Genetic and epigenetic mechanisms collaborate to control SERPINA3 expression and its association with placental diseases.

SERPINA3 (Serpin peptidase inhibitor clade A member 3), also known as a1-antichymotrypsin, is a serine protease inhibitor involved in a wide range of biological processes. Recently, it has been shown to be up-regulated in human placental diseases in association with a hypomethylation of the 5' region of the gene. In the present study, we show that the promoter of SERPINA3 is transcriptionally activated by three transcription factors (TFs) (SP1, MZF1 and ZBTB7B), the level of induction being dependent on the rs1884082 single nucleotide polymorphism (SNP) located inside the promoter, the T allele being consistently induced to a higher level than the G, with or without added TFs. When the promoter was methylated, the response to ZBTB7B was allele specific (the G allele was strongly induced, while the T allele was strongly down-regulated). We propose an adaptive model to explain the interest of such a regulation for placental function and homeostasis. Overexpression of SERPINA3 in JEG-3 cells, a trophoblast cell model, decreased cell adhesion to the extracellular matrix and to neighboring cells, but protects them from apoptosis, suggesting a way by which this factor could be deleterious at high doses. In addition, we show in different human populations that the T allele appears to predispose to Intra Uterine Growth Restriction (IUGR), while a G allele at a second SNP located in the second exon (rs4634) increases the risk of preeclampsia. Our results provide mechanistic views inside the involvement of SERPINA3 in placental diseases, through its regulation by a combination of epigenetic, genetic and TF-mediated regulations.

Combination of promoter hypomethylation and PDX1 overexpression leads to TBX15 decrease in vascular IUGR placentas.

Preeclampsia (PE) and vascular intra-uterine growth restriction (vIUGR) are two pathological obstetrical conditions originating from placental dysfunction. Recently, methylation changes at the placental level have been shown to be indicative of these diseases. The alteration of such epigenetic marks is therefore a novel pathway that might be critical for these pathologies. Here, we identified a region located in the distal promoter of the T-box-containing transcription factor TBX15 that is differentially methylated in pathological placentas. The level of methylation correlated significantly with the weight and stature of the newborn. The promoter was found to be hypomethylated in vIUGR coinciding with the down-regulation of its expression. PDX1, a transcription factor important for the regulation of insulin metabolism regulation was able to repress the TBX15 promoter in a methylation-dependent manner, which might, at least partially, explain the specific mRNA decrease of TBX15 observed in vIUGR placentas. Overall, the data presented herein suggest that TBX15 might be involved in the pathophysiology of placental diseases.

Serum profile in preeclampsia and intra-uterine growth restriction revealed by iTRAQ technology.

In order to identify new protein markers modified in placental diseases, high-throughput analysis of proteins in the plasma of pregnant women was carried out for normal and pathological pregnancies (Preeclampsia and/or Intra-Uterine Growth Restriction) using iTRAQ technology. We could identify 166 proteins that were modified (p<0.05) and the technique used allowed the detection of previously undetected factors, such as various members of the SERPINA clade. The modifications of two proteins (C reactive protein and antichymotrypsin, SERPINA3) were validated on individual samples. Complement and coagulation cascades proteins were significantly enriched among modified protein clusters in the case of intra-uterine growth restriction (p<2.6.10(-11)). Several proteins were specifically enriched in isolated preeclampsia and depleted when preeclampsia was complicated by intra-uterine growth restriction. These findings suggest that the growth restricted foeto-placental unit is able to moderate some changes in maternal plasma composition. Overall, the use of iTRAQ technology, for the first time on this subject, enabled us to provide a new list of proteins modified in placental diseases, among which proteins expressed at a low level that were not accessible by other methods.

Re-evaluation of the role of STOX1 transcription factor in placental development and preeclampsia.

Preeclampsia is a common disease of pregnancy, characterized by high blood pressure and proteinuria appearing from the second trimester of gestation. Preeclampsia has been shown to have a strong genetic component. In 2005 a positional cloning project led to the discovery of the STOX1 transcription factor, and mutations of this gene were proposed as causal for preeclampsia in Dutch families. Despite the publication of three contradictory studies, we have shown by analyzing the functional effects of STOX1 that its overexpression in choriocarcinoma cells recapitulates several transcriptomic aspects of preeclampsia. In this review, the current literature is analyzed to evaluate the possible involvement of STOX1 in the pathogenesis of this disease. While preeclampsia obviously cannot be considered as a disease caused by mutation in a single gene, we argue that STOX1 may be at the center of common pathways leading to preeclampsia.

STOX1 overexpression in choriocarcinoma cells mimics transcriptional alterations observed in preeclamptic placentas.

BACKGROUND: Mutations in STOX1 were proposed to be causal for predisposing to preeclampsia, a hypertensive disorder originating from placental defects, affecting up to 10% of human pregnancies. However, after the first study published in 2005 three other groups have dismissed the polymorphism described in the first paper as a causal mutation. METHODOLOGY AND PRINCIPAL FINDINGS: In the present study, we have produced a choriocarcinoma cell line overexpressing STOX1. This overexpression results in transcriptional modification of 12.5% of the genes, some of them being direct targets as shown by chromatin immunoprecipitation. STOX1 overexpression correlates strongly and specifically with transcriptomic alterations in preeclamptic placentas (r = 0.30, p = 9.10(-7)). Numerous known key modulators of preeclampsia (such as Endoglin, Syncytin, human chorionic gonadotrophin -hCG-, and Glial Cell Missing Homolog -GCM1-) were modified in these transformed choriocarcinoma cells. CONCLUSIONS: Our results contribute to reconcile contradictory data concerning the involvement of STOX1 in preeclampsia. In addition, they strongly suggest that anomalies in STOX1 expression are associated with the onset of preeclampsia, thus indicating that this gene should be the target of future studies. Our cellular model could constitute an invaluable resource for studying specific aspects of this human disease.

Gene expression profile for ectopic versus eutopic endometrium provides new insights into endometriosis oncogenic potential.

Endometriosis is a common gynecological disorder characterized by pain and infertility, where the lesions disseminate everywhere in the body with a preference for the pelvis. In that, it could be regarded as a benign metastatic disease, because its issue is not fatal. However, the molecular bases of this intriguing clinical condition are not well known. The objective of this study is to characterize the transcriptome differences between eutopic vs. ectopic endometrium with a special interest in pathways involved in cancerogenesis. We performed two hybridizations in technical replicate on highly specific long oligonucleotides microarrays (NimbleGen), with cDNA prepared from six-patients pools, where the same patient provided both eutopic and ectopic endometrium (endometriomas). To confirm the expression microarrays data, quantitative RT-PCR validation was performed on 12 individuals for 20 genes. Over 8000 transcripts were significantly modified (more than twice) in the lesions corresponding to 5600 down- or up-regulated genes. These were clustered through DAVID Bioinformatics Resources into 55 functional groups. The data are presented in a detailed and visual way on 24 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways implemented with induction ratios for each differentially expressed gene. An outstanding control of the cell cycle and a very specific modulation of the HOX genes were observed and provide some new evidence on why endometriosis only very rarely degenerates into cancer. The study constitutes a noteworthy update of gene profiling in endometriosis, by delivering the most complete and reliable list of dysregulated genes to date.

Expressional and epigenetic alterations of placental serine protease inhibitors: SERPINA3 is a potential marker of preeclampsia.

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.

Non-random, individual-specific methylation profiles are present at the sixth CTCF binding site in the human H19/IGF2 imprinting control region.

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.

Hypoxia-activated genes from early placenta are elevated in preeclampsia, but not in Intra-Uterine Growth Retardation.

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.

Corticotropin-releasing hormone effects on human pregnant vs. nonpregnant myometrium explants estimated from a mathematical model of uterine contraction.

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.

Profiling of oxygen-modulated gene expression in early human placenta by systematic sequencing of suppressive subtractive hybridization products.

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.

The placental-umbilical unit in sickle cell disease pregnancy: a model for studying in vivo functional adjustments to hypoxia in humans.

The placental-umbilical unit in sickle cell disease (SCD) pregnancy was used to explore hypoxia in vivo, an important factor in the pathophysiology of this disease. Gross examination and microscopic analysis of the placentas, taken immediately after delivery, indicate good concordance between maturity and term as controls, but higher frequency of vascular injuries such as excess syncytial knots, excess fibrin deposits, congestion and villous necroses. Unexpectedly, neither leukocyte recruitment nor alteration in extraplacental membrane was observed, suggesting the absence of inflammation. Additionally, interleukin (IL)-6 and IL-8 concentrations, measured by enzyme-linked immunosorbent assay (ELISA), were similar in the placental maternal blood from controls and SCD. There were also no significant differences found in IL-6 vein blood concentrations between controls and SCD, IL-8 being not detected. Immunostaining of umbilical vein endothelium in SCD pregnancies showed redistribution of PECAM-1 (CD31), von Willebrand factor (vWF), and P-selectin to the cell surface, controls exhibiting the classical pattern. Staining quantification indicated increases in vWF (+36.2%; P=.006) and vascular endothelial growth factor (VEGF) expression (+96.0%; P=.006) over control, but a reduction in endothelial nitric oxide synthase (eNOS) (-45.5%; P=.029). These results document, for the first time, direct functional adjustments in response to hypoxia in human in vivo. The mechanism for these changes has not been clearly established, but it may reflect increased tolerance to SCD hypoxic conditions and hypoxia in general.

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.