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1.
Hypertension ; 49(1): 76-83, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17088445

ABSTRACT

Preeclampsia is the major pregnancy-induced hypertensive disorder. It modifies the expression profile of placental genes, including several serine protease inhibitors (SERPINs). The objective of this study was to perform a systematic expression analysis of these genes in normal and pathological placentas and to pinpoint epigenetic alterations inside their promoter regions. Expression of 18 placental SERPINs was analyzed by quantitative RT-PCR on placentas from pregnancies complicated by preeclampsia, intrauterine growth restriction, or both and was compared with normal controls. SERPINA3, A5, A8, B2, B5, and B7 presented significant differences in expression in >or=1 pathological situation. In parallel, the methylation status of the CpG islands located in their promoter regions was studied on a sample of control and preeclamptic placentas. Ten SERPIN promoters were either totally methylated or totally unmethylated, whereas SERPINA3, A5, and A8 presented complex methylation profiles. For SERPINA3, the analysis was extended to 81 samples and performed by pyrosequencing. For the SERPINA3 CpG island, the average methylation level was significantly diminished in preeclampsia and growth restriction. The hypomethylated CpGs were situated at putative binding sites for developmental and stress response (hypoxia and inflammation) factors. Our results provide one of the first observations of a specific epigenetic alteration in human placental diseases and provide new potential markers for an early diagnosis.


Subject(s)
Epigenesis, Genetic , Placenta/metabolism , Pre-Eclampsia/metabolism , Serine Proteinase Inhibitors/metabolism , Serpins/metabolism , Adult , Biomarkers/metabolism , CpG Islands , DNA Methylation , Female , Gene Expression Regulation , Humans , Placenta Diseases/genetics , Placenta Diseases/metabolism , Pregnancy , Promoter Regions, Genetic , RNA, Messenger/blood , Serpins/genetics
3.
Nucleic Acids Res ; 34(19): 5438-48, 2006.
Article in English | MEDLINE | ID: mdl-17012269

ABSTRACT

Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR--target of the CTCF insulator--is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.


Subject(s)
CpG Islands , DNA-Binding Proteins/metabolism , Genomic Imprinting , Proteins/genetics , RNA, Untranslated/genetics , Repressor Proteins/metabolism , Binding Sites , CCCTC-Binding Factor , DNA Methylation , Female , Gene Expression , Genotype , Humans , Infant, Newborn , Inheritance Patterns , Insulin-Like Growth Factor II , Male , Models, Genetic , Pedigree , Placenta/metabolism , Polymerase Chain Reaction , Proteins/metabolism , RNA, Long Noncoding , RNA, Untranslated/metabolism , Regulatory Sequences, Nucleic Acid
4.
BMC Genomics ; 6: 111, 2005 Aug 29.
Article in English | MEDLINE | ID: mdl-16129025

ABSTRACT

BACKGROUND: As a first step to explore the possible relationships existing between the effects of low oxygen pressure in the first trimester placenta and placental pathologies developing from mid-gestation, two subtracted libraries totaling 2304 cDNA clones were constructed. For achieving this, two reciprocal suppressive/subtractive hybridization procedures (SSH) were applied to early (11 weeks) human placental villi after incubation either in normoxic or in hypoxic conditions. The clones from both libraries (1440 hypoxia-specific and 864 normoxia-specific) were spotted on nylon macroarrays. Complex cDNAs probes prepared from placental villi (either from early pregnancy, after hypoxic or normoxic culture conditions, or near term for controls or pathological placentas) were hybridized to the membranes. RESULTS: Three hundred and fifty nine clones presenting a hybridization signal above the background were sequenced and shown to correspond to 276 different genes. Nine of these genes are mitochondrial, while 267 are nuclear. Specific expression profiles characteristic of preeclampsia (PE) could be identified, as well as profiles specific of Intra-Uterine Growth Retardation (IUGR). Focusing on the chromosomal distribution of the fraction of genes that responded in at least one hybridization experiment, we could observe a highly significant chromosomal clustering of 54 genes into 8 chromosomal regions, four of which containing imprinted genes. Comparative mapping data indicate that these imprinted clusters are maintained in synteny in mice, and apparently in cattle and pigs, suggesting that the maintenance of such syntenies is requested for achieving a normal placental physiology in eutherian mammals. CONCLUSION: We could demonstrate that genes induced in PE were also genes highly expressed under hypoxic conditions (P = 5 x 10(-5)), which was not the case for isolated IUGR. Highly expressed placental genes may be in syntenies conserved interspecifically, suggesting that the maintenance of such clusters is requested for achieving a normal placental physiology in eutherian mammals.


Subject(s)
Fetal Growth Retardation/genetics , Fetal Growth Retardation/metabolism , Hypoxia , Placenta/metabolism , Pre-Eclampsia/metabolism , Promoter Regions, Genetic , Adult , Animals , Chromosomes/metabolism , Chromosomes/ultrastructure , Cluster Analysis , Cytogenetics , DNA, Complementary/metabolism , Female , Gene Expression Regulation , Gene Library , Humans , Kinetics , Mitochondria/metabolism , Models, Genetic , Models, Statistical , Multigene Family , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Poly A/metabolism , Pregnancy , RNA/metabolism , Swine
5.
J Appl Physiol (1985) ; 99(3): 1157-63, 2005 Sep.
Article in English | MEDLINE | ID: mdl-15860688

ABSTRACT

In this paper, we applied a new theoretical model of uterine contraction to a large panel of human pregnant and nonpregnant myometrial strips, treated or not by corticotrophin-releasing hormone (CRH). This model is based on a fine analysis of the contraction curves. This analysis yielded four mathematical parameters (beta, theta, tau 1, and tau 2) related to excitability, duration of plateau phase, and time constants for relaxation describing, respectively, the different portions of the contraction cycle. This leads to specific differences in spontaneous contractile activity between pregnant and nonpregnant states. The relaxing effect of CRH in the pregnant state is presumably correlated with the origin of the strips (the lower uterine segment). Besides our observation of a specific receptor-dependent relaxing effect of CRH in both pregnant and nonpregnant myometrium, we could identify highly significant effects at given CRH concentration for beta in nonpregnant myometrium and for theta, tau 1, and tau 2 in pregnant myometrium. In addition, highly significant differences were found between pregnant and nonpregnant myometrium. Also, we discovered a strong correlation between theta and tau 1, specifically in the pregnant state. Although the biochemical signification of these results remains to be elucidated, they contribute to emphasize the complex network of CRH action at the myometrial level. Furthermore, our approach could pave the way toward a better analysis of the efficacy of the uterine contractile behavior.


Subject(s)
Corticotropin-Releasing Hormone/administration & dosage , Models, Biological , Myometrium/drug effects , Myometrium/physiology , Pregnancy/physiology , Uterine Contraction/drug effects , Uterine Contraction/physiology , Adult , Computer Simulation , Dose-Response Relationship, Drug , Female , Humans , In Vitro Techniques , Stress, Mechanical
6.
Physiol Genomics ; 22(1): 99-107, 2005 Jun 16.
Article in English | MEDLINE | ID: mdl-15797968

ABSTRACT

Villi from first-trimester human placenta were exposed to oxygen concentrations of either 2 or 20% during 3 h to construct two reciprocally subtracted libraries using the suppressive subtractive hybridization (SSH) methodology. After cloning, sequencing, and gene identification, the genes (1,071 clones corresponding to 822 different sequences) were classified according to 1) the subtracted library from which they originated and 2) within 58 groups of gene functions. We then developed a logarithm of the odds (LOD) test to identify a possible excess of genes in each group. We show that genes involved in angiogenesis are significantly overrepresented in the "hypoxic" condition (2% O2), whereas apoptotic genes are overrepresented in the "normoxic" condition (20% O2). Furthermore, we observed an excess of kinases relative to phosphatases and an excess of genes involved in proliferation over genes involved in cell growth in the hypoxic condition. To validate our results, we used quantitative RT-PCR to analyze the set of eight genes involved in angiogenesis on six independent placentas. Finally, we studied the distribution of gene clusters on human chromosomes to check whether their chromosomal distribution was random or not. We observed on human chromosome 11 a clear clustering of genes regulated similarly by O2 tension, and we also discovered indications that such clustering exists on chromosomes 6 and 12.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Gene Library , Oxygen/pharmacology , Placenta/drug effects , Placenta/metabolism , Cell Hypoxia , Chromosomes, Human, Pair 11/genetics , Cluster Analysis , Female , Humans , Neovascularization, Physiologic/genetics , Nucleic Acid Hybridization , Oxygen/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA
7.
Biol Reprod ; 70(2): 458-64, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14561639

ABSTRACT

The anti-inflammatory and utero-relaxant effects of two potent phosphodiesterase 4 (PDE4) inhibitors of the latest generation: cilomilast (one of the most advanced PDE4 inhibitors in clinical development, reportedly more selective for PDE4D) and compound A (which displays 12-fold greater selectivity toward PDE4B and/or PDE4A than toward PDE4D) were evaluated in human uterine smooth muscle. We first established that these compounds exhibit greater efficacy in inhibiting total cAMP-PDE activity in pregnant versus nonpregnant myometrium (E(max) = 78.0% +/- 3.6% and 80.3% +/- 2.2% in pregnant versus 57% +/- 4.7% and 70.5% +/- 5.9% in nonpregnant women for compound A and cilomilast, respectively; P < 0.05 for both compounds), confirming the prominent participation of PDE4 isoforms in cAMP hydrolysis in the near-term pregnant myometrium. Using pregnant myometrial explants, we have shown that both these drugs and also rolipram, the prototype PDE4 inhibitor, produce concentration-dependent inhibition of lipopolysaccharide (LPS) induced tumor necrosis factor alpha (TNFalpha) release with similar potency in each case (pD2 = 8.0 +/- 0.5, 7.9 +/- 0.2, and 7.6 +/- 0.2 for compound A, cilomilast, and rolipram, respectively). The maximum inhibition produced is 65%. Pretreatment with forskolin or 8-bromo-cAMP mimics the PDE4 inhibitor effect. Furthermore, compound A and cilomilast both produce concentration-dependent inhibition of the spontaneous contractions of myometrial strips and are more potent in pregnant than in nonpregnant myometrium (pD2 = 7.3 +/- 0.7 and 8.1 +/- 0.3 in pregnant versus 6.2 +/- 0.9 and 6.6 +/- 0.1 in nonpregnant myometrium for compound A and cilomilast, respectively; P < 0.05 for both compounds). This demonstrates that the PDE4 isoforms involved in the mechanism of contraction are different in the pregnant and nonpregnant myometrium. Our study highlights the importance of developing PDE4 inhibitors for the pharmacological management of infection-induced preterm labor.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Bronchodilator Agents/pharmacology , Myometrium/drug effects , Phosphodiesterase Inhibitors/pharmacology , Uterine Contraction/drug effects , Anti-Inflammatory Agents/pharmacology , Carboxylic Acids , Cyclic AMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 3 , Cyclic Nucleotide Phosphodiesterases, Type 4 , Cyclohexanecarboxylic Acids , Ethers/pharmacology , Female , Humans , Hydrocarbons, Fluorinated/pharmacology , In Vitro Techniques , Lipopolysaccharides/pharmacology , Myometrium/immunology , Myometrium/metabolism , Nitriles , Pregnancy , Rolipram/pharmacology , Tumor Necrosis Factor-alpha/metabolism
8.
Eur J Obstet Gynecol Reprod Biol ; 108(2): 152-6, 2003 Jun 10.
Article in English | MEDLINE | ID: mdl-12781403

ABSTRACT

OBJECTIVE: Our goal was to study the effect of amniotic fluid obtained at 16 and 39 weeks of gestation in normal human pregnancies upon prostaglandin production by human myometrial cells in culture. STUDY DESIGN: Amniotic fluid, sampled either at 16 weeks, during amniocentesis, or at 39 weeks, during caesarean section before labor, was fractionated by molecular-weight and then incubated with human myometrial cells in culture. We then used radioimmunoassay to measure PGE(2) and PGI(2) production. RESULTS: The "3-30 kDa" fraction of amniotic fluid sampled at 16 weeks significantly inhibited PGE(2) and PGI(2) production by human myometrial cells. When amniotic fluid was sampled at 39 weeks, it stimulated both PGE(2) and PGI(2) production, and the ">30 kDa" fraction increased levels of PGE(2) considerably more than of PGI(2) (420.0+/-88.0 ng/10(6)cells versus 188.2+/-21.4 ng/10(6)cells, P<0.001). CONCLUSION: Amniotic fluid contains substances whose effects in cultured myometrial cells vary according to gestational age and type of prostaglandin. These data suggest that the fetus plays a role in the regulation of myometrial activity during pregnancy.


Subject(s)
Amniotic Fluid/physiology , Dinoprostone/biosynthesis , Epoprostenol/biosynthesis , Myometrium/metabolism , Amniocentesis , Amniotic Fluid/chemistry , Cells, Cultured , Cesarean Section , Female , Gestational Age , Humans , Kinetics , Molecular Weight , Pregnancy
9.
Acta Biotheor ; 51(1): 19-34, 2003.
Article in English | MEDLINE | ID: mdl-12765250

ABSTRACT

The in vitro spontaneous contractions of human myometrium samples can be described using a phenomenological model involving different cell states and adjustable parameters. In patients not receiving hormone treatment, the dynamic behavior could be described using a three-state model similar to the one we have already used to explain the oscillations of intrauterine pressure during parturition. However, the shape of the spontaneous contractions of myometrium from patients on progestin treatment was different, due to a two-step relaxation regime including a latched phase which cannot be simulated using the previous model without introducing an ad hoc mechanism to account for the extra energy involved in this sustained contraction. One way to do this is to introduce an anomalous rate of ATP consumption, the biochemical reasons for which have not yet been elucidated and which cannot be mathematically simulated using our experimental data. An alternative explanation is the reduced cycling rate of actin-myosin cross-bridges known to occur during the latch-phase. Our experimental findings suggest a third possibility, namely a sol-gel transition with a specific relaxation time constant, which would maintain a significant part of the cell population in the contracted-state until the intracellular-medium returns to its normal fluid behavior.


Subject(s)
Models, Biological , Myometrium/drug effects , Progestins/pharmacology , Uterine Contraction/drug effects , Adenosine Triphosphate/metabolism , Adult , Computer Simulation , Female , Humans , In Vitro Techniques , Labor, Obstetric/physiology , Middle Aged , Myometrium/physiology , Pregnancy , Uterine Contraction/physiology
10.
Biol Reprod ; 68(3): 937-42, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12604645

ABSTRACT

To our knowledge, the problem of how to maintain isolated smooth cells in a "contractile" phenotypic state without deviation after subculturing has yet to be resolved. The present study characterized the in vitro contractile response of human uterine smooth muscle cell to endothelin-1, which induces contractions in isolated uterine strips. Contractile effects were qualitatively investigated using silicone rubber substrata. Endothelin-1 was able to distort and reduce the wrinkles in the silicone surface. Contractions were also quantified by measuring the resulting change in the collagen lattice area. Endothelin-1 significantly increased the contractile response in a dose-dependent manner by selectively activating endothelin A receptors. When myometrial cells were cultured within collagen lattices, a microfilament-disrupting agent, cytochalasin B, abolished contractions, and no change was observed in smooth muscle alpha-actin immunostaining. Taken together, these observations show that the uterine smooth muscle cells are contractile and respond appropriately to a potent uterotonic agent. Based on these findings, a cultured uterine smooth muscle cell model, which could be used to elucidate the mechanisms controlling uterine activity, is proposed.


Subject(s)
Endothelin-1/pharmacology , Myometrium/drug effects , Myometrium/physiology , Uterine Contraction/drug effects , Uterine Contraction/physiology , Collagen/physiology , Cytochalasin B/pharmacology , Female , Humans , In Vitro Techniques , Microscopy, Fluorescence , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/physiology , Myometrium/cytology , Receptors, Endothelin/physiology , Silicone Elastomers
11.
J Clin Endocrinol Metab ; 87(12): 5524-31, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12466348

ABSTRACT

Intrauterine infections are important etiological factors of preterm labor. They trigger an increase in proinflammatory cytokines, in particular IL-1beta, that induces a cascade of events resulting in the production of potent effectors of myometrial contractility, such as the prostaglandin E(2) (PGE(2)). Within the smooth muscle cells, contractility is under the control of cAMP content, partly regulated by cAMP-phosphodiesterase 4 (PDE4), the predominant family of PDEs expressed in human myometrium. In the present study, using a model of inflammation of human myometrial cells in culture, we demonstrated that exposing the cells to IL-1beta resulted in a significant up-regulation of PDE4 activity through an increase in PDE4B2 mRNA and protein levels. The IL-1beta-induced PDE4 activity occurs after an increase in PGE(2) production and subsequent cAMP augmentation. Pretreatment with indomethacin or NS 398 completely blocked this long-term effect of IL-1beta, revealing a PGE(2)-dependent pathway. Accordingly, our results demonstrated that the PDE4B2 variant can participate in the regulation of the inflammatory reaction that occurs at term or in preterm labor and leads to myometrial contractions. Knowing the myorelaxant effect of PDE4 inhibitors and the implication of the PDE4B2 in the inflammatory process, this isoform may be an appropriate target for discovering antiinflammatory drugs to manage infection-induced preterm deliveries.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/metabolism , Cyclic AMP/physiology , Dinoprostone/physiology , Interleukin-1/pharmacology , Myometrium/enzymology , 3',5'-Cyclic-AMP Phosphodiesterases/genetics , Cells, Cultured , Cyclic Nucleotide Phosphodiesterases, Type 4 , Enzyme Inhibitors/pharmacology , Female , Humans , Indomethacin/pharmacology , Isoenzymes/genetics , Isoenzymes/metabolism , Myometrium/cytology , Prostaglandin-Endoperoxide Synthases/drug effects , RNA, Messenger/metabolism , Up-Regulation
12.
Biol Reprod ; 66(1): 44-9, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11751262

ABSTRACT

The role of protein kinase C (PKC)-alpha in endothelin-1 (ET-1)-induced proliferation of human myometrial cells was investigated. Inhibition of conventional PKC with Gö 6976 eliminated the proliferative effect of ET-1. Treatment of myometrial cells with an antisense oligonucleotide against PKCalpha efficiently reduced PKCalpha protein expression without effect on other PKC isoforms and resulted in the loss of ET-1-induced cell growth. Immunocytochemistry using an antibody against PKCalpha revealed that there was no PKCalpha immunoreactivity in the nuclei of quiescent nonconfluent untreated cells, whereas it is evenly distributed throughout the cytoplasm. Exposure of myometrial cells to ET-1 for 15 min caused the PKCalpha to shift towards the perinuclear area, and incubation for 60 min caused a shift towards the nucleus. These results reveal that PKCalpha is required for ET-1-induced human myometrial cell growth and suggest that targeting of PKCalpha by antisense nucleotides might be an important approach for the development of anticancer treatments.


Subject(s)
Endothelin-1/pharmacology , Isoenzymes/physiology , Myometrium/cytology , Protein Kinase C/physiology , Blotting, Western , Cell Division/drug effects , Cell Nucleus/enzymology , Cells, Cultured , Cytoplasm/enzymology , DNA/biosynthesis , DNA/genetics , Female , Fluorescent Antibody Technique , Humans , Isoenzymes/antagonists & inhibitors , Myometrium/drug effects , Oligonucleotides, Antisense/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C-alpha
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