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.
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 mechanisms that lead to the onset of human parturition are still unknown, although selected critical factors have been identified. To investigate the changes in myometrial gene expression associated with parturition, we used two macroarrays each containing 1176 different complementary human cDNA clones. Methods involving hierarchical clustering and conventional statistical analysis allowed us to generate a profile of genes expression at three stages of late pregnancy: preterm (29 wk amenorrhea); full term, not in labor (38 wk amenorrhea); and full term in labor (39 wk amenorrhea). Only 4% of the genes investigated were differentially expressed between the preterm and term groups (P < 0.05). These genes could be clustered as groups of either down-regulated or up-regulated transcripts. The changes in transcript abundance were particularly marked between the preterm and term stages of gestation, whereas the differences between term not in labor and term in labor were less pronounced. The parturition was characterized by a massive down-regulation of a large panel of developmental, cell adhesion molecule and proliferation-related genes, along with the up-regulation of inflammatory, contraction and apoptosis associated genes. We propose that the mechanisms of parturition consist primarily in the arrest of the processes of myometrial development, a step that might be essential to allow the uterus to recover appropriate contractile function before delivery.
To assess whether pregnancy might influence the functionality and expression of human myometrial beta(2)- and beta(3)-adrenoceptors (beta(2)- and beta(3)-AR), we performed functional, binding, Western blot, and molecular biology experiments in human nonpregnant and near-term pregnant myometrium. Inhibition of spontaneous contractions induced by a beta(3)-AR agonist, SR 59119A, was significantly greater in pregnant, compared with nonpregnant, myometrial strips (E'(max) = 61 +/- 5% vs. 44 +/- 5% for pregnant and nonpregnant myometrium, respectively), whereas salbutamol, a beta(2)-AR agonist, was significantly less efficient in pregnant, compared with nonpregnant, myometrium (E(max) = 29 +/- 4 vs. 54 +/- 8%). Although two populations of binding sites corresponding to beta(2)- and beta(3)-AR were identified in both nonpregnant and pregnant myometrium, we found a clear predominance of the beta(3)-AR subtype. Moreover, beta(3)-AR binding sites were up-regulated 2-fold in myometrium at the end of pregnancy. Both beta(2)- and beta(3)-AR mRNA were expressed in human nonpregnant and pregnant myometrium. Contrary to beta(2)-AR, the expression of the beta(3)-AR transcripts and immunoreactive proteins was increased in pregnant, compared with nonpregnant, myometrium. Such compelling data suggest a predominant role for beta(3)-AR in the regulation of human myometrium contractility, especially at the end of pregnancy, which might have important consequences for the clinical management of preterm 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.
1 In order to compare the b 2 -and b 3 -adrenoceptor (b-AR) desensitisation process in human nearterm myometrium, we examined the influence of a pretreatment of myometrial strips with either a b 2 -or a b 3 -AR agonist (salbutamol or SR 59119A, respectively, both at 10 mM, for 5 and 15 h) on the relaxation and the cyclic adenosine monophosphate (cAMP) production induced by these agonists. 2 To assess some of the mechanisms potentially implicated in the b-AR desensitisation process, we studied the influence of such treatment on the number of b 2 -and b 3 -AR binding sites, the b 2 -and b 3 -AR transcripts expression and the phosphodiesterase 4 (PDE4) activity. 3 Salbutamol, but not SR 59119A, concentration-response curve (CRC) was shifted by a 15 h salbutamol preincubation, with a significant difference in Àlog EC 20 values (6.3170.13 vs 5.5870.24, for control and 15 h salbutamol pretreatment, respectively, Po0.05). Neither salbutamol nor SR 59119A CRCs were modified after a 15 h preincubation with SR 59119A. 4 A 15 h exposure of myometrial strips to salbutamol significantly reduced the salbutamol-induced (0.6070.26 vs 1.5470.24 pmol mg À1 protein, Po0.05), but not the SR 59119A-induced, cAMP production. No decrease in cAMP production was observed after a 15 h SR 59119A exposure. 5 A 15 h salbutamol exposure of myometrial strips significantly reduced the b 2 -but not the b 3 -AR binding site density, whereas no decrease in the number of b 2 -and b 3 -AR binding sites was observed after a 15 h SR 59119A treatment. 6 Neither PDE4 activity nor the b 2 -and b 3 -AR mRNA expression levels were affected by salbutamol or SR 59119A treatments. 7 Our results indicate that b 3 -AR, but not b 2 -AR, are resistant to the agonist-induced desensitisation. In our model, b 2 -AR desensitisation is mediated by a decreased number of b 2 -AR that was not explained by transcriptional regulation of the receptor.
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.
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.
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