Immune Modifications in Fetal Membranes Overlying the Cervix Precede Parturition in Humans.

In humans, parturition is currently viewed as an intrauterine outbreak of inflammation, accompanied by a massive release of proinflammatory cytokines at the maternal-fetal interface that comprises the maternal decidua, placenta, and fetal membranes. At term, fetal membranes overlying the cervix, the future site of rupture, show altered morphology and are termed the zone of altered morphology (ZAM). These alterations occur in normal fetal membranes during late pregnancy, in preparation for labor. In this study, transcriptome, flow cytometry, electron microscopy, and immunohistochemistry analyses collectively highlight a local shift in gene expression and lymphocyte activation in the ZAM. Just before labor, we show that highly polymorphic HLA-A, -B, and -C determinants of fetal origin are selectively exposed in the ZAM to the maternal immune system. A graft rejection-like program occurs in the ZAM, which involves 1) the activation of cytotoxic decidual NK cells, and 2) the decline of decidual immunotolerant M2-like macrophages. Comparison with a prior cohort of fetal membranes shows that acute inflammation only takes place after these first steps of immune modifications. Our results therefore strongly argue in favor of local immune remodeling at the onset of parturition.

Functional screening of TLRs in human amniotic epithelial cells.

Intrauterine infection is a major cause of spontaneous preterm birth. Amniotic epithelial cells represent the first line of defense against intra-amniotic bacteria. We hypothesize that this epithelial cell barrier is able to recognize and respond to pathogens through the function of TLRs, which are crucial regulators of the innate immune system. In this study, we describe the expression of transcripts for TLR1-TLR10 in human amniotic epithelial cells. We show that amniotic epithelial cells express functional TLR5, TLR6/2, and TLR4. Activation by TLR5 and TLR6/2 agonists produces IL-6 and IL-8, concomitantly with the activation of NF-κB signaling pathway, matrix metalloproteinase-9 induction, and PTGS2 expression. In contrast, TLR4 activation reduced amniotic epithelial cell viability and induced cell apoptosis evidenced by an elevated Bax/Bcl-2 ratio and cleavage of caspase-3. These data suggest specific TLR-mediated functions in human amniotic epithelial cells for initiating different immune responses, which ultimately may lead to preterm birth.

The endothelin axis in uterine leiomyomas: new insights.

The endothelin axis, comprising endothelin-1 (ET-1) and its receptors (ETA and ETB), is involved in the pathophysiology of different human tumors. Here we review conventional approaches and gene expression profiling indicating the association of ET-1 and its cognate receptors with human and rat leiomyomas, the most common benign tumors of myometrium. Specifically, ET-1/ETA interactions affect human and rat leiomyoma cell proliferation through protein kinase C and mitogen-activated protein kinase-dependent signaling pathways. Recent experiments demonstrate that the ET-1 axis exerts a potent antiapoptotic effect involving sphingolipid metabolism and prostaglandin-endoperoxide synthase 2/prostaglandin system in the rat Eker leiomyoma tumor-derived ELT3 cell line. Evidence supports that steroid hormones, growth factors, and extracellular matrix are key regulators of the leiomyoma growth. Interestingly, the ET-1 axis is under steroid hormones and can cooperate with these growth factors. Therefore, ET-1 alone or in association with these factors could contribute to the complex regulation of uterine tumor growth, such as proliferation, survival, and extracellular matrix production. This review summarizes current knowledge and emerging data on ET-1 in uterine leiomyoma pathology.

Secreted surfactant protein A from fetal membranes induces stress fibers in cultured human myometrial cells.

In the present study, we investigated the ability of human fetal membranes (amnion and choriodecidua) to regulate human maternal uterine cell functions through the secretion of surfactant protein (SP)-A and SP-D at the end of pregnancy. We detected the expression of both SP-A (SP-A1 and SP-A2) and SP-D by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry revealed that human fetal membranes expressed both SP-A and SP-D. By Western blot analysis, we demonstrated that SP-A protein expression was predominant in choriodecidua, whereas the amnion predominantly expressed SP-D. Only the secretion of SP-A was evidenced in the culture supernatants of amnion and choriodecidua explants by immunodot blot and confirmed by Western blot. Exogenous human purified SP-A induced stress fiber formation in cultured human myometrial cells via a pathway involving Rho-kinase. Conditioned medium from choriodecidua and amnion explants mimicked the SP-A effect. Treatment of myometrial cells with SP-A-depleted conditioned medium from choriodecidua or amnion explants failed to change the actin dynamic. These data indicate that SP-A released by human fetal membranes is able to exert a paracrine regulation of F-actin filament organization in myometrial cells.

Differential expression of the enzymatic system controlling synthesis, metabolism, and transport of PGF2 alpha in human fetal membranes.

The present study investigated the expression of genes and proteins associated with PGF2alpha biosynthesis, catabolism, and transport in matched amnion and choriodecidua of human term placenta. The concentration of PGF2alpha within fetal membranes depends on the balance between complex enzymatic systems responsible for, respectively, its synthesis-by prostaglandin-endoperoxide synthase 2 (PTGS2) and members of the aldo-keto reductase (AKR) family, AKR1C3 and AKR1B1-and its catabolic inactivation-through hydroxy-prostaglandin-dehydrogenase (HPGD). We observed that AKR1C3 shows equal basal expression (mRNA and protein) in choriodecidua and amnion but that AKR1B1 exhibits preferential expression in the choriodecidua. Expression of HPGD and solute carrier organic anion transporter family member 2A1 (SLCO2A1) was found primarily in the choriodecidua. We also evaluated whether an inflammatory environment induced by the gram-negative bacterial endotoxin lipopolysaccharide (LPS) affects expression of each candidate enzymes. The amnion responded to LPS with a small but significant decrease of AKR1B1 mRNA expression. In contrast, we found a significant increase in PTGS2 and AKR1C3 mRNA expression in choriodecidua after LPS challenge, but such regulation was confirmed only at protein levels for PTGS2 and not for AKR1C3. Our results suggest that the choriodecidua appears to be the main tissue, which expresses maximally all the components (synthesis, degradation, and transport) controlling PGF2alpha levels.

Protein kinase C and human uterine contractility.

Abnormalities in uterine contractility are thought to contribute to several clinical problems, including preterm labor. A better understanding of the mechanisms controlling uterine activity would make it possible to propose more appropriate and effective management practices than those currently in use. Recent advances point to a role of the protein kinase C (PRKC) family in the regulation of uterine smooth muscle contraction at the end of pregnancy. In this review, we highlight recent work that explores the involvement of individual PRKC isoforms in cellular process, with an emphasis on the properties of PRKCZ isoform.

Should phosphodiesterase 5 selective inhibitors be used for uterine relaxation?

Sildenafil citrate is a phosphodiesterase 5-selective inhibitor used successfully in treating erectile dysfunction. High doses of sildenafil can inhibit myometrial contractions. However, no study has demonstrated a role for phosphodiesterase 5 in myometrial contractility. No clinical trial using sildenafil to promote uterine relaxation should be initiated based on the currently available data.

Regulation of the endothelin/endothelin receptor system by interleukin-1{beta} in human myometrial cells.

Proinflammatory cytokines produced at the fetomaternal interface, such as IL-1beta, have been implicated in preterm and term labor. The present study was performed to evaluate the influence of IL-1beta on the endothelin (ET)/ET receptor system in human myometrial cells. We report that myometrial cells under basal conditions not only respond to but also secrete ET-1, one of the main regulators of uterine contractions. Prolonged exposure of the cells to IL-1beta led to a decrease in prepro-ET-1 and ET-3 mRNA correlated with a decrease in immunoreactive ET-1 and ET-3 levels in the culture medium. Whereas ETA receptor expression at both protein and mRNA levels was not affected by IL-1beta treatment, we demonstrated an unexpected predominance of the ETB receptor subtype under this inflammatory condition. Whereas the physiological function of ETB remains unclear, we confirmed that only ETA receptors mediate ET-1-induced myometrial cell contractions under basal conditions. By contrast, prolonged exposure of the cells to IL-1beta abolished the contractile effect induced by ET-1. Such a regulation of IL-1beta on the ET release and the balance of ETA to ETB receptors leading to a loss of ET-1-induced myometrial cell contractions suggest that complex regulatory mechanisms take place to constraint the onset of infection-induced premature contractions.

A role for PKCzeta in the LPS-induced translocation NF-kappaB p65 subunit in cultured myometrial cells.

Human myometrial cells respond to the endotoxin lipopolysaccharide (LPS) by activation of protein kinase C (PKC) zeta and nuclear translocation of the p65 subunit of NF-kB. Our first objective was to determine the expression of TLR4 in cultured myometrial cells. Positive immunoreactivity observed for TLR4 suggests that myometrial cells have the potential to respond to LPS. To confirm that LPS signals via TLR4, the ability of an anti-TLR4 neutralizing antibody to block LPS-induced translocation of p65 was demonstrated. To determine whether LPS-induced nuclear translocation of p65 is mediated through the PKC pathway, myometrial cells were treated with various inhibitors of the PKC isoforms already characterized in human myometrium. Neither the selective conventional PKC inhibitor nor the inhibitor of PKCdelta affected NF-kB activation. By contrast, we found that treatment of myometrial cells with an antisense against PKCzeta affect LPS-induced nuclear translocation of the p65 subunit of NF-kB. Accordingly, our data support the notion that PKCzeta is essential for LPS-induced NF-kB p65 subunit nuclear translocation in human myometrial cells.

Preeclampsia-like symptoms induced in mice by fetoplacental expression of STOX1 are reversed by aspirin treatment.

Preeclampsia (PE) is a common human-specific pregnancy disorder defined by hypertension and proteinuria during gestation and responsible for maternal and fetal morbimortality. STOX1, encoding a transcription factor, was the first gene associated with PE as identified by positional cloning approaches. Its overexpression in choriocarcinoma cells mimics the transcriptional consequences of PE in the human placenta. Here, we created transgenic mouse strains overexpressing human STOX1. Wild-type female mice crossed with transgenic male mice reproduce accurately the symptoms of severe PE: gestational hypertension, proteinuria, and elevated plasma levels of soluble fms-like tyrosine kinase 1 and soluble endoglin. Placental and kidney histology were altered. Symptoms were prevented or alleviated by aspirin treatment. STOX1-overexpressing mice constitute a unique model for studying PE, allow testing therapeutic approaches, and assessing the long-term effects of the preeclamptic syndrome.

Endothelin-1: physiological and pathological roles in myometrium.

Endothelin-1 (ET-1), a member of endothelin peptide family is released by many different tissues including uterine smooth muscle. ET-1 acts through ETA and ETB receptors and is implicated in a wide range of biological and pathological functions that explain the great attention of the pharmacological industry for ET-1 receptors as potential therapeutic targets in vascular pathologies and cancers. It is now well established that ET-1 is also able to regulate myometrial functions. In the present review, we focused on ET axis and related signaling pathways involved in the regulation of myometrial contraction, as well as cell proliferation and survival. Such ET-1-mediated cellular functions play a critical role in normal pregnancy, preterm birth and uterine leiomyoma.

Surfactant protein A: an immunoregulatory molecule involved in female reproductive biology.

Surfactant protein A (SP-A), a member of the collectin family originally described as a major component of lung surfactant, plays an important role in the modulation of lung host defense. A new interest in SP-A is provided by the link between fetal lung development and the timing of labor in the mouse. In the present review, we discuss some of the known features of SP-A such as biological functions, signaling pathways involved and the recent developments showing that SP-A bind and serve as a signal in the female genital tract. Therefore, such reports support a new paradigm involving SP-A as a multifunctional protein in the parturition process.

Surfactant protein A signaling pathways in human uterine smooth muscle cells.

The present study investigated the ability of surfactant associated protein A1 (SFTPA1), a major component of lung surfactant, to bind and serve as a signal in human cultured myometrial cells. By using ligand blot analysis with 125I-SFTPA1, we consistently identified two myometrial SFTPA1 interacting proteins (55 and 200 kDa). We found that the SFTPA1 immunoreactive protein was present in myometrial cells. We also showed by indirect immunofluorescence the nuclear translocation of RELA (also known as NFkappaB p65 subunit) after activation of myometrial cells by SFTPA1. Neutralization of TLR4 did not reverse this effect. Moreover, SFTPA1 rapidly activated mitogen-activated protein kinase 1/3 (MAPK1/3) and protein kinase C zeta (PRKCZ). The prolonged treatment of myometrial cells with SFTPA1 upregulated PTGS2 (COX2) protein levels. We next evaluated whether SFTPA1 affected the actin dynamic. Stimulation of myometrial cells with SFTPA1 markedly enhanced the intensity of the filamentous-actin pool stained with fluorescein isothiocyanate-phalloidin. Inhibition of PRKC or Rho-associated, coiled-coil containing protein kinase 1 (ROCK) reduced the SFTPA1-mediated stress fiber formation. Our data support the hypothesis that human myometrial cells express functional SFTPA1 binding sites and respond to SFTPA1 to initiate activation of signaling events related to human parturition.

ADRB3 adrenergic receptor is a key regulator of human myometrial apoptosis and inflammation during chorioamnionitis.

The pathophysiology underlying preterm labor triggered by inflammatory conditions such as chorioamnionitis remains largely unclear. It has already been suggested that beta-3 adrenergic (ADRB3) agonists might be of interest in the pharmacological management of preterm labor. Although there is evidence implicating ADRB receptors in the control of inflammation, there are minimal data relating specifically to ADRB3. To explore the cellular consequences of chorioamnionitis and detect apoptosis, we first performed immunostaining and Western blot experiments on human myometrial samples obtained from women with confirmed chorioamnionitis. We then developed an in vitro model of chorioamnionitis by incubating the myometrial samples obtained from uncomplicated pregnancies with Escherichia coli lipopolysaccharide (LPS). We observed that chorioamnionitis was associated with a significant increase in cleaved CASP3 protein expression, as well as chromatin condensation, which were reproduced experimentally by LPS stimulation (10 microg/ml, 48 h). Lipopolysaccharide stimulation of normal human myometrium also induced CASP3 transcripts, increased the proapoptotic marker BAX, and decreased the antiapoptotic marker BCL2. Lipopolysaccharide-induced apoptosis was antagonized by neutralization of secreted tumor necrosis factor by a specific antibody. Furthermore, LPS stimulation increased medium culture levels of proinflammatory cytokines interleukin 6 (IL6) and IL8. Lipopolysaccharide-induced apoptosis and cytokine production were prevented by the new and potent ADRB3 agonist SAR150640 in a concentration-dependent manner. SAR150640 by itself did not exhibit any effect on apoptosis or cytokine production in control tissues. This study shows that chorioamnionitis is associated with apoptosis of human myometrial cells. It emphasizes the potential therapeutic interest of ADRB3 agonists in the field of preterm labor and other inflammatory conditions.

Inflammation of choriodecidua induces tumor necrosis factor alpha-mediated apoptosis of human myometrial cells.

The present study investigated the ability of human choriodecidua to induce myometrial cell apoptosis through the secretion of tumor necrosis factor alpha (TNF). The secretion of TNF was evaluated in the culture supernatants of amnion and choriodecidua explants that were exposed to the bacterial endotoxin lipopolysaccharide (LPS) to mimic inflammation. The choriodecidua explants produced more TNF than the amnion explants in response to LPS stimulation, despite the fact that the choriodecidua had lower levels of TLR4 expression. Moreover, conditioned medium obtained from LPS-treated choriodecidua explants, but not that from amnion explants, decreased the number of viable cultured myometrial cells and induced cell apoptosis by inducing the overexpression of the proapoptotic protein BAX and by decreasing the expression of the anti-apoptotic protein BCL2. Neutralization of TNF in the choriodecidua-conditioned medium reversed this effect. Exogenous TNF mimicked LPS-treated choriodecidua-conditioned medium in that it induced myometrial cell apoptosis, reduced BCL2 expression, and increased BAX expression. Using neutralizing antibodies against both subtypes of TNF receptors, we found that only TNFRSF1A participates in TNF-induced myometrial cell apoptosis. Our in vitro model of LPS-induced inflammation of human fetal membrane explants suggests a mechanism by which TNF secreted by choriodecidua governs human myometrial cell apoptosis at the end of pregnancy. These data support the hypothesis that TNF participates in the complex network of signaling processes associated with uterine involution.

In vitro and in vivo pharmacological characterization of ethyl-4-[trans-4-[((2S)-2-hydroxy-3-[4-hydroxy-3[(methylsulfonyl)amino]-phenoxy]propyl) amino]cyclohexyl]benzoate hydrochloride (SAR150640), a new potent and selective human beta3-adrenoceptor agonist for the treatment of preterm labor.

Ethyl-4-[trans-4-[((2S)-2-hydroxy-3-[4-hydroxy-3[(methylsulfonyl)amino] phenoxy]propyl) amino]cyclohexyl]benzoate hydrochloride (SAR150640) was characterized as a new potent and selective beta(3)-adrenoceptor agonist for the treatment of preterm labor. SAR150640 and its major metabolite, the corresponding acid 4-[trans-4-[((2S)-2-hydroxy-3-[4-hydroxy-3[(methylsulfonyl) amino] phenoxy]propyl)amino]cyclohexyl]benzoic acid (SSR500400), showed high affinity for beta(3)-adrenoceptors (K(i) = 73 and 358 nM) and greater potency than (-)-isoproterenol in increasing cAMP production in membrane preparations from human neuroblastoma cells (SKNMC), which express native beta(3)-adrenoceptors (pEC(50) = 6.5, 6.2, and 5.1, respectively). SAR150640 and SSR500400 also increased cAMP production in membrane preparations from human uterine smooth muscle cells (UtSMC), which also express native beta(3)-adrenoceptors (pEC(50) = 7.7 and 7.7, respectively). In these cells, SAR150640 dose-dependently inhibited oxytocin-induced intracellular Ca(2+) mobilization and extracellular signal-regulated kinase 1/2 phosphorylation. SAR150640 and SSR500400 had no beta(1)- or beta(2)-agonist or antagonist activity in guinea pig atrium and trachea, or in human isolated atrium and bronchus preparations. Both compounds concentration-dependently inhibited spontaneous contractions in human near-term myometrial strips, with greater potency than salbutamol and 4-[3-[(1,1-dimethylethyl)-amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP12177) (pIC(50) = 6.4, 6.8, 5.9, and 5.8, respectively), but with similar potency to (-)-isoproterenol and atosiban (oxytocin/vasopressin V(1)a receptor antagonist). SAR150640 also inhibited the contractions induced by oxytocin and prostaglandin F(2alpha). In vivo, after intravenous administration, SAR150640 (1 and 6 mg/kg), but not atosiban (6 mg/kg), dose-dependently inhibited myometrial contractions in conscious unrestrained female cynomolgus monkeys, with no significant effects on heart rate or blood pressure. In contrast, salbutamol (50 and 250 microg/kg) had no inhibitory effect on uterine contractions, but it dose-dependently increased heart rate. These findings indicate a potential for the therapeutic use of SAR150640 in mammals during preterm labor.

Gene and protein expression in the myometrium in pregnancy and labor.

Microarray technologies widen our comprehension of the major structural and metabolic transformations which affect the myometrium from the very beginning of pregnancy until parturition. The results are coherent with the mass of information which was accumulated previously, primarily on the basis of studies of selected critical factors. They highlight the activation of precise signaling pathways, some of which may have been previously under evaluated. The remodelling and maturation processes that the myometrium undergoes in pregnancy appear clearly as phenomena which last during the full course of gestation. Comparatively, the onset of labor is perhaps the phenomenon which remains the least well described by these methods of analysis. Nevertheless, genomic studies constitute a necessary first step of orientation and help establishing new links between the generic signaling pathways that are activated during the normal or pathological gestation. These studies also represent an indicative step that will have to be paralleled, in the future, with the results of the systematic proteomic analysis of the myometrium.

Protein kinase Calpha is required for endothelin-1-induced proliferation of human myometrial cells.

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.

Contraction of cultured human uterine smooth muscle cells after stimulation with endothelin-1.

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.

A mathematical model for the spontaneous contractions of the isolated uterine smooth muscle from patients receiving progestin treatment.

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.