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

Can magnesium gluconate be used as an alternative therapy for preeclampsia?

Magnesium (Mg+2) in the body plays a structural and regulatory role and it is involved in fundamental cellular reactions. It is known that Mg+2 blood levels decrease during pregnancy, which has been related to preeclampsia and premature delivery, as well as other pathologies such as cardiovascular alterations and renal, gastrointestinal, neurological, and muscular dysfunctions among others. Mg+2 salts are used to treat its deficiency, and parenteral magnesium sulfate (MgSO4) is relatively effective in preeclampsia and eclampsia. The use of MgSO4 has the main disadvantage that it is mainly administered intravenously which leads to significant toxicity risks. Currently, other magnesium salts are being studied as alternative treatments. Magnesium gluconate (Mg-gluconate) has been used to prevent pregnancy-induced hypertension, showing a greater antioxidant capacity than MgSO4. Mg-gluconate can scavenge hydroxyl and alkoxyl radicals and it has been shown that it can inhibit lipid peroxidation in microsomal membranes treated in vitro with the Fenton reaction. Mg-gluconate seems to be an excellent candidate to replace MgSO4 as a therapy for preeclampsia with severe features.El magnesio (Mg+2) en el organismo, juega un papel estructural y regulador, y participa en reacciones celulares fundamentales. Se sabe que los niveles séricos de Mg+2 disminuyen durante el embarazo, lo cual se ha relacionado con la preeclampsia y el parto prematuro, así como con otras patologías como alteraciones cardiovasculares y disfunciones renales, gastrointestinales, neurológicas, musculares, entre otras. Las sales de Mg+2 se utilizan para tratar su deficiencia, y el sulfato de magnesio parenteral (MgSO4) ha demostrado ser relativamente eficaz en la preeclampsia y la eclampsia. El uso de MgSO4 tiene el principal inconveniente de que se administra principalmente por vía intravenosa, lo cual conlleva a riesgos importantes de toxicidad. Actualmente, se están estudiando otras sales de magnesio como tratamientos alternativos. El gluconato de magnesio (Mg-gluconato) se ha utilizado para prevenir la hipertensión inducida por el embarazo, mostrando una mayor capacidad antioxidante que el MgSO4. El Mg-gluconato es capaz de eliminar radicales hidroxilo y alcoxilo e inhibir la peroxidación lipídica en membranas microsomales tratadas in vitro con la reacción de Fenton. El Mg-gluconato parece ser un excelente candidato para sustituir al MgSO4 como terapia para la preeclampsia con características graves.Sociedad Argentina de Fisiologí

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

Oxidative stress and mitochondrial dysfunction in early-onset and late-onset preeclampsia

Preeclampsia is a pregnancy-specific syndrome with multisystem involvement which leads to foetal, neonatal, and maternal morbidity and mortality. This syndrome is characterized by the onset of clinical signs and symptoms and delivery before (early-onset preeclampsia, eoPE), or after (late-onset preeclampsia, loPE), the 34 weeks of gestation. Preeclampsia is a mitochondrial disorder where its differential involvement in eoPE and loPE is unclear. Mitochondria regulate cell metabolism and are a significant source of reactive oxygen species (ROS). The syncytiotrophoblast in eoPE and loPE show altered mitochondrial structure and function resulting in ROS overproduction, oxidative stress, and cell damage and death. Mitochondrial dysfunction in eoPE may result from altered expression of several molecules, including dynamin-related protein 1 and mitofusins, compared with loPE where these factors are either reduced or unaltered. Equally, mitochondrial fusion/fission dynamics seem differentially modulated in eoPE and loPE. It is unclear whether the electron transport chain and oxidative phosphorylation are differentially altered in these two subgroups of preeclampsia. However, the activity of complex IV (cytochrome c oxidase) and the expression of essential proteins involved in the electron transport chain are reduced, leading to lower oxidative phosphorylation and mitochondrial respiration in the preeclamptic placenta. Interventional studies in patients with preeclampsia using the coenzyme Q, a key molecule in the electron transport chain, suggest that agents that increase the antioxidative capacity of the placenta may be protective against preeclampsia development. In this review, the mitochondrial dysfunction in both eoPE and loPE is summarized. Therapeutic approaches are discussed in the context of contributing to the understanding of mitochondrial dysfunction in eoPE and loPE

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

Magnesium salts in pregnancy

Background: Magnesium is one of the most abundant elements in the body. Although the total serum magnesium content does not represent more than 1% of total body magnesium, serum magnesium determinations have been routinely used in clinical practice to assess body magnesium status. Thus, serum magnesium concentration ranges from 1.8 to 2.2 mg.dl−1 (0.75–0.95 mmol.l − 1 or 1.5–1.9 mEq.l − 1). Consequently, when serum magnesium levels fall below the range considered normal, the patient is diagnosed with hypomagnesemia. This deficiency has been associated with low-grade systemic inflammation, increased levels of proinflammatory molecules, mitochondrial dysfunction, increased reactive oxygen species production, and hypertriglyceridemia leading to an increase in the number of easily oxidizable lipoproteins in the circulation. Results: Several magnesium salts have been used to treat hypomagnesemia during pregnancy, with magnesium sulfate (MgSO4) being the most commonly used magnesium salt in current obstetric practice. However, the exact mechanism of action of MgSO4 remains largely an enigma, and its parenteral use poses a significant toxicological risk at high doses. In this review, we summarize the use of magnesium salts during pregnancy not only from a clinical point of view but also, with the use of computational simulations, discuss advances in the understanding of the molecular mechanisms of action of magnesium salts, with emphasis on MgSO4. These molecular simulations are required to unveil the pharmacological action of the magnesium salts during pregnancy. Conclusions: MgSO4 plays a role as an antioxidant agent at the plasma membrane level which can explain the mechanism of action of this salt in current obstetric practice

Oxidative stress: Normal pregnancy versus preeclampsia.

The role of oxidative stress in the physiopathology of human pregnancy is of particular interest. Pregnancy is well-known to increase the oxidative stress, mainly produced by a normal systemic inflammatory response, which results in high amounts of circulating reactive oxygen species (ROS) and reactive nitrogen species (RNS). Both ROS and RNS play an important role as secondary messengers in many intracellular signalling cascades. However, they can also exert critical effects on pathological processes involving the pregnant woman. ROS, RNS and antioxidants establish a balance that determines the oxidation status of animals and humans. This review focuses on the mechanism of oxidative stress in pregnancy as well as its involvement and consequences on the human pregnancy-specific clinical syndrome preeclampsia

Oxidative stress: normal pregnancy versus preeclampsia

The role of oxidative stress in the physiopathology of human pregnancy is of particular interest. Pregnancy is well-known to increase the oxidative stress, mainly produced by a normal systemic inflammatory response, which results in high amounts of circulating reactive oxygen species (ROS) and reactive nitrogen species (RNS). Both ROS and RNS play an important role as secondary messengers in many intracellular signalling cascades. However, they can also exert critical effects on pathological processes involving the pregnant woman. ROS, RNS and antioxidants establish a balance that determines the oxidation status of animals and humans. This review focuses on the mechanism of oxidative stress in pregnancy as well as its involvement and consequences on the human pregnancy-specific clinical syndrome preeclampsia

Characterization of a human placental clearance system to regulate serotonin levels in the fetoplacental unit

Abstract Background Serotonin (5-HT) is a biogenic monoamine with diverse functions in multiple human organs and tissues. During pregnancy, tightly regulated levels of 5-HT in the fetoplacental unit are critical for proper placental functions, fetal development, and programming. Despite being a non-neuronal organ, the placenta expresses a suite of homeostatic proteins, membrane transporters and metabolizing enzymes, to regulate monoamine levels. We hypothesized that placental 5-HT clearance is important for maintaining 5-HT levels in the fetoplacental unit. We therefore investigated placental 5-HT uptake from the umbilical circulation at physiological and supraphysiological levels as well as placental metabolism of 5-HT to 5-hydroxyindoleacetic acid (5-HIAA) and 5-HIAA efflux from trophoblast cells. Methods We employed a systematic approach using advanced organ-, tissue-, and cellular-level models of the human placenta to investigate the transport and metabolism of 5-HT in the fetoplacental unit. Human placentas from uncomplicated term pregnancies were used for perfusion studies, culturing explants, and isolating primary trophoblast cells. Results Using the dually perfused placenta, we observed a high and concentration-dependent placental extraction of 5-HT from the fetal circulation. Subsequently, within the placenta, 5-HT was metabolized to 5-hydroxyindoleacetic acid (5-HIAA), which was then unidirectionally excreted to the maternal circulation. In the explant cultures and primary trophoblast cells, we show concentration- and inhibitor-dependent 5-HT uptake and metabolism and subsequent 5-HIAA release into the media. Droplet digital PCR revealed that the dominant gene in all models was MAO-A, supporting the crucial role of 5-HT metabolism in placental 5-HT clearance. Conclusions Taken together, we present transcriptional and functional evidence that the human placenta has an efficient 5-HT clearance system involving (1) removal of 5-HT from the fetal circulation by OCT3, (2) metabolism to 5-HIAA by MAO-A, and (3) selective 5-HIAA excretion to the maternal circulation via the MRP2 transporter. This synchronized mechanism is critical for regulating 5-HT in the fetoplacental unit; however, it can be compromised by external insults such as antidepressant drugs