Christophe Buffat

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.

A genome-wide approach reveals novel imprinted genes expressed in the human placenta

Genomic imprinting characterizes genes with a monoallelic expression, which is dependent on the parental origin of each allele. Approximately 150 imprinted genes are known to date, in humans and mice but, though computational searches have tried to extract intrinsic characteristics of these genes to identify new ones, the existing list is probably far from being comprehensive. We used a high-throughput strategy by diverting the classical use of genotyping microarrays to compare the genotypes of mRNA/cDNA vs. genomic DNA to identify new genes presenting monoallelic expression, starting from human placental material. After filtering of data, we obtained a list of 1,082 putative candidate monoallelic SNPs located in more than one hundred candidate genes. Among these, we found known imprinted genes, such as IPW, GRB10, INPP5F and ZNF597, which contribute to validate the approach. We also explored some likely candidates of our list and identified seven new imprinted genes, including ZFAT, ZFAT-AS1, GLIS3, NTM, MAGI2, ZC3H12Cand LIN28B, four of which encode zinc finger transcription factors. They are, however, not imprinted in the mouse placenta, except for Magi2. We analyzed in more details the ZFAT gene, which is paternally expressed in the placenta (as ZFAT-AS1, a non-coding antisense RNA) but biallelic in other tissues. The ZFAT protein is expressed in endothelial cells, as well as in syncytiotrophoblasts. The expression of this gene is, moreover, downregulated in placentas from complicated pregnancies. With this work we increase by about 10% the number of known imprinted genes in humans.

Effects Of Maternally Administered Drugs On The Fetal And Neonatal Kidney

The number of pregnant women and women of childbearing age who are receiving drugs is increasing. A variety of drugs are prescribed for either complications of pregnancy or maternal diseases that existed prior to the pregnancy. Such drugs cross the placental barrier, enter the fetal circulation and potentially alter fetal development, particularly the development of the kidneys. Increased incidences of intrauterine growth retardation and adverse renal effects have been reported. The fetus and the newborn infant may thus experience renal failure, varying from transient oligohydramnios to severe neonatal renal insufficiency leading to death. Such adverse effects may particularly occur when fetuses are exposed to NSAIDs, ACE inhibitors and specific angiotensin II receptor type 1 antagonists. In addition to functional adverse effects, in utero exposure to drugs may affect renal structure itself and produce renal congenital abnormalities, including cystic dysplasia, tubular dysgenesis, ischaemic damage and a reduced nephron number. Experimental studies raise the question of potential long-term adverse effects, including renal dysfunction and arterial hypertension in adulthood. Although neonatal data for many drugs are reassuring, such findings stress the importance of long-term follow-up of infants exposed in utero to certain drugs that have been administered to the mother.

Adverse consequences of accelerated neonatal growth: cardiovascular and renal issues

Epidemiological and experimental studies show that the risk of cardiovascular and metabolic diseases at adulthood is inversely related to the weight at birth. Although with less evidence, low birth weight has been suggested to increase the risk of chronic kidney disease (CKD). It is well established that the developmental programming of arterial hypertension and of renal disease involves in particular renal factors, especially nephron endowment, which is reduced in low birth weight and maternal diabetes situations. Experimental studies, especially in rodents, have demonstrated the long-term influence of postnatal nutrition and/or postnatal growth on cardiovascular, metabolic and renal functions, while human data are scarce on this issue. Vascular and renal diseases appear to have a “multihits” origin, with reduced nephron number the initial hit and rapid postnatal growth the second hit. This review addresses the current understanding of the role of the kidney, both as a mechanism and as a target, in the developmental origins of adult disease theory, with a particular focus on the long-term effects of postnatal growth and nutrition.

Gs alpha overexpression and loss of Gs alpha imprinting in human somatotroph adenomas: association with tumor size and response to pharmacologic treatment.

Gsα, the α‐subunit of the heterotrimeric GTP‐binding protein, is coded from the GNAS gene, which is imprinted in a tissue‐specific manner. Gsα is paternally silenced in normal pituitary, but Gsα imprinting relaxation is found in some tumoral tissue. In addition, Gsα mRNA levels are high in some somatotroph adenomas not bearing the active Gsα mutant, the gsp oncogene. In this study, the impact of loss of imprinting on Gsα expression level and on tumoral phenotype has been investigated. We compared the expression and imprinting of 4 transcripts of GNAS locus (NESP55, XLαs, exon 1A, Gsα) of 60 somatotroph adenomas with those of 23 lactotroph adenomas. The paternal and maternal transcripts were quantified using allele‐specific real‐time PCR and FokI polymorphism. Moreover, the methylation of exon 1A DMR was analyzed. As is the case for the gsp oncogene, high Gsα expression in gsp‐ tumors was associated with smaller tumor size and better octreotide sensitivity. A strong imprinting relaxation (percentage of paternal Gsα expression ≥7.5%) was found only in gsp‐ tumors. The loss of Gsα imprinting was associated with a decrease in exon 1A mRNA expression. Unexpectedly, the methylation status of exon 1A DMR was not modified in relaxed tumors. Maternal Gsα mRNA level decreased with exon 1A level, and consequently the loss of Gsα imprinting did not induce the expected Gsα overexpression. Finally, XLαs mRNA level correlated with that of paternal Gsα and of NESP55 showing the complexity of gene regulation in the GNAS locus.

Renal effects of ibuprofen for the treatment of patent ductus arteriosus in premature infants

In recent years ibuprofen has been proposed for the treatment of patent ductus arteriosus (PDA) as it has been proved to be equally as effective as indomethacin and shows fewer cerebral blood flow, intestinal and renal hemodynamic effects. A number of studies and several meta-analyses comparing both drugs are now available that debate whether indomethacin or ibuprofen should be used for PDA prophylaxis or closure. This review examines the available knowledge on the specific issue of the effects of ibuprofen on kidney function, as improved renal tolerance is a major argument in favor of its use in the routine treatment of PDA. There is sufficient evidence to consider that ibuprofen, at the currently proposed dosing regimen, has a similar efficacy to indomethacin but is better tolerated by the neonatal kidney when employed for the treatment of established PDA. However, adverse effects of ibuprofen have been evidenced both in trials on the use of ibuprofen for the prevention of PDA and of intraventricular hemorrhage–periventricular hemorrhage (IVH–PVH), and in experimental studies on a neonatal, anesthetized animal model. Thus ibuprofen, as with other cyclooxygenase (COX) inhibitors, may not be exempt from causing renal adverse effects, especially in circumstances when renal prostaglandin activation is maximal (i.e., when administrated early after birth, in more immature patients and in certain situations such as in the anesthetized rabbit). However, although the issue has been addressed extensively in the last decades, there is insufficient evidence that therapeutic intervention in PDA is beneficial in terms of mortality or clinically significant morbidity outcomes. Studies aimed at resolving this key issue are still needed.

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.

Transcriptomic analysis of human placenta in intrauterine growth restriction

Background:Intrauterine growth restriction (IUGR) is a frequent complication of pregnancy defined as a restriction of fetal growth. The objective of this work was to improve the knowledge on the pathophysiology of IUGR using a genome-wide method of expression analysis.Methods:We analyzed differentially expressed genes in pooled placental tissues from vascular IUGR (four pools of three placentas) and normal pregnancies (four pools of three placentas) using a long nucleotide microarray platform (Nimblegen). We first did a global bioinformatics analysis based only on P value without any a priori. We secondly focused on “target” genes among the most modified ones. Finally, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed on an extended panel of tissue samples (n = 62) on selected “target”.Results:We identified 636 modified genes among which 206 were upregulated (1.5 and higher; P < 0.05). Groups of patients were classified unambiguously. Genes involved in mitochondrial function and oxidative phosphorylation were decreased affecting three out of five complexes of the respiratory chain of the mitochondria, and thus energy production and metabolism. Among the most induced genes, we identified LEP, IGFBP1, and RBP4.Conclusion:Complementary studies on the role and function of LEP, IGFBP1, and RBP4 in IUGR pathophysiology and also in fetal programming remain necessary.

Altered angiogenesis in low birth weight individuals: a role for anti-angiogenic circulating factors

Abstract Objective: Low birth weight (LBW) is a risk factor for hypertension at adulthood. Endothelial progenitor cells (EPCs) dysfunction has been characterized in LBW neonates. We hypothesized that changes in soluble, plasma pro- or anti-angiogenic factors are associated with EPCs dysfunction and impaired angiogenesis in LBW neonates. Method: Venous umbilical cord blood was collected from 42 normal, term neonates and 75 LBW neonates. Cord blood endothelial colony forming cells (ECFC) from control patients were cultured in the presence of 10% of serum obtained from both groups. Results: The proliferation and the migration of ECFC were significantly reduced when cultured with 10% of serum of LBW neonates compared to serum of control neonates. Matrigel invasion assay was not significantly altered. Umbilical vein plasma VEGF concentration was significantly reduced in LBW neonates while that of sVEGFR and PF4 were significantly higher. Addition of VEGF corrected the inhibitory effect of LBW serum on normal ECFC proliferation. Conclusions: Serum obtained from LBW babies contains factors that exhibit an antiangiogenic effect on ECFC proliferation and migration. VEGF/sVEGF/PF4 pathway seems to be involved in the EPCs dysfunction in LBW neonates.

Preeclamptic Plasma Induces Transcription Modifications Involving the AP-1 Transcriptional Regulator JDP2 in Endothelial Cells

Preeclampsia is a pregnancy disorder characterized by hypertension and proteinuria. In preeclampsia, the placenta releases factors into the maternal circulation that cause a systemic endothelial dysfunction. Herein, we investigated the effects of plasma from women with preeclamptic and normal pregnancies on the transcriptome of an immortalized human umbilical vein endothelial cell line. The cells were exposed for 24 hours to preeclamptic or normal pregnancy plasma and their transcriptome was analyzed using Agilent microarrays. A total of 116 genes were found differentially expressed: 71 were up-regulated and 45 were down-regulated. In silico analysis revealed significant consistency and identified four functional categories of genes: mitosis and cell cycle progression, anti-apoptotic, fatty acid biosynthesis, and endoplasmic reticulum stress effectors. Moreover, several genes involved in vasoregulation and endothelial homeostasis showed modified expression, including EDN1, APLN, NOX4, and CBS. Promoter analysis detected, among the up-regulated genes, a significant overrepresentation of genes containing activation protein-1 regulatory sites. This correlated with down-regulation of JDP2, a gene encoding a repressor of activation protein-1. The role of JDP2 in the regulation of a subset of genes in the human umbilical vein endothelial cells was confirmed by siRNA inhibition. We characterized transcriptional changes induced by preeclamptic plasma on human umbilical vein endothelial cells, and identified, for the first time to our knowledge, JDP2 as a regulator of a subset of genes modified by preeclamptic plasma.