Antiphospholipid syndrome (APS) is associated with recurrent pregnancy morbidity, yet the underlying mechanisms remain elusive. We performed multifaceted characterization of the biological and transcriptomic signatures of mouse placenta and uterine natural killer (uNK) cells in APS. Histological analysis of APS placentas unveiled placental abnormalities, including disturbed angiogenesis, occasional necrotic areas, fibrin deposition, and nucleated red blood cell enrichment. Analyses of APS placentas showed a reduced cell proliferation, lower protein content and thinning of endothelial cells. Disturbances in APS trophoblast cells were linked to a cell cycle shift in cytotrophoblast cells, and a reduced number of spiral artery-associated trophoblast giant cells (SpA-TGC). Transcriptomic profiling of placental tissue highlighted disruptions in cell cycle regulation with notable downregulation of genes involved in developmental or signaling processes. Cellular senescence, metabolic and p53-related pathways were also enriched, suggesting potential mechanisms underlying placental dysfunction in APS. Thrombotic events, though occasionally detected, appeared to have no significant impact on the overall pathological changes. The increased number of dysfunctional uNK cells was not associated with enhanced cytotoxic capabilities. Transcriptomic data corroborated these findings, showing prominent suppression of NK cell secretory capacity and cytokine signaling pathways. Our study highlights the multifactorial nature of APS-associated placental pathologies, which involve disrupted angiogenesis, cell cycle regulation, and NK cell functionality.
- MeSH
- antifosfolipidový syndrom * imunologie patologie MeSH
- buňky NK * imunologie metabolismus MeSH
- modely nemocí na zvířatech * MeSH
- myši MeSH
- placenta * metabolismus patologie MeSH
- proliferace buněk MeSH
- stanovení celkové genové exprese MeSH
- těhotenství MeSH
- transkriptom MeSH
- trofoblasty metabolismus patologie imunologie MeSH
- uterus * patologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Syncytin-1, a human fusogenic protein of retroviral origin, is crucial for placental syncytiotrophoblast formation. To mediate cell-to-cell fusion, Syncytin-1 requires specific interaction with its cognate receptor. Two trimeric transmembrane proteins, Alanine, Serine, Cysteine Transporters 1 and 2 (ASCT1 and ASCT2), were suggested and widely accepted as Syncytin-1 cellular receptors. To quantitatively assess the individual contributions of human ASCT1 and ASCT2 to the fusogenic activity of Syncytin-1, we developed a model system where the ASCT1 and ASCT2 double knockout was rescued by ectopic expression of either ASCT1 or ASCT2. We demonstrated that ASCT2 was required for Syncytin-1 binding, cellular entry, and cell-to-cell fusion, while ASCT1 was not involved in this receptor interaction. We experimentally validated the ASCT1-ASCT2 heterotrimers as a possible explanation for the previous misidentification of ASCT1 as a receptor for Syncytin-1. This redefinition of receptor specificity is important for proper understanding of Syncytin-1 function in normal and pathological pregnancy.
- MeSH
- antigeny CD98 - těžký řetězec MeSH
- fúze buněk * MeSH
- genové produkty env * metabolismus genetika MeSH
- lidé MeSH
- placenta * metabolismus MeSH
- těhotenské proteiny * metabolismus genetika MeSH
- těhotenství MeSH
- transportní systém ASC pro aminokyseliny * metabolismus genetika MeSH
- transportní systémy pro neutrální aminokyseliny metabolismus genetika MeSH
- trofoblasty metabolismus cytologie MeSH
- vedlejší histokompatibilní antigeny metabolismus genetika MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Proper fetal development requires tight regulation of serotonin concentrations within the fetoplacental unit. This homeostasis is partly maintained by the placental transporter OCT3/SLC22A3, which takes up serotonin from the fetal circulation. Metformin, an antidiabetic drug commonly used to treat gestational diabetes mellitus, was shown to inhibit OCT3. We, therefore, hypothesized that its use during pregnancy could disrupt placental serotonin homeostasis. This hypothesis was tested using three experimental model systems: primary trophoblast cells isolated from the human term placenta, fresh villous human term placenta fragments, and rat term placenta perfusions. Inhibition of serotonin transport by metformin at three concentrations (1 μM, 10 μM, and 100 μM) was assessed in all three models. The OCT3 inhibitor decynium-22 (100 μM) and paroxetine (100 μM), a dual inhibitor of SERT and OCT3, were used as controls. In primary trophoblasts, paroxetine exhibited the strongest inhibition of serotonin uptake, followed by decynium-22. Metformin showed a concentration-dependent effect, reducing serotonin uptake by up to 57 % at the highest concentration. Its inhibitory effect was less pronounced in fresh villous fragments but remained statistically significant at all concentrations. In the perfused rat placenta, metformin demonstrated a concentration-dependent effect, reducing placental serotonin uptake by 44 % at the highest concentration tested. Our findings across all experimental models show inhibition of placental OCT3 by metformin, resulting in reduced serotonin uptake by the trophoblast. This sheds light on mechanisms that may underpin metformin-mediated effects on fetal development.
- MeSH
- biologický transport účinky léků MeSH
- hypoglykemika farmakologie MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- lidé MeSH
- metformin * farmakologie MeSH
- oktamerní transkripční faktor 3 metabolismus MeSH
- placenta * metabolismus účinky léků MeSH
- potkani Wistar MeSH
- proteiny přenášející organické kationty MeSH
- serotonin * metabolismus MeSH
- těhotenství MeSH
- trofoblasty * metabolismus účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The placenta plays a critical role in maternal-fetal nutrient transport and fetal protection against drugs. Creating physiological in vitro models to study these processes is crucial, but technically challenging. This study introduces an efficient cell model that mimics the human placental barrier using co-cultures of primary trophoblasts and primary human umbilical vein endothelial cells (HUVEC) on a Transwell®-based system. Monolayer formation was examined over 7 days by determining transepithelial electrical resistance (TEER), permeability of Lucifer yellow (LY) and inulin, localization of transport proteins at the trophoblast membrane (immunofluorescence), and syncytialization markers (RT-qPCR/ELISA). We analysed diffusion-based (caffeine/antipyrine) and transport-based (leucine/Rhodamine-123) processes to study the transfer of physiologically relevant compounds. The latter relies on the adequate localization and function of the amino-acid transporter LAT1 and the drug transporter P-glycoprotein (P-gp) which were studied by immunofluorescence microscopy and application of respective inhibitors (2-Amino-2-norbornanecarboxylic acid (BCH) for LAT1; cyclosporine-A for P-gp). The formation of functional monolayer(s) was confirmed by increasing TEER values, low LY transfer rates, minimal inulin leakage, and appropriate expression/release of syncytialization markers. These results were supported by microscopic monitoring of monolayer formation. LAT1 was identified on the apical and basal sides of the trophoblast monolayer, while P-gp was apically localized. Transport assays confirmed the inhibition of LAT1 by BCH, reducing both intracellular leucine levels and leucine transport to the basal compartment. Inhibiting P-gp with cyclosporine-A increased intracellular Rhodamine-123 concentrations. Our in vitro model mimics key aspects of the human placental barrier. It represents a powerful tool to study nutrient and drug transport mechanisms across the placenta, assisting in evaluating safer pregnancy therapies.
- MeSH
- biologické modely MeSH
- biologický transport MeSH
- endoteliální buňky pupečníkové žíly (lidské) * metabolismus MeSH
- inulin metabolismus MeSH
- isochinoliny MeSH
- kokultivační techniky MeSH
- leucin metabolismus MeSH
- lidé MeSH
- maternofetální výměna látek * MeSH
- P-glykoprotein metabolismus MeSH
- placenta * metabolismus MeSH
- rhodamin 123 metabolismus MeSH
- těhotenství MeSH
- trofoblasty * metabolismus MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The development of preeclampsia (PE) is associated with the impaired trophoblast motility. MicroRNAs (miRs) contribute to the modulation of trophoblast invasion. In the current study, the role of miR-206/AGTR1 in the TNF-alpha-induced invasion defect of trophoblasts was explored. The levels of miR-206 and ATGR1 in clinical placenta tissues were investigated. Trophoblasts were treated with TNF-alpha, and the levels of miR-206 and ATGR1 were modulated. Changes in cell viability, invasion, and inflammation in trophoblasts were detected. The level of miR-206 was induced, while the level of AGTR1 was suppressed in placenta tissues. In in vitro assays, TNF-alpha suppressed viability, induced inflammatory response, inhibited invasion, upregulated miR-206, and down-regulated AGTR1. The inhibited expression of miR-206 or the overexpression of AGTR1 counteracted the effects of TNF-alpha, indicating the key role of the miR-206/AGTR1 in progression of PE. Collectively, miR-206 suppressed viability, induced inflammatory response, and decreased invasion of trophoblasts by inhibiting AGTR1.
- MeSH
- lidé MeSH
- mikro RNA * genetika metabolismus MeSH
- placenta metabolismus MeSH
- pohyb buněk MeSH
- preeklampsie * genetika metabolismus MeSH
- proliferace buněk MeSH
- receptor angiotensinu typ 1 genetika metabolismus MeSH
- těhotenství MeSH
- TNF-alfa farmakologie MeSH
- trofoblasty metabolismus MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The placenta represents a non-neuronal organ capable of transporting and metabolizing monoamines. Since these bioactive molecules participate in numerous processes essential for placental and fetal physiology, any imbalance in their levels during pregnancy may affect brain development, projecting a higher risk of behavioral disorders in childhood or adulthood. Notably, the monoamine system in the placenta is a target of various psychoactive drugs and can be disrupted in several pregnancy pathologies. As research in pregnant women poses significant ethical restrictions, animal models are widely employed to study monoamine homeostasis as a mechanism involved in fetal programming. However, detailed knowledge of monoamine transport in the rat placenta is still lacking. Moreover, relatability to the human placental monoamine system is not examined. The present study provides insights into the transplacental monoamine dynamics between maternal and fetal circulation. We show that norepinephrine maternal-to-fetal transport is <4% due to high metabolism within the trophoblast. In contrast, dopamine maternal-to-fetal transport exceeds 25%, likely through passive transport across the membrane. In addition, we show high clearance of norepinephrine and dopamine from the fetal circulation mediated by the organic cation transporter 3 (OCT3). Altogether, we present transcriptional and functional evidence that the in situ rat placenta perfusion represents a suitable model for (patho)physiological investigation of dopamine and norepinephrine homeostasis in the fetoplacental unit. With the rapid advancements in drug discovery and environmental toxicity, the use of rat placenta as a preclinical model could facilitate screening of possible xenobiotic effects on monoamine homeostasis in the placenta.
- MeSH
- biologický transport MeSH
- dopamin * metabolismus farmakologie MeSH
- krysa rodu rattus MeSH
- maternofetální výměna látek MeSH
- noradrenalin metabolismus MeSH
- placenta * metabolismus MeSH
- těhotenství MeSH
- trofoblasty metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Three primary monoamines-serotonin, norepinephrine, and dopamine-play major roles in the placenta-fetal brain axis. Analogously to the brain, the placenta has transport mechanisms that actively take up these monoamines into trophoblast cells. These transporters are known to play important roles in the differentiated syncytiotrophoblast layer, but their status and activities in the undifferentiated, progenitor cytotrophoblast cells are not well understood. Thus, we have explored the cellular handling and regulation of monoamine transporters during the phenotypic transitioning of cytotrophoblasts along the villous pathway. METHODS: Experiments were conducted with two cellular models of syncytium development: primary trophoblast cells isolated from the human term placenta (PHT), and the choriocarcinoma-derived BeWo cell line. The gene and protein expression of membrane transporters for serotonin (SERT), norepinephrine (NET), dopamine (DAT), and organic cation transporter 3 (OCT3) was determined by quantitative PCR and Western blot analysis, respectively. Subsequently, the effect of trophoblast differentiation on transporter activity was analyzed by monoamine uptake into cells. RESULTS: We present multiple lines of evidence of changes in the transcriptional and functional regulation of monoamine transporters associated with trophoblast differentiation. These include enhancement of SERT and DAT gene and protein expression in BeWo cells. On the other hand, in PHT cells we report negative modulation of SERT, NET, and OCT3 protein expression. We show that OCT3 is the dominant monoamine transporter in PHT cells, and its main functional impact is on serotonin uptake, while passive transport strongly contributes to norepinephrine and dopamine uptake. Further, we show that a wide range of selective serotonin reuptake inhibitors affect serotonin cellular accumulation, at pharmacologically relevant drug concentrations, via their action on both OCT3 and SERT. Finally, we demonstrate that BeWo cells do not well reflect the molecular mechanisms and properties of healthy human trophoblast cells. CONCLUSIONS: Collectively, our findings provide insights into the regulation of monoamine transport during trophoblast differentiation and present important considerations regarding appropriate in vitro models for studying monoamine regulation in the placenta.
The human placenta represents a unique non-neuronal site of monoamine transporter expression, with pathophysiological relevance during the prenatal period. Monoamines (serotonin, dopamine, norepinephrine) are crucial neuromodulators for proper placenta functions and fetal development, including cell proliferation, differentiation, and neuronal migration. Accumulating evidence suggests that even a transient disruption of monoamine balance during gestation may lead to permanent changes in the fetal brain structures and functions, projecting into adulthood. Nonetheless, little is known about the transfer of dopamine and norepinephrine across the placental syncytiotrophoblast. Employing the method of isolated membranes from the human term placenta, here we delineate the transport mechanisms involved in dopamine and norepinephrine passage across the apical microvillous (MVM) and basal membranes. We show that the placental uptake of dopamine and norepinephrine across the mother-facing MVM is mediated via the high-affinity and low-capacity serotonin (SERT/SLC6A4) and norepinephrine (NET/SLC6A2) transporters. In the fetus-facing basal membrane, however, the placental uptake of both monoamines is controlled by the organic cation transporter 3 (OCT3/SLC22A3). Our findings thus provide insights into physiological aspects of dopamine and norepinephrine transport across both the maternal and fetal sides of the placenta. As monoamine transporters represent targets for several neuroactive drugs such as antidepressants, our findings are pharmacologically relevant to ensure the safety of drug use during pregnancy.
- MeSH
- buněčná membrána metabolismus MeSH
- dopamin metabolismus MeSH
- dospělí MeSH
- lidé MeSH
- membránové transportní proteiny pro serotonin metabolismus MeSH
- noradrenalin metabolismus MeSH
- placenta * metabolismus MeSH
- serotonin metabolismus MeSH
- těhotenství MeSH
- trofoblasty * metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Steroid hormones play a crucial role in supporting a successful pregnancy and ensuring proper fetal development. The placenta is one of the principal tissues in steroid production and metabolism, expressing a vast range of steroidogenic enzymes. Nevertheless, a comprehensive characterization of steroidogenic pathways in the human placenta and potential developmental changes occurring during gestation are poorly understood. Furthermore, the specific contribution of trophoblast cells in steroid release is largely unknown. Thus, this study aimed to (i) identify gestational age-dependent changes in the gene expression of key steroidogenic enzymes and (ii) explore the role of trophoblast cells in steroid biosynthesis and metabolism. Quantitative and Droplet Digital PCR analysis of 12 selected enzymes was carried out in the first trimester (n = 13) and term (n = 20) human placentas. Primary trophoblast cells (n = 5) isolated from human term placentas and choriocarcinoma-derived cell lines (BeWo, BeWo b30 clone, and JEG-3) were further screened for gene expression of enzymes involved in placental synthesis/metabolism of steroids. Finally, de novo steroid synthesis by primary human trophoblasts was evaluated, highlighting the functional activity of steroidogenic enzymes in these cells. Collectively, we provide insights into the expression patterns of steroidogenic enzymes as a function of gestational age and delineate the cellular origin of steroidogenesis in the human placenta.
- MeSH
- choriokarcinom metabolismus patologie MeSH
- dospělí MeSH
- gestační stáří MeSH
- kultivované buňky MeSH
- lidé MeSH
- novorozenec MeSH
- placenta cytologie metabolismus MeSH
- první trimestr těhotenství metabolismus MeSH
- regulace genové exprese * MeSH
- steroidhydroxylasy genetika metabolismus MeSH
- steroidy metabolismus MeSH
- těhotenství MeSH
- trofoblasty cytologie metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- novorozenec MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Preeclampsia (PE) is a major cause of the pregnancy morbidity and mortality over the world. Disorganized placentation caused by trophoblast cell abnormity is one of main risk factors to induce PE. MiR-133a-3p has been shown to contain regulatory effects on oxidative stress in the cardiomyocytes. But the effects of miR-133a-3p on oxidative stress-induced apoptosis in the trophoblast cells remain unknown. In this study, trophoblast HTR-8/SVneo cells were transfected with miR-133a-3p mimics and inhibitor. H2O2 (250 microM) treatment of cells was adopted to induce oxidative stress. A series of typical molecular and cellular experiments was subsequently performed in order to investigate this issue. It was found that miR-133a-3p overexpression attenuated the oxidative stress induced by H2O2 through reduced ROS and MDA levels and enhanced antioxidase activities in the trophoblast cells. Overexpressed miR-133a-3p was shown to relieve the oxidative stress-induced apoptosis of HTR-8/SVneo cells. At molecular levels, a direct binding effect of miR-133a-3p on BACH1 was verified. Moreover, miR-133a-3p overexpression also enhanced BACH1 downstream Nrf2/HO-1 signaling to activate antioxidant genes. It is collectively demonstrated that miR-133a-3p can relieve the oxidative stress-induced apoptosis in the trophoblast cells through the BACH1/Nrf2/HO-1 signaling pathway via targeting BACH1 directly. This regulatory mechanism of miR-133a-3p in the trophoblast cells under oxidative stress may give a new perspective for oxidative stress-induced trophoblast cell abnormality and be useful to study more pathological mechanisms of PE.
- MeSH
- apoptóza účinky léků MeSH
- buněčné linie MeSH
- faktor 2 související s NF-E2 metabolismus MeSH
- hemoxygenasa-1 metabolismus MeSH
- lidé MeSH
- mikro RNA aplikace a dávkování genetika MeSH
- oxidační stres účinky léků MeSH
- preeklampsie metabolismus patologie prevence a kontrola MeSH
- signální transdukce MeSH
- těhotenství MeSH
- transkripční faktory bZIP metabolismus MeSH
- trofoblasty účinky léků metabolismus patologie MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
