Exosomes are extracellular vesicles that mediate intercellular communication and are involved in several biological processes. The objective of our study was to determine whether endogenous retrovirus group WE, member l (ERVWE1)/syncytin-1 and endogenous retrovirus group FRD, member 1 (ERVFRDE1)/syncytin-2, encoded by human endogenous retrovirus (HERV) envelope (env) genes, are present at the surface of exosomes produced by placenta-derived villous cytotrophoblasts and whether they play a role in cellular uptake of exosomes. In addition, we sought to determine whether these proteins are present in various abundances in serum-derived exosomes from normal pregnant women vs. women with preeclampsia (PE). Isolated exosomes were analyzed for their content by Western blot, a bead-associated flow cytometry approach, and a syncytin-2 ELISA. Binding and uptake were tested through confocal and electron microscopy using the BeWo choriocarcinoma cell line. Quality control of exosome preparations consisted of detection of exosomal and nonexosomal markers. Exosome-cell interactions were compared between cells incubated in the presence of control exosomes, syncytin-1 or syncytin-2-deprived exosomes, or exosomes solely bearing the uncleaved forms of these HERV env proteins. From our data, we conclude that villous cytotrophoblast exosomes are positive for both env proteins and are rapidly taken up by BeWo cells in a syncytin-1- and syncytin-2-dependent manner and that syncytin-2 is reduced in serum-derived exosomes from women with PE when compared to exosomes from normal pregnant women.
Knowledge of the consequences of maternal obesity in human placental fatty acids (FA) transport and metabolism is limited. Animal studies suggest that placental uptake of maternal FA is altered by maternal overnutrition. We hypothesized that high maternal body mass index (BMI) affects human placental FA transport by modifying expression of key transporters. Full-term placentas were obtained by vaginal delivery from normal weight (BMI, 18.5-24.9 kg/m(2)) and obese (BMI > 30 kg/m(2)) women. Blood samples were collected from the mother at each trimester and from cord blood at delivery. mRNA and protein expression levels were evaluated with real-time RT-PCR and Western blotting. Lipoprotein lipase (LPL) activity was evaluated using enzyme fluorescence. In vitro linoleic acid transport was studied with isolated trophoblasts. Our results demonstrated that maternal obesity is associated with increased placental weight, decreased gestational age, decreased maternal high-density lipoprotein (HDL) levels during the first and third trimesters, increased maternal triglyceride levels during the second and third trimesters, and increased maternal T3 levels during all trimesters, and decreased maternal cholesterol (CHOL) and low-density lipoprotein (LDL) levels during the third trimester; and increased newborn CHOL, LDL, apolipoprotein B100, and T3 levels. Increases in placental CD36 mRNA and protein expression levels, decreased SLC27A4 and FABP1 mRNA and protein and FABP3 protein expression, and increased LPL activity and decreased villus cytotrophoblast linoleic acid transport were also observed. No changes were seen in expression of PPARA, PPARD, or PPARG mRNA and protein. Overall this study demonstrated that maternal obesity impacts placental FA uptake without affecting fetal growth. These changes, however, could modify the fetus metabolism and its predisposition to develop diseases later in life.
Maternal hypercholesterolemia (HC) during pregnancy and gestational diabetes mellitus (GDM) are associated with disturbance of fetal development which may also modify key features of placental functions. In this study, we evaluated the impact of maternal hypercholesterolemia on placental cholesterol and lipid metabolism in 59 women classified in two groups according to the median concentration of plasma total cholesterol (6.42 mM). The impact of GDM was also evaluated on the metabolism of placentas obtained from 7 insulin-treated GDM and 7 non-GDM women. We showed that high maternal circulating cholesterol is associated with a significant increase in the LDL-cholesterol, ApoB-100 and triglyceride concentrations in the maternal blood. However the level of cholesterol in the venous cord blood and placenta remains unchanged in response to modification in maternal cholesterol profile. The levels of Fatty acid synthase (FAS) and SREBP-2 expressions in placenta are significantly increased in the HC group while expression of both sterol regulatory element-binding proteins-1 (SREBP-1) and HMG-CoA reductase (HMGR) are not modified. GDM is not associated with modification in the maternal lipid profile but it increases the concentration of inflammatory cytokines (IL-1beta and TNF-alpha) in placenta which correlates with a dramatic induction of FAS expression without affecting the expression of mature SREBPs proteins. In conclusion, our study suggests that in placenta, expressions of key proteins involved in de novo lipid synthesis are affected by changes in maternal metabolism (HC and GDM) that may subsequently affect fetal development.
Gestational diabetes mellitus (GDM) is a common complication of pregnancy that is characterized by glucose intolerance, leads to dyslipidemia, and is aggravated by obesity. Cholesterol is taken up by the placenta as part of lipoproteins through the scavenger receptor class B type I receptor (SRBI), low-density lipoprotein receptor (LDLR), and very low density lipoprotein receptor (VLDLR), and its efflux is then mediated by ABCA1 and ABCG1. PCSK9 is involved in the degradation of LDLR and VLDLR. The goal of this study was to evaluate the impact of GDM and prepregnancy body mass index (BMI) on cholesterol transport through the modulation of the expression of several key players. Human full-term placenta, maternal, and venous cord blood samples were obtained at delivery from normal-weight women without GDM (n = 10), normal-weight women with GDM (n = 6), and overweight/obese women with GDM (n = 6). Lipids (total cholesterol, high-density lipoprotein, low-density lipoprotein, triglycerides, free fatty acids, apolipoprotein A1, apolipoprotein B100) levels were evaluated in blood samples. Messenger RNA and protein expression levels (LDLR, VLDLR, SRBI, ABCA1, ABCG1, proprotein convertase subtilisin/kexin type 9, liver x receptors, peroxisome proliferator-activated receptors) were assessed in human full-term placenta, respectively, by real-time RT-PCR and Western blots. Lipoprotein lipase activity was evaluated using a commercial kit on tissue homogenates. Overall, our study demonstrates that GDM affects the maternal and neonatal lipid profiles as well as different key players of placental cholesterol transfer from the maternal to the fetal circulation, depending on the maternal BMI. These changes could affect the fetal metabolism and predispose the fetus to future metabolic diseases.
Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is a rapid and high throughput gene expression quantification technology. In order to obtain accurate results, several key experimental design and standardization steps must be rigorously followed as previously described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. This study investigates the effect of reference gene normalization and the impact of RNA degradation on gene expression of 8-oxoguanine DNA glycosylase in human placenta from pregnancies complicated by preeclampsia and gestational diabetes mellitus and their gestation-matched controls. The data presented here show how RNA quality and appropriate reference gene selection is not only important to obtain accurate and reproducible RT-qPCR data but how different and even opposite results can be reported if the key steps outlined in the MIQE guidelines are not followed. The procedures and associated results presented in this study provide the first practical application of the MIQE guidelines to placental analysis in normal and pathological pregnancies.
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