Fyziologické a farmakologické aspekty homeostázy tryptofanu a serotoninu ve fetoplacentární jednotce

Univerzita Karlova, Farmaceutická fakulta v Hradci Králové Katedra Katedra farmakologie a toxikologie Kandidát Mgr. Rona Karahoda Školitel Prof. PharmDr. Frantisek Staud, Ph.D. Název disertační práce Fyziologické a farmakologické aspekty homeostázy tryptofanu a serotoninu ve fetoplacentární jednotce Placenta je dočasný orgán, zajišťující spojení mezi matkou a plodem. Po dobu těhotenství vykonává řadu funkcí, včetně endokrinních, transportních a imunoprotektivních, které jsou zcela zásadní pro zdárný průběh gestace, normální růst a vývoj embrya/plodu. Nejnovější výzkumy poukazují na spojitost mezi endogenními (např. onemocnění, stres nebo zánět) a exogenními (např. farmakoterapie) faktory a změnami ve fyziologických funkcích placentárních trofoblastů. Příkladem může být narušení homeostázy látek s neuroaktivními, imunosupresivními nebo antioxidačními vlastnostmi. To může vyústit v nesprávné programování plodu a s tím spojené vyšší riziko závažných onemocnění v dospělosti. Jedním ze zdrojů takových metabolitů je esenciální aminokyselina, tryptofan. Je známo, že metabolismus tryptofanu probíhá serotoninovou a kynureninovou cestou, nicméně komplexní charakterizace enzymů a transportérů ovlivňujících placentární homeostázu tryptofanu, serotoninu a kynureninu je stále nedostatečná. V rámci řešení této...Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate Mgr. Rona Karahoda Supervisor Prof. PharmDr. Frantisek Staud, Ph.D. Title of Doctoral Thesis Physiological and pharmacological aspects of tryptophan and serotonin homeostasis in the fetoplacental unit The placenta is an ephemeral organ inevitable for the successful course of pregnancy. As the main link between the mother and the fetus, the placenta fulfills numerous roles during gestation, including endocrine, transport, and immunoprotective processes. Proper functioning of the placenta is critical for the normal growth and development of the embryo/fetus. Importantly, the latest research has associated perturbations of maternal conditions (such as pharmacotherapy, malnutrition, diseases, stress, or inflammation) with alterations of the trophoblasts' endocrine, transport, and metabolic functions. Of note is the placental utilization of the essential amino acid tryptophan, suggested as a potential mechanism contributing to fetal programming of adulthood diseases. Tryptophan flux along the serotonin and kynurenine pathways generates metabolites with neuroactive, immunosuppressive, and antioxidant properties. Current literature suggests that fine-tuning of tryptophan metabolite concentrations...Department of Pharmacology and ToxicologyKatedra farmakologie a toxikologieFarmaceutická fakulta v Hradci KrálovéFaculty of Pharmacy in Hradec Králov

Fyziologické a farmakologické aspekty homeostázy tryptofanu a serotoninu ve fetoplacentární jednotce

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate Mgr. Rona Karahoda Supervisor Prof. PharmDr. Frantisek Staud, Ph.D. Title of Doctoral Thesis Physiological and pharmacological aspects of tryptophan and serotonin homeostasis in the fetoplacental unit The placenta is an ephemeral organ inevitable for the successful course of pregnancy. As the main link between the mother and the fetus, the placenta fulfills numerous roles during gestation, including endocrine, transport, and immunoprotective processes. Proper functioning of the placenta is critical for the normal growth and development of the embryo/fetus. Importantly, the latest research has associated perturbations of maternal conditions (such as pharmacotherapy, malnutrition, diseases, stress, or inflammation) with alterations of the trophoblasts' endocrine, transport, and metabolic functions. Of note is the placental utilization of the essential amino acid tryptophan, suggested as a potential mechanism contributing to fetal programming of adulthood diseases. Tryptophan flux along the serotonin and kynurenine pathways generates metabolites with neuroactive, immunosuppressive, and antioxidant properties. Current literature suggests that fine-tuning of tryptophan metabolite concentrations...Univerzita Karlova, Farmaceutická fakulta v Hradci Králové Katedra Katedra farmakologie a toxikologie Kandidát Mgr. Rona Karahoda Školitel Prof. PharmDr. Frantisek Staud, Ph.D. Název disertační práce Fyziologické a farmakologické aspekty homeostázy tryptofanu a serotoninu ve fetoplacentární jednotce Placenta je dočasný orgán, zajišťující spojení mezi matkou a plodem. Po dobu těhotenství vykonává řadu funkcí, včetně endokrinních, transportních a imunoprotektivních, které jsou zcela zásadní pro zdárný průběh gestace, normální růst a vývoj embrya/plodu. Nejnovější výzkumy poukazují na spojitost mezi endogenními (např. onemocnění, stres nebo zánět) a exogenními (např. farmakoterapie) faktory a změnami ve fyziologických funkcích placentárních trofoblastů. Příkladem může být narušení homeostázy látek s neuroaktivními, imunosupresivními nebo antioxidačními vlastnostmi. To může vyústit v nesprávné programování plodu a s tím spojené vyšší riziko závažných onemocnění v dospělosti. Jedním ze zdrojů takových metabolitů je esenciální aminokyselina, tryptofan. Je známo, že metabolismus tryptofanu probíhá serotoninovou a kynureninovou cestou, nicméně komplexní charakterizace enzymů a transportérů ovlivňujících placentární homeostázu tryptofanu, serotoninu a kynureninu je stále nedostatečná. V rámci řešení této...Katedra farmakologie a toxikologieDepartment of Pharmacology and ToxicologyFaculty of Pharmacy in Hradec KrálovéFarmaceutická fakulta v Hradci Králov

Trophoblast Differentiation Affects Crucial Nutritive Functions of Placental Membrane Transporters.

Cytotrophoblasts are progenitor cells that proliferate and fuse to form the multinucleated syncytiotrophoblast layer, implicated in placental endocrine and transport functions. While membrane transporters play a critical role in the distribution of nutrients, hormones, and xenobiotics at the maternal-fetal interface, their selectivity to the syncytiotrophoblast layer is poorly characterized. We aimed to evaluate the regulation of placental transporters in response to trophoblast differentiation in vitro. Experiments were carried out in isolated primary human trophoblast cells before and after syncytialization. Gene expression of six molecular markers and thirty membrane transporters was investigated by qPCR analysis. Subsequently, functional expression was evaluated for proteins involved in the transplacental transfer of essential nutrients i.e., cholesterol (ABCA1, ABCG1), glucose (SLC2A1), leucine (SLC3A2, SLC7A5), and iron (transferrin receptor, TfR1). We identified that human chorionic gonadotropin, placental lactogen, endoglin, and cadherin-11 serve as optimal gene markers for the syncytialization process. We showed that trophoblast differentiation was associated with differential gene expression (mostly up-regulation) of several nutrient and drug transporters. Further, we revealed enhanced protein expression and activity of ABCG1, SLC3A2, SLC7A5, and TfR1 in syncytialized cells, with ABCA1 and GLUT1 displaying no change. Taken together, these results indicate that the syncytiotrophoblast has a dominant role in transporting essential nutrients cholesterol, leucine, and iron. Nonetheless, we present evidence that the cytotrophoblast cells may also be linked to transport functions that could be critical for the cell fusion processes. Our findings collectively yield new insights into the cellular functions associated with or altered by the trophoblast fusion. Importantly, defective syncytialization could lead to nutrient transfer imbalance, ultimately compromising fetal development and programming

Functional reorganization of monoamine transport systems during villous trophoblast differentiation: evidence of distinct differences between primary human trophoblasts and BeWo cells.

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

Revisiting Steroidogenic Pathways in the Human Placenta and Primary Human Trophoblast Cells

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

Physiological and pharmacological aspects of tryptophan and serotonin homeostasis in the fetoplacental unit

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate Mgr. Rona Karahoda Supervisor Prof. PharmDr. Frantisek Staud, Ph.D. Title of Doctoral Thesis Physiological and pharmacological aspects of tryptophan and serotonin homeostasis in the fetoplacental unit The placenta is an ephemeral organ inevitable for the successful course of pregnancy. As the main link between the mother and the fetus, the placenta fulfills numerous roles during gestation, including endocrine, transport, and immunoprotective processes. Proper functioning of the placenta is critical for the normal growth and development of the embryo/fetus. Importantly, the latest research has associated perturbations of maternal conditions (such as pharmacotherapy, malnutrition, diseases, stress, or inflammation) with alterations of the trophoblasts' endocrine, transport, and metabolic functions. Of note is the placental utilization of the essential amino acid tryptophan, suggested as a potential mechanism contributing to fetal programming of adulthood diseases. Tryptophan flux along the serotonin and kynurenine pathways generates metabolites with neuroactive, immunosuppressive, and antioxidant properties. Current literature suggests that fine-tuning of tryptophan metabolite concentrations..

Physiological and pharmacological aspects of tryptophan and serotonin homeostasis in the fetoplacental unit

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate Mgr. Rona Karahoda Supervisor Prof. PharmDr. Frantisek Staud, Ph.D. Title of Doctoral Thesis Physiological and pharmacological aspects of tryptophan and serotonin homeostasis in the fetoplacental unit The placenta is an ephemeral organ inevitable for the successful course of pregnancy. As the main link between the mother and the fetus, the placenta fulfills numerous roles during gestation, including endocrine, transport, and immunoprotective processes. Proper functioning of the placenta is critical for the normal growth and development of the embryo/fetus. Importantly, the latest research has associated perturbations of maternal conditions (such as pharmacotherapy, malnutrition, diseases, stress, or inflammation) with alterations of the trophoblasts' endocrine, transport, and metabolic functions. Of note is the placental utilization of the essential amino acid tryptophan, suggested as a potential mechanism contributing to fetal programming of adulthood diseases. Tryptophan flux along the serotonin and kynurenine pathways generates metabolites with neuroactive, immunosuppressive, and antioxidant properties. Current literature suggests that fine-tuning of tryptophan metabolite concentrations..

Revisiting Steroidogenic Pathways in the Human Placenta and Primary Human Trophoblast Cells.

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

Dynamics of Tryptophan Metabolic Pathways in Human Placenta and Placental-Derived Cells: Effect of Gestation Age and Trophoblast Differentiation.

L-Tryptophan is an essential amino acid and a precursor of several physiologically active metabolites. In the placenta, the serotonin and kynurenine metabolic pathways of tryptophan metabolism have been identified, giving rise to various molecules of neuroactive or immunoprotective properties, such as serotonin, melatonin, kynurenine, kynurenic acid, or quinolinic acid. Current literature suggests that optimal levels of these molecules in the fetoplacental unit are crucial for proper placenta functions, fetal development and programming. Placenta is a unique endocrine organ that, being equipped with a battery of biotransformation enzymes and transporters, precisely orchestrates homeostasis of tryptophan metabolic pathways. However, because pregnancy is a dynamic process and placental/fetal needs are continuously changing throughout gestation, placenta must adapt to these changes and ensure proper communication in the feto-placental unit. Therefore, in this study we investigated alterations of placental tryptophan metabolic pathways throughout gestation. Quantitative polymerase chain reaction (PCR) analysis of 21 selected genes was carried out in first trimester (n = 13) and term (n = 32) placentas. Heatmap analysis with hierarchical clustering revealed differential gene expression of serotonin and kynurenine pathways across gestation. Subsequently, digital droplet PCR, Western blot, and functional analyses of the rate-limiting enzymes suggest preferential serotonin synthesis early in pregnancy with a switch to kynurenine production toward term. Correspondingly, increased function and/or protein expression of serotonin degrading enzyme and transporters at term indicates efficient placental uptake and metabolic degradation of serotonin. Lastly, gene expression analysis in choriocarcinoma-derived cell lines (BeWo, BeWo b30, JEG-3) revealed dissimilar expression patterns and divergent effect of syncytialization compared to primary trophoblast cells isolated from human term placentas; these findings show that the commonly used in vitro placental models are not suitable to study placental handling of tryptophan. Altogether, our data provide the first comprehensive evidence of changes in placental homeostasis of tryptophan and its metabolites as a function of gestational age, which is critical for proper placental function and fetal development

Profiling of Tryptophan Metabolic Pathways in the Rat Fetoplacental Unit during Gestation

Placental homeostasis of tryptophan is essential for fetal development and programming. The two main metabolic pathways (serotonin and kynurenine) produce bioactive metabolites with immunosuppressive, neurotoxic, or neuroprotective properties and their concentrations in the fetoplacental unit must be tightly regulated throughout gestation. Here, we investigated the expression/function of key enzymes/transporters involved in tryptophan pathways during mid-to-late gestation in rat placenta and fetal organs. Quantitative PCR and heatmap analysis revealed the differential expression of several genes involved in serotonin and kynurenine pathways. To identify the flux of substrates through these pathways, Droplet Digital PCR, western blot, and functional analyses were carried out for the rate-limiting enzymes and transporters. Our findings show that placental tryptophan metabolism to serotonin is crucial in mid-gestation, with a subsequent switch to fetal serotonin synthesis. Concurrently, at term, the close interplay between transporters and metabolizing enzymes of both placenta and fetal organs orchestrates serotonin homeostasis and prevents hyper/hypo-serotonemia. On the other hand, the placental production of kynurenine increases during pregnancy, with a low contribution of fetal organs throughout gestation. Any external insult to this tightly regulated harmony of transporters and enzymes within the fetoplacental unit may affect optimal in utero conditions and have a negative impact on fetal programming