- Published
- 2016
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5. The Effect of Progestins on Tumor Necrosis Factor α-Induced Matrix Metalloproteinase-9 Activity and Gene Expression in Human Primary Amnion and Chorion Cells In Vitro.
- Author
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Allen TK, Feng L, Nazzal M, Grotegut CA, Buhimschi IA, and Murtha AP
- Subjects
- 17 alpha-Hydroxyprogesterone Caproate, Amnion cytology, Amnion enzymology, Cells, Cultured, Chorion cytology, Chorion enzymology, Female, Gene Expression Regulation, Enzymologic drug effects, Humans, Matrix Metalloproteinase 9 genetics, Pregnancy, RNA, Messenger biosynthesis, Amnion drug effects, Chorion drug effects, Hydroxyprogesterones pharmacology, Matrix Metalloproteinase 9 metabolism, Medroxyprogesterone Acetate pharmacology, Progesterone pharmacology, Progestins pharmacology, Tumor Necrosis Factor-alpha pharmacology
- Abstract
Background: Current treatment modalities for preventing preterm premature rupture of membranes are limited, but progestins may play a role. Tumor necrosis factor α (TNFα) enhances matrix metalloproteinase-9 (MMP-9) gene expression and activity in fetal membranes, contributing to membrane weakening and rupture. We previously demonstrated that progestins attenuate TNFα-induced MMP-9 activity in a cytotrophoblast cell line. However, whether they have a similar effect in primary amnion and chorion cells of fetal membranes is unknown. In this study, we evaluated the effect of progestins on basal and TNFα-induced MMP-9 activity and gene expression in primary chorion and amnion cells harvested from the fetal membranes of term nonlaboring patients., Methods: Primary amnion and chorion cells were isolated from fetal membranes obtained from term uncomplicated nonlaboring patients following elective cesarean delivery (n = 11). Confluent primary amnion and chorion cell cultures were both pretreated with vehicle (control), progesterone (P4), 17α-hydroxyprogesterone caproate (17P), or medroxyprogesterone acetate (MPA) at 10 M concentration for 6 hours followed by stimulation with TNFα at 10 ng/mL for an additional 24 hours. Cell cultures pretreated with the vehicle only served as the unstimulated control and the vehicle stimulated with TNFα served as the stimulated control. Both controls were assigned a value of 100 units. Cell culture medium was harvested for MMP-9 enzymatic activity quantification using gelatin zymography. Total RNA was extracted for quantifying MMP-9 gene expression using real-time quantitative PCR. Basal MMP-9 activity and gene expression data were normalized to the unstimulated control. TNFα-stimulated MMP-9 activity and gene expression were normalized to the stimulated control. The primary outcome was the effect of progestins on TNFα-induced MMP-9 enzymatic activity in term human primary amnion and chorion cells in vitro. Secondary outcomes included the effect of progestin therapy on TNFα-induced MMP-9 gene expression and on basal MMP-9 activity and gene expression in primary amnion and chorion cells in vitro., Results: Primary cells were harvested from 11 patients. Compared with the unstimulated control, TNFα increased MMP-9 activity (P = 0.005 versus control in primary amnion cells and P < 0.001 versus control in primary chorion cells) and MMP-9 gene expression (P = 0.030 versus control in primary amnion cells, P < 0.001 versus control in primary chorion cells). Compared with the unstimulated controls, MPA, but not P4 or 17P, reduced basal MMP-9 activity [mean difference (95% CI) -49.6 (-81.9, -17.3) units, P = 0.001] and gene expression [mean difference (95% CI) -53.4 (-105.9, -0.9) units, P = 0.045] in primary amnion cells. Compared with the stimulated control, MPA also reduced TNFα-induced MMP-9 activity [mean difference (95% CI) -69.0 (-91.8, -46.3) units, P < 0.001] and gene expression [mean difference (95% CI) -86.0 (-120.7, -51.3) units, P < 0.001] in primary amnion cells. Progestin pretreatment had no significant effect on basal or TNFα-induced MMP-9 activity and gene expression in primary chorion cells., Conclusions: The inhibitory effect of MPA on both basal and TNFα-induced MMP-9 activity and gene expression in primary amnion cells demonstrate a possible mechanism by which progestins may prevent fetal membrane weakening leading to preterm premature rupture of membranes.
- Published
- 2015
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6. Amniotic Mesenchymal Stem Cells Can Enhance Angiogenic Capacity via MMPs In Vitro and In Vivo.
- Author
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Jiang F, Ma J, Liang Y, Niu Y, Chen N, and Shen M
- Subjects
- Amnion cytology, Animals, Coculture Techniques, Female, Human Umbilical Vein Endothelial Cells cytology, Humans, Mesenchymal Stem Cells cytology, Mice, Mice, Inbred BALB C, Mice, Nude, Pregnancy, Amnion enzymology, Human Umbilical Vein Endothelial Cells metabolism, Matrix Metalloproteinase 2 metabolism, Matrix Metalloproteinase 9 metabolism, Mesenchymal Stem Cells enzymology, Neovascularization, Physiologic
- Abstract
The aim of this study was to evaluate the angiogenic capacity and proteolytic mechanism of coculture using human amniotic mesenchymal stem cells (hAMSCs) with human umbilical vein endothelial cells (HUVECs) in vivo and in vitro by comparing to those of coculture using bone marrow mesenchymal stem cells with HUVEC. For the in vivo experiment, cells (HUVEC-monoculture, HUVEC-hAMSC coculture, and HUVEC-BMMSC coculture) were seeded in fibrin gels and injected subcutaneously in nude mice. The samples were collected on days 7 and 14 and histologically analyzed by H&E and CD31 staining. CD31-positive staining percentage and vessel-like structure (VLS) density were evaluated as quantitative parameters for angiogenesis. The increases of CD31-positive staining area and VLS density in both HUVEC-hAMSC group and HUVEC-BMMSC group were found between two time points, while obvious decline of those was observed in HUVEC-only group. For the in vitro experiment, we utilized the same 3D culture model to investigate the proteolytic mechanism related to capillary formation. Intensive vascular networks formed by HUVECs were associated with hAMSCs or BMMSCs and related to MMP2 and MMP9. In conclusion, hAMSCs shared similar capacity and proteolytic mechanism with BMMSCs on neovascularization.
- Published
- 2015
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7. Class I to III histone deacetylases differentially regulate inflammation-induced matrix metalloproteinase 9 expression in primary amnion cells.
- Author
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Poljak M, Lim R, Barker G, and Lappas M
- Subjects
- Amnion drug effects, Cells, Cultured, Female, Gene Expression Regulation, Enzymologic, Group III Histone Deacetylases antagonists & inhibitors, Histone Deacetylase Inhibitors pharmacology, Humans, Inflammation enzymology, Pregnancy, RNA, Small Interfering pharmacology, Amnion enzymology, Group III Histone Deacetylases physiology, Histone Deacetylases physiology, Matrix Metalloproteinase 9 biosynthesis
- Abstract
Matrix metalloproteinase (MMP) 9 plays an important role in the degradation of the extracellular matrix in fetal membranes, and pathological activation of MMP-9 can lead to preterm birth. In nongestational tissues, modulation of histone deacetylases (HDACs) regulates MMP-9 expression. The aim of this study was to determine whether class I to III HDACs regulate MMP-9 expression and activity in primary amnion cells. Class I and II HDAC regulation of MMP-9 was assessed using the general class I and II HDAC inhibitors (HDACi) trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), the class I HDACi MS-275, and the class II HDACi MC1568. Class III HDAC regulation of MMP-9 was assessed using the SIRT1 activators resveratrol and SRT1720 as well as SIRT1 small interfering RNA (siRNA). Primary amnion epithelial cells were incubated with 1 ng/mL interleukin (IL) 1β in the absence or presence of 0.3 μmol/L TSA, 5 μmol/L SAHA, 2.5 μmol/L MS-275, 2.5 μmol/L MC1568, 50 μmol/L resveratrol, or 10 μmol/L SRT1720 for 20 hours. We found that the class I and II HDACi TSA and SAHA and the class II HDACi MC1568 significantly decreased IL-β-induced MMP-9 gene and pro-MMP-9 expression in primary amnion cells. There was, however, no effect of the class I HDACi MS-275 on IL-β-induced MMP-9 expression. On the other hand, inhibition of class III HDAC SIRT1 using siRNA significantly augmented IL-1β-induced MMP-9, and SIRT1 activation using resveratrol and SRT1720 inhibited IL-1β-induced MMP-9 expression. In summary, class I to III HDACs differentially regulate inflammation-induced MMP-9 expression in primary amnion cells.
- Published
- 2014
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8. Altered expression of enzymes regulating the activity of endothelin-1 in the lower segment of the human amnion during labor.
- Author
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Kotani T, Iwase A, Tsuda H, Mano Y, Yamamoto E, Nakano T, Hasegawa Y, Li H, Sumigama S, Itakura A, and Kikkawa F
- Subjects
- Adult, Amnion metabolism, Cells, Cultured, Cytokines physiology, Endothelin-Converting Enzymes, Epithelial Cells drug effects, Epithelial Cells metabolism, Female, Humans, Immunohistochemistry, Infant, Newborn, Lysophospholipids pharmacology, Mesenchymal Stem Cells drug effects, Mesenchymal Stem Cells metabolism, Pregnancy, Sphingosine analogs & derivatives, Sphingosine pharmacology, Amnion enzymology, Aspartic Acid Endopeptidases metabolism, Endothelin-1 metabolism, Labor, Obstetric metabolism, Metalloendopeptidases metabolism, Neprilysin metabolism
- Abstract
The level of endothelin (ET)-1, a uterotonin, increases in amniotic fluid during labor. The known metallopeptidases include ET-converting enzyme (ECE), which converts inactive precursor to potent ET-1, and neutral endopeptidase (NEP), which inactivates ET-1. These enzymes are present in fetal membranes, and the aims of this study were to establish the protein expression of the enzymes within the amnion of human fetal membranes. Expressions were compared between amnions obtained before and after term labor using a Western blot analysis and enzyme-linked immunosorbent assay, respectively. The localization of these enzymes was determined using immunohistochemistry. The protein expression of the enzymes and output of bioactive ET-1 in human amnion epithelial cells (HAECs) and mesenchymal cells (HAMCs) were investigated with and without proinflammatory cytokines, oxytocin, and prostaglandin treatment. The effects of sphingosine-1-phosphate (S1P), a bioactive lipid, were also examined. The protein expression of ECE-1 was significantly increased (P < 0.01), whereas that of NEP was significantly decreased, followed by increased ET-1 (P < 0.01), in the amnion obtained after labor (P < 0.01). HAECs and HAMCs primarily expressed ECE-1 and NEP, respectively. The protein expression of ECE-1 was significantly induced (P < 0.01). However, the NEP levels were significantly reduced (P < 0.05) by treatment with TNFalpha and IL1beta followed by the 7.5-fold and 6.5-fold increase of ET-1 (P < 0.01), respectively, in the HAECs. ET-1 was increased 2-fold by S1P (P < 0.01). These results suggest that the altered expression of enzymes regulating the activity of ET-1 during parturition is controlled by inflammatory cytokines.
- Published
- 2013
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9. High aminopeptidase N/CD13 levels characterize human amniotic mesenchymal stem cells and drive their increased adipogenic potential in obese women.
- Author
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Iaffaldano L, Nardelli C, Raia M, Mariotti E, Ferrigno M, Quaglia F, Labruna G, Capobianco V, Capone A, Maruotti GM, Pastore L, Di Noto R, Martinelli P, Sacchetti L, and Del Vecchio L
- Subjects
- Adult, Amnion growth & development, Amnion pathology, Body Mass Index, CD13 Antigens antagonists & inhibitors, CD13 Antigens genetics, Case-Control Studies, Female, Gene Expression, Humans, Immunophenotyping, Infant, Newborn, Mesenchymal Stem Cells cytology, Obesity enzymology, Obesity pathology, PPAR gamma genetics, PPAR gamma metabolism, Pregnancy, RNA, Messenger antagonists & inhibitors, RNA, Messenger genetics, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Risk Factors, Adipogenesis genetics, Amnion enzymology, CD13 Antigens metabolism, Mesenchymal Stem Cells enzymology, Obesity genetics, RNA, Messenger metabolism
- Abstract
Maternal obesity is associated to increased fetal risk of obesity and other metabolic diseases. Human amniotic mesenchymal stem cells (hA-MSCs) have not been characterized in obese women. The aim of this study was to isolate and compare hA-MSC immunophenotypes from obese (Ob-) and normal weight control (Co-) women, to identify alterations possibly predisposing the fetus to obesity. We enrolled 16 Ob- and 7 Co-women at delivery (mean/SEM prepregnancy body mass index: 40.3/1.8 and 22.4/1.0 kg/m2, respectively), and 32 not pregnant women. hA-MSCs were phenotyped by flow cytometry; several maternal and newborn clinical and biochemical parameters were also measured. The expression of membrane antigen CD13 was higher on Ob-hA-MSCs than on Co-hA-MSCs (P = 0.005). Also, serum levels of CD13 at delivery were higher in Ob- versus Co-pregnant women and correlated with CD13 antigen expression on Ob-hA-MSCs (r2 = 0.84, P < 0.0001). Adipogenesis induction experiments revealed that Ob-hA-MSCs had a higher adipogenic potential than Co-hA-MSCs as witnessed by higher peroxisome proliferator-activated receptor gamma and aP2 mRNA levels (P = 0.05 and P = 0.05, respectively), at postinduction day 14 associated with increased CD13 mRNA levels from baseline to day 4 postinduction (P < 0.05). Adipogenesis was similar in the two sets of hA-MSCs after CD13 silencing, whereas it was increased in Co-hA-MSCs after CD13 overexpression. CD13 expression was high also in Ob-h-MSCs from umbilical cords or visceral adipose tissue of not pregnant women. In conclusion, antigen CD13, by influencing the adipogenic potential of hA-MSCs, could be an in utero risk factor for obesity. Our data strengthen the hypothesis that high levels of serum and MSC CD13 are obesity markers.
- Published
- 2013
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10. Increased expression of sphingosine kinase in the amnion during labor.
- Author
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Erkhembaatar LO, Kotani T, Sumigama S, Tsuda H, Mano Y, Hua L, Hasegawa Y, Wang J, Sugiyama C, Nakahara T, Iwase A, and Kikkawa F
- Subjects
- Aldehyde-Lyases biosynthesis, Cesarean Section, Female, Humans, Lysophospholipids biosynthesis, Lysophospholipids pharmacology, Pregnancy, Sphingosine analogs & derivatives, Sphingosine biosynthesis, Sphingosine pharmacology, Amnion enzymology, Cyclooxygenase 2 metabolism, Labor, Obstetric metabolism, Phosphotransferases (Alcohol Group Acceptor) biosynthesis
- Abstract
Introduction: Sphingosine-1-phosphate (S1P), a bioactive lipid, has been reported to regulate inflammation processes. The onset of labor is thought to be related to inflammation. We therefore hypothesized that S1P might be involved in the onset of labor., Methods: The expression of sphingosine kinase (SPHK)-1, which produces S1P, and S1P lyase (SPL)-1, which irreversibly inactivates S1P, were examined in the fetal membranes. The expression levels were compared between amnions from cases of elective Caesarean deliveries (pre-labor) and those from vaginal deliveries (post-labor). In primary cultured human amnion cells, the expression levels of prostaglandin-endoperoxide synthase (PTGS)-2 were examined in the presence or absence of S1P treatment., Results: SPHK-1 and SPL-1 were both expressed in the amnion. The expression of SPHK-1 in the post-labor amnions increased compared with that in the pre-labor amnions. The expression of PTGS-2, a key regulator of labor, also increased in the post-labor amnion. However, the SPL-1 expression in the pre-labor amnion was not significantly different from that in the post-labor amnion. S1P1-3 and 5, which were coupled with Gi protein, were consistently found in the amnion cells. The treatment with S1P increased the expression of PTGS-2, and this was completely suppressed by a Gi inhibitor in the amnion cells., Discussion: We are herein provide the first evidence of increased SPHK-1 expression in post-labor amnions, and that S1P increases the PTGS-2 expression in amnion cells., Conclusions: Our results suggest that S1P might play a role in the onset of labor via the induction of PTGS-2., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
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11. Autophagy can promote but is not required for epithelial cell extrusion in the amnioserosa of the Drosophila embryo.
- Author
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Cormier O, Mohseni N, Voytyuk I, and Reed BH
- Subjects
- Amnion enzymology, Amnion ultrastructure, Animals, Apoptosis, Biosensing Techniques, Caspases metabolism, Cell Communication, Cell Polarity, Cell Shape, Drosophila melanogaster metabolism, Embryo, Nonmammalian enzymology, Embryo, Nonmammalian ultrastructure, Enzyme Activation, Epithelial Cells ultrastructure, Green Fluorescent Proteins metabolism, Mutation genetics, Proteolysis, Amnion cytology, Amnion embryology, Autophagy, Drosophila melanogaster cytology, Drosophila melanogaster embryology, Embryo, Nonmammalian cytology, Epithelial Cells cytology
- Abstract
During Drosophila embryogenesis the majority of the extra-embryonic epithelium known as the amnioserosa (AS) undergoes programmed cell death (PCD) following the completion of the morphogenetic process of dorsal closure. Approximately ten percent of AS cells, however, are eliminated during dorsal closure by extrusion from the epithelium. Using biosensors that report autophagy and caspase activity in vivo, we demonstrate that AS cell extrusion occurs in the context of elevated autophagy and caspase activation. Furthermore, we evaluate AS extrusion rates, autophagy, and caspase activation in embryos in which caspase activity or autophagy are altered by genetic manipulation. This includes using the GAL4/UAS system to drive expression of p35, reaper, dINR (ACT) and Atg1 in the AS; we also analyze embryos lacking both maternal and zygotic expression of Atg1. Based on our results we suggest that autophagy can promote, but is not required for, epithelial extrusion and caspase activation in the amnioserosa.
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- 2012
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12. Molecular evidence of a (pro)renin/ (pro)renin receptor system in human intrauterine tissues in pregnancy and its association with PGHS-2.
- Author
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Pringle KG, Zakar T, Yates D, Mitchell CM, Hirst JJ, and Lumbers ER
- Subjects
- Amnion drug effects, Amnion enzymology, Chorion drug effects, Chorion enzymology, Cyclooxygenase 2 genetics, Female, Humans, Labor, Obstetric metabolism, Placenta cytology, Placenta enzymology, Pregnancy, Protein Transport, RNA, Messenger genetics, RNA, Messenger metabolism, Receptors, Cell Surface genetics, Renin genetics, Renin pharmacology, Uterus cytology, Uterus enzymology, Prorenin Receptor, Cyclooxygenase 2 metabolism, Receptors, Cell Surface metabolism, Renin metabolism, Uterus metabolism
- Abstract
Prorenin stimulates decidual prostaglandin (PG) production in vitro, the (pro)renin receptor ((P)RR) may mediate this action. The role of prorenin in amnion PG synthesis has not been examined, despite this being the key site of PG synthesis. To determine if (P)RR, prorenin and PGHS-2 are co-localized in gestational tissues and if expression is altered by labour, term amnion, chorion, decidua and placenta were collected during elective caesarean section or after spontaneous labour. Prorenin, (P)RR and PGHS-2 mRNA abundance was determined by real-time RT-PCR. (P)RR protein was examined by immunohistochemistry. The effect of recombinant human (rh) prorenin on PGHS-2 mRNA abundance in amnion explants was determined. Prorenin and (P)RR mRNA were highest in decidua and placenta, respectively. Decidual prorenin, (P)RR and placental (P)RR mRNA abundance decreased with labour. (P)RR protein was present in all gestational tissues. After labour, decidual prorenin was positively correlated with amnion PGHS-2 mRNA and rh-prorenin significantly increased PGHS-2 mRNA abundance in amnion explants. We conclude that the decidua is the principal source of prorenin and is downregulated with labour. All gestational tissues are targets for prorenin. Decidual prorenin may be involved in the labour-associated increase in amnion PGHS-2 abundance via the (P)RR.
- Published
- 2011
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13. Osteogenic differentiation of intact human amniotic membrane.
- Author
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Lindenmair A, Wolbank S, Stadler G, Meinl A, Peterbauer-Scherb A, Eibl J, Polin H, Gabriel C, van Griensven M, and Redl H
- Subjects
- Alkaline Phosphatase metabolism, Amnion drug effects, Amnion enzymology, Biomarkers metabolism, Calcium metabolism, Cell Survival drug effects, Cells, Cultured, Culture Media pharmacology, Gene Expression Regulation drug effects, Humans, Intracellular Space drug effects, Intracellular Space enzymology, Reverse Transcriptase Polymerase Chain Reaction, Amnion cytology, Cell Differentiation drug effects, Osteogenesis drug effects
- Abstract
Tissue engineering strategies usually require cell isolation and combination with a suitable biomaterial. Human amniotic membrane (AM) represents a natural two-layered sheet comprising cells with proven stem cell characteristics. In our approach, we evaluated the differentiation potential of AM in toto with its sessile stem cells as alternative to conventional approaches requiring cell isolation and combination with biomaterials. For this, AM-biopsies were differentiated in vitro using two osteogenic media compared with control medium (CM) for 28 days. Mineralization and osteocalcin expression was demonstrated by (immuno)histochemistry. Alkaline phosphatase (AP) activity, calcium contents and mRNA expression of RUNX2, AP, osteopontin, osteocalcin, BMP-2 (bone morphogenetic protein), and BMP-4 were quantified and AM viability was evaluated. Under osteogenic conditions, AM-biopsies mineralized successfully and by day 28 the majority of cells expressed osteocalcin. This was confirmed by a significant rise in calcium contents (up to 27.4 ± 6.8 mg/dl d28), increased AP activity, and induction of RUNX2, AP, BMP-2 and BMP-4 mRNA expression. Relatively high levels of viability were retained, especially in osteogenic media (up to 78.3 ± 19.0% d14; 62.9 ± 22.3% d28) compared to CM (42.2 ± 15.2% d14; 35.1 ± 8.6% d28). By this strategy, stem cells within human AM can successfully be driven along the osteogenic pathways while residing within their natural environment., (Copyright © 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
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14. Direct analysis reveals an absence of gamma-carboxyglutamic acid in cancer procoagulant from human tissues.
- Author
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Kaplinska K and Mielicki WP
- Subjects
- 1-Carboxyglutamic Acid immunology, Antibodies, Monoclonal immunology, Anticoagulants pharmacology, Cell Line, Tumor enzymology, Cysteine Endopeptidases pharmacology, Enzyme Activation drug effects, Factor X drug effects, Female, Humans, Melanoma pathology, Neoplasm Proteins pharmacology, Pregnancy, Warfarin pharmacology, 1-Carboxyglutamic Acid analysis, Amnion enzymology, Chorion enzymology, Cysteine Endopeptidases chemistry, Melanoma enzymology, Neoplasm Proteins chemistry
- Abstract
Additional carboxylation of glutamic acid by vitamin K-dependent gamma-carboxylase is a common posttranslational modification of many proteins, including some of blood clotting factors. Vitamin K-antagonists, such as warfarin, are often included in the therapy of malignant disease, decreasing the blood coagulation potential. Cancer procoagulant, a direct blood coagulation factor X activator from malignant tissue, is considered as a vitamin K-dependent protein, so it could serve as one of possible targets for the therapy with warfarin. However, there is still no experimental data demonstrating directly the presence of gamma-carboxyglutamic acid (Gla) in a cancer procoagulant molecule. The presence of Gla in cancer procoagulant isolated from human amnion-chorion membranes and from human malignant melanoma WM 115 cell line was analyzed directly, using specific anti-Gla monoclonal antibodies. There was no detectable amount of Gla in cancer procoagulant isolated from fetal or malignant tissue. Cancer procoagulant from human tissues does not contain Gla-rich domain. The finding indicates that cancer procoagulant is rather a poor target for warfarin therapy of malignant disease.
- Published
- 2009
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15. Protein kinase PKR-dependent activation of mitogen-activated protein kinases occurs through mitochondrial adapter IPS-1 and is antagonized by vaccinia virus E3L.
- Author
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Zhang P, Langland JO, Jacobs BL, and Samuel CE
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Amnion enzymology, Cell Line, Enzyme Activation, Humans, Mitogen-Activated Protein Kinases genetics, Phosphorylation, RNA-Binding Proteins genetics, Signal Transduction, Vaccinia virus genetics, Viral Proteins genetics, eIF-2 Kinase genetics, Adaptor Proteins, Signal Transducing metabolism, Mitochondria metabolism, Mitogen-Activated Protein Kinases metabolism, RNA-Binding Proteins metabolism, Vaccinia virus metabolism, Viral Proteins metabolism, eIF-2 Kinase metabolism
- Abstract
The p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) play important roles in the host innate immune response. The protein kinase regulated by RNA (PKR) is implicated in p38 MAPK activation in response to proinflammatory signals in mouse embryonic fibroblasts. To test the role of PKR in the activation of p38 and JNK MAPKs in human cells following viral infection, HeLa cells made stably deficient in PKR by using an RNA interference strategy were compared to cells with sufficient PKR. The phosphorylation of both p38 and JNK in cells with sufficient PKR was activated following either infection with an E3L deletion (DeltaE3L) mutant of vaccinia virus or transfection with double-stranded RNA (dsRNA) in the absence of infection with wild-type vaccinia virus. The depletion of PKR by stable knockdown impaired the phosphorylation of both p38 and JNK induced by either the DeltaE3L mutant virus or dsRNA but not that induced by tumor necrosis factor alpha. The PKR-dependent activation of MAPKs in DeltaE3L mutant-infected cells was abolished by treatment with cytosine beta-d-arabinoside. The complementation of PKR-deficient cells with the human PKR wild-type protein, but not with the PKR catalytic mutant (K296R) protein, restored p38 and JNK phosphorylation following DeltaE3L mutant virus infection. Transient small interfering RNA knockdown established that the p38 and JNK kinase activation following DeltaE3L infection was dependent upon RIG-I-like receptor signal transduction pathway components, including the mitochondrial adapter IPS-1 protein.
- Published
- 2009
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16. Direct contribution of inducible nitric oxide synthase expression to apoptosis induction in primary smooth chorion trophoblast cells of human fetal membrane tissues.
- Author
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Yuan B, Ohyama K, Takeichi M, and Toyoda H
- Subjects
- Amnion cytology, Amnion enzymology, Cells, Cultured, Down-Regulation, Enzyme Activation, Female, Heme Oxygenase-1 biosynthesis, Heme Oxygenase-1 genetics, Humans, Immunohistochemistry, MAP Kinase Signaling System, Nitric Oxide Synthase Type II genetics, Nitric Oxide Synthase Type II metabolism, Oxidative Stress, Transfection, p38 Mitogen-Activated Protein Kinases metabolism, Apoptosis physiology, Chorion cytology, Chorion enzymology, Nitric Oxide Synthase Type II biosynthesis, Trophoblasts cytology, Trophoblasts enzymology
- Abstract
We have previously demonstrated that apoptosis induction is observed only in smooth chorion laeve trophoblast cells, and not in amnion epithelial cells of human fetal membrane tissues prepared at the term. Apoptosis induction was suppressed by the presence of an inhibitor specific for inducible nitric oxide synthase (iNOS), suggesting that intracellular oxidative stress plays a critical role in this process. In this study, we transfected the iNOS gene into primary cultured chorion and amnion cells to examine the direct contribution of iNOS gene expression to the apoptosis induction in these cells. We identified a significant increase in the levels of iNOS protein expression and nitrite accumulation in both chorion and amnion cells after the iNOS gene transfection. However, the induction of apoptosis was observed in an approximately 70% of chorion cells transfected with iNOS gene. Transfection of the iNOS gene into chorion cells resulted in the activation of p38 mitogen-activated protein kinase (MAPK) and downregulation of hemeoxygenase-1 protein expression, whereas no such events were observed in the transfected amnion cells. These results suggest that apoptosis induced in the chorion trophoblast cells by the iNOS gene expression is closely linked to a physiological consequence, such as the rupture of fetal membranes.
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- 2009
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17. The involvement of human amnion in histologic chorioamnionitis is an indicator that a fetal and an intra-amniotic inflammatory response is more likely and severe: clinical implications.
- Author
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Park CW, Moon KC, Park JS, Jun JK, Romero R, and Yoon BH
- Subjects
- Adult, Amniocentesis, Amnion enzymology, Amniotic Fluid chemistry, Amniotic Fluid microbiology, C-Reactive Protein analysis, Chorioamnionitis microbiology, Female, Fetal Blood chemistry, Gestational Age, Humans, Infant, Newborn, Infant, Newborn, Diseases etiology, Male, Matrix Metalloproteinase 8 analysis, Predictive Value of Tests, Pregnancy, Premature Birth, Sepsis complications, Young Adult, Amnion pathology, Chorioamnionitis diagnosis, Chorion pathology, Infant, Newborn, Diseases pathology, Sepsis pathology
- Abstract
Objective: Amnionitis (inflammation of the amnion) is the final stage of extra-placental chorioamniotic inflammation. We propose that patients with "amnionitis", rather than "chorionitis" have a more advanced form of intra-uterine inflammation/infection and, thus, would have a more intense fetal and intra-amniotic inflammatory response than those without "amnionitis"., Study Design: The relationship between the presence of amnionitis, and a fetal and an intra-amniotic inflammatory response was examined in 290 singleton preterm births (
- Published
- 2009
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18. Tocilizumab inhibits interleukin-6-mediated matrix metalloproteinase-2 and -9 secretions from human amnion cells in preterm premature rupture of membranes.
- Author
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Mano Y, Shibata K, Sumigama S, Hayakawa H, Ino K, Yamamoto E, Kajiyama H, Nawa A, and Kikkawa F
- Subjects
- Amnion enzymology, Amnion metabolism, Antibodies, Monoclonal, Humanized, Blotting, Western, Cell Line, Epithelial Cells drug effects, Epithelial Cells enzymology, Epithelial Cells metabolism, Female, Humans, Immunohistochemistry, Interleukin-1beta pharmacology, Interleukin-6 pharmacology, Interleukin-8 pharmacology, Matrix Metalloproteinase 2 biosynthesis, Matrix Metalloproteinase 2 genetics, Matrix Metalloproteinase 2 metabolism, Matrix Metalloproteinase 9 biosynthesis, Matrix Metalloproteinase 9 genetics, Matrix Metalloproteinase 9 metabolism, Peptide Fragments pharmacology, Pregnancy, RNA, Messenger biosynthesis, RNA, Messenger genetics, Reverse Transcriptase Polymerase Chain Reaction, Tumor Necrosis Factor-alpha pharmacology, Amnion drug effects, Antibodies, Monoclonal pharmacology, Fetal Membranes, Premature Rupture enzymology, Interleukin-6 antagonists & inhibitors, Matrix Metalloproteinase Inhibitors
- Abstract
Background/aims: In the present study, we investigated the participation of inflammatory cytokine-induced mediated matrix metalloproteinase (MMP) expressions and inhibition of interleukin (IL)-6-induced MMP secretion in amniotic epithelial cells by tocilizumab., Methods: To investigate the role of MMP expressions, immunohistochemical staining was performed using membranes obtained from 10 patients with preterm premature rupture of membranes (PPROM) and from 10 patients who underwent a nonlabor cesarean section. We also investigated the regulation of MMP expression by inflammatory cytokines in human amnion cells., Results: Immunohistochemical staining showed a significantly higher expression of MMP-2 and -9 in PPROM. Treatment of cultured WISH and primary amniotic epithelial cells with 10(-8) or 10(-7)M IL-6 or tumor necrosis factor (TNF)-alpha clearly increased the secretion of MMP-2 and -9. Treatment with 10(-8)M TNF-alpha or IL-6 significantly increased the invasion of WISH or primary amniotic epithelial cells, respectively, compared with the control. At a low concentration of 1 microg/ml, tocilizumab (anti-human IL-6 receptor monoclonal antibody) inhibited the IL-6-induced MMP secretion., Conclusions: This paper is the 1st report of tocilizumab inhibiting IL-6-induced MMP-2 and MMP-9 secretions from human amnion cells in PPROM.
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- 2009
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19. [Fetal membranes: embryological development, structure and the physiopathology of the preterm premature rupture of membranes].
- Author
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Pasquier JC and Doret M
- Subjects
- Amnion enzymology, Chorion enzymology, Decidua enzymology, Female, Fetal Membranes, Premature Rupture enzymology, Humans, Matrix Metalloproteinases metabolism, Pregnancy, Extraembryonic Membranes embryology, Extraembryonic Membranes enzymology, Fetal Membranes, Premature Rupture physiopathology
- Abstract
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.
- Published
- 2008
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20. Role of glucocorticoid receptor and CCAAT/enhancer-binding protein alpha in the feed-forward induction of 11beta-hydroxysteroid dehydrogenase type 1 expression by cortisol in human amnion fibroblasts.
- Author
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Yang Z, Guo C, Zhu P, Li W, Myatt L, and Sun K
- Subjects
- 11-beta-Hydroxysteroid Dehydrogenase Type 1 genetics, Base Sequence, Benzimidazoles pharmacology, Blotting, Western, Cells, Cultured, Chromatin Immunoprecipitation, Dose-Response Relationship, Drug, Electrophoretic Mobility Shift Assay, Enzyme Induction drug effects, Feedback, Physiological, Female, Genes, Dominant, Genes, Reporter, Glucocorticoids, Hormone Antagonists pharmacology, Humans, Hydrocortisone administration & dosage, Luciferases genetics, Mifepristone pharmacology, Mutation, Plasmids, Polymerase Chain Reaction, Promoter Regions, Genetic drug effects, Promoter Regions, Genetic physiology, Receptors, Glucocorticoid antagonists & inhibitors, Receptors, Glucocorticoid metabolism, Response Elements genetics, Transfection, 11-beta-Hydroxysteroid Dehydrogenase Type 1 biosynthesis, Amnion enzymology, CCAAT-Enhancer-Binding Protein-alpha physiology, Fibroblasts enzymology, Hydrocortisone pharmacology, Receptors, Glucocorticoid physiology
- Abstract
The amount of cortisol available to its receptors is increased by the pre-receptor enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) which converts cortisone to cortisol. We examined the molecular mechanisms of the feedback effect of cortisol on 11beta-HSD1 mRNA expression in human amnion fibroblasts. Our data showed that cortisol-induced 11beta-HSD1 mRNA expression dose dependently in amnion fibroblasts, which could be completely blocked both by the mRNA transcription inhibitor 5,6-dichlorobenzimidazole riboside and by the glucocorticoid receptor (GR) antagonist RU486, and partially blocked by global inhibition of CCAAT/enhancer-binding proteins (C/EBPs) with transfection of C/EBP-specific dominant-negative expression CMV500 plasmid (AC/EBP) into the cells. Likewise, the induction of the promoter activity by cortisol could also be completely blocked by RU486 and partially by AC/EBP transfection. Progressive 5' deletion of the 11beta-HSD1promoter located the region responsible for cortisol's induction within -204 bp upstream to the transcription start site. Specific nucleotide mutations of the putative glucocorticoid responsive element or CCAAT in this promoter region attenuated the induction by cortisol. Moreover, chromatin immunoprecipitation assay and electrophoretic mobility shift assay showed that GR and C/EBPalpha but not C/EBPbeta could bind this promoter region upon cortisol stimulation of amnion fibroblasts. In conclusion, we demonstrated that GR and C/EBPalpha were involved in cortisol-induced 11beta-HSD1 mRNA expression via binding to 11beta-HSD1 promoter in amnion fibroblasts, which may cast a feed-forward production of cortisol in the fetal membranes at the end of gestation.
- Published
- 2007
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21. The effect of progesterone on gelatinase expression in the decidua and fetal membranes before and after contractions.
- Author
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Goldman S, Weiss A, and Shalev E
- Subjects
- 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, Transfection, Decidua enzymology, Extraembryonic Membranes enzymology, Gelatinases metabolism, Progesterone pharmacology, Progestins pharmacology, Uterine Contraction drug effects, Uterine Contraction physiology
- Abstract
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.
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- 2007
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22. Evidence of in vitro differential secretion of 72 and 92 kDa type IV collagenases after selective exposure to lipopolysaccharide in human fetal membranes.
- Author
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Garcia-Lopez G, Vadillo-Ortega F, Merchant-Larios H, Maida-Claros R, Osorio M, Soriano-Becerril D, Flores-Herrera H, Beltran-Montoya J, Garfias-Becerra Y, and Zaga-Clavellina V
- Subjects
- Amnion drug effects, Amnion enzymology, Amnion ultrastructure, Decidua drug effects, Decidua enzymology, Decidua ultrastructure, Enzyme-Linked Immunosorbent Assay, Extracellular Matrix metabolism, Extraembryonic Membranes drug effects, Extraembryonic Membranes ultrastructure, Female, Humans, Tissue Inhibitor of Metalloproteinase-1 metabolism, Tissue Inhibitor of Metalloproteinase-2 metabolism, Tissue Inhibitor of Metalloproteinases metabolism, Tissue Inhibitor of Metalloproteinase-4, Extraembryonic Membranes enzymology, Lipopolysaccharides pharmacology, Matrix Metalloproteinase 2 metabolism, Matrix Metalloproteinase 9 metabolism
- Abstract
Premature rupture of chorioamniotic membranes complicated with intrauterine infection has been associated to degradation of extracellular matrix (ECM), which could explain local morphological changes. We used a culture system in which the chorioamniotic membranes form two independent chambers, allowing for the selective stimulation of either the amnion (AMN) and/or the choriodecidua (CHD) regions. Lipopolysaccharide (500 ng/ml) was added to the AMN and/or the CHD; secretions and gelatinolytic activity of matrix metalloproteinase (MMP)-2 and MMP-9 were measured in both compartments by enzyme-linked immunosorbent assay (ELISA) and zymography. Secretions of TIMP-1, TIMP-2 and TIMP-4 were measured by ELISA. Both metalloproteinases were immunolocalized in tissue sections. All stimulation modalities induced a similar proMMP-2 and proMMP-9 secretion pattern in the CHD with concentrations of 2.49 ng/ml and 90.91 pg/ml, respectively; the AMN showed no significant changes. The active forms of both enzymes did not change with any stimulation modality. TIMP-1, TIMP-2 and TIMP-4 secretions remained without significant changes (P = 0.41). ECM degradation and structural disarrangement were evident after stimulation. Secretion of proMMP-2 and proMMP-9 mainly in the CHD, presence of active forms associated to the tissue and minor changes in TIMPs secretion could favor ECM degradation and explain the weakening and thinning associated with the pathological rupture of chorioamniotic membranes.
- Published
- 2007
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23. 15-Hydroxyprostaglandin dehydrogenase protein expression in human fetal membranes with and without subclinical inflammation.
- Author
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Rizek RM, Watson CS, Keating S, Tai HH, Challis JR, and Bocking AD
- Subjects
- Acute Disease, Adult, Amnion pathology, Analysis of Variance, Antibodies, Blotting, Western, Chorioamnionitis enzymology, Chorioamnionitis pathology, Chorion pathology, Female, Humans, Immunohistochemistry, Inflammation pathology, Isoenzymes metabolism, Mesoderm enzymology, Mesoderm pathology, Obstetric Labor, Premature enzymology, Pregnancy, Trophoblasts enzymology, Trophoblasts pathology, Amnion enzymology, Chorion enzymology, Fetal Membranes, Premature Rupture enzymology, Hydroxyprostaglandin Dehydrogenases metabolism, Inflammation enzymology, Premature Birth enzymology
- Abstract
Prostaglandins play a central role in the stimulation and maintenance of both term and preterm labor. 15-Hydroxyprostaglandin dehydrogenase (PGDH), localized primarily to chorion trophoblasts, is the key enzyme responsible for the metabolism of prostaglandins. In preterm chorion, levels of PGDH protein and activity were lower when compared to term and were further reduced with the presence of infection, but effects of subclinical inflammation and membrane rupture on PGDH expression are not known. Our objectives were (1) to determine the relative expression of PGDH in amnion and chorion and (2) to determine the effect of preterm premature rupture of membranes (PPROM) and (3) subclinical inflammation on PGDH protein expression in preterm fetal membranes. Fetal membranes were collected from women with idiopathic preterm labor. Patients were divided into preterm birth (1) <32 weeks with PPROM (n = 6), (2) <32 weeks with intact membranes (n = 11), (3) >or=32 and <37 weeks with PPROM (n = 10), and (4) >or=32 and <37 weeks with intact membranes (n = 10). Different antibodies were used to detect protein expression and localization of PGDH in amnion and chorion from these patients using both Western blotting and immunohistochemistry. Antibody T (AbT) localized PGDH to chorion trophoblasts, whereas antibody C (AbC) detected immunoreactive (ir) PGDH predominantly in the amnion mesenchyme. By Western blot, AbT showed a stronger 29-kDa ir-PGDH band whereas with AbC, a stronger 55-kDa ir-PGDH signal was detected. 55-kDa ir-PGDH was significantly higher in PPROM amnion, specifically in the <32 weeks group (P < .05) and with PPROM >24 hours (P < .05). No change was detected in the 29-kDa ir-PGDH in either amnion or chorion with gestational age or the presence and absence of PPROM. In addition, neither form of ir-PGDH was altered significantly with or without subclinical inflammation. ir-PGDH is detectable in both chorion trophoblasts and amnion, especially in the mesenchyme; however, the predominant form of the enzyme differs in the 2 tissues. PPROM and subclinical inflammation do not appear to affect the levels of 29-kDa ir-PGDH protein in the fetal membranes. The differential expression of 55-kDa ir-PGDH in preterm amnion with and without PPROM supports the need for a better understanding of the different forms of PGDH.
- Published
- 2007
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24. The proteolytic profile of prelabour ruptured amnion at term: a case-control study.
- Author
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Stuart EL, Evans GS, and Powers HJ
- Subjects
- Adult, Case-Control Studies, Electrophoresis, Polyacrylamide Gel, Female, Fetal Membranes, Premature Rupture etiology, Fluorescence, Gelatinases genetics, Gene Expression Regulation, Enzymologic, Humans, Peptide Hydrolases metabolism, Pregnancy, Serine Endopeptidases genetics, Amnion enzymology, Fetal Membranes, Premature Rupture enzymology, Gelatinases metabolism, Serine Endopeptidases metabolism
- Abstract
Objectives: The aim of this study was to investigate whether prelabour ruptured membranes at term display increased proteolytic activity and to determine whether regional structural alterations within membranes are reflective of regional variations in proteolytic activity., Study Design: Multiple amnion samples were collected from 37 women with prelabour membrane rupture and 37 women whose membranes ruptured spontaneously during labour. In all cases the gestation was greater than 37 weeks. Substrate zymography was used to qualitatively assess gelatinase and serine protease involvement. General protease activity (metallo, serine, acid and sulfhydryl) was measured quantitatively by fluorescent substrate cleavage., Results: Substrate zymography revealed no active gelatinases or serine proteases. Fluorescent studies of general protease activity showed no significant difference between the groups and no significant regional variation., Conclusions: Gelatinase and serine protease activity do not play a major role in the formation of a membrane rupture initiation site or in prelabour membrane rupture at term.
- Published
- 2007
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25. [Identification of extracellular matrix metalloprotease 3 in the fetal membrane of the rat and its possible implication in the rupture of chorioamniotic membranes].
- Author
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Meraz Cruz N, Beltrán Montoya J, Estrada Gutiérrez G, and Vadillo Ortega F
- Subjects
- Amnion metabolism, Amnion ultrastructure, Animals, Basement Membrane enzymology, Enzyme Precursors analysis, Epithelial Cells enzymology, Epithelial Cells metabolism, Extracellular Matrix enzymology, Extracellular Matrix Proteins analysis, Female, Gelatinases analysis, Matrix Metalloproteinase 2 analysis, Matrix Metalloproteinase 3 analysis, Matrix Metalloproteinase 3 metabolism, Matrix Metalloproteinase 9 analysis, Metalloendopeptidases analysis, Microscopy, Confocal, Pregnancy, Rats, Wistar, Rupture, Spontaneous, Amnion enzymology, Matrix Metalloproteinase 3 physiology, Rats metabolism
- Abstract
Objective: Human corioamniotic membranes, or their equivalent in the rat, function as selective barrier during gestation and their rupture is part of the mechanisms implied in the labor. Molecular mechanisms carried out in this process are unknown., Type of Study: Experimental animal model., Material and Methods: Corioamniotic membranes (obtained at the beginning of the labor) of rats with programmed and synchronous pregnancies were analized. The coexistence and distribution of metalloproteinase of extracellular-3 matrix (estromelisine) in these tissues were determined., Results: Secretion and tissue location of metalloproteinase of extracellular-3 matrix in fetal membranes were identified for the first time. Metalloproteinase of extracellular-3 matrix was immunolocated in the compact layer of the amnion and its secretion (by the membranes) was confirmed through electrophoresis, zimography and Western blot. By confocal microscopy it was verified that metalloproteinase of extracellular-3 matrix is located in the same places of that of extracellular-9 matrix., Conclusions: Rupture of corioamniotic membranes relates to the expression and local activity of the metalloproteinases of extracellular matrix. The coexistence of metalloproteinase of extracellular-3 matrix in the amnion of the rat has been identified; this element is added to the biochemical process of rupture, since metalloproteinase of extracelular-3 matrix is an activator of that of extracellular-9 matrix. It is possible that the physiological function of this enzyme is implied, of a main way, in the process of rupture of corioamniotic membranes during the childbirth.
- Published
- 2006
26. In situ immunolabeling allows for detailed localization of prostaglandin synthesizing enzymes within amnion epithelium.
- Author
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Ackerman WE 4th, Hughes LH, Robinson JM, and Kniss DA
- Subjects
- Animals, Cells, Cultured, Cryoultramicrotomy, Epithelium enzymology, Female, Humans, Amnion enzymology, Fluorescent Antibody Technique, Indirect, Prostaglandin-Endoperoxide Synthases analysis, Prostaglandins E biosynthesis
- Abstract
Detailed information regarding the subcellular distribution of proteins within amnion epithelial cells is a goal of numerous placental biologists. In this report, we describe a versatile technique for in situ immunolabeling in amnion that is as technically permissible as traditional immunolabeling of cultured cells and, when coupled with confocal laser scanning microscopy, is similarly capable of providing detailed information regarding subcellular protein distribution. Using antibodies directed against sequential enzymes of the prostaglandin E biosynthesis cascade, we compared this novel method with immunofluorescent labeling using amnion cells in primary culture and cryosections of reflected fetal membrane rolls. By several criteria, we observed morphological variation between the cells cultured in vitro and the tissue specimens. Despite general consistencies in immunostaining patterns between the cryosectioned specimens and those labeled in situ, morphological preservation was superior using the latter technique. Relative to the cryosectioned specimens, in situ immunostaining was advantageous in that it permitted improved sampling efficiency, and allowed regional variations in labeling to be observed in a more global context within the tissue. Our results demonstrate that in situ immunolabeling provides a useful adjunct or alternative to immunolabeling using membrane roll preparations.
- Published
- 2006
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27. Changes in matrix metalloproteinase (MMP)-2 and MMP-9 in the fetal amnion and chorion during gestation and at term and preterm labor.
- Author
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Yonemoto H, Young CB, Ross JT, Guilbert LL, Fairclough RJ, and Olson DM
- Subjects
- Adult, Blotting, Western, Female, Humans, Pregnancy, Premature Birth, Amnion enzymology, Chorion enzymology, Labor, Obstetric metabolism, Matrix Metalloproteinase 2 metabolism, Matrix Metalloproteinase 9 metabolism, Obstetric Labor, Premature metabolism
- Abstract
Increased matrix metalloproteinase (MMP)-9 proteolytic activity is associated with term birth, preterm birth and premature rupture of membranes. However, most studies show no changes with MMP-2, which binds tightly to cell and matrix proteins. We hypothesized better protein extraction would reveal new MMP patterns. Human amnion and chorion were collected from 25 patients at preterm or term, extracted with 2% SDS (a high concentration), and the MMP protein levels and pro-enzyme activities were determined by Western immunoblotting and zymography. MMP-2 protein and MMP-2 and -9 pro-enzyme activities in the amnion increased significantly (p<0.05) with labor at term, and were higher than at preterm labor (p<0.05), when extracted with high SDS concentration. There were no changes in chorion MMPs under any condition. These associations suggest MMP-2 may be another regulator of membrane rupture and other labor-associated mechanisms at term parturition, and its role(s) should be examined further.
- Published
- 2006
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28. Encapsulation cell therapy for mucopolysaccharidosis type VII using genetically engineered immortalized human amniotic epithelial cells.
- Author
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Nakama H, Ohsugi K, Otsuki T, Date I, Kosuga M, Okuyama T, and Sakuragawa N
- Subjects
- Amnion enzymology, Amnion transplantation, Animals, Brain enzymology, Cell Line, Transformed, Epithelial Cells enzymology, Fibroblasts, Glucuronidase biosynthesis, Glucuronidase genetics, Glucuronidase therapeutic use, Humans, Lysosomes enzymology, Mice, Mice, Inbred C3H, Amnion cytology, Epithelial Cells transplantation, Genetic Engineering, Genetic Therapy, Mucopolysaccharidosis VII therapy
- Abstract
Mucopolysaccharidosis type VII (MPSVII) is a lysosomal storage disease resulted from a deficiency of the enzyme beta-glucuronidase (GUSB), which is necessary for degradation of glycosaminoglycans (GAGs). The deficiency of GUSB causes progressive accumulation of GAGs and subsequent lysosomal distension in multiple tissues, including the central nervous system (CNS). In murine experiments, bone marrow transplant, enzyme replacement, viral vectors, and genetically modified cells were successfully used for correction of the visceral accumulation of GAGs, but little improvement was seen in the brain, because these therapeutic agents cannot cross the blood-brain barrier (BBB). Although direct intracerebral injection of GUSB-encoding viral vectors has been developed to bypass the BBB, the possibility of tumor formation and the toxicity of over-expressed GUSB have been reported. In this study, we generated immortalized human amniotic epithelial (IHAE) cells to maintain the effect of implantation, and encapsulated these cells to prevent harmful immunological response and tumor formation and to regulate the level of GUSB expression within the host. Moreover, we generated IHAE cells that over-express and secrete human GUSB following transduction with an adenoviral vector encoding human GUSB. Therapeutic efficacy for MPSVII was evaluated in and ex vivo experiments using these encapsulated genetically engineered GUSB-encoding IHAE cells. We confirmed that encapsulated genetically engineered IHAE cells could secrete significant amounts of GUSB outside the capsule in vitro and into the cerebral parenchyma of C3H mice seven days after the capsule implantation. Thus, encapsulation cell therapy using genetically engineered IHAE cells is an effective armamentarium for the treatment of MPSVII.
- Published
- 2006
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29. Novel targeting of cyclooxygenase-2 (COX-2) pre-mRNA using antisense morpholino oligonucleotides directed to the 3' acceptor and 5' donor splice sites of exon 4: suppression of COX-2 activity in human amnion-derived WISH and myometrial cells.
- Author
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Tyson-Capper AJ and Europe-Finner GN
- Subjects
- Amnion cytology, Amnion drug effects, Amnion enzymology, Exons drug effects, Exons genetics, Female, Humans, Lipopolysaccharides pharmacology, Myometrium cytology, Myometrium drug effects, Myometrium enzymology, RNA Precursors genetics, RNA Splice Sites genetics, Cyclooxygenase 2 genetics, Drug Delivery Systems, Oligodeoxyribonucleotides, Antisense pharmacology, RNA Precursors antagonists & inhibitors, RNA Splice Sites drug effects
- Abstract
Increased expression of cyclooxygenase-2 (COX-2) has been implicated in the onset of both term and preterm labor. In this context, both selective and nonselective COX-2 inhibitors have been used in clinical trials to determine their efficacy in delaying preterm labor. However, recent evidence indicates that these tocolytics may have potentially adverse fetal and maternal side effects. Therefore, the development of more specific and nontoxic agents to inhibit COX-2 needs to be considered. We have evaluated whether antisense morpholino oligonucleotides have therapeutic potential in inhibiting COX-2 by specifically targeting both the 3' and 5' acceptor and donor sites of exon 4 of COX-2's pre-mRNA sequence. Confocal microscopy on "live" cells illustrated high levels of penetrance of antisense morpholino oligonucleotides using the Endo-Porter formula (Gene-Tools, LLC, Philomath, OR), with delivery efficiencies of 82 and 78%, respectively, in amnion-derived WISH and myometrial cells. Substantial inhibition by the morpholino oligonucleotides of COX-2 expression, induced by lipopolysaccharide administration, was observed at both the mRNA and protein levels. Loss of enzymic activity of COX-2 was confirmed using a sensitive COX enzyme activity assay, which reflects the rate of conversion of arachidonic acid to prostaglandin H2. Our results indicate that antisense morpholino oligonucleotides significantly inhibit expression and activity of this enzyme in in vitro cultures of amnion-WISH and myometrial cells. The potential thus exists that a similar approach can be mimicked in vivo to produce a highly specific and nontoxic strategy to inhibit COX-2 activity with its subsequent effects on the better management of preterm labor and other inflammatory conditions.
- Published
- 2006
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30. Use of amnion and placenta in neonatal screening for canine GM1-gangliosidosis and the risk of diagnostic misclassifications.
- Author
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Yamato O, Jo EO, Satoh H, Yamauchi T, Kobayashi A, Yamasaki M, and Maede Y
- Subjects
- Animals, Animals, Newborn, Dog Diseases genetics, Dog Diseases pathology, Dogs, Gangliosidosis, GM1 diagnosis, Gangliosidosis, GM1 genetics, Genotype, Pedigree, Risk, Umbilical Cord, beta-Galactosidase genetics, beta-Galactosidase metabolism, Amnion enzymology, Diagnostic Errors veterinary, Dog Diseases diagnosis, Gangliosidosis, GM1 veterinary, Placenta enzymology
- Abstract
Background: A closed breeding colony of Shiba dogs with GM1-gangliosidosis is maintained at Hokkaido University (Sapporo, Japan). Neonatal genotyping is essential to control the breeding colony genetically as an animal model for the human disease., Objectives: The purpose of the present study was to determine the utility of amnion and placenta in the neonatal screening or diagnosis for canine GM1-gangliosidosis., Methods: Twenty neonatal Shiba dogs of a pedigree with GM1- gangliosidosis were differentiated into 3 genotypes--normal, heterozygous, and affected dogs--by using a previously reported DNA mutation assay. Acid beta-galactosidase activity was measured in amnion and placenta and compared among the 3 genotypes., Results: The level of beta-galactosidase activity in the amnion of affected dogs was negligible and <2% of the mean activity in normal dogs; there was no significant difference among the 3 genotypes. In placenta, beta-galactosidase activity was significantly different among all the genotypes; however, there was wide overlap in enzyme activity between normal and heterozygous dogs. The level of activity in affected dogs was relatively high and >10% of the mean activity in normal dogs. The DNA mutation assay gave correct information about genotype with genomic DNA extracted from amnion but ambiguous information with DNA from placenta., Conclusions: Amnion and placenta were not useful as enzyme sources in neonatal screening in canine GM1-gangliosidosis because of the risk of misdiagnosis. DNA from amnion is applicable as a template for genotyping, whereas placenta should not be used because canine placenta contains maternal cells.
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- 2006
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31. Mechanisms regulating prostaglandin H2 synthase-2 mRNA level in the amnion and chorion during pregnancy.
- Author
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Johnson RF, Mitchell CM, Giles WB, Bisits A, and Zakar T
- Subjects
- Cyclooxygenase 2 metabolism, Female, Gene Expression, Humans, Labor, Obstetric metabolism, Pregnancy, Pregnancy Trimester, First, Pregnancy Trimester, Third, RNA, Messenger analysis, Reverse Transcriptase Polymerase Chain Reaction, Amnion enzymology, Chorion enzymology, Cyclooxygenase 2 genetics, RNA, Messenger metabolism
- Abstract
Increasing prostaglandin H(2) synthase (PGHS)-2 expression in the fetal membranes is implicated in the production of prostaglandins (PGs) that stimulate labour. We have determined the activity of the PGHS-2 gene in the amnion and chorion throughout gestation and defined the contribution of transcriptional and post-transcriptional mechanisms to the increase of PGHS-2 mRNA levels. We also measured PGHS-1 mRNA abundance to assess the participation of the two isoenzymes in fetal membrane PG-production during pregnancy. Amnion and chorion were collected from non-labouring women at 10-19 weeks (early), at 28-36 weeks (preterm) and at term (37-41 weeks). We determined PGHS-1 and -2 mRNA abundance and assessed PGHS-2 gene activity by measuring PGHS-2 heterogeneous nuclear RNA levels using real-time RT-PCR. PGHS-2 gene activity and mRNA levels were up-regulated in both tissues with advancing gestation. Path analysis demonstrated that the PGHS-2 mRNA up-regulation involved both transcriptional and post-transcriptional components. PGHS-2 mRNA abundance increased 9-11 fold between the early (10-19 weeks) and preterm (28-36 weeks) groups and remained high at term. The underlying mechanism was predominantly transcriptional in the amnion and post-transcriptional in the chorion. PGHS-1 mRNA expression precipitously decreased between early gestation and term. Thus, PGHS-2 mRNA abundance is up-regulated well in advance of term and is not a trigger for labour. There is a switch in PGHS mRNA expression during pregnancy with PGHS-1 dominating in the early period and PGHS-2 dominating at term.
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- 2006
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32. Dexamethasone fails to inhibit the induction of cytosolic phospholipase A(2) expression by interleukin-1beta in cultured primary human amnion fibroblasts.
- Author
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Sun K, Qu X, Gao L, and Myatt L
- Subjects
- Amnion enzymology, Cells, Cultured, Cytosol enzymology, Dinoprostone metabolism, Female, Fibroblasts drug effects, Fibroblasts enzymology, Genes, Reporter, Humans, Luciferases analysis, Luciferases genetics, Phospholipases A antagonists & inhibitors, Phospholipases A genetics, Promoter Regions, Genetic, Amnion cytology, Amnion drug effects, Dexamethasone pharmacology, Interleukin-1 pharmacology, Phospholipases A metabolism
- Abstract
Objective: To investigate the interaction of dexamethasone and interleukin-1beta (IL-1beta) on the expression of cytosolic phospholipase A(2) (cPLA(2)), the enzyme catalyzing the first reaction in the formation of prostaglandins, in cultured primary human amnion fibroblasts., Design and Methods: Human amnion fibroblasts were prepared from fetal amnion collected at term and were treated with dexamethasone with or without interleukin-1beta for 24h. Prostaglandin E(2) (PGE(2)) output and cPLA(2) expression in cultured amnion fibroblasts were measured with radioimmunoassay, quantitative real-time polymerase chain reaction, Western blotting and cPLA(2) promoter-driven luciferase reporter gene activity., Results: Both dexamethasone and IL-1beta caused a significant increase in prostaglandin E(2) output, cPLA(2) mRNA and protein expression in cultured human amnion fibroblasts. Both dexamethasone and IL-1beta stimulated cPLA(2) promoter-driven luciferase reporter gene activity. There was no obvious antagonistic or synergistic effect of combined treatment of dexamethasone and IL-1beta on PGE(2) output, cPLA(2) expression or cPLA(2) promoter-driven luciferase reporter gene activity in cultured human amnion fibroblasts., Conclusion: The above findings suggest that paradoxically dexamethasone is a stimulator for both prostaglandin synthesis and cPLA(2) expression in human amnion fibroblasts. The interaction between dexamethasone and IL-1beta on prostaglandin synthesis and cPLA(2) expression is neither synergistic nor conventionally antagonistic.
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- 2006
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33. Prostaglandin H2 synthase-1 and -2 expression in guinea pig gestational tissues during late pregnancy and parturition.
- Author
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Welsh T, Mitchell CM, Walters WA, Mesiano S, and Zakar T
- Subjects
- Amnion enzymology, Animals, Cyclooxygenase 1 biosynthesis, Cyclooxygenase 1 genetics, Cyclooxygenase 2 biosynthesis, Cyclooxygenase 2 genetics, Cyclooxygenase 2 metabolism, Cyclooxygenase Inhibitors pharmacology, Endometrium enzymology, Female, Guinea Pigs, Labor, Obstetric metabolism, Models, Animal, Piroxicam pharmacology, Placenta enzymology, Pregnancy, Prostaglandins metabolism, RNA, Messenger metabolism, Time Factors, Amnion metabolism, Cyclooxygenase 1 metabolism, Endometrium metabolism, Parturition metabolism, Placenta metabolism
- Abstract
Increased intrauterine prostaglandin (PG) production is crucial for the initiation of parturition. To investigate the mechanisms controlling intrauterine PG synthesis, we examined the expression of the key PG biosynthetic isoenzymes, PG-H2 synthase (PTGS)-1 and -2, in the amnion, visceral yolk sac (VYS), placenta and myo-endometrium of pregnant guinea pigs. This animal model was chosen because the hormonal milieu of pregnancy and the role of PGs in the hormonal control of parturition are similar to those in the human. PTGS1 mRNA abundance, measured by real-time RT-PCR, increased in the amnion and the placenta during the last third of gestation. During labour, PTGS1 mRNA levels decreased precipitously in all four tissues. PTGS1 protein abundance, assessed by immunoblotting, increased to high levels in the amnion and the placenta by the end of pregnancy and remained high during labour. PTGS2 mRNA expression was higher in the placenta than in the other tissues, but did not change before and during labour. PTGS2 protein expression decreased in the placenta and remained low in the other tissues during labour. Immunohistochemistry showed pervasive PTGS1 protein expression in the amnion and strong expression in the parietal yolk sac membrane (PYS) covering the placenta. PTGS2 was expressed in the PYS and the endometrium. The PTGS inhibitor piroxicam, administered in doses that inhibited PTGS1 but not PTGS2, significantly prolonged gestation. These data suggest that PGs generated by intrauterine PTGS1 are involved in the timing of birth in guinea pigs. The induction of PTGS1 in the amnion and the PYS is a critical event leading to labour in guinea pigs and models analogous changes in the human gestational tissues before labour.
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- 2005
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34. The role of CCAAT/enhancer-binding protein beta in the transcriptional regulation of COX-2 in human amnion.
- Author
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Lee YS, Terzidou V, Lindstrom T, Johnson M, and Bennett PR
- Subjects
- Amnion enzymology, Base Sequence, Binding Sites, Cesarean Section, DNA Primers, Female, Gene Expression Regulation, Enzymologic, Genes, Reporter, Humans, Mutagenesis, Site-Directed, Pregnancy, Promoter Regions, Genetic, Transfection, Amnion physiology, CCAAT-Binding Factor physiology, Cyclooxygenase 2 genetics, Transcription, Genetic
- Abstract
Human labour is associated with increased prostaglandin synthesis within the uterus by the action of the inducible type-2 cyclo-oxygenase enzyme (COX-2). A major source of prostaglandin is the fetal membranes, in particular the amnion, in which expression of COX-2 increases in late pregnancy and with labour. The COX-2 gene promoter contains several putative transcription factor binding sites including those for NF-kappaB, AP-1 and C/EBP and therefore has the features of a rapid response gene. We have previously shown that, in amnion, the NF-kappaB DNA-binding sites in the COX-2 promoter are essential for gene expression and that there is an increase in NF-kappaB activity in amnion with the onset of labour. In this study, we demonstrate that in primary human amnion cells, CCAAT/enhancer-binding protein beta (C/EBPbeta) DNA-binding sites are crucial for the function of the COX-2 gene promoter. Three potential C/EBPbeta DNA-binding sites were identified within the COX-2 promoter which were shown to bind to C/EBPbeta but not to C/EBPalpha, C/EBPdelta, CREB (cAMP responsive element modulator) or CREM. Luciferase reporter constructs with site-directed mutagenesis of the three C/EBPbeta sites in the COX-2 promoter showed reduced expression of luciferase in transient transfection studies. However, comparison of C/EBPbeta protein levels and their DNA-binding activity from cells obtained before and after labour showed no significant differences. This suggests that although C/EBPbeta plays an essential constitutive role in the expression of COX-2, C/EBPbeta may not be directly involved in its regulation in association with human labour.
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- 2005
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35. 5 Beta-dihydroprogesterone and steroid 5 beta-reductase decrease in association with human parturition at term.
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Sheehan PM, Rice GE, Moses EK, and Brennecke SP
- Subjects
- 5-alpha-Dihydroprogesterone blood, Amnion enzymology, Chorion enzymology, Female, Humans, Myometrium enzymology, Oxidoreductases genetics, Placenta enzymology, Pregnancy, RNA, Messenger metabolism, 5-alpha-Dihydroprogesterone metabolism, Labor, Obstetric metabolism, Oxidoreductases metabolism, Parturition metabolism
- Abstract
The role of progesterone withdrawal in human parturition continues to provoke controversy. One possible mechanism by which functional progesterone withdrawal may be achieved is by a decrease in the circulating concentration of its bioactive metabolites. The progesterone metabolite 5beta-dihydroprogesterone (5betaDHP) has been shown to be a potent tocolytic in vitro. We quantified plasma concentrations of 5betaDHP in association with the onset of spontaneous labour in women at term and steroid 5beta-reductase mRNA expression in placenta, myometrium, chorion and amnion in relation to parturition, using real time RT-PCR. Serial blood samples were obtained from patients late in pregnancy, before term labour, during term labour and within the first 24 h postpartum. Following organic solvent extraction, steroids including 5betaDHP were separated by high-performance liquid chromatography (HPLC) and then quantified by radioimmunoassay (RIA). 5betaDHP concentration decreased two-fold (P = 0.00001, n = 25) from 0.317 +/- 0.039 nmol/ml to 0.178 +/- 0.017 nmol/ml in association with active labour. Tissue 5beta-reductase mRNA-relative abundance was determined in placenta, myometrium, chorion and amnion obtained from labouring and non-labouring women. In placenta and myometrium, relative expression decreased significantly in association with labour, by about two-fold and 10-fold, respectively. These data are consistent with a possible role for 5betaDHP in the onset of spontaneous human labour. Further studies exploring this hitherto unrecognized endocrinological pathway are indicated.
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- 2005
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36. Evidence for a role of phosphodiesterase 4 in lipopolysaccharide-stimulated prostaglandin E2 production and matrix metalloproteinase-9 activity in human amniochorionic membranes.
- Author
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Oger S, Méhats C, Dallot E, Cabrol D, and Leroy MJ
- Subjects
- 3',5'-Cyclic-AMP Phosphodiesterases antagonists & inhibitors, Adjuvants, Immunologic pharmacology, Adjuvants, Immunologic physiology, Amnion drug effects, Amnion metabolism, Chorion drug effects, Chorion metabolism, Cyclic AMP antagonists & inhibitors, Cyclic AMP physiology, Cyclic Nucleotide Phosphodiesterases, Type 4, Cyclooxygenase 2, Enzyme Activation drug effects, Enzyme Activation physiology, Enzyme Precursors metabolism, Female, Humans, Immune Sera pharmacology, Interleukin-10 immunology, Interleukin-10 metabolism, Membrane Proteins, Phosphodiesterase Inhibitors pharmacology, Pregnancy, Prostaglandin-Endoperoxide Synthases biosynthesis, Rolipram pharmacology, Tissue Culture Techniques, Tumor Necrosis Factor-alpha antagonists & inhibitors, Tumor Necrosis Factor-alpha metabolism, Up-Regulation drug effects, Up-Regulation immunology, 3',5'-Cyclic-AMP Phosphodiesterases physiology, Amnion enzymology, Amnion immunology, Chorion enzymology, Chorion immunology, Dinoprostone biosynthesis, Lipopolysaccharides immunology, Matrix Metalloproteinase 9 metabolism
- Abstract
Chorioamniotic infection is a leading cause of preterm premature rupture of fetal membranes (amnion and chorion). Bacterial infection induces an inflammatory response characterized by elevated production of proinflammatory cytokines; the latter activate the production of both PGs that stimulate uterine contractions, and matrix metalloproteinases (MMPs) that degrade the extracellular matrix of the chorioamniotic membranes. The inflammatory response is under the control of cAMP content, which is partly regulated by phosphodiesterases (PDE). In this study, we investigated the role of the PDE4 family in the inflammatory process triggered by LPS in a model of amniochorionic explants. We found that PDE4 family is the major cAMP-PDE expressed in human fetal membranes and that PDE4 activity is increased by LPS treatment. Selective inhibition of PDE4 activity affected LPS signaling, because PDE4 inhibitors (rolipram and/or cilomilast) reduced the release of the proinflammatory cytokine TNF-alpha and increased the release of the anti-inflammatory cytokine IL-10. PDE4 inhibition reduced cyclooxygenase-2 protein expression and PGE(2) production and also modulated MMP-9, a key mediator of the membrane rupture process, by inhibiting pro-MMP-9 mRNA expression and pro-MMP-9 activity. These results demonstrate that the PDE4 family participates in the regulation of the inflammatory response associated with fetal membrane rupture during infection. The PDE4 family may be an appropriate pharmacological target for the management of infection-induced preterm delivery.
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- 2005
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37. Initial characterization of the microenvironment that regulates connective tissue degradation in amniochorion during normal human labor.
- Author
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Estrada-Gutierrez G, Zaga V, Gonzalez-Jimenez MA, Beltran-Montoya J, Maida-Claros R, Giono-Cerezo S, and Vadillo-Ortega F
- Subjects
- Amnion cytology, Amnion enzymology, Chorion cytology, Chorion enzymology, Connective Tissue enzymology, Female, Humans, Leukocyte Count, Matrix Metalloproteinase 2 genetics, Matrix Metalloproteinase 2 metabolism, Matrix Metalloproteinase 9 genetics, Matrix Metalloproteinase 9 metabolism, Pregnancy, Tissue Culture Techniques, Amnion metabolism, Chorion metabolism, Connective Tissue metabolism, Labor, Obstetric metabolism
- Abstract
Extracellular matrix degradation in fetal membranes leading to its rupture is coupled to myometrial activity and cervical ripening during human normal labor. Mechanisms which modulate collagen degradation in amniochorion during labor have not been elucidated. Initial characterization of the effect of different blood compartments on connective tissue degradation in amniochorion during human labor was explored. Amniochorion explants were stimulated with plasma of maternal venous blood, umbilical cord blood or placental blood, obtained from women with pregnancies at term, with or without labor. MMP-2 and MMP-9 activities were quantified in conditioned media by gelatin-zymography as an index of connective tissue degradation. Collagen content was measured in tissue explants and collagen fibrils distribution was examined by electron microscopy. Placental plasma from term pregnancies, with or without labor, is enriched with soluble signals that enhance the in vitro MMP-9 production by amniochorion. Accompanying ultrastructural distortion of collagen fibers and demonstration of collagen degradation fragments confirmed induction of extracellular matrix degradation. Control experiments in which MMP-9 activity was blocked with TIMP-1 resulted in inhibition of all the above mentioned changes. These results suggest that placental intervillous space is a functional compartment in which mediators capable to induce collagen degradation in amniochorion are selectively expressed during human labor.
- Published
- 2005
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38. Amnioserosa is required for dorsal closure in Drosophila.
- Author
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Scuderi A and Letsou A
- Subjects
- Amnion drug effects, Amnion enzymology, Animals, Animals, Genetically Modified, Cell Movement, Cell Shape, Drosophila cytology, Drosophila enzymology, Drosophila Proteins genetics, Embryo, Nonmammalian, Epidermal Cells, Epidermis drug effects, Epidermis embryology, Gene Expression Regulation, Developmental, Genes, Reporter, Green Fluorescent Proteins metabolism, JNK Mitogen-Activated Protein Kinases genetics, Models, Biological, Mutation, Ricin genetics, Ricin metabolism, Ricin pharmacology, Signal Transduction, Transcription, Genetic, Transgenes, beta-Galactosidase metabolism, Amnion embryology, Body Patterning, Drosophila embryology, Drosophila genetics, JNK Mitogen-Activated Protein Kinases metabolism
- Abstract
Dorsal closure in the fruit fly Drosophila melanogaster is a complex morphogenetic process, driven by sequential signaling cascades and involving multiple forces, which contribute to cell movements and rearrangements as well as to changes in cell shape. During closure, lateral epidermal cells elongate along the dorsoventral axis and subsequently spread dorsally to cover the embryonic dorsal surface. Amnioserosal cells, which are the original occupants of the most dorsal position in the developing embryo, constrict during closure; thus, the increase in epidermal surface area is accommodated by a reduction in the amnioserosal surface area. Several of the epidermal requirements for closure have been established in functional assays. In contrast, amnioserosal requirements for closure have remained elusive, in part because laser ablation and clonal approaches are limited to only subsets of amnioserosal cells. Here, we report our use of the UAS-GAL4 system to target expression of the cell autonomous toxin Ricin-A to all cells of the amnioserosa. We show that ablation of the amnioserosa leads to clear defects in dorsal closure and, thus, directly demonstrate a role for the amnioserosa in dorsal closure. We also show that DJNK (Drosophila Jun N-terminal kinase) signaling, an epidermal trigger of closure, is unaffected by amnioserosal ablation. These data, together with our demonstration that amnioserosal ablated and Dpp signaling mutant embryos exhibit shared loss-of-function phenotypes, point to a requirement for the amnioserosa in dorsal closure that is downstream of Dpp, perhaps as part of a paracrine response to this signaling cascade., (Copyright (c) 2005 Wiley-Liss, Inc.)
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- 2005
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39. Epidermal growth factor and interleukin-1beta utilize divergent signaling pathways to synergistically upregulate cyclooxygenase-2 gene expression in human amnion-derived WISH cells.
- Author
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Ackerman WE 4th, Rovin BH, and Kniss DA
- Subjects
- Amnion cytology, Amnion enzymology, Cell Line, Cyclooxygenase 2, Drug Synergism, Enzyme Inhibitors pharmacology, Epidermal Growth Factor pharmacology, Female, Gene Expression, Humans, Interleukin-1 pharmacology, Leupeptins pharmacology, Membrane Proteins, NF-kappa B antagonists & inhibitors, Promoter Regions, Genetic drug effects, Prostaglandin-Endoperoxide Synthases genetics, RNA, Messenger metabolism, Recombinant Proteins pharmacology, Time Factors, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors, Amnion metabolism, Epidermal Growth Factor metabolism, Interleukin-1 metabolism, Prostaglandin-Endoperoxide Synthases metabolism, Signal Transduction physiology, Up-Regulation
- Abstract
In human parturition, uterotonic prostaglandins (PGs) arise predominantly via increased expression of cyclooxygenase-2 (COX-2 [also known as prostaglandin synthase 2]) within intrauterine tissues. Interleukin-1 (IL-1) and epidermal growth factor (EGF), both inducers of COX-2 transcription, are among numerous factors that accumulate within amniotic fluid with advancing gestation. It was previously demonstrated that EGF could potentiate IL-1beta-driven PGE(2) production in amnion and amnion-derived (WISH) cells. To define the mechanism for this observation, we hypothesized that EGF and IL-1beta might exhibit synergism in regulating COX-2 gene expression. In WISH cells, combined treatment with EGF and IL-1beta resulted in a greater-than-additive increase in COX-2 mRNA relative to challenge with either agent independently. Augmentation of IL-1beta-induced transactivation by EGF was not observed in cells harboring reporter plasmids bearing nuclear factor-kappa B (NFkappaB) regulatory elements alone, but was evident when a fragment (-891/ +9) of the COX-2 gene 5'-promoter was present. Both agents transiently activated intermediates of multiple signaling pathways potentially involved in the regulation of COX-2 gene expression. The 26 S proteasome inhibitor, MG-132, selectively abrogated IL-1beta-driven NFkappaB activation and COX-2 mRNA expression. Only pharmacologic blockade of the p38 mitogen-activated protein kinase eliminated COX-2 expression following EGF stimulation. We conclude that EGF and IL-1beta appear to signal through different signaling cascades leading to COX-2 gene expression. IL-1beta employs the NFkappaB pathway predominantly, while the spectrum of EGF signaling is broader and includes p38 kinase. The synergism observed between IL-1beta and EGF does not rely on augmented NFkappaB function, but rather, occurs through differential use of independent response elements within the COX-2 promoter.
- Published
- 2004
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40. The effect of insulin-like growth factor-I on production of vascular endothelial growth factor by amnion-derived (WISH) cells.
- Author
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Kawano Y, Nakamura S, Fukuda J, and Miyakawa I
- Subjects
- Amnion cytology, Cells, Cultured, Enzyme Inhibitors metabolism, Humans, Insulin-Like Growth Factor I pharmacology, Mitogen-Activated Protein Kinases metabolism, Phosphatidylinositol 3-Kinases metabolism, Vascular Endothelial Growth Factor A antagonists & inhibitors, Amnion enzymology, Insulin-Like Growth Factor I physiology, Vascular Endothelial Growth Factor A biosynthesis
- Abstract
Objective: Our objective was to clarify the modulation of vascular endothelial growth factor (VEGF) by amniotic cells., Design: Amnion-derived (WISH) cells were cultured, and the effect of insulin-like growth factor (IGF), mitogen-activated protein (MAP) kinase kinase and/or extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors (U0126), and phosphatidylinositol (PI) 3-kinase inhibitors (wortmannin) on the production of VEGF was examined. VEGF was assayed using ELISA. The activations of MAP kinase and akt, which is phosphorylated by PI 3-kinase, were detected by Western blot analysis using anti-phosphorylated MAP kinase antibody and anti-phosphorylated akt antibody., Results: In the time course of VEGF production following IGF-I treatment, VEGF production showed significant increases only at 16 and 32 h (p < 0.01). Also, IGF-I increased the production of VEGF by WISH cells in a dose-dependent manner. The MAP kinase and akt activities were recognized by treatment with IGF-I and suppressed by U0126 and wortmannin, respectively. When WISH cells were pretreated for 2 h with U0126 and wortmannin and then treated with IGF-I for 16 h, the production of VEGF was significantly decreased in a dose-dependent manner (p < 0.01, p < 0.01, respectively)., Conclusions: WISH cells appeared to produce VEGF via a mechanism involving tyrosine kinase interaction with IGF-I receptor, resulting in MAP kinase and PI 3-kinase activation. It is suggested that VEGF may contribute to the neovascularization and proliferation of the placenta and gestational tissue, and IGF-I may play an important role in the modulation of VEGF production in the placenta.
- Published
- 2004
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41. Hypothetical proteins with putative enzyme activity in human amnion, lymphocyte, bronchial epithelial and kidney cell lines.
- Author
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Afjehi-Sadat L, Krapfenbauer K, Slavc I, Fountoulakis M, and Lubec G
- Subjects
- Amino Acid Sequence, Amnion cytology, Amnion enzymology, Bronchi enzymology, Cell Line, Electrophoresis, Gel, Two-Dimensional, Endopeptidases metabolism, Enzymes chemistry, Enzymes metabolism, Epithelial Cells enzymology, Humans, Kidney cytology, Kidney enzymology, Lymphocytes enzymology, Molecular Sequence Data, Protein Binding, Proteins chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Ubiquitin metabolism, Amnion metabolism, Bronchi metabolism, Epithelial Cells metabolism, Kidney metabolism, Lymphocytes metabolism, Proteins metabolism
- Abstract
A myriad of predicted proteins have been described based upon nucleic acid sequences but the existence of these structures has not been confirmed at the protein level. The aim of the study was therefore to show expression of hypothetical proteins in several cell lines and to provide the analytical basis for their identification and characterisation. We used two-dimensional gel electrophoresis with in-gel digestion of high protein spots and subsequent MALDI-TOF analysis of cell lysates from human amnion, lymphocyte, bronchial epithelial and kidney cell lines. A pI range from 3 to 10 was selected and second dimension was run using 9-16% gradient gels. A series of structures that have not been described before at the protein level were identified in several cell lines and were assigned to major enzyme systems including proteolysis (proteases, peptidases, ubiquitin), intermediary metabolism and oxidoreductases. We conclude that the proteomic approach used serves as a suitable tool to verify the existence of predicted/hypothetical proteins. The herein identified enzymes may contribute to several pathways/cascades in the human organism. Furthermore, analytical data given are of major relevance as pIs, a prerequisite to find proteins in a map, cannot be predicted from nucleic acid sequences.
- Published
- 2004
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- View/download PDF
42. Regulation of phospholipase isozymes by nuclear factor-kappaB in human gestational tissues in vitro.
- Author
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Lappas M, Permezel M, Georgiou HM, and Rice GE
- Subjects
- Amnion enzymology, Amnion metabolism, Chorion enzymology, Chorion metabolism, Cyclooxygenase 1, Cyclooxygenase 2, Cytosol enzymology, DNA metabolism, Decidua enzymology, Decidua metabolism, Dinoprost metabolism, Electrophoresis, Enzyme-Linked Immunosorbent Assay, Extraembryonic Membranes enzymology, Female, Humans, In Vitro Techniques, Isoenzymes metabolism, Membrane Proteins, NF-kappa B genetics, Phospholipases A metabolism, Placenta enzymology, Pregnancy, Prostaglandin-Endoperoxide Synthases metabolism, Sulfasalazine pharmacology, Transcription Factor RelA, Dinoprost analogs & derivatives, Extraembryonic Membranes metabolism, NF-kappa B physiology, Phospholipases metabolism, Placenta metabolism
- Abstract
Phospholipid-derived mediators are implicated in the initiation and progression of human labor and delivery, particularly in relation to infection-induced preterm labor. We previously demonstrated that, in human intrauterine tissues, lipopolysaccharide (LPS)-stimulated nuclear factor-kappaB (NF-kappaB) DNA binding activity, and subsequent cytokine release can be suppressed by sulfasalazine (SASP) concentrations greater than 5 mM. The aim of this study was to elucidate the effect the SASP on secretory type II phospholipase A(2) (PLA(2)), cytosolic PLA(2) (cPLA(2)), cyclooxygenase (COX) isozymes, and subsequent prostaglandin F(2alpha) (PGF(2alpha)) production in human gestational tissues. Human placenta, amnion, and choriodecidua (n = 4-9 separate placentas) were incubated in the presence of SASP (0.1, 1, 5, and/or 10 mM) under either basal or LPS (10 microg/ml) conditions. After 6 h incubation, the tissues were collected and assayed for type II PLA(2) by ELISA and cPLA(2), COX-1, and COX-2 content by Western blotting. The incubation medium was collected and assayed for type II PLA(2) and 13,14-dihydro-15-keto PGF(2alpha) release by ELISA and PGF(2alpha) by RIA. Treatment of placenta, amnion, and choriodecidua with SASP concentrations greater than 5 mM significantly inhibited basal and/or LPS-stimulated type II PLA(2) content and release, 13,14-dihydro-15-keto PGF(2alpha) release, and cPLA(2) protein content (ANOVA, P < 0.05); however, no effect of SASP was observed on basal or LPS-stimulated COX-1 or COX-2 protein. Although no effect of SASP was observed on basal and LPS-stimulated PGF(2alpha) release from placenta and amnion, it significantly increased both basal and LPS-stimulated PGF(2alpha) release from choriodecidua. In addition, SASP concentrations of 5 mM or greater significantly suppressed NF-kappaB DNA binding activity. These data are consistent with the hypothesis that NF-kappaB regulates the expression and release of phospholipase isozymes.
- Published
- 2004
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43. [Properties of the chorioamnios zone inducing premature membranes rupture].
- Author
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Meraz Cruz MC, Beltrán Montoya J, Bustos López H, Flores Pliego A, Espejel A, Buendía Díaz G, and Vadillo-Ortega F
- Subjects
- Adult, Culture Techniques, Female, Humans, Matrix Metalloproteinase 9 metabolism, Pregnancy, Amnion enzymology, Amnion pathology, Chorion enzymology, Chorion pathology, Fetal Membranes, Premature Rupture etiology
- Abstract
Premature membrane rupture (PMR) is one of the most serious public health problems in the world, ocurring in 10% of all pregnancies. PMR has important adverse effects on maternofetal morbidity-mortality, as it has been estimated that it accounts on the whole for 70% and 40% of neonatal morbidity and mortality, respectively. PMR treatment is empirical, as its aetiology is unknown and its physiopathogenic description has just been initiated. This work analyzes the possibility of documenting functional differences in human chorio-amnios, comparing the zone where rupture most frequently occurs in PMR with some other distant chorio-amnionic zones and with equivalent zones of fetal membranes obtained from nine month pregnancies which have not undergone labor. The membrane zone which was nearest to the cervical os was identified and marked to be analyzed later for extracellular matrix metalloprotease (MMP) activity, histology and topographical MMP distribution. The MMP expression was quantitatively determined in explant culture media from membrane fragments using specific immuno-enzymatic essays (ELISA) and zymography. In addition, immuno-histochemistry methods were used to reveal MMP expression in the different tissues. This methods allowed us to show the existence of a decreasing MMP activity gradient, with the greatest value corresponding to the zone nearest to the cervical os in the membranes obtained from PMR cases. In membranes obtained from cesarean operations no characteristic pattern was documented and values were always lower than those obtained for PMR tissues. We conclude that there is a chorio-amnionic zone in which connective tissue degradation is specifically induced and which coincides with the membrane zone in contact with the cervical os.
- Published
- 2003
44. Late gestation increase in 11beta-hydroxysteroid dehydrogenase 1 expression in human fetal membranes: a novel intrauterine source of cortisol.
- Author
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Alfaidy N, Li W, MacIntosh T, Yang K, and Challis J
- Subjects
- 11-beta-Hydroxysteroid Dehydrogenase Type 1, Female, Fetus enzymology, Humans, Labor, Obstetric physiology, Pregnancy, Pregnancy Trimester, Third, Amnion enzymology, Chorion enzymology, Hydrocortisone metabolism, Hydroxysteroid Dehydrogenases metabolism, Placenta enzymology
- Abstract
Late human gestation is associated with an increase in the concentration of cortisol (F) in the fetal circulation and amniotic fluid. It had been assumed that most of the F measured in the amniotic fluid came from the fetal adrenal gland. However, local production of F can also occur in human intrauterine tissues from inactive cortisone under the influence of the enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1. Recent studies have shown that 11beta-HSD 1 activity is up-regulated by prostaglandins (PG) E2 and F2alpha, hormones that are produced in the fetal membranes (FM) at term. In the present study, we hypothesized that 11beta-HSD 1 expression would increase in FM during pregnancy and at labor, creating the potential for local increase in F production at term. We examined 11beta-HSD 1 expression in placenta and FM obtained during normal pregnancy from nonlaboring women [26-28 wk (n = 3); 29-30 wk (n = 3); 32-33 wk (n = 3); 35-36 wk (n = 3)] and from uncomplicated term pregnancies after elective cesarean section (n = 6). 11beta-HSD 1 expression was also examined in amnion and chorionic tissues in relation to term labor (n = 12). Immunohistochemistry and Western blot analysis were used to examine 11beta-HSD 1 localization and expression. 11beta-HSD 1 activity was also measured in microsomal fractions prepared from whole fetal membranes. At term, immunoreactive 11beta-HSD 1 expression was localized predominantly to the chorion trophoblast cells, attached decidua, and amnion epithelial cells. 11beta-HSD 1 expression in FM increased with gestational age and reflected increased enzyme reductase activity. No change in 11beta-HSD 1 expression was found in placental tissue from the same patients. There was a significant increase in 11beta-HSD 1 expression in amnion but not in chorion with the onset of labor. We suggest that increases in 11beta-HSD 1 expression/activity by intrauterine membranes during late gestation may result in increased potential for a local increase in F production and that FM should be considered as an extraadrenal source of F during late gestation. This local F production may be involved in different pathways contributing to the regulation of parturition.
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- 2003
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45. Sphingosine 1-phosphate in amniotic fluid modulates cyclooxygenase-2 expression in human amnion-derived WISH cells.
- Author
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Kim JI, Jo EJ, Lee HY, Cha MS, Min JK, Choi CH, Lee YM, Choi YA, Baek SH, Ryu SH, Lee KS, Kwak JY, and Bae YS
- Subjects
- Amnion cytology, Base Sequence, Blotting, Western, Cell Line, Cyclooxygenase 2, DNA Primers, Dinoprostone biosynthesis, Humans, Membrane Proteins, Reverse Transcriptase Polymerase Chain Reaction, Amnion enzymology, Amniotic Fluid metabolism, Isoenzymes metabolism, Lysophospholipids, Prostaglandin-Endoperoxide Synthases metabolism, Sphingosine analogs & derivatives, Sphingosine metabolism
- Abstract
The metabolism of arachidonic acid, in particular the generation of prostaglandins (PGs), has been proposed to play a key role in the regulation of labor. Moreover, several extracellular proteins have been reported to modulate PG synthesis in amnion cells. In this study, we found that lipid components dissolved in the amniotic fluid modulate PG synthesis in WISH human amnion cells and identified one of these components as a sphingosine 1-phosphate (S1P). WISH cells express several S1P receptors including S1P1, S1P2, and S1P3. When WISH cells were stimulated with S1P, PGE2 synthesis increased in a concentration-dependent manner, showing maximal activity at around 100 nM. S1P treatment also caused the up-regulation of cyclooxygenase-2 (COX-2) mRNA and protein, which was apparent within 3-12 h of stimulation. In terms of the intracellular signaling pathway of S1P-induced WISH cell activation, we found that S1P stimulated two kinds of MAPK, ERK, and p38 kinase. We examined the roles of these two MAPKs in S1P-induced COX-2 expression. S1P-induced COX-2 expression was blocked completely by PD-98059 but not by SB-203580, suggesting that ERK has a critical role in the process. Transfection of S1P1 or S1P3 but not of S1P2 antisense oligonucleotide inhibited S1P-induced COX-2 expression and PGE2 production in WISH cells, indicating the involvements of S1P1 and S1P3 in the processes. This study demonstrates the physiological role of S1P in amniotic fluid and its effect on the modulation of COX-2 expression and PGs synthesis in WISH cells.
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- 2003
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46. Progesterone represses interleukin-8 and cyclo-oxygenase-2 in human lower segment fibroblast cells and amnion epithelial cells.
- Author
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Loudon JA, Elliott CL, Hills F, and Bennett PR
- Subjects
- Amnion enzymology, Amnion immunology, Cells, Cultured, Cyclooxygenase 2, Dinoprostone biosynthesis, Epithelial Cells drug effects, Epithelial Cells enzymology, Epithelial Cells immunology, Female, Fibroblasts drug effects, Fibroblasts enzymology, Fibroblasts immunology, Humans, Interleukin-1 pharmacology, Interleukin-8 genetics, Isoenzymes genetics, Labor, Obstetric genetics, Labor, Obstetric immunology, Labor, Obstetric metabolism, Membrane Proteins, Pregnancy, Promoter Regions, Genetic drug effects, Prostaglandin-Endoperoxide Synthases genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Amnion drug effects, Interleukin-8 biosynthesis, Isoenzymes metabolism, Progesterone pharmacology, Prostaglandin-Endoperoxide Synthases metabolism
- Abstract
Labor is preceded by cervical ripening through upregulation of interleukin (IL)-1beta, IL-8, and increased prostaglandin synthesis via inducible type 2 cyclooxygenase (COX-2). Progesterone maintains myometrial quiescence during pregnancy. In this study, we examined the effects of IL-1beta and progesterone on IL-8 and prostaglandin E2 (PGE2) synthesis and IL-8 and COX-2 mRNA and promoter activity in amnion cells and lower segment fibroblast (LSF) cells. In both cell types, progesterone had no effect on basal IL-8 or PGE2 synthesis. In LSF cells, IL-1beta significantly increased IL-8 and PGE2 synthesis and COX-2 and IL-8 mRNA expression, but progesterone significantly attenuated these effects. In prelabor amnion cells, IL-1beta also increased IL-8 and PGE2 synthesis and both COX-2 and IL-8 mRNA and promoter expression; however, progesterone significantly attenuated these effects on IL-8 and PGE2 synthesis and COX-2 expression. In postlabor amnion cells, IL-1beta increased IL-8 and PGE2 synthesis and COX-2 expression, but progesterone did not attenuate the effect of IL-1beta upon IL-8 synthesis. Progesterone repression of IL-8 and COX-2 in LSF cells suggests that IL-8 and COX-2 have similar regulatory mechanisms in LSF cells and that progesterone may play a role in maintenance of cervical competence. The lack of effect of progesterone on IL-8 in postlabor cells may be the result of downregulation of the progesterone receptor during labor.
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- 2003
- Full Text
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47. Expression of membrane prostaglandin E synthase in human placenta and fetal membranes and effect of labor.
- Author
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Alfaidy N, Sun M, Challis JR, and Gibb W
- Subjects
- Adult, Amnion enzymology, Autoradiography, Blotting, Western, Chorion enzymology, Decidua enzymology, Female, Gene Expression Regulation, Enzymologic genetics, Humans, Immunohistochemistry, In Situ Hybridization, Pregnancy, Prostaglandin-E Synthases, RNA, Messenger biosynthesis, Extraembryonic Membranes enzymology, Intramolecular Oxidoreductases biosynthesis, Labor, Obstetric physiology, Placenta enzymology
- Abstract
Initiation and maintenance of labor in humans is associated with an increase in prostaglandin synthesis by intrauterine tissues. The objective of the present study was to characterize the distribution of membrane-bound PGES (mPGES) protein and mPGES mRNA in human placenta, fetal membranes, and decidua at term and to determine whether any changes occurred with labor. Immunoreactive mPGES was found to be highly concentrated in amnion epithelial cells and the chorion laeve trophoblasts, with lower levels in the mesenchymal layers. The enzyme was at very low levels or undetectable in the decidual tissue. Much lower levels of mPGES protein and mRNA were found in placenta than in fetal membranes. mPGES was associated with the syncytiotrophoblast and in cells surrounding blood vessels. The expression of mPGES mRNA did not change with labor in full membranes or placenta, but Western analysis showed an increase in mPGES protein in chorion laeve and a decrease in mPGES protein in placenta during labor, with no change in the amnion. The differences in expression found among placenta, chorion, and amnion before and after labor would indicate that this enzyme is differentially regulated in these tissues at this time.
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- 2003
- Full Text
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48. Effects of sex hormones, forskolin, and nicotine on choline acetyltransferase activity in human isolated placenta.
- Author
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Wessler I, Schwarze S, Brockerhoff P, Bittinger F, Kirkpatrick CJ, and Kilbinger H
- Subjects
- Amnion drug effects, Amnion enzymology, Chorionic Villi drug effects, Chorionic Villi enzymology, Female, Follicle Stimulating Hormone pharmacology, Humans, In Vitro Techniques, Luteinizing Hormone pharmacology, Pregnancy, Choline O-Acetyltransferase metabolism, Colforsin pharmacology, Gonadal Steroid Hormones pharmacology, Nicotine pharmacology, Nicotinic Agonists pharmacology, Placenta drug effects, Placenta enzymology
- Abstract
The activity of choline acetyltransferase (ChAT) was investigated in the human placenta before and after long-term incubation (24 h) to test the effects of sex hormones, nicotine and forskolin. ChAT activity differed considerably between the amnion (0.03 micromol/mg protein/h) and the villus (0.56). After long-term incubation, ChAT activity persisted in the latter but declined in the amnion. Neither sex hormones (beta-estradiol, testosterone, progesterone; 10 or 100 nM each) nor follicle stimulating hormone and luteinizing hormone (FSH/LH; 8.4 U/ml each) modified ChAT activity. Also nicotine (1 nM-100 microM) did not affect ChAT activity. Forskolin, an activitor of adenylyl cyclase, reduced ChAT activity in the villus but not in amnion. The present model offers the possibility to investigate ChAT regulation in intact tissue under long-term incubation. The risks of maternal smoking during pregnancy cannot be attributed to an effect of nicotine on placental ChAT activity. Differences in the regulation of ChAT appear to exist between neuronal and nonneuronal cells.
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- 2003
- Full Text
- View/download PDF
49. Changes in the isoforms of the sodium pump in the placenta and myometrium of women in labor.
- Author
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Esplin MS, Fausett MB, Faux DS, and Graves SW
- Subjects
- Adult, Amnion enzymology, Chorion enzymology, Female, Gestational Age, Humans, Pregnancy, Tissue Distribution, Isoenzymes metabolism, Labor, Obstetric metabolism, Myometrium enzymology, Placenta enzymology, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
Objective: We determined whether changes in sodium pump isoform abundance accompanied active human labor., Study Design: Specimens of placenta, amniochorion, and myometrium were collected from women in active spontaneous labor and from those not in labor. The abundance of the three sodium pump alpha-isoforms was determined by Western blot analysis., Results: Levels of the alpha1 and alpha2 isoforms were comparable in the three tissues for women in labor and not in labor. However, alpha3 isoform abundance in placenta and myometrium (but not amniochorion) was significantly decreased in women in active labor compared with women not in labor (sodium pump alpha3 in placenta: no labor 91.2 +/- 27.6 vs labor 46.9 +/- 3.6 density units, P =.002. Sodium pump alpha3 in myometrium: no labor 52.3 +/- 7.7 vs labor 19.8 +/- 1.6 density units, P =.0002)., Conclusion: Because reductions in sodium pump number can result in hormone release from secretory tissues and in contraction of muscle, this suggests that the sodium pump may play a significant role in the initiation or maintenance of human labor.
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- 2003
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50. Human amniotic epithelial cells are morphologically homogeneous: enzymehistochemical, tracer, and freeze-substitution fixation study.
- Author
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Iwasaki R, Matsubara S, Takizawa T, Takayama T, Yashiro T, and Suzuki M
- Subjects
- Adenosine Triphosphatases metabolism, Amnion ultrastructure, Cell Size, Electron Transport Complex IV metabolism, Epithelial Cells ultrastructure, Female, Freeze Substitution, Humans, Infant, Newborn, Phosphoric Monoester Hydrolases metabolism, Pregnancy, Tissue Fixation, Amnion cytology, Amnion enzymology, Epithelial Cells cytology, Epithelial Cells enzymology
- Abstract
We examined the fine subcellular morphology of human amniotic epithelial cells and attempted to answer the question as to whether amniotic epithelial cells consist of heterogeneous or homogeneous cells, which has long been controversial. Study subjects were fetal membranes from pregnant women (n=18) who abdominally gave birth to healthy infants at term (37.9+/-0.7 weeks of gestation, mean+/-sd). The methods employed were transmission electron microscopy, enzymehistochemistry, tracer permeability analysis, and freeze-substitution fixation. The labelings for acid phosphatase, cytochrome c oxidase, and CA++ATPase were seen in the lysosomes, mitochondria, and lateral plasma membranes, respectively. The staining distribution pattern of these three enzymes and the morphology of the organelle highlighted by these enzymehistochemistry did not differ among cells. Freeze-substitution fixation revealed that intercellular spaces in the amniotic epithelial cells were narrower than previously thought, but the tracers (horse radish peroxidase and lanthanum nitrate) fully entered these spaces. There were no variations in the tracer permeability among cells. All cells from freeze-substitution fixation exhibited the same morphological features. From these morphological viewpoints, we conclude that human term amniotic epithelial cells consist of a homogeneous cell population.
- Published
- 2003
- Full Text
- View/download PDF
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