Apical Membrane Of Syncytiotrophoblast Research Articles

Contributions of Drug Transporters to Blood-Placental Barrier.

The placenta is the only organ linking two different individuals, mother and fetus, termed as blood-placental barrier. The functions of the blood-placental barrier are to regulate material transfer between the maternal and fetal circulation. The main functional units are the chorionic villi within which fetal blood is separated by only three or four cell layers (placental membrane) from maternal blood in the surrounding intervillous space. A series of drug transporters such as P-glycoprotein (P-GP), breast cancer resistance protein (BCRP), multidrug resistance-associated proteins (MRP1, MRP2,MRP3,MRP4,andMRP5), organic anion-transporting polypeptides (OATP4A1, OATP1A2, OATP1B3, and OATP3A1), organic anion transporter 4 (OAT4), organic cation transporter 3 (OCT3), organic cation/carnitine transporters (OCTN1andOCTN2), multidrug and toxin extrusion 1 (MATE1), and equilibrative nucleoside transporters (ENT1 and ENT2) have been demonstrated on the apical membrane of syncytiotrophoblast, some of which also expressed on the basolateral membrane of syncytiotrophoblast or fetal capillary endothelium. These transporters are involved in transport of most drugs in the placenta, in turn, affecting drug distribution in fetus. Moreover, expressions of these transporters in the placenta often vary along with the gestational ages and are also affected by pathophysiological factor. This chapter will mainly illustrate function and expression of these transporters in placentas, their contribution to drug distribution in fetus, and their clinical significance.

High Levels of Human Chorionic Gonadotropin (hCG) Correlate With Increased Aquaporin-9 (AQP9) Expression in Explants From Human Preeclamptic Placenta

Trophoblastic abnormalities have a central role in the pathophysiology of preeclampsia, and some placental hormones, such as human chorionic gonadotropin (hCG), could affect the placental function. Here, we hypothesized that the elevated serum levels of hCG may be involved in the increased aquaporin-9 (AQP9) protein expression in preeclamptic placentas via adenosine 3('),5(')-cyclic phosphate (cAMP) pathways. Normal placental explants were cultured with different concentrations of recombinant hCG or 8-Br-cAMP, a potent analogue of cAMP. We evaluated AQP9 protein expression and localization. After both treatments, we localized AQP9 in the apical membrane of syncytiotrophoblast and in the cytoplasm. We also observed a concentration-dependent effect on AQP9 protein expression. In addition, water uptake increased 1.6-fold in explants treated with hCG. Our results suggest that hCG may increase AQP9 protein expression and functionality via cAMP pathways. Although, in preeclamptic placentas high levels of hCG may upregulate AQP9 protein expression, AQP9 functionality was reduced possibly by other factors.

Non-classical MHC-E (Mamu-E) Expression in the Rhesus Monkey Placenta

The aim of this study was to characterize the expression of the rhesus HLA-E ortholog Mamu-E, particularly at the maternal–fetal interface. Mamu-E expression was confirmed by locus-specific RT-PCR in the placenta as well as in peripheral blood mononuclear cells (PBMC) and other organs. We evaluated the utility of antibodies recognizing HLA-E (MEM-E/06 against native HLA-E, MEM-E/02 against denatured HLA-E) to detect Mamu-E by flow cytometry/immunofluorescence, Western blot, and immunohistochemistry (IHC). Western blot analysis of cells and selected transfectants confirmed the recognition of Mamu-E but not Mamu-AG by antibodies MEM-E/06 and HC10 but not MEM-E/02. Immunohistochemical staining of frozen sections of rhesus placenta with the MEM-E/06 antibody demonstrated expression in most populations of rhesus monkey trophoblast cells, including villous cytotrophoblasts (strong positive staining), apical membrane of syncytiotrophoblasts (light to moderate staining) and extravillous cytotrophoblasts (moderate to strong staining, especially endovascular trophoblasts in early pregnancy). Expression was not trophoblast cell-specific, especially at term, when endothelial cells in both the chorionic plate and placental villi showed strong staining for Mamu-E. Staining of rhesus extravillous trophoblast cells suggested the co-expression of Mamu-E and Mamu-AG (the rhesus HLA-G homolog) on these cells. MEM-E/06 was shown also to react with differentiating rhesus placental syncytiotrophoblasts in primary culture, detecting intracellular and weak surface expression of Mamu-E. We conclude that the gestation-dependent co-expression of Mamu-E with Mamu-AG in villous and extravillous trophoblast cells suggests important and perhaps complementary but distinct roles of these two non-classical MHC class I loci in pregnancy at the maternal–fetal interface. In addition, the MEM-E/06 antibody will be useful for the detection of Mamu-E at the maternal–fetal interface in the rhesus monkey.

Mucin 15 is expressed in human placenta and suppresses invasion of trophoblast-like cells in vitro

Trophoblast invasion is crucial for the development of normal placentas. Mucins are suggested to be involved in cancer invasion. However, the function of mucins in trophoblast invasion has never been reported. This study was to investigate the expression of mucin (MUC) 15 in human placenta and its role in trophoblast invasion. MUC15 mRNA in human tissues was analyzed by Northern blot. MUC15 mRNA and protein in human placenta were detected by real-time RT-PCR and Western blot, respectively. The distribution of MUC15 was revealed by immunohistochemistry. The effects of MUC15 on trophoblast invasion in vitro were analyzed by matrigel invasion assay in human choriocarcinoma JAR and JEG-3 cells. MUC15 was expressed most highly in human placenta. MUC15 mRNA and protein increased with gestational age (P < 0.05, first versus third trimester). Immunohistochemistry showed that MUC15 protein was expressed by both cytotrophoblasts and syncytiotrophoblasts, especially at the apical membrane of syncytiotrophoblasts. In addition, MUC15 was found to be present in the glandular epithelium of the decidua. Overexpression of MUC15 substantially decreased matrigel invasion of JAR and JEG-3 cells by 87.5 +/- 1.1 and 83.8 +/- 5.7%, respectively, versus control, which was closely associated with an increase in mRNA expression of tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2. Knockdown of MUC15 with small interfering RNA significantly reversed these effects (P < 0.05). Differential expression of MUC15 in human placentas may play a critical role in the regulation of trophoblast invasion.

VARIABLE EXPRESSION OF MRP2 (ABCC2) IN HUMAN PLACENTA: INFLUENCE OF GESTATIONAL AGE AND CELLULAR DIFFERENTIATION

MRP2 (ABCC2) is an ATP-binding cassette (ABC)-type membrane protein involved in transport of conjugates of various drugs and endogenous compounds. MRP2 has been localized to the apical membrane of syncytiotrophoblasts and is assumed to be involved in diaplacental transfer of the above substances. It has been shown that both genetic and environmental factors can influence MRP2 expression. We therefore investigated whether gestational age, cellular differentiation, and genetic polymorphisms influence expression and localization of MRP2 in 58 human placenta samples. We detected a significant increase of transporter-mRNA with gestational age by quantitative real-time polymerase chain reaction (MRP2 mRNA/18S rRNA ratio x 1000 +/- S.D.; 0.43 +/- 0.13 in early preterms versus 1.18 +/- 0.44 in late preterms versus 2.1 +/- 0.63 in terms; p < 0.05). MRP2 protein followed the mRNA amount as shown by Western blotting (mean relative band intensity +/- S.D.; 0.56 +/- 0.1 versus 0.7 +/- 0.18 versus 0.92 +/- 0.19; early preterms versus terms p < 0.05). In cultured cytotrophoblasts, MRP2 expression increased with differentiation to syncytiotrophoblasts, with a peak on day 2 (MRP2 mRNA/18S rRNA ratio x 1000 +/- S.D.; 0.06 +/- 0.01 versus 0.88 +/- 0.27 versus 0.24 +/- 0.02 on days 0, 2, and 4). Moreover, we studied the effect of single nucleotide polymorphisms (C-24T; G1249A, and C3972T) in the MRP2 gene on placental expression. One of these polymorphisms (G1249A) resulted in a significantly reduced expression of MRP2 mRNA in preterms. In summary, the expression of MRP2 in human placenta is influenced by gestational age, cellular differentiation, and genetic factors.

Annexin 6 Modulates the Maxi-chloride Channel of the Apical Membrane of Syncytiotrophoblast Isolated from Human Placenta

The syncytiotrophoblast separates the maternal and fetal blood and constitutes the primary barrier for maternal-fetal transport. The Maxi-chloride channel from the apical membrane of the syncytiotrophoblast plays a role in the chloride conductance. Annexins can play an important role in the regulation of membrane events. In this study we evaluate the role of annexin 6 in the Maxichloride channel properties. The results showed that annexin 6 is bound in the apical placenta membranes in a calcium-dependent phospholipid-binding manner but also in a calcium-independent fashion. The neutralization of annexin 6 decreased the total current by 39 +/- 1.9% in the range of +/-80 mV, and the currents decrease with the time. The single-channel slope conductance was decreased from 253 +/- 7.4 pS (control) to 105 +/- 13 pS, and the amplitude decreased by 50%. The open probability was also affected when higher voltage steps were used, changes in either the positive or negative direction induced the channel to close, and the open probability (P(o)) did not decrease. In channels with neutralized annexin 6, it was maintained at 1 at +/-40 mV and at +/-80 mV. These results suggest that endogenous annexin 6 could regulate the Maxi-chloride channel. The results obtained with normal placentae, in which annexin 6 was neutralized, are similar to those described for the Maxi-chloride channel isolated from pre-eclamptic placenta. Together these data suggest that annexin 6 could play an important role in ion transport of the placenta.

Differential Distribution of Placental Leucine Aminopeptidase/Oxytocinase and Aminopeptidase A in Human Trophoblasts of Normal Placenta and Complete Hydatidiform Mole

Placental leucine aminopeptidase (P-LAP)/oxytocinase (OTase) degrades several small peptides such as oxytocin (OT), arginine vasopressin (AVP) and angiotensin III (ANGIII), and aminopeptidase A (AP-A) converts angiotensin II (ANGII) to ANGIII. These proteases play an important role in foetal growth and the maintenance of human homeostasis during pregnancy. In this study, we confirmed the distribution of P-LAP and AP-A proteins and messenger RNAs in human trophoblasts in normal placenta and complete hydatidiform mole by immunohistochemical and in-situ hybridization techniques. Immunoreactivity of P-LAP was mainly noted in the apical membrane of syncytiotrophoblasts, and the expression of messenger RNA (mRNA) for P-LAP was predominantly noted in the cytoplasm of syncytiotrophoblastic cells. However, immunoreactivity of AP-A was mainly noted in the apical membrane of cytotrophoblasts and in the basal zone of the syncytiotrophoblasts, and the expression of mRNA for AP-A was predominantly noted in cytoplasm of cytotrophoblastic cells and a little in cytoplasm of syncytiotrophoblastic cells. Thereby, the two proteases were differentially distributed both in normal placenta and hydatidiform mole throughout the gestational age. These results are useful for the further understanding of not only the pathophysiology of pregnancy, but also the pathogenesis of trophoblastic diseases.

Leptin Receptor in Human Term Placenta: in Situ Hybridization and Immunohistochemical Localization

In the present study, we investigated the expression and localization of leptin receptors in human term placentae. On human term placenta tissue slices, digoxigenin-UTP labelled RNA-probe detected the long form of the leptin receptor ObRLmRNA in syncytiotrophoblasts of the villi, whereas the haematological subtype of the leptin receptor ObR/B219.1 was detected in blood cells of the intervillous space and fetal vessels. Immunohistochemistry, with two polyclonal antibodies to the N-terminus recognizing ObRLand ObRSof the leptin receptors and one to the C-terminus recognizing the long form of the leptin receptor ObRL, localized leptin receptor protein at the apical membrane of the syncytiotrophoblasts. Our results show that the long form of the leptin receptor ObRLis expressed in human term placentae. We localized the long form of leptin receptor mRNA to the cytoplasm of syncytiotrophoblasts and leptin receptor proteins in human term placentae to the apical membrane of syncytiotrophoblasts. We conclude that in term placentae, leptin could mediate a growth promoting effect in the fetoplacental unit through the long form of the leptin receptor localized in the syncytiotrophoblasts. In contrast, the haematological subtype of the leptin receptor is not expressed in placental cells, but solely by blood cells in the intervillous space and fetal vessels.

Cyclic AMP-dependent protein kinases and human trophoblast cell differentiation in vitro.

Human trophoblast cells offer a unique in vitro model for the study of aspects of the dynamic processes occurring during cell fusion and syncytium formation. In the human placenta, mononuclear cytotrophoblasts aggregate and fuse to form a multinucleated syncytiotrophoblast. In vitro, the addition of cyclic AMP analogs, 8-bromo-cyclic-AMP or Sp-8-bromo-cyclic AMPS, promotes syncytiotrophoblast formation, as shown by the disappearance of immunostained E-cadherin and desmoplakin, and increased numbers of nuclei per syncytium. An antagonist of cyclic AMP, Rp-8-bromo-cyclic AMPS, and an inhibitor of the cyclic AMP-dependent protein kinase catalytic subunit, H-89, impair cell fusion. This led us to study the pattern of expression and subcellular localization of cyclic-AMP-dependent protein kinase subunits during syncytium formation. Cytotrophoblasts expressed the RIalpha and RIIalpha regulatory subunits and the Calpha and Cbeta catalytic subunits. RIalpha was down-regulated during syncytium formation. No change in RIIalpha protein levels was observed, but there was a drastic subcellular redistribution. RIIalpha located in the Golgi-centrosomal area of cytotrophoblasts was scattered throughout the cytoplasm of the syncytiotrophoblast. Interestingly, an accumulation of RIIalpha was observed underneath the apical membrane of syncytiotrophoblast in vitro and in situ. This suggests a key role of cyclic AMP-dependent protein kinase type IIalpha during cell fusion and microvilli formation, both of which are essential for the secretory and transfer functions of the syncytiotrophoblast.

Role of cyclic AMP-dependent protein kinases in human villous cytotrophoblast differentiation: A review

Summary Human trophoblast cells offer a unique in vitro model to study aspects of the dynamic processes occuring during cell fusion and syncytium formation. In the human placenta, mononuclear cytotrophoblasts aggregate and fuse to form a multinucleated syncytiotrophoblast. In vitro , the presence of 8-bromo-cAMP promotes syncytiotrophoblast formation, while an inhibitor of the cAMP dependent protein kinase (PKA) catalytic subunit, H-89, impairs cell fusion. This led us to review the pattern of expression and the subcellular localization of PKA subunits during syncytium formation as well as the role of specific A-kinase anchoring proteins in cytotrophoblast differentiation. Cytotrophoblasts expressed the RIα and RIIα regulatory subunits and Cα and Cβ catalytic subunits. RIα was down-regulated during syncytium formation. No change in RIIα mRNA or protein expression was observed, but there was a drastic subcellular redistribution. RIIα, located in the centrosomal Golgi area of cytotrophoblasts, was scattered throughout the cytoplasm in syncytiotrophoblast. Interestingly, an accumulation of RIIα was observed underneath the apical membrane of syncytiotrophoblast in vitro and in situ . As shown by overlay assay, RIIα appeared to bind to two major binding proteins, one of them being ezrin, an actin-binding protein. This suggests a key role of PKA type IIα in the secretory and transfer functions of the syncytiotrophoblast, but also in membrane reorganization associated with cell fusion and microvilli formation.

Location of insulin receptors in the placenta and its progenitor tissues.

The insulin receptor gene is constitutively expressed, so the presence of insulin receptor proteins might be expected on all mammalian tissues, with the plasma membrane as the predominant site of receptor location. Results reviewed here indicate that insulin receptors are also present in all placental tissues and the placenta's progenitor tissues and cells, i.e., oocytes, spermatozoa, and preimplantation embryos, in most of the species studied. Receptor densities, however, vary among individual cells and cell types and at various developmental stages. Three aspects deserve emphasis. 1) In human placenta, the insulin receptor distribution pattern is characterized by a spatiotemporal change between first trimester and term. At the beginning of pregnancy, insulin receptors are found predominantly on the maternal side (apical membrane of syncytiotrophoblast, low density on cytotrophoblast); at term, however, they are on the fetal side (lining the fetal vessels). This suggests that, in the first trimester, maternal insulin regulates insulin-dependent processes, whereas, at term, it must be fetal insulin mainly controlling these processes. 2) The majority of insulin receptors is expressed on structures that are currently assumed to drive placental growth, i.e., syncytial sprouts and mesenchymal villi in first-trimester placentas and fetal endothelium at term. Therefore, we hypothesize a growth-promoting function, among others, of insulin on the placenta. 3) At present, no histologic evidence is available to demonstrate insulin receptors in structures commonly associated with receptor-mediated endocytosis. Whether placental insulin receptors are internalized, therefore, awaits clarification.

Re-association of human and ovine placental eluate immunoglobulins with acidified trophoblast vesicles

The aim of this study was to characterize the expression of the rhesus HLA-E ortholog Mamu-E, particularly at the maternal–fetal interface. Mamu-E expression was confirmed by locus-specific RT-PCR in the placenta as well as in peripheral blood mononuclear cells (PBMC) and other organs. We evaluated the utility of antibodies recognizing HLA-E (MEM-E/06 against native HLA-E, MEM-E/02 against denatured HLA-E) to detect Mamu-E by flow cytometry/immunofluorescence, Western blot, and immunohistochemistry (IHC). Western blot analysis of cells and selected transfectants confirmed the recognition of Mamu-E but not Mamu-AG by antibodies MEM-E/06 and HC10 but not MEM-E/02. Immunohistochemical staining of frozen sections of rhesus placenta with the MEM-E/06 antibody demonstrated expression in most populations of rhesus monkey trophoblast cells, including villous cytotrophoblasts (strong positive staining), apical membrane of syncytiotrophoblasts (light to moderate staining) and extravillous cytotrophoblasts (moderate to strong staining, especially endovascular trophoblasts in early pregnancy). Expression was not trophoblast cell-specific, especially at term, when endothelial cells in both the chorionic plate and placental villi showed strong staining for Mamu-E. Staining of rhesus extravillous trophoblast cells suggested the co-expression of Mamu-E and Mamu-AG (the rhesus HLA-G homolog) on these cells. MEM-E/06 was shown also to react with differentiating rhesus placental syncytiotrophoblasts in primary culture, detecting intracellular and weak surface expression of Mamu-E. We conclude that the gestation-dependent co-expression of Mamu-E with Mamu-AG in villous and extravillous trophoblast cells suggests important and perhaps complementary but distinct roles of these two non-classical MHC class I loci in pregnancy at the maternal–fetal interface. In addition, the MEM-E/06 antibody will be useful for the detection of Mamu-E at the maternal–fetal interface in the rhesus monkey.