Placental cartography of NADPH oxidase (NOX) family proteins: Involvement in the pathophysiology of preeclampsia.

The placenta is an essential organ for fetal development. During the first trimester, it undergoes dramatic changes as it develops in an environment poor in oxygen (around 2-3%). From about 10 gestational weeks, oxygen levels increase to 8% in the intervillous chamber. These changes are accompanied by modulation of the activity of NADPH oxidase, a major source of production of reactive oxygen species in the first trimester of pregnancy. The NOX complex is composed of seven different proteins (NOX1-5 and DUOX1-2) whose placental involvements during physiological and pathological pregnancies are largely unknown. The aim of the study was to produce a cartography of NOX family proteins, in terms of RNA, protein expression, and localization during physiological pregnancy and in the case of preeclampsia (PE), in a cohort of early-onset PE (n = 11) and late-onset PE (n = 7) cases. NOX family proteins were mainly expressed in trophoblastic cells (NOX4-5, DUOX1) and modulated during physiological pregnancy. NOX4 underwent an unexpected and hitherto unreported nuclear translocation at term. In the case of PE, two groups stood out: NOX1-3, superoxide producers, were down-regulated (p < 0.05) while NOX4-DUOX1, hydrogen peroxide producers, were up-regulated (p < 0.05), compared to the control group. Mapping of placental NOX will constitute a reference and guide for future investigations concerning its involvement in the pathophysiology of PE.

Benzo(a)pyrene and Cerium Dioxide Nanoparticles in Co-Exposure Impair Human Trophoblast Cell Stress Signaling.

Human placenta is a multifunctional interface between maternal and fetal blood. Studying the impact of pollutants on this organ is crucial because many xenobiotics in maternal blood can accumulate in placental cells or pass into the fetal circulation. Benzo(a)pyrene (BaP) and cerium dioxide nanoparticles (CeO2 NP), which share the same emission sources, are found in ambient air pollution and also in maternal blood. The aim of the study was to depict the main signaling pathways modulated after exposure to BaP or CeO2 NP vs. co-exposure on both chorionic villi explants and villous cytotrophoblasts isolated from human term placenta. At nontoxic doses of pollutants, BaP is bioactivated by AhR xenobiotic metabolizing enzymes, leading to DNA damage with an increase in γ-H2AX, the stabilization of stress transcription factor p53, and the induction of its target p21. These effects are reproduced in co-exposure with CeO2 NP, except for the increase in γ-H2AX, which suggests a modulation of the genotoxic effect of BaP by CeO2 NP. Moreover, CeO2 NP in individual and co-exposure lead to a decrease in Prx-SO3, suggesting an antioxidant effect. This study is the first to identify the signaling pathways modulated after co-exposure to these two pollutants, which are common in the environment.

Human Placental NADPH Oxidase Mediates sFlt-1 and PlGF Secretion in Early Pregnancy: Exploration of the TGF-ß1/p38 MAPK Pathways.

Preeclampsia, a hypertensive disorder occurring during pregnancy, is characterized by excessive oxidative stress and trophoblast dysfunction with dysregulation of soluble Fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF) production. Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase (Nox) is the major source of placental superoxide in early pregnancy and its activation with the subsequent formation of superoxide has been demonstrated for various agents including Transforming Growth Factor beta-1 (TGF-ß1), a well-known p38 MAPK pathway activator. However, the bridge between Nox and sFlt-1 remains unknown. The purpose of this study was to explore the possible signaling pathway of TGF-ß1/Nox/p38 induced sFlt-1 production in human chorionic villi (CV). METHODS: Human chorionic villi from first trimester placenta (7-9 Gestational Weeks (GW)) were treated with TGF-ß1 or preincubated with p38 inhibitor, SB203580. For NADPH oxidase inhibition, CV were treated with diphenyleneiodonium (DPI). The protein levels of phospho-p38, p38, phospho-Mothers Against Decapentaplegic homolog 2 (SMAD2), and SMAD2 were detected by Western blot. The secretion of sFlt-1 and PlGF by chorionic villi were measured with Electrochemiluminescence Immunologic Assays, and NADPH oxidase activity was monitored by lucigenin method. RESULTS: We demonstrate for the first time that NADPH oxidase is involved in sFlt-1 and PlGF secretion in first trimester chorionic villi. Indeed, the inhibition of Nox by DPI decreases sFlt-1, and increases PlGF secretions. We also demonstrate the involvement of p38 MAPK in sFlt-1 secretion and Nox activation as blocking the p38 MAPK phosphorylation decreases both sFlt-1 secretion and superoxide production. Nevertheless, TGF-ß1-mediated p38 activation do not seem to be involved in regulation of the first trimester placental angiogenic balance and no crosstalk was found between SMAD2 and p38 MAPK pathways. CONCLUSIONS: Thus, the placental NADPH oxidase play a major role in mediating the signal transduction cascade of sFlt-1 production. Furthermore, we highlight for the first time the involvement of p38 activation in first trimester placental Nox activity.

Uptake of Cerium Dioxide Nanoparticles and Impact on Viability, Differentiation and Functions of Primary Trophoblast Cells from Human Placenta.

The human placenta is at the interface between maternal and fetal circulations, and is crucial for fetal development. The nanoparticles of cerium dioxide (CeO2 NPs) from air pollution are an unevaluated risk during pregnancy. Assessing the consequences of placenta exposure to CeO2 NPs could contribute to a better understanding of NPs' effect on the development and functions of the placenta and pregnancy outcome. We used primary villous cytotrophoblasts purified from term human placenta, with a wide range of CeO2 NPs concentrations (0.1-101 µg/cm2) and exposure time (24-72 h), to assess trophoblast uptake, toxicity and impact on trophoblast differentiation and endocrine function. We have shown the capacity of both cytotrophoblasts and syncytiotrophoblasts to internalize CeO2 NPs. CeO2 NPs affected trophoblast metabolic activity in a dose and time dependency, induced caspase activation and a LDH release in the absence of oxidative stress. CeO2 NPs decreased the fusion capacity of cytotrophoblasts to form a syncytiotrophoblast and disturbed secretion of the pregnancy hormones hCG, hPL, PlGF, P4 and E2, in accordance with NPs concentration. This is the first study on the impact of CeO2 NPs using human primary trophoblasts that decrypts their toxicity and impact on placental formation and functions.

NADPH oxidase is the major source of placental superoxide in early pregnancy: association with MAPK pathway activation.

First-trimester placenta (<10 gestational weeks (GW)) develops in a low oxygen environment (≈2%). Early oxygen exposure can cause oxidative damage leading to pregnancy disorders. The aim of this work was to determine the major sources of placental superoxide during early pregnancy - more specifically before 10 GW - and to study redox adaptation to increased oxygen pressure after 12 GW. Our results show that NADPH oxidase (Nox) is the main source of superoxide in first-trimester chorionic villi. Its activity is higher before 10 GW and concomitant with the location on the syncytiotrophoblast apical pole of p47phox, the Nox organizer subunit. After the increase in pO2 pressure (12-14 GW), the activities of the antioxidant enzymes SOD1, catalase and GPX1 are increased. The redox-sensitive MAPK pathways show increased phosphorylated-p38 expression, but no variation in the phosphorylation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) during first trimester, suggesting a physiological redox adaptation, whilst ERK1/2 phosphorylation is higher after 12 GW. Nox is the major superoxide source in early pregnancy (<10 GW). Increased superoxide production at 7-9 GW is associated with p38 MAPK pathway activation, suggesting that it is involved in physiological placental function and healthy early development of the placenta, through MAPK pathways.

Analysis of aquaporin expression in liver with a focus on hepatocytes.

A deeper understanding of aquaporins (AQPs) expression and transcriptional regulation will provide useful information for liver pathophysiology. We established a complete AQPs mRNA expression profile in human and mouse liver, as well as protein localization of expressed AQPs. Additionally, the modulation of AQPs mRNA levels in response to various agents was determined in human HuH7 cells and in primary culture of mouse hepatocytes. AQP1, AQP3, AQP7, AQP8, and AQP9 mRNA and protein expressions were detected in human liver, while only AQP6 and AQP11 mRNAs were detected. We reported for the first time the localization of AQP3 in Kupffer cells, AQP7 in hepatocytes and endothelial cells, and AQP9 in cholangiocytes. In addition, we confirmed the localization of AQP1 in endothelial cells, and of AQP8 and AQP9 in hepatocytes. On HuH7 cells, we reported the presence of AQP4 mRNA, confirmed the presence of AQP3, AQP7, and AQP11 mRNAs, but not of AQP8 mRNA. On primary culture of murine hepatocytes, AQP1 and AQP7 mRNAs were identified, while the presence of AQP3, AQP8, AQP9, and AQP11 mRNAs was confirmed. At the protein level, murine endothelial liver cells expressed AQP1 and AQP9, while hepatocytes expressed AQP3, AQP7, AQP8, and AQP9, and macrophages expressed AQP3. Dexamethasone, forskolin, AICAR, rosiglitazone, octanoylated, and non-octanoylated ghrelin regulated some AQP expression in primary culture of murine hepatocytes and human HuH7 cells. Additional studies will be required to further assess the role of AQPs expression in human and murine liver and understand the transcriptional regulation of AQPs in hepatocytes under pathophysiological conditions.

GH receptor plays a major role in liver regeneration through the control of EGFR and ERK1/2 activation.

GH is a pleiotropic hormone that plays a major role in proliferation, differentiation, and metabolism via its specific receptor. It has been previously suggested that GH signaling pathways are required for normal liver regeneration but the molecular mechanisms involved have yet to be determined. The aim of this study was to identify the mechanisms by which GH controls liver regeneration. We performed two thirds partial hepatectomies in GH receptor (GHR)-deficient mice and wild-type littermates and showed a blunted progression in the G(1)/S transition phase of the mutant hepatocytes. This impaired liver regeneration was not corrected by reestablishing IGF-1 expression. Although the initial response to partial hepatectomy at the priming phase appeared to be similar between mutant and wild-type mice, cell cycle progression was significantly blunted in mutant mice. The main defect in GHR-deficient mice was the deficiency of the epidermal growth factor receptor activation during the process of liver regeneration. Finally, among the pathways activated downstream of GHR during G(1) phase progression, namely Erk1/2, Akt, and signal transducer and activator of transcription 3, we only found a reduced Erk1/2 phosphorylation in mutant mice. In conclusion, our results demonstrate that GH signaling plays a major role in liver regeneration and strongly suggest that it acts through the activation of both epidermal growth factor receptor and Erk1/2 pathways.

HDL3-mediated inactivation of LDL-associated phospholipid hydroperoxides is determined by the redox status of apolipoprotein A-I and HDL particle surface lipid rigidity: relevance to inflammation and atherogenesis.

OBJECTIVE: Small dense HDL3 particles of defined lipidome and proteome potently protect atherogenic LDL against free radical-induced oxidation; the molecular determinants of such antioxidative activity in these atheroprotective, antiinflammatory particles remain indeterminate. METHODS AND RESULTS: Formation of redox-active phosphatidylcholine hydroperoxides (PCOOH) and redox-inactive phosphatidylcholine hydroxides (PCOH) was initiated in LDL by free radical-induced oxidation. Human HDL3 inactivated LDL-derived PCOOH (-62%, P<0.01) and enhanced accumulation of PCOH (2.1-fold, P<0.05); in parallel, HDL3 accumulated minor amounts of PCOOH. Enzyme-deficient reconstituted dense HDL potently inactivated PCOOH (-43%, P<0.01). HDL3-mediated reduction of PCOOH to PCOH occurred concomitantly with oxidation of methionine residues in HDL3-apolipoprotein AI (apoAI). Preoxidation of methionine residues by chloramine T markedly attenuated PCOOH inactivation (-35%); by contrast, inhibition of HDL3-associated enzymes was without effect. PCOOH transfer rates from oxidized LDL to phospholipid liposomes progressively decreased with increment in the rigidity of the phospholipid monolayer. CONCLUSIONS: The redox status of apoAI and surface lipid rigidity represent major determinants of the potent HDL3-mediated protection of LDL against free radical-induced oxidation. Initial transfer of PCOOH to HDL3 is modulated by the surface rigidity of HDL3 particles with subsequent reduction of PCOOH to PCOH by methionine residues of apoAI.