Myeloperoxidase enhances the migration and invasion of human choriocarcinoma JEG-3 cells.

Myeloperoxidase (MPO) is one of the most abundant proteins in neutrophil granules. It catalyzes the production of reactive oxygen species, which are important in inflammation and immune defense. MPO also binds to several proteins, lipids, and DNA to alter their function. MPO is present at the feto-maternal interface during pregnancy, where neutrophils are abundant. In this study, we determined the effect of MPO on JEG-3 human choriocarcinoma cells as a model of extravillous trophoblasts (EVTs) during early pregnancy. We found that MPO was internalized by JEG-3 cells and localized to the cytoplasm and nuclei. MPO internalization and activity enhanced JEG-3 cell migration and invasion, whereas this effect was impaired by pre-treating cells with heparin, to block cellular uptake, and MPO-activity inhibitor 4-ABAH. This study identifies a novel mechanism for the effect of MPO on EVT function during normal pregnancy and suggests a potential role of MPO in abnormal pregnancies.

Evidence from the very beginning: endoglandular trophoblasts penetrate and replace uterine glands in situ and in vitro.

STUDY QUESTION: How is histiotrophic nutrition of the embryo secured during the first trimester of pregnancy? SUMMARY ANSWER: Rather than specifically focusing on invasion into spiral arteries, extravillous trophoblasts also invade into uterine glands (endoglandular trophoblast) from the very beginning and open them toward the intervillous space. WHAT IS KNOWN ALREADY: Extravillous trophoblasts can be found in close contact and within the lumen of uterine glands, sometimes replacing glandular epithelial cells. STUDY DESIGN, SIZE, DURATION: As well as extensive screening of specimens from first trimester placentation sites in situ we used a previously established three-dimensional co-culture in vitro model system of first trimester villous explants with non-invaded decidua parietalis. PARTICIPANTS/MATERIALS, SETTING, METHODS: First trimester placentas were obtained from elective terminations of pregnancies (n = 48) at 5-11 weeks of gestational age. A subset was processed for confrontation co-culture (n = 31). Invaded decidua basalis was obtained from 20 placentas. All tissues were sectioned, subsequently immunostained and immunodoublestained with antibodies against keratin 7 (KRT7), major histocompatibility complex, class I, G (HLA-G), matrix metallopeptidase 9 (MMP9), von Willebrand factor (VWF) and the appropriate Immunoglobulin G (IgG) negative controls. Replacement of endothelial/epithelial cells by extravillous trophoblasts was quantified semi-quantitatively. Additionally, hematoxylin and eosin-stained archival specimens from early implantation sites were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: The earliest available specimen was from around Day 10 after conception; already at this stage trophoblasts had penetrated into uterine glands and had started to replace the epithelium of the glands. Endoglandular trophoblasts replaced uterine glands in vitro and in situ and could be found in the lumen of invaded glands. Quantitative analysis revealed significantly more replacement of epithelial cells in glands (63.8 ± 22.1%) compared with endothelial cells in vessels (26.4 ± 8.8%). Accumulated detached glandular epithelial cells could be repeatedly observed in the lumen of invaded glands. Additionally, in areas of trophoblast invasion the glandular epithelium seemed to be completely disintegrated compared with glandular epithelium in the non-invaded parts of the decidua. Whole tissue specimens were used in vitro and in situ instead of cell lines; these systems mostly maintain the context of the in vivo situation. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study supported by in vitro experiments. However, a histological section will always only be a snapshot and quantification from histological sections has its limitations. WIDER IMPLICATIONS OF THE FINDINGS: This study further strengthens the hypothesis of histiotrophic nutrition of the embryo prior to the establishment of the maternal blood flow toward the placenta. Invasion of uterine glands by endoglandular trophoblasts may have more impact on the outcome of early pregnancy than assumed up to now.

Adhering maternal platelets can contribute to the cytokine and chemokine cocktail released by human first trimester villous placenta.

Placental villous explant culture has been increasingly recognized as suitable model to study secretion of inflammatory and immune modulating factors by human placenta. Most of these factors likely derive from the syncytiotrophoblast, whereas extraplacental sources such as maternal peripheral blood cells are rarely considered. Due to their small size and absence of a nucleus, platelets adhering to perivillous fibrinoid of normal placenta are frequently ignored in routine immunohistochemistry. Here we demonstrate adhering maternal platelets on first trimester placental villi after explant culture and point out that platelet-derived factors must be considered when analyzing the inflammatory secretion profile of human placenta.

The placenta and gestational diabetes mellitus.

By its location between maternal and fetal bloodstreams the human placenta not only handles the materno-fetal transport of nutrients and gases, but may also be exposed to intrauterine conditions adversely affecting placental and fetal development. Such adverse conditions exist in pregnancies complicated by gestational diabetes mellitus (GDM), and have been associated with alterations in placental anatomy and physiology. These alterations are mainly based on changes on the micro-anatomical and/or even molecular level including aberrant villous vascularization, a disbalance of vasoactive molecules, and enhanced oxidative stress. The consequence thereof may be impaired fetal oxygenation and changes in transplacental nutrient supply. Although transplacental glucose flux is flow limited and independent of glucose transporter availability, transport of essential and nonessential amino acids and expression of genes involved in lipid transport and metabolism are significantly affected by GDM.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.

The art of identification of extravillous trophoblast.

Immunohistochemical staining with specific markers for the respective cell type facilitates tracking and identification of cells such as extravillous trophoblast in the uterine wall. Cytokeratin has been recommended as a marker for all kinds of trophoblasts and is commonly used as a marker to identify interstitial as well as endovascular trophoblast. With immunohistochemical double staining of specimens of first trimester placental bed we show that staining with anti-cytokeratin alone is not sufficient to track all routes of trophoblast invasion. Endovascular trophoblasts can be easily mixed up with endoglandular trophoblasts. Thus, additional application of specific markers for extravillous trophoblast such as anti-HLA-G is strongly recommended, ideally in combination with other markers in immunohistochemical or immunofluorescence double staining.

Effects of vitamins C and E, acetylsalicylic acid and heparin on fusion, beta-hCG and PP13 expression in BeWo cells.

Preeclampsia is one of the leading causes for maternal and fetal morbidity. Placental protein 13 (PP13) is a placenta specific protein and with its decreased maternal serum levels in the first trimester it is one of the most promising markers to predict the syndrome in early pregnancy. In clinical trials attempts to prevent preeclampsia have already been made using low-dose aspirin, low-molecular-weight heparin, and antioxidants such as vitamins C and E. Here we investigated the effect of these agents on PP13 and beta-hCG levels using choriocarcinoma cell lines as surrogates for primary villous trophoblast. Five different cell lines were triggered with forskolin and cultured for 48 h. Amongst the five tested cell lines BeWo cells showed the strongest increase in PP13 mRNA after forskolin treatment compared to controls. Hence these cells were used to investigate the effect of varying concentrations of vitamin C, acetylsalicylic acid (ASA), Trolox) and heparin on cell fusion and PP13 and beta-hCG levels. The response to vitamin C was a dose-dependent increase in protein expression, while the other drugs showed only modest effects. Since first trimester PP13 has been shown to be significantly decreased in women subsequently developing preeclampsia, this data might point to a beneficial effect of very early vitamin C treatment of such women already in the early first trimester of pregnancy.

Endoglandular trophoblast, an alternative route of trophoblast invasion? Analysis with novel confrontation co-culture models.

BACKGROUND: Routes of trophoblast invasion seem to be clear, whereas specific invasive pathways need further elucidation. Extravillous trophoblasts (EVTs) transform spiral arteries to guarantee appropriate blood flow to the placenta in the second trimester. Embryo nutrition during the first trimester is thought to be histiotrophic, whereas proof that EVTs also invade uterine glands is lacking. We developed novel three-dimensional confrontation co-culture models to elucidate invasion of EVTs into uterine glands. METHODS: First trimester decidua parietalis and placental villous explants were directly confronted and co-cultured for 72 h, or confronted indirectly after 72 h pre-culture for re-epithelialization of decidua pieces. Cryosections were stained by immunohistochemistry or immunofluorescent/immunohistochemical double labelling and compared with first trimester placentation sites in situ. RESULTS: EVTs deeply invaded decidual tissues in direct confrontation assays and were found between the decidual epithelial cells and epithelial basement membrane. EVTs were also detected in the decidual stroma in direct proximity to glands, sometimes even replacing glandular epithelial cells. Similar observations were made in sections from the first trimester decidua/placental bed. In the invaded parts of sections of decidua basalis, 55% +/- 7% (mean +/- SEM; n = 10, range 6-11 weeks) of glandular cross sections were associated with or infiltrated by EVTs. CONCLUSIONS: Using novel confrontation co-culture assays, a potential new route of EVT invasion was detected. EVTs appear to break through the basement membrane of uterine glands to open their lumen towards the intervillous space. These data support the hypothesis of histiotrophic nutrition of the embryo prior to onset of maternal blood flow within the placenta.

The paradox of caspase 8 in human villous trophoblast fusion.

Differentiation and subsequent fusion of villous cytotrophoblasts with the overlying syncytiotrophoblast is an essential process for growth and maintenance of the villous trophoblast layer in the human placenta. The understanding of intrinsic mechanisms behind this process is in its infancy, while the list of suggested factors, involved in intercellular fusion of trophoblasts, rapidly increased in the recent past and promises progress on this issue. The early stages of the apoptosis cascade, in particular caspase 8, was suggested to trigger differentiation of cytotrophoblasts, priming them for upcoming fusion. This may sound paradoxical, especially for those who still associate caspase activity with apoptosis only. Here, we summarize data on caspase 8 in the villous trophoblast layer, with a specific focus on localization of pro- and active forms, the sites of its activation and deactivation, and its role and regulation during fusion. Moreover, we revisit the knowledge on fusogens in the villous trophoblast, compare in vitro models for trophoblast fusion and discuss methods to quantify fusion.

Caspases rather than calpains mediate remodelling of the fodrin skeleton during human placental trophoblast fusion.

Fusion of cytotrophoblasts with the overlying syncytiotrophoblast is an integral step in differentiation of the human placental villous trophoblast. Multiple factors, such as growth factors, hormones, cytokines, protein kinases, transcription factors and structural membrane proteins, were described to modulate trophoblast fusion. However, the knowledge on remodelling of the membrane-associated cytoskeleton during trophoblast fusion is very limited. This study describes the link between remodelling of spectrin-like alpha-fodrin and intercellular trophoblast fusion. Experiments with primary trophoblasts isolated from term placentas and the choriocarcinoma cell line BeWo revealed a biphasic strategy of the cells to achieve reorganization of alpha-fodrin. Syncytialization of trophoblasts was accompanied by down-regulation of alpha-fodrin mRNA, whereas the full-length alpha-fodrin protein was cleaved into 120 and 150 kDa fragments. Application of calpeptin and calpain inhibitor III did not affect alpha-fodrin fragmentation in primary term trophoblasts and forskolin-treated BeWo cells, but decreased secretion of beta human chorionic gonadotropin. In contrast, inhibitors of caspases 3, 8 and 9 attenuated generation of the 120 kDa fragment and a general caspase inhibitor completely blocked fragmentation, suggesting an exclusive function of caspases in alpha-fodrin remodelling. Immunofluorescence double staining of human placenta revealed co-localization of active caspase 8 with alpha-fodrin positive vesicles in fusing villous cytotrophoblasts. These results suggest that caspase-dependent fragmentation of alpha-fodrin may be important for reorganization of the sub-membranous cytoskeleton during trophoblast fusion.

Fusion of villous trophoblast can be visualized by localizing active caspase 8.

Villous cytotrophoblast differentiation and subsequent fusion with the overlying syncytiotrophoblast depend on multiple factors such as growth factors, cytokines, hormones, protein kinases, transcription factors, structural membrane proteins and proteases. Caspase 8, an aspartate-specific cysteine protease, is mainly known for its role in programmed cell death, but was also demonstrated to be crucial for villous trophoblast differentiation. This study aimed to localize active caspase 8 in the villous trophoblast layer of human first trimester placenta. To this end, immunofluorescence double staining was performed, using a monoclonal rabbit antibody against cleaved caspase 8 in combination with antibodies against cytokeratin 7, chorionic gonadotropin beta subunit (beta hCG), beta-actin, placental protein 13 (PP13), alpha-fodrin and Ki-67. Immunofluorescence revealed cleaved caspase 8 in one out of 422 villous cytotrophoblasts resting on the basement membrane, in one out of 759 perinuclear regions within the syncytiotrophoblast and in few trophoblasts located between these two layers. Double staining of cleaved caspase 8 and Ki-67 antigen revealed that caspase 8 is activated only in cytotrophoblasts which have left the cell cycle. The staining suggests that caspase 8 is activated in villous cytotrophoblasts just prior to fusion of these cells and escorts the nuclei from the mononucleated to the syncytial state.

Factors involved in regulating trophoblast fusion: potential role in the development of preeclampsia.

In the human placenta, turnover of villous trophoblast involves proliferation, differentiation and fusion of mononucleated cytotrophoblasts with the overlying syncytiotrophoblast. In this way the syncytiotrophoblast is continuously supplied with compounds derived from the fusing cytotrophoblasts. Acquisition of fresh cellular components is balanced by a concomitant release of apoptotic material as syncytial knots from the syncytiotrophoblast to the maternal circulation. In the turnover of villous trophoblast, fusion is an essential step and has been shown to be regulated by multiple factors, such as cytokines, hormones, protein kinases, transcription factors, proteases and membrane proteins. Dysregulation of one or more of these fusion factors entails aberrant fusion of the cytotrophoblast with the syncytiotrophoblast, which adversely affects the maintenance and integrity of the placental barrier. Unbalanced trophoblast fusion and release of apoptotic material into the intervillous space may provoke a massive systemic inflammatory response by the mother and thus lead to preeclampsia.

Dysregulation of placental endothelial lipase and lipoprotein lipase in intrauterine growth-restricted pregnancies.

CONTEXT: Fetal supply of maternally derived fatty acids requires lipase-mediated hydrolysis of lipoprotein-borne triglycerides and phospholipids at the placental surface. OBJECTIVE: The objective of the study was to test the hypothesis that members of the triglyceride lipase gene (TLG) family are expressed in the human placenta at the maternoplacental (syncytiotrophoblast) and fetoplacental (endothelial cells) interface and that their expression is altered in pregnancy pathologies. DESIGN AND SETTING: Expression of TLG family members in primary placental cells (trophoblast and endothelial cells) and tissues of first-trimester and term human placenta was analyzed by microarrays, RT-PCR, Western blotting, and immunohistochemistry. Their expression was compared between normal pregnancies and those complicated with intrauterine growth restriction (IUGR). PARTICIPANTS: Participants included women with uncomplicated pregnancies and pregnancies complicated by IUGR. RESULTS: Endothelial lipase (EL) and lipoprotein lipase (LPL) were the only lipases among the TLG family expressed in key cells of the human placenta. In first trimester, EL and LPL were expressed in trophoblasts. At term, EL was detected in trophoblasts and endothelial cells, whereas LPL was absent in these cells. Both lipases were found at placental blood vessels, EL in vascular endothelial cells and LPL in the surrounding smooth muscle cells. In total placental tissue EL expression prevails in first trimester and at term. Compared with normal placentas, EL mRNA was decreased (30%; P < 0.02), whereas LPL mRNA expression was increased (2.4-fold; P < 0.015) in IUGR. CONCLUSION: EL is the predominant TLG family member in the human placenta present at both interfaces. EL and LPL are dysregulated in IUGR.

Endothelial lipase releases saturated and unsaturated fatty acids of high density lipoprotein phosphatidylcholine.

We assessed the ability of endothelial lipase (EL) to hydrolyze the sn-1 and sn-2 fatty acids (FAs) from HDL phosphatidylcholine. For this purpose, reconstituted discoidal HDLs (rHDLs) that contained free cholesterol, apolipoprotein A-I, and either 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or 1-palmitoyl-2-arachidonylphosphatidylcholine were incubated with EL- and control (LacZ)-conditioned media. Gas chromatography analysis of the reaction mixtures revealed that both the sn-1 (16:0) and sn-2 (18:1, 18:2, and 20:4) FAs were liberated by EL. The higher rate of sn-1 FA cleavage compared with sn-2 FA release generated corresponding sn-2 acyl lyso-species as determined by MS analysis. EL failed to release sn-2 FA from rHDLs containing 1-O-1'-hexadecenyl-2-arachidonoylphosphatidylcholine, whose sn-1 position contained a nonhydrolyzable alkyl ether linkage. The lack of phospholipase A(2) activity of EL and its ability to liberate [(14)C]FA from [(14)C]lysophosphatidylcholine (lyso-PC) led us to conclude that EL-mediated deacylation of phosphatidylcholine (PC) is initiated at the sn-1 position, followed by the release of the remaining FA from the lyso-PC intermediate. Thin-layer chromatography analysis of cellular lipids obtained from EL-overexpressing cells revealed a pronounced accumulation of [(14)C]phospholipid and [(14)C]triglyceride upon incubation with 1-palmitoyl-2-[1-(14)C]linoleoyl-PC-labeled HDL(3), indicating the ability of EL to supply cells with unsaturated FAs.

Adenovirus-mediated apo(a)-antisense-RNA expression efficiently inhibits apo(a) synthesis in vitro and in vivo.

Apo(a) is a very atherogenic plasma protein without apparent function, which is highly expressed in humans. The variation in plasma Lp(a) concentration among individuals is considerable. Approximately 10-15% of the white population exhibit plasma Lp(a) concentrations above the atherogenic cut-off value of approximately 30 mg/dl. Since there is currently no safe way of treating those patients with drugs, we have tested the possibility of interfering with apo(a) biosynthesis by adenovirus-mediated expression of antisense apo(a) mRNA comprising the 5' UTR, the signal sequence and the first three kringles of native apo(a). Transduction of rat hepatoma McA RH 7777 cells which stably expressed apo(a) with 18 kringle IV (KIV) domains with apo(a)-antisense adenovirus (AS-Ad) at multiplicity of infection (MOI) of 30 reduced apo(a) synthesis to 23% as compared with control cells. As apo(a) is not synthesized in laboratory animals, we induced biosynthesis of the N-terminal fragments of apo(a) in mice by adenovirus-mediated gene transfer. Cotransduction of these mice with AS-Ad, which expressed up to eight times higher amounts of apo(a) than stable transgenic apo(a) mice, led to an almost complete disappearance of apo(a) from plasma. We conclude that the proposed AS-construct is very efficient in interfering with apo(a) biosynthesis in vivo. The strategy of inducing the synthesis of a nonexpressed protein followed by knocking it out by AS technology may also be applicable to other systems.