Thierry Fournier

Diesel exhaust particles are taken up by human airway epithelial cells in vitro and alter cytokine production

The involvement of diesel exhaust particles (DEPs) in respiratory diseases was evaluated by studying their effects on two in vitro models of human airway epithelial cells. The cytotoxicity of DEPs, their phagocytosis, and the resulting immune response were investigated in a human bronchial epithelial cell line (16HBE14o-) as well as in human nasal epithelial cells in primary culture. DEP exposure induced a time- and dose-dependent membrane damage. Transmission electron microscopy showed that DEPs underwent endocytosis by epithelial cells and translocated through the epithelial cell sheet. Flow cytometric measurements allowed establishment of the time and dose dependency of this phagocytosis and its nonspecificity with different particles (DEPs, carbon black, and latex particles). DEPs also induced a time-dependent increase in interleukin-8, granulocyte-macrophage colony-stimulating factor, and interleukin-1beta release. This inflammatory response occurred later than phagocytosis, and its extent seems to depend on the content of adsorbed organic compounds because carbon black had no effect on cytokine release. Furthermore, exhaust gas posttreatments, which diminished the adsorbed organic compounds, reduced the DEP-induced increase in granulocyte-macrophage colony-stimulating factor release. These results suggest that DEPs could 1) be phagocytosed by airway epithelial cells and 2) induce a specific inflammatory response.The involvement of diesel exhaust particles (DEPs) in respiratory diseases was evaluated by studying their effects on two in vitro models of human airway epithelial cells. The cytotoxicity of DEPs, their phagocytosis, and the resulting immune response were investigated in a human bronchial epithelial cell line (16HBE14o-) as well as in human nasal epithelial cells in primary culture. DEP exposure induced a time- and dose-dependent membrane damage. Transmission electron microscopy showed that DEPs underwent endocytosis by epithelial cells and translocated through the epithelial cell sheet. Flow cytometric measurements allowed establishment of the time and dose dependency of this phagocytosis and its nonspecificity with different particles (DEPs, carbon black, and latex particles). DEPs also induced a time-dependent increase in interleukin-8, granulocyte-macrophage colony-stimulating factor, and interleukin-1β release. This inflammatory response occurred later than phagocytosis, and its extent seems to depend on the content of adsorbed organic compounds because carbon black had no effect on cytokine release. Furthermore, exhaust gas posttreatments, which diminished the adsorbed organic compounds, reduced the DEP-induced increase in granulocyte-macrophage colony-stimulating factor release. These results suggest that DEPs could 1) be phagocytosed by airway epithelial cells and 2) induce a specific inflammatory response.

Placenta-specific Methylation of the Vitamin D 24-Hydroxylase Gene IMPLICATIONS FOR FEEDBACK AUTOREGULATION OF ACTIVE VITAMIN D LEVELS AT THE FETOMATERNAL INTERFACE

Plasma concentrations of biologically active vitamin D (1,25-(OH)2D) are tightly controlled via feedback regulation of renal 1α-hydroxylase (CYP27B1; positive) and 24-hydroxylase (CYP24A1; catabolic) enzymes. In pregnancy, this regulation is uncoupled, and 1,25-(OH)2D levels are significantly elevated, suggesting a role in pregnancy progression. Epigenetic regulation of CYP27B1 and CYP24A1 has previously been described in cell and animal models, and despite emerging evidence for a critical role of epigenetics in placentation generally, little is known about the regulation of enzymes modulating vitamin D homeostasis at the fetomaternal interface. In this study, we investigated the methylation status of genes regulating vitamin D bioavailability and activity in the placenta. No methylation of the VDR (vitamin D receptor) and CYP27B1 genes was found in any placental tissues. In contrast, the CYP24A1 gene is methylated in human placenta, purified cytotrophoblasts, and primary and cultured chorionic villus sampling tissue. No methylation was detected in any somatic human tissue tested. Methylation was also evident in marmoset and mouse placental tissue. All three genes were hypermethylated in choriocarcinoma cell lines, highlighting the role of vitamin D deregulation in this cancer. Gene expression analysis confirmed a reduced capacity for CYP24A1 induction with promoter methylation in primary cells and in vitro reporter analysis demonstrated that promoter methylation directly down-regulates basal promoter activity and abolishes vitamin D-mediated feedback activation. This study strongly suggests that epigenetic decoupling of vitamin D feedback catabolism plays an important role in maximizing active vitamin D bioavailability at the fetomaternal interface.

Induction of diaphragmatic nitric oxide synthase after endotoxin administration in rats: role on diaphragmatic contractile dysfunction.

Nitric oxide (NO), a free radical that is negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by an NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to examine iNOS induction in the rat diaphragm after Escherichia Coli LPS inoculation (1.6 mg/kg i.p.), and its involvement in diaphragmatic contractile dysfunction. Inducible NOS protein and activity could be detected in the diaphragm as early as 6 h after LPS inoculation. 6 and 12 h after LPS, iNOS was expressed in inflammatory cells infiltrating the perivascular spaces of the diaphragm, whereas 12 and 24 h after LPS it was expressed in skeletal muscle fibers. Inducible NOS was also expressed in the left ventricular myocardium, whereas no expression was observed in the abdominal, intercostal, and peripheral skeletal muscles. Diaphragmatic force was significantly decreased 12 and 24 h after LPS. This decrease was prevented by inhibition of iNOS induction by dexamethasone or by inhibition of iNOS activity by N(G)-methyl-L-arginine. We conclude that iNOS was induced in the diaphragm after E. Coli LPS inoculation in rats, being involved in the decreased muscular force.

Tumor Necrosis Factor-α Inversely Regulates Prostaglandin D2 and Prostaglandin E2 Production in Murine Macrophages SYNERGISTIC ACTION OF CYCLIC AMP ON CYCLOOXYGENASE-2 EXPRESSION AND PROSTAGLANDIN E2 SYNTHESIS

Increased synthesis of insulin-like growth factor-1 is induced in murine macrophages by prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNFα). Accordingly, we have investigated mechanisms regulating synthesis of PGE2 that might contribute to autocrine/paracrine effects on insulin-like growth factor-1 production. In response to zymosan, TNFα specifically induced a 5-fold increase in PGE2 synthesis, at the same time decreasing PGD2 production in a reciprocal fashion. Activators of cyclic AMP-dependent protein kinase (PKA), such as PGE2 itself or dibutyryl cyclic AMP, did not modify PGE2 production by themselves but potentiated the TNFα-induced increase in PGE2; this effect required both RNA and protein synthesis. No significant change in arachidonate release or production of other eicosanoids was observed. The inducible form of cyclooxygenase-2 (COX2) but not of the constitutive form COX1 was implicated in the generation of both PGE2 and PGD2 in these cells by use of specific inhibitors and effects of dexamethasone. Neither COX1 nor COX2 protein levels were affected by TNFα or PKA activators used alone, whereas in association, marked up-regulation of COX2 mRNA and protein was observed. Incubations of cells carried out with PGH2demonstrated that PGE2 synthase activity was increased after a TNFα pretreatment. Taken together, our results suggest that TNFα induced a switch from the PGD2 to PGE2synthesis pathway by regulating PGE2 synthase expression and/or activity and that activators of PKA markedly potentiated the TNFα-induced increase in PGE2 through up-regulation of COX2 gene expression.

Review: hCGs: Different sources of production, different glycoforms and functions

Human chorionic gonadotropin (hCG) is the first hormonal message from the placenta to the mother. It is detectable in maternal blood two days after implantation and behaves like an agonist of LH stimulating progesterone secretion by the corpus luteum. hCG has also a role in quiescence of the myometrium and local immune tolerance. Specific to humans, hCG is a complex glycoprotein composed of two glycosylated subunits. The α-subunit is identical to the pituitary gonadotropin hormones (LH, FSH, TSH), contains two N-glycosylation sites, and is encoded by a single gene (CGA). By contrast the β-subunits are distinct in each of the hormones and confer receptor and biological specificity. The hCG β-subunit contains two sites of N-glycosylation and four sites of O-glycosylation and is encoded by a cluster of genes (CGB). In this review, we will stress the importance of hCG glycosylation state, which varies with the stage of pregnancy, its source of production and in the pathology. It is well established that hCG is mainly secreted by the syncytiotrophoblast into maternal blood where it peaks around 8-10 weeks of gestation (WG). The invasive extravillous trophoblast also secretes hCG, and in particular like choriocarcinoma cells, hyperglycosylated forms of hCG (hCG-H). In maternal blood hCG-H is high during early first trimester. In addition to its endocrine role, hCG has autocrine and paracrine roles. It promotes formation of the syncytiotrophoblast and angiogenesis through LHCG receptor. In contrast, hCG-H stimulates trophoblast invasion and angiogenesis by interacting with the TGFβ receptor 2. hCG is largely used in antenatal screening and hCG-H represents a serum marker of early trophoblast invasion. Other abnormally glycosylated hCG are described in aneuploidies. In conclusion, hCG is the major pregnancy glycoprotein hormone, whose maternal concentration and glycan structure change all along pregnancy. Depending on its source of production, glycoforms of hCG display different biological activities and functions that are essential for pregnancy outcome.

Statins and Pregnancy

Cardiovascular diseases are the leading cause of mortality in industrialized countries. Treatment with statins is effective in primary prevention in patients at high cardiovascular risk. Statins are inhibitors of hydroxymethylglutarylcoenzyme A (HMG-CoA) reductase and are classed as lipid-lowering drugs. In 2010, atorvastatin was the biggest-selling drug in the world (

The Role of PPAR‐γ/RXR‐α Heterodimers in the Regulation of Human Trophoblast Invasion

US10.73 billion). Increases in the average age of pregnant women and in the prevalence of morbid obesity have inevitably led to exposure to statins in certain women during the first trimester of pregnancy. The teratogenic risk attendant upon use of statins is unclear because the available data are contradictory, but statins remain contraindicated in pregnant women.The benefits of statins in prevention of cardiovascular risk may not be solely due to their cholesterol-lowering effects: the so-called pleiotropic effects of vascular protection lead some experts to posit a potential benefit in the management of preeclampsia.In this review we evaluate the theoretical benefits and supposed risks of statins in pregnant women. After a brief overview of the pharmacodynamic properties of statins, we address the question of the teratogenic risk of statins, and then detail the rationale for the therapeutic potential of statins in preeclampsia.

Activation of Peroxisome Proliferator-Activated Receptor Gamma by Human Cytomegalovirus for De Novo Replication Impairs Migration and Invasiveness of Cytotrophoblasts from Early Placentas

Abstract: PPAR‐γ/RXR heterodimers play a key regulatory role in murine placental development. We report here that PPAR‐γ and RXR‐α are highly expressed in cultured primary extravillous cytotrophoblasts (EVCT) isolated from human first‐trimester placenta. Using this cell culture model, we showed that PPAR‐γ agonists inhibit EVCT invasion, whereas PPAR‐γ or pan‐RXR antagonists promoted EVCT invasion by themselves and abolished PPAR‐γ agonist‐mediated effect. Together these data underscore an important function of PPAR/RXR heterodimers in the modulation of trophoblast invasion.

Development and hormonal functions of the human placenta

ABSTRACT Human cytomegalovirus (HCMV) contributes to pathogenic processes in immunosuppressed individuals, in fetuses, and in neonates. In the present report, by using reporter gene activation assays and confocal microscopy in the presence of a specific antagonist, we show for the first time that HCMV infection induces peroxisome proliferator-activated receptor gamma (PPARγ) transcriptional activity in infected cells. We demonstrate that the PPARγ antagonist dramatically impairs virus production and that the major immediate-early promoter contains PPAR response elements able to bind PPARγ, as assessed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Due to the key role of PPARγ in placentation and its specific trophoblast expression within the human placenta, we then provided evidence that by activating PPARγ human cytomegalovirus dramatically impaired early human trophoblast migration and invasiveness, as assessed by using well-established in vitro models of invasive trophoblast, i.e., primary cultures of extravillous cytotrophoblasts (EVCT) isolated from first-trimester placentas and the EVCT-derived cell line HIPEC. Our data provide new clues to explain how early infection during pregnancy could impair implantation and placentation and therefore embryonic development.

PPARγ and human trophoblast differentiation

The human placenta is characterized by the intensity of the trophoblast invasion into the uterus wall and the specificity of its hormonal functions. Placental hormones are required for the establishment and maintenance of pregnancy, adaptation of the maternal organism to pregnancy and fetal growth. In the early placenta at the maternofetal interface, the human trophoblast differentiates along two pathways: 1/ the villous trophoblast pathway including the cytotrophoblastic cells which differentiate by fusion to form the syncytiotrophoblast that covers the entire surface of the villi; 2/ the extravillous trophoblast pathway. The cytotrophoblastic cells of the anchoring villi in contact with the uterus wall proliferate and then migrate into the decidua and the myometrium but also participate to the remodeling of the spiral arteries. During the first trimester of pregnancy the spiral arteries are plugged by trophoblastic cells, allowing the development of the fetoplacental unit in low oxygen environment. At this stage of pregnancy the extravillous trophoblast secretes a large amount of hormones such as particular hyperglycosylated forms of hCG directly involved in the quality of the placentation. At 10-12 weeks of pregnancy, the trophoblastic plugs are progressively dislocated and the syncytiotrophoblast starts to bath in maternal blood. It secretes the major part of its polypeptide hormones in maternal circulation taking over the maternal metabolism in order to increase the energetic flux to the fetus. As example the placental GH (growth hormone) secreted continuously by the syncytiotrophoblast is directly involved in the insulino-resistance of pregnancy. Capturing the cholesterol from the maternal lipoproteins, the syncytiotrophoblast synthesizes also large amount of progesterone essential for the uterine quiescence. Deprived of cytochrome P450 17alpha-hydroxylase-17:20 lyase, it uses the maternal and fetal adrenal androgens to synthesize estrogens. The differentiation and hormonal functions of the human trophoblast are regulated by the environmental O2 and reflect mammalian evolution.