NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation.

Failures in growth and differentiation of the early human placenta are associated with severe pregnancy disorders such as pre-eclampsia and fetal growth restriction. However, regulatory mechanisms controlling development of placental epithelial cells, the trophoblasts, remain poorly elucidated. Using trophoblast stem cells (TSCs), trophoblast organoids (TB-ORGs) and primary cytotrophoblasts (CTBs) of early pregnancy, we herein show that autocrine NOTCH3 signalling controls human placental expansion and differentiation. The NOTCH3 receptor was specifically expressed in proliferative CTB progenitors and its active form, the nuclear NOTCH3 intracellular domain (NOTCH3-ICD), interacted with the transcriptional co-activator mastermind-like 1 (MAML1). Doxycycline-inducible expression of dominant-negative MAML1 in TSC lines provoked cell fusion and upregulation of genes specific for multinucleated syncytiotrophoblasts, which are the differentiated hormone-producing cells of the placenta. However, progenitor expansion and markers of trophoblast stemness and proliferation were suppressed. Accordingly, inhibition of NOTCH3 signalling diminished growth of TB-ORGs, whereas overexpression of NOTCH3-ICD in primary CTBs and TSCs showed opposite effects. In conclusion, the data suggest that canonical NOTCH3 signalling plays a key role in human placental development by promoting self-renewal of CTB progenitors.

Hippo signaling cofactor, WWTR1, at the crossroads of human trophoblast progenitor self-renewal and differentiation.

Healthy progression of human pregnancy relies on cytotrophoblast (CTB) progenitor self-renewal and its differentiation toward multinucleated syncytiotrophoblasts (STBs) and invasive extravillous trophoblasts (EVTs). However, the underlying molecular mechanisms that fine-tune CTB self-renewal or direct its differentiation toward STBs or EVTs during human placentation are poorly defined. Here, we show that Hippo signaling cofactor WW domain containing transcription regulator 1 (WWTR1) is a master regulator of trophoblast fate choice during human placentation. Using human trophoblast stem cells (human TSCs), primary CTBs, and human placental explants, we demonstrate that WWTR1 promotes self-renewal in human CTBs and is essential for their differentiation to EVTs. In contrast, WWTR1 prevents induction of the STB fate in undifferentiated CTBs. Our single-cell RNA sequencing analyses in first-trimester human placenta, along with mechanistic analyses in human TSCs revealed that WWTR1 fine-tunes trophoblast fate by directly regulating WNT signaling components. Importantly, our analyses of placentae from pathological pregnancies show that extreme preterm births (gestational time ≤28 wk) are often associated with loss of WWTR1 expression in CTBs. In summary, our findings establish the critical importance of WWTR1 at the crossroads of human trophoblast progenitor self-renewal versus differentiation. It plays positive instructive roles in promoting CTB self-renewal and EVT differentiation and safeguards undifferentiated CTBs from attaining the STB fate.

Transforming growth factor-ß signaling governs the differentiation program of extravillous trophoblasts in the developing human placenta.

Abnormal placentation has been noticed in a variety of pregnancy complications such as miscarriage, early-onset preeclampsia, and fetal growth restriction. Defects in the developmental program of extravillous trophoblasts (EVTs), migrating from placental anchoring villi into the maternal decidua and its vessels, is thought to be an underlying cause. Yet, key regulatory mechanisms controlling commitment and differentiation of the invasive trophoblast lineage remain largely elusive. Herein, comparative gene expression analyses of HLA-G-purified EVTs, isolated from donor-matched placenta, decidua, and trophoblast organoids (TB-ORGs), revealed biological processes and signaling pathways governing EVT development. In particular, bioinformatics analyses and manipulations in different versatile trophoblast cell models unraveled transforming growth factor-ß (TGF-ß) signaling as a crucial pathway driving differentiation of placental EVTs into decidual EVTs, the latter showing enrichment of a secretory gene signature. Removal of Wingless signaling and subsequent activation of the TGF-ß pathway were required for the formation of human leukocyte antigen-G+ (HLA-G+) EVTs in TB-ORGs that resemble in situ EVTs at the level of global gene expression. Accordingly, TGF-ß-treated EVTs secreted enzymes, such as DAO and PAPPA2, which were predominantly expressed by decidual EVTs. Their genes were controlled by EVT-specific induction and genomic binding of the TGF-ß downstream effector SMAD3. In summary, TGF-ß signaling plays a key role in human placental development governing the differentiation program of EVTs.

MSX2 safeguards syncytiotrophoblast fate of human trophoblast stem cells.

Multiple placental pathologies are associated with failures in trophoblast differentiation, yet the underlying transcriptional regulation is poorly understood. Here, we discovered msh homeobox 2 (MSX2) as a key transcriptional regulator of trophoblast identity using the human trophoblast stem cell model. Depletion of MSX2 resulted in activation of the syncytiotrophoblast transcriptional program, while forced expression of MSX2 blocked it. We demonstrated that a large proportion of the affected genes were directly bound and regulated by MSX2 and identified components of the SWItch/Sucrose nonfermentable (SWI/SNF) complex as strong MSX2 interactors and target gene cobinders. MSX2 cooperated specifically with the SWI/SNF canonical BAF (cBAF) subcomplex and cooccupied, together with H3K27ac, a number of differentiation genes. Increased H3K27ac and cBAF occupancy upon MSX2 depletion imply that MSX2 prevents premature syncytiotrophoblast differentiation. Our findings established MSX2 as a repressor of the syncytiotrophoblast lineage and demonstrated its pivotal role in cell fate decisions that govern human placental development and disease.

WNT and NOTCH signaling in human trophoblast development and differentiation.

Correct development of the human placenta and its differentiated epithelial cells, syncytial trophoblasts (STBs) and extravillous trophoblasts (EVTs), is crucial for a successful pregnancy outcome. STBs develop by cell fusion of mononuclear cytotrophoblasts (CTBs) in placental floating villi, whereas migratory EVTs originate from specialized villi anchoring to the maternal decidua. Defects in trophoblast differentiation have been associated with severe pregnancy disorders such as early-onset preeclampsia and fetal growth restriction. However, the evolutionary pathways underlying normal and adverse placentation are poorly understood. Herein, we discuss Wingless (WNT) and NOTCH signaling, two pathways that play pivotal roles in human placenta and trophoblast development. Whereas WNT is necessary for expansion of trophoblast progenitors and stem cells, NOTCH1 is required for proliferation and survival of EVT precursors. Differentiation of the latter is orchestrated by a switch in NOTCH receptor expression as well as by changes in WNT ligands and their downstream effectors.

Pivotal role of the transcriptional co-activator YAP in trophoblast stemness of the developing human placenta.

Various pregnancy complications, such as severe forms of preeclampsia or intrauterine growth restriction, are thought to arise from failures in the differentiation of human placental trophoblasts. Progenitors of the latter either develop into invasive extravillous trophoblasts, remodeling the uterine vasculature, or fuse into multinuclear syncytiotrophoblasts transporting oxygen and nutrients to the growing fetus. However, key regulatory factors controlling trophoblast self-renewal and differentiation have been poorly elucidated. Using primary cells, three-dimensional organoids, and CRISPR-Cas9 genome-edited JEG-3 clones, we herein show that YAP, the transcriptional coactivator of the Hippo signaling pathway, promotes maintenance of cytotrophoblast progenitors by different genomic mechanisms. Genetic or chemical manipulation of YAP in these cellular models revealed that it stimulates proliferation and expression of cell cycle regulators and stemness-associated genes, but inhibits cell fusion and production of syncytiotrophoblast (STB)-specific proteins, such as hCG and GDF15. Genome-wide comparisons of primary villous cytotrophoblasts overexpressing constitutively active YAP-5SA with YAP KO cells and syncytializing trophoblasts revealed common target genes involved in trophoblast stemness and differentiation. ChIP-qPCR unraveled that YAP-5SA overexpression increased binding of YAP-TEAD4 complexes to promoters of proliferation-associated genes such as CCNA and CDK6 Moreover, repressive YAP-TEAD4 complexes containing the histone methyltransferase EZH2 were detected in the genomic regions of the STB-specific CGB5 and CGB7 genes. In summary, YAP plays a pivotal role in the maintenance of the human placental trophoblast epithelium. Besides activating stemness factors, it also directly represses genes promoting trophoblast cell fusion.

PRG2 and AQPEP are misexpressed in fetal membranes in placenta previa and percreta†.

The obstetrical conditions placenta accreta spectrum (PAS) and placenta previa are a significant source of pregnancy-associated morbidity and mortality, yet the specific molecular and cellular underpinnings of these conditions are not known. In this study, we identified misregulated gene expression patterns in tissues from placenta previa and percreta (the most extreme form of PAS) compared with control cases. By comparing this gene set with existing placental single-cell and bulk RNA-Seq datasets, we show that the upregulated genes predominantly mark extravillous trophoblasts. We performed immunofluorescence on several candidate molecules and found that PRG2 and AQPEP protein levels are upregulated in both the fetal membranes and the placental disk in both conditions. While this increased AQPEP expression remains restricted to trophoblasts, PRG2 is mislocalized and is found throughout the fetal membranes. Using a larger patient cohort with a diverse set of gestationally aged-matched controls, we validated PRG2 as a marker for both previa and PAS and AQPEP as a marker for only previa in the fetal membranes. Our findings suggest that the extraembryonic tissues surrounding the conceptus, including both the fetal membranes and the placental disk, harbor a signature of previa and PAS that is characteristic of EVTs and that may reflect increased trophoblast invasiveness.

Genome amplification and cellular senescence are hallmarks of human placenta development.

Genome amplification and cellular senescence are commonly associated with pathological processes. While physiological roles for polyploidization and senescence have been described in mouse development, controversy exists over their significance in humans. Here, we describe tetraploidization and senescence as phenomena of normal human placenta development. During pregnancy, placental extravillous trophoblasts (EVTs) invade the pregnant endometrium, termed decidua, to establish an adapted microenvironment required for the developing embryo. This process is critically dependent on continuous cell proliferation and differentiation, which is thought to follow the classical model of cell cycle arrest prior to terminal differentiation. Strikingly, flow cytometry and DNAseq revealed that EVT formation is accompanied with a genome-wide polyploidization, independent of mitotic cycles. DNA replication in these cells was analysed by a fluorescent cell-cycle indicator reporter system, cell cycle marker expression and EdU incorporation. Upon invasion into the decidua, EVTs widely lose their replicative potential and enter a senescent state characterized by high senescence-associated (SA) ß-galactosidase activity, induction of a SA secretory phenotype as well as typical metabolic alterations. Furthermore, we show that the shift from endocycle-dependent genome amplification to growth arrest is disturbed in androgenic complete hydatidiform moles (CHM), a hyperplastic pregnancy disorder associated with increased risk of developing choriocarinoma. Senescence is decreased in CHM-EVTs, accompanied by exacerbated endoreduplication and hyperploidy. We propose induction of cellular senescence as a ploidy-limiting mechanism during normal human placentation and unravel a link between excessive polyploidization and reduced senescence in CHM.

Barusiban, a selective oxytocin receptor antagonist: placental transfer in rabbit, monkey, and human†.

The use of drugs in pregnancy always raises concerns regarding potential fetal exposure and possible adverse effects through their accumulation in fetal tissues and organs. Barusiban is an oxytocin antagonist under development for potential use as tocolytic in preterm-labor patients. It displays greater affinity for the oxytocin receptor compared to vasopressin V1A receptor and would thus not interfere with vasopressin-induced effects of the V1A receptor. Barusiban placental transfer was determined in the rabbit and cynomolgus monkey and in an ex vivo human cotyledon model. In the rabbit, there was an approximately 5% transfer of barusiban from the maternal to the fetal blood, without significant accumulation in any of the investigated fetal tissues. In the cynomolgus monkeys, the mean fetal plasma barusiban concentration was 9.1% of the maternal level. This was similar to the percentage of barusiban transfer in the human placental single cotyledon, which once equilibrated ranged between 9.3 and 11.0% over the observation period. The transfer of the small-molecule antipyrine as a comparator in this human model was approximately three times greater. The similarity in the degree of transfer in the cynomolgus monkey and human cotyledon, while being less in the rabbit, may reflect the species-specific placental barrier structure between the maternal and fetal compartments. In conclusion, limited placental transfer of barusiban occurred in all three models. The similarity of barusiban transfer in the cynomolgus and the human placental single cotyledon suggests the latter ex vivo model to be useful in assessing future drug candidates to be used in pregnant women.

Densities of decidual high endothelial venules correlate with T-cell influx in healthy pregnancies and idiopathic recurrent pregnancy losses.

STUDY QUESTION: Do high endothelial venules (HEVs) appear in the uterus of healthy and pathological pregnancies? SUMMARY ANSWER: Our study reveals that HEVs are present in the non-pregnant endometrium and decidua parietalis (decP) but decline upon placentation in decidua basalis (decB) and are less abundant in decidual tissues from idiopathic, recurrent pregnancy losses (RPLs). WHAT IS KNOWN ALREADY: RPL is associated with a compromised decidual vascular phenotype. STUDY DESIGN, SIZE, DURATION: Endometrial (n = 29) and first trimester decidual (n = 86, 6-12th week of gestation) tissue samples obtained from endometrial biopsies or elective pregnancy terminations were used to determine the number of HEVs and T cells. In addition, quantification of HEVs and immune cells was performed in a cohort of decidual tissues from RPL (n = 25). PARTICIPANTS/MATERIALS, SETTING, METHODS: Position and frequency of HEVs were determined in non-pregnant endometrial as well as decidual tissue sections using immunofluorescence (IF) staining with antibodies against E-selectin, intercellular adhesion molecule, von Willebrand factor, ephrin receptor B4, CD34 and a carbohydrate epitope specific to HEVs (MECA-79). Immune cell distribution and characterization was determined by antibodies recognizing CD45 and CD3 by IF staining- and flow cytometry-based analyses. Antibodies against c-c motif chemokine ligand 21 (CCL21) and lymphotoxin-beta were used in IF staining and Western blot analyses of decidual tissues. MAIN RESULTS AND THE ROLE OF CHANCE: Functional HEVs are found in high numbers in the secretory endometrium and decP but decline in numbers upon placentation in decB (P ≤ 0.001). Decidua parietalis tissues contain higher levels of the HEV-maintaining factor lymphotoxin beta and decP-associated HEVs also express CCL21 (P ≤ 0.05), a potent T-cell chemoattractant. Moreover, there is a positive correlation between the numbers of decidual HEVs and the abundance of CD3+ cells in decidual tissue sections (P ≤ 0.001). In-depth analysis of a RPL tissue collection revealed a decreased decB (P ≤ 0.01) and decP (P ≤ 0.01) HEV density as well as reduced numbers of T cells in decB (P ≤ 0.05) and decP (P ≤ .001) sections when compared with age-matched healthy control samples. Using receiver-operating characteristics analyses, we found significant predictive values for the ratios of CD3/CD45 (P < 0.001) and HEVs/total vessels (P < 0.001) for the occurrence of RPL. LIMITATIONS, REASONS FOR CAUTION: Analyses were performed in first trimester decidual tissues from elective terminations of pregnancy or non-pregnant endometrium samples from patients diagnosed with non-endometrial pathologies including cervical polyps, ovarian cysts and myomas. First trimester decidual tissues may include pregnancies which potentially would have developed placental disorders later in gestation. In addition, our cohort of non-pregnant endometrium may not reflect the endometrial vascular phenotype of healthy women. Finally, determination of immune cell distributions in the patient cohorts studied may be influenced by the different modes of tissue derivation. Pregnancy terminations were performed by surgical aspiration, endometrial tissues were obtained by biopsies and RPL tissues were collected after spontaneous loss of pregnancy. WIDER IMPLICATIONS OF THE FINDINGS: In this study, we propose an inherent mechanism by which the endometrium and in particular the decidua control T-cell recruitment. By demonstrating reduced HEV densities and numbers of T cells in decB and decP tissues of RPL samples we further support previous findings reporting an altered vascular phenotype in early pregnancy loss. Altogether, the findings provide important information to further decipher the etiologies of unexplained RPL. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Austrian Science Fund (P31470 B30 to M.K.) and by the Austrian National Bank (17613ONB to J.P.). There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Ropivacaine Activates Multiple Proapoptotic and Inflammatory Signaling Pathways That Might Subsume to Trigger Epidural-Related Maternal Fever.

BACKGROUND: Epidural-related maternal fever (ERMF) is an adverse effect of epidural analgesia during labor and is associated with perinatal and neonatal morbidity. Local anesthetics have been proposed to trigger ERMF via sterile inflammation. Ropivacaine is currently the most frequently used epidural anesthetic and considered least toxic. This study investigates molecular effects of ropivacaine on human umbilical vein endothelial cells (HUVECs) as model system for endothelial cells and human placental trophoblasts (TBs), compares the effects to the putative anti-inflammatory lidocaine and investigates the partially alleviating impact of the anti-inflammatory corticosteroid dexamethasone. METHODS: HUVECs and TBs were exposed to ropivacaine (35 µM-7 mM) or lidocaine (21 mM) with or without dexamethasone (1 µM). AnnexinV/propidium iodide staining and lactate dehydrogenase release were used to analyze apoptosis and cytotoxicity. Proinflammatory interleukins-6 (IL-6) and IL-8 as well as prostaglandin E2 (PGE2) were measured by enzyme-linked immunosorbent assay (ELISA), while activation of signaling pathways was detected by Western blotting. Oxidative stress was visualized by live cell imaging and quantification of antioxidant proteins, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, platelet endothelial cell adhesion molecule 1, cyclooxygenase 2, and mitochondrial deoxyribonucleic acid by real-time polymerase chain reaction. Dissipation of the mitochondrial membrane potential was assessed with cytofluorimetric analysis using the J-Aggregate (JC-1 staining [cytofluorimetric analysis using the J-Aggregate]). RESULTS: Ropivacaine exposure dose-dependently induced apoptosis and an increased release of IL-6, IL-8, and PGE2 from HUVECs and TBs. Furthermore, caspase-3, nuclear factor-κB, and p38 mitogen-activated protein kinase pathways were activated, while extracellular signal-regulated kinase 1/2 and protein kinase B (Akt) were dephosphorylated. Downregulation of antioxidative proteins induced oxidative stress and upregulation of ICAM1, VCAM1, and PECAM1 possibly facilitate leukocyte transmigration. Mitochondrial effects included increased release of the proinflammatory mitochondrial DNA damage-associated molecular patterns, but no significant dissipation of the mitochondrial membrane potential. Conversely, lidocaine exhibited repression of IL-6 and IL-8 release over all time points, and early downregulation of COX2 and cell adhesion molecules, which was followed by a late overshooting reaction. Dexamethasone reduced especially inflammatory effects, but as an inducer of mitophagy, had negative long-term effects on mitochondrial function. CONCLUSIONS: This study suggests that ropivacaine causes cellular injury and death in HUVECs and TBs via different signaling pathways. The detrimental effects induced by ropivacaine are only partially blunted by dexamethasone. This observation strengthens the importance of inflammation in ERMF.

Human placenta and trophoblast development: key molecular mechanisms and model systems.

Abnormal placentation is considered as an underlying cause of various pregnancy complications such as miscarriage, preeclampsia and intrauterine growth restriction, the latter increasing the risk for the development of severe disorders in later life such as cardiovascular disease and type 2 diabetes. Despite their importance, the molecular mechanisms governing human placental formation and trophoblast cell lineage specification and differentiation have been poorly unravelled, mostly due to the lack of appropriate cellular model systems. However, over the past few years major progress has been made by establishing self-renewing human trophoblast stem cells and 3-dimensional organoids from human blastocysts and early placental tissues opening the path for detailed molecular investigations. Herein, we summarize the present knowledge about human placental development, its stem cells, progenitors and differentiated cell types in the trophoblast epithelium and the villous core. Anatomy of the early placenta, current model systems, and critical key regulatory factors and signalling cascades governing placentation will be elucidated. In this context, we will discuss the role of the developmental pathways Wingless and Notch, controlling trophoblast stemness/differentiation and formation of invasive trophoblast progenitors, respectively.

Placenta-Specific Genes, Their Regulation During Villous Trophoblast Differentiation and Dysregulation in Preterm Preeclampsia.

The human placenta maintains pregnancy and supports the developing fetus by providing nutrition, gas-waste exchange, hormonal regulation, and an immunological barrier from the maternal immune system. The villous syncytiotrophoblast carries most of these functions and provides the interface between the maternal and fetal circulatory systems. The syncytiotrophoblast is generated by the biochemical and morphological differentiation of underlying cytotrophoblast progenitor cells. The dysfunction of the villous trophoblast development is implicated in placenta-mediated pregnancy complications. Herein, we describe gene modules and clusters involved in the dynamic differentiation of villous cytotrophoblasts into the syncytiotrophoblast. During this process, the immune defense functions are first established, followed by structural and metabolic changes, and then by peptide hormone synthesis. We describe key transcription regulatory molecules that regulate gene modules involved in placental functions. Based on transcriptomic evidence, we infer how villous trophoblast differentiation and functions are dysregulated in preterm preeclampsia, a life-threatening placenta-mediated obstetrical syndrome for the mother and fetus. In the conclusion, we uncover the blueprint for villous trophoblast development and its impairment in preterm preeclampsia, which may aid in the future development of non-invasive biomarkers for placental functions and early identification of women at risk for preterm preeclampsia as well as other placenta-mediated pregnancy complications.

Metabolism of cholesterol and progesterone is differentially regulated in primary trophoblastic subtypes and might be disturbed in recurrent miscarriages.

During pregnancy, extravillous trophoblasts (EVTs) invade the maternal decidua and remodel the local vasculature to establish blood supply for the growing fetus. Compromised EVT function has been linked to aberrant pregnancy associated with maternal and fetal morbidity and mortality. However, metabolic features of this invasive trophoblast subtype are largely unknown. Using primary human trophoblasts isolated from first trimester placental tissues, we show that cellular cholesterol homeostasis is differentially regulated in EVTs compared with villous cytotrophoblasts. Utilizing RNA-sequencing, gene set-enrichment analysis, and functional validation, we provide evidence that EVTs display increased levels of free and esterified cholesterol. Accordingly, EVTs are characterized by increased expression of the HDL-receptor, scavenger receptor class B type I, and reduced expression of the LXR and its target genes. We further reveal that EVTs express elevated levels of hydroxy-delta-5-steroid dehydrogenase 3 beta- and steroid delta-isomerase 1 (HSD3B1) (a rate-limiting enzyme in progesterone synthesis) and are capable of secreting progesterone. Increasing cholesterol export by LXR activation reduced progesterone secretion in an ABCA1-dependent manner. Importantly, HSD3B1 expression was decreased in EVTs of idiopathic recurrent spontaneous abortions, pointing toward compromised progesterone metabolism in EVTs of early miscarriages. Here, we provide insights into the regulation of cholesterol and progesterone metabolism in trophoblastic subtypes and its putative relevance in human miscarriage.

Notch1 controls development of the extravillous trophoblast lineage in the human placenta.

Development of the human placenta and its different epithelial trophoblasts is crucial for a successful pregnancy. Besides fusing into a multinuclear syncytium, the exchange surface between mother and fetus, progenitors develop into extravillous trophoblasts invading the maternal uterus and its spiral arteries. Migration into these vessels promotes remodelling and, as a consequence, adaption of blood flow to the fetal-placental unit. Defects in remodelling and trophoblast differentiation are associated with severe gestational diseases, such as preeclampsia. However, mechanisms controlling human trophoblast development are largely unknown. Herein, we show that Notch1 is one such critical regulator, programming primary trophoblasts into progenitors of the invasive differentiation pathway. At the 12th wk of gestation, Notch1 is exclusively detected in precursors of the extravillous trophoblast lineage, forming cell columns anchored to the uterine stroma. At the 6th wk, Notch1 is additionally expressed in clusters of villous trophoblasts underlying the syncytium, suggesting that the receptor initiates the invasive differentiation program in distal regions of the developing placental epithelium. Manipulation of Notch1 in primary trophoblast models demonstrated that the receptor promotes proliferation and survival of extravillous trophoblast progenitors. Notch1 intracellular domain induced genes associated with stemness of cell columns, myc and VE-cadherin, in Notch1- fusogenic precursors, and bound to the myc promoter and enhancer region at RBPJκ cognate sequences. In contrast, Notch1 repressed syncytialization and expression of TEAD4 and p63, two regulators controlling self-renewal of villous cytotrophoblasts. Our results revealed Notch1 as a key factor promoting development of progenitors of the extravillous trophoblast lineage in the human placenta.

Neuregulin-1-mediated ErbB2-ErbB3 signalling protects human trophoblasts against apoptosis to preserve differentiation.

During placentation, foetal trophoblasts invade deeply into maternal tissue to establish a foeto-maternal circulation. We have previously shown that extravillous trophoblast (EVT) lineage cells express ErbB2 and ErbB3, of which the potential as an oncogenic unit is well established. However, a physiological function of this receptor combination in humans remains a puzzling question. Here, we demonstrate neuregulin 1 (NRG1) expression and secretion by human decidual stromal cells. Stimulation of human primary trophoblasts with exogenous NRG1 induced phosphorylation of ErbB2, ErbB3 and related downstream effectors. Co-immunoprecipitation experiments confirmed the formation of ErbB2-ErbB3 dimers upon ligand engagement. Along this line, receptor knockdown and ErbB3 neutralization strongly diminished NRG1-dependent activation of the signalling complex. Functional studies revealed that NRG1 promotes EVT formation in placental explant cultures. Although, in the presence of NRG1, basal and camptothecin-induced trophoblast apoptosis was significantly repressed, this effect was abolished upon ErbB3 inhibition. Notably, camptothecin provoked a strong reduction of trophoblast cell column size, whereas NRG1-treated explants were refractory to the compound. Taken together, our findings newly identify a physiological function of the NRG1-ErbB2-ErbB3 axis in trophoblast survival during human placental development.

Extravillous trophoblast invasion of venous as well as lymphatic vessels is altered in idiopathic, recurrent, spontaneous abortions.

STUDY QUESTION: Do extravillous trophoblasts (EVTs) invade non-arterial decidual vessels in healthy and pathological pregnancies? SUMMARY ANSWER: Our results reveal that trophoblast invasion of venous and lymphatic vessels is a frequent event during the first trimester of pregnancy and is compromised in  recurrent spontaneous abortion (RSA). In addition, the present data suggest that EVTs populate regional lymph nodes during pregnancy. WHAT IS ALREADY KNOWN: Human trophoblasts remodel and invade decidual spiral arteries. In addition, a recent report demonstrates that trophoblasts contact and invade decidual veins. STUDY DESIGN, SIZE, DURATION: Tissue samples of human first trimester deciduae basalis (n = 54, 6th-13th weeks of gestation) obtained from elective pregnancy terminations were used to study trophoblast invasion into veins and lymphatics, in comparison to arteries. Age-matched cases of idiopathic, recurrent spontaneous abortions tissue samples (n = 23) were assessed for cell numbers of EVTs in these decidual vessels. In addition, lymph nodes of four pregnant women were analysed for the presence of EVTs. PARTICIPANTS/MATERIALS, SETTING, METHODS: Localization, frequency and EVT-mediated targeting and invasion of arterial, venous as well as lymphatic vessels were determined in first trimester decidua basalis tissue sections using immunofluorescence staining with antibodies against CD31, CD34, ephrin B2 (EFNB2), ephrin receptor B4 (EPHB4), HLA-G, podoplanin, prospero-related homeobox 1 (Prox-1), alpha-smooth muscle actin 2 (ATCTA2), von willebrand factor (vWF) and proteoglycan 2 (PRG2). Arterial, venous and lymphatic-associated EVTs were further characterized according to their position in the vascular structure and classified as intramural (im) or intraluminal (il). MAIN RESULTS AND THE ROLE OF CHANCE: EVTs, specifically expressing PRG2, target and invade veins and lymphatics in first trimester decidua basalis since HLA-G+ trophoblast were detected in the vascular wall (intramural EVT, imEVTs) and in the lumen of these vessels (intraluminal EVT, ilEVTs). In total, 276 arteries, 793 veins and 113 lymphatics were analysed. While EVTs contact and invade arteries and veins to a similar extent we found that lymphatics are significantly less affected by EVTs (P = 0.001). Moreover, ilEVTs were detected in the lumen of venous and lymphatic vessels, whereas ilEVTs were only found occasionally in the lumen of arteries. Interestingly, RSA tissue sections contained significantly more arterial (P = 0.037), venous (P = 0.002) and lymphatic vessels (P < 0.001), compared to healthy controls. However, while RSA-associated arterial remodeling was unchanged (P = 0.39) the ratios of EVT-affected versus total number of veins (P = 0.039) and lymphatics (P < 0.001) were significantly lower in RSA compared to age-matched healthy decidual sections. Finally, HLA-G+/PRG2+/CD45-EVTs can be detected in regional lymph nodes of pregnant women diagnosed with cervical cancer. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, first trimester decidual tissues from elective terminations of pregnancies have been examined and used as a reference for healthy pregnancy. However, this collective may also include pregnancies which would have developed placental disorders later in gestation. Due to limitations in tissue availability our staining results for EVT-specific marker expression in regional lymph nodes of pregnant women are based on four cases only. WIDER IMPLICATIONS OF THE FINDINGS: In this study, we propose migration of HLA-G+ cells into regional lymph nodes during pregnancy suggesting that the human EVT is capable of infiltrating maternal tissues via the blood stream. Moreover, the description of compromised EVT invasion into the venous and lymphatic vasculature in RSA may help to better understand the pathological characteristics of idiopathic recurrent pregnancy loss. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Austrian Science Fund (grant P-25187-B13 to J.P. and grant P-28417-B30 to M.K.). There are no competing interests to declare.

Endothelin-1 down-regulates matrix metalloproteinase 14 and 15 expression in human first trimester trophoblasts via endothelin receptor type B.

STUDY QUESTION: Does endothelin-1 (ET-1) regulate matrix metalloproteinase (MMP) 14 and 15 production and invasion of human first trimester trophoblasts? SUMMARY ANSWER: ET-1 in pathophysiological concentrations down-regulates MMP14 and MMP15 expression via endothelin receptor (ETR) type B and decreases trophoblast migration and invasion. WHAT IS KNOWN ALREADY: MMP14 and MMP15 are involved in trophoblast invasion. Impairment of invasion has been linked to pregnancy complications such as pre-eclampsia (PE). ET-1 is up-regulated in PE. STUDY DESIGN, SIZE, DURATION: In vitro study using primary human trophoblasts from 50 first trimester placentas (gestational week 7-12). PARTICIPANTS/MATERIALS, SETTING, METHODS: Trophoblasts were cultured in the absence or presence of 10-100 nM ET-1. MMP14 and MMP15 mRNA and protein were quantified by RT-qPCR and Western blotting, respectively. Selective antagonists for ETRA (BQ-123) or ETRB (BQ-788) were used to identify ETR subtypes involved. Functional ET-1 effects were tested in first trimester chorionic villous explants and transwell invasion assays. The roles of tumor necrosis factor (TNF)-α (25 ng/ml) and oxygen (1%) in ET-1 regulation of MMP14 and 15 expression were assessed by Western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: ET-1 down-regulated MMP14 and MMP15 mRNA (-21% and -26%, respectively, P < 0.05) and protein levels (-18% and -22%, respectively, P < 0.05). This effect was mediated via ETRB. ET-1 decreased trophoblast outgrowth in placental explants (-24%, P < 0.05) and trophoblast invasion (-26%, P ≤ 0.01). TNF-α enhanced ET-1 mediated MMP15 down-regulation (by 10%, P < 0.05), whereas hypoxia abolished the effect of ET-1 on both MMPs. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Only primary trophoblasts were used in this study. Since trophoblast yield from first trimester placental material is limited, further aspects of MMP14 and 15 regulation could not be characterized. Other anti-invasive factors may be altered by ET-1 in trophoblasts and, thus, contribute to the reduced invasion, but have not been investigated. Oxygen levels similar to those found in the decidua (5-8% O2) were not analyzed in this study. WIDER IMPLICATIONS OF THE FINDINGS: ET-1 modifies placental function already during the first trimester of pregnancy, the time-window when the placental changes implicated in PE occur. Thus, our results improve the understanding of the placental mechanisms underlying trophoblast invasion and PE. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Oesterreichische Nationalbank (Anniversary Fund, project number: 14796) and the Herzfelder'sche Familienstiftung (to J.P.; number: 00685). AMM received funding from the Austrian Science Fund FWF (W1241) and the Medical University Graz through the PhD Program Molecular Fundamentals of Inflammation (DK-MOLIN). The authors have no conflict of interest.

Autocrine fibroblast growth factor 18 signaling mediates Wnt-dependent stimulation of CD44-positive human colorectal adenoma cells.

Expansion of a stem-like subpopulation with increased growth and survival potential is thought to drive colorectal tumor growth and progression. We investigated a CD44-positive (CD44((+))) subpopulation with extended growth and survival capacity in the human colon adenoma cell line LT97. This subpopulation expressed elevated levels of fibroblast growth factor 18 (FGF18) and fibroblast growth factor receptor FGFR3-IIIc. Expression levels of the FGFR3-IIIb, which does not bind FGF18, were similar in CD44((+)) and CD44((-)). Addition of FGF18 to the medium or its overexpression from an adenoviral vector increased the colony formation capacity of CD44((+)) threefold, and stimulated phosphorylation of ERK and GSK3ß in both total LT97 populations and CD44((+)) cells. FGFR3 signaling blockade by expression of a dominant-negative FGFR3-IIIc mutant led to inhibition of both colony formation and down-stream signaling in the CD44((+)) cells. CD44((-)) cells did not respond. Blockade of the wnt-pathway by a dominant-negative Tcf4-mutant inhibited FGFR3 activation in LT97 cells as well as in HT29 colorectal cancer cells. The chemical wnt-inhibitor sulindac sulfide amide inhibited expression of FGF18 and FGFR3-IIIc and led to inhibition of receptor activation to less than 30% of control treated cells, both in LT97 and HT29 cultures. Our results demonstrate that an FGF18/FGFR3-IIIc autocrine growth and survival loop is up-regulated in a wnt-dependent manner and drives tumor cell growth in a subpopulation of colon adenoma cells. This subpopulation can be regarded as a precursor of colon cancer development and can be targeted for CRC-prevention by blocking either wnt- or FGFR3-signaling.

Trophoblast subtype-specific EGFR/ERBB4 expression correlates with cell cycle progression and hyperplasia in complete hydatidiform moles.

STUDY QUESTION: Do trophoblast subtypes differ in their expression of erythroblastic leukaemia viral oncogene homologue (ERBB) receptor family members and responsiveness towards specific growth factor ligands? SUMMARY ANSWER: Our data reveal a reciprocal expression pattern of epidermal growth factor receptor (EGFR)/ERBB4 in proliferative and ERBB2/ERBB3 in invasive trophoblast subtypes, as well as a restricted responsiveness to epidermal growth factor (EGF) and heparin-binding (HB)-EGF. WHAT IS KNOWN ALREADY: EGFR is expressed by villous cytotrophoblasts (vCTBs), but absent from extravillous trophoblasts (EVTs), which specifically up-regulate ERBB2. STUDY DESIGN, SIZE, DURATION: Tissue samples of human first trimester placentae (n = 50) and deciduae (n = 5) obtained from elective pregnancy terminations were used to study trophoblast subtype-specific ERBB receptor expression and responsiveness to recombinant human EGF and HB-EGF. Age-matched complete hydatidiform mole (CHM) placentae (n = 12) were assessed for EGFR and ERBB4 expression in proliferation-competent regions. PARTICIPANTS/MATERIALS, SETTING, METHODS: ERBB receptor expression was analysed in primary trophoblast cell isolates by means of microarray, quantitative real-time PCR and western blotting, as well as immunofluorescence stainings of placental and decidual tissue sections. EGF and HB-EGF were tested for their potential to activate ERBB receptors in purified EGFR(+) and HLA-G(+) trophoblasts. 5-Ethynyl-2'-deoxyuridine incorporation assays were performed to study the effect of both ligands on the proliferative capacity of primary trophoblasts as well as of vCTBs and proximal cell column trophoblasts (pCCTs) in placental floating explants. Finally, the average number of EGFR(+) vCTB and pCCT layers was determined in CHM placentae and compared with healthy age-matched controls. MAIN RESULTS AND THE ROLE OF CHANCE: Proliferative vCTBs and pCCTs co-express EGFR and ERBB4, but are devoid of ERBB2 and ERBB3. In contrast, HLA-G(+) trophoblast subtypes exhibit an EGFR/ERBB4(-) and ERBB2/ERBB3(+) phenotype. EGF and HB-EGF activate EGFR, ERBB4, AKT and extracellular signal-regulated kinase 1/2 in EGFR(+) primary trophoblasts; however, they do not show an effect on HLA-G(+) EVTs. Both ligands strongly induce cell cycle progression in primary trophoblasts (P < 0.05) and placental explant-associated vCTBs (P < 0.05) and pCCTs (P < 0.05). Notably, EGFR(+) vCTB (P < 0.0001) and pCCT (P < 0.0001) layers are significantly expanded in CHM placentae when compared with healthy controls. LIMITATIONS, REASONS FOR CAUTION: Cells were removed from their physiological context and may therefore respond differently to various stimuli. WIDER IMPLICATIONS OF THE FINDINGS: In this study we define EGFR as a marker for proliferative trophoblast subtypes within the human placenta. Manipulation of EGFR signalling might thus offer a promising therapeutic avenue for the treatment of molar pregnancies associated with trophoblast hyperplasia. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the Austrian Science Fund (grant P-25187-B13 to J.P.). There are no competing interests to declare.