Fig 9 - available via license: Creative Commons Attribution 4.0 International
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Effect of lithium chloride on metalloproteinase activity and cell invasion. (A) Cells were incubated in the presence of lithium chloride or sodium chloride (control) for 7 days after which MMP9 and MMP2 activities were measured by zymography as described in Methods. (B) To test for effects of lithium chloride on proliferation, equal numbers of trophoblasts were incubated in the presence of lithium chloride or sodium chloride for 7 days after which cell numbers were measured as described in Methods. (C) To assess invasive activity, trophoblasts were cultured in chamber inserts in the presence of lithium chloride or sodium chloride for 7 days after which invasion to the lower chamber was measured as described in Methods. Results are means ± SEM (n = 4).
Source publication
Trophoblast differentiation during early placental development is critical for successful pregnancy and aberrant differentiation causes preeclampsia and early pregnancy loss. During the first trimester, cytotrophoblasts are exposed to low oxygen tension (equivalent to~2%-3% O 2) and differentiation proceeds along an extravillous pathway (giving ris...
Citations
... The decrease in butyric acid levels in faeces may promote immune inflammation through LPS-related mechanisms, thereby promoting the development of PE (Chang et al., 2020). Kumar P, et al. reported that NaB induced trophoblast differentiation in early gestation rhesus monkey trophoblasts through activation of the Wnt/β-catenin pathway (Kumar et al., 2015). In our study, we found that the composition of SCFAs in PE rats was changed. ...
Aims:
Preeclampsia (PE) affects pregnant patients worldwide, but there is no effective treatment for this condition. We aimed to explore the effect of sodium butyrate (NaB) on PE.
Methods and results:
In this study, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) was used to induce PE in pregnant rats. We found that NaB significantly decreased the levels of blood pressure, 24-h protein urine and inflammatory factors (IL-1β, IL-6, and TGF-β), increased the fetal and placental weights and intestinal barrier markers (ZO-1, claudin-5, and occludin) expression. In addition, NaB intervention reduced the levels of soluble fms-like tyrosine kinase 1 (sFlt1) and soluble endoglin (sEng) and increased placental growth factor (PlGF) level. Meanwhile, after NaB treatment, the Treg/Th17 ratio of immune cells in the spleen and small intestine of pregnant rats decreased, while the level of pregnancy-related DAO increased. Notably, the PE rat treatment with NaB improved gut microbiota compositions, especially for the abundances of Firmicutes and Bacteroides, and significantly increased butyric acid and pentanoic acid levels, which might help to alleviate PE in pregnant rats.
Conclusion:
In the PE rat model, exogenous NaB improved intestinal barrier function and reduced adverse outcomes, which might be associated with the gut microbiota and its production of SCFA metabolites.
Significance and impact of the study:
NaB might alleviate the adverse outcomes of PE by regulating gut microbiota and its metabolite SCFA, which revealed that NaB might be a potential regulator of gut microbiota and a therapeutic substance for PE.
... Short-chain fatty acids (SCFAs), products of intestinal microbiota, not only serve as an energy source for hosts (Zhi et al., 2019), but also play a vital role in placental development. For instance, butyrate induces trophoblast differentiation and syncytiotrophoblast formation in early gestation (Kumar et al., 2015); and propionate blocks the secretion of pro-inflammatory cytokines and chemokines in placenta (Roy et al., 2020). Given SCFAs function as a link between the microbiota and the host, understanding the dynamic changes of SCFAs in obesity will help us have a better understanding of the relationship between intestinal microbiota and placental function. ...
... However, the mechanism needs further investigation. SCFAs are a possible link between the intestinal microbiota and placenta (Kumar et al., 2015). Therefore, the concentration of SCFAs in feces was determined in the present study. ...
Maternal obesity induces placental dysfunction and intestinal microbial dysbiosis. However, the associations between intestinal microbiota and placental dysfunction are still unclear. In the present study, a gilt model was used to investigate the role of maternal obesity on placental oxidative stress, mitochondrial function, and fecal microbiota composition, meanwhile identifying microbiota markers associated with placental oxidative stress. Twenty gilts were divided into two groups based on their backfat thickness on parturition day: namely Con group (average backfat thickness = 33 mm), and Obese group (average backfat thickness = 39 mm). The results showed that Obese group was lower than Con group in the birth weight of piglets. Compared with the Con group, the Obesity group exhibited an increased oxidative damage and inflammatory response in placenta, as evidenced by the increased concentrations of placental reactive oxygen species (ROS), protein carboxyl, and interleukin-6 (IL-6). Obesity group was lower than Con group in the concentrations of placental adenosine triphosphate, citrate synthase, and complex I activity. In addition, lower propionate level and Bacteroidetes abundance in feces were seen in the Obese Group. Furthermore, the concentrations of placental ROS, protein carboxyl, and IL-6 were positively correlated with the abundance of Christensenellaceae_R-7_group and negatively correlated with that of norank_f_Bacteroidales_S24-7_group. In conclusion, these findings suggest that maternal obesity might impair oxidative and inflammatory response in placenta through modulating intestinal microbiota composition.
... Because the placenta is a heterogeneous organ comprised of many cell types in addition to trophoblasts, such as immune, stromal, and vascular cells [46], we next sought to isolate primary trophoblasts from bulk rhesus placentas for immortalization and characterization, including a comparison of gene expression. While previous studies have successfully isolated and cultured primary trophoblasts from first and third trimester rhesus placenta [47][48][49][50], as well as generated rhesus blastocyst-and placentaderived trophoblast stem cells [51,52], no rhesus immortalized trophoblast cell lines currently exist for in vitro investigations. Using the strategy described in Fig. 2a, we isolated primary trophoblast cells from rhesus placental tissues collected at gestational day 26 (~6 weeks human pregnancy), day 28 (~7 weeks human pregnancy), day 50 (~12 weeks human pregnancy), day 141 (~34 weeks human pregnancy), and day 149 (~35 weeks human pregnancy). ...
... Pellets were resuspended in DMEM and repelleted. Cells were carefully layered over a preformed Percoll gradient layered at 60,55,50,45,35,30, and 25%, prior to centrifugation at 2800 rpm for 30 min without brake. The CTB cells between 35% and 55% were collected, counted and resuspended in CCM. ...
Background
Proper placentation, including trophoblast differentiation and function, is essential for the health and well-being of both the mother and baby throughout pregnancy. Placental abnormalities that occur during the early stages of development are thought to contribute to preeclampsia and other placenta-related pregnancy complications. However, relatively little is known about these stages in humans due to obvious ethical and technical limitations. Rhesus macaques are considered an ideal surrogate for studying human placentation, but the unclear translatability of known human placental markers and lack of accessible rhesus trophoblast cell lines can impede the use of this animal model.
Results
Here, we performed a cross-species transcriptomic comparison of human and rhesus placenta and determined that while the majority of human placental marker genes (HPGs) were similarly expressed, 952 differentially expressed genes (DEGs) were identified between the two species. Functional enrichment analysis of the 447 human-upregulated DEGs, including ADAM12 , ERVW-1 , KISS1 , LGALS13 , PAPPA2 , PGF , and SIGLEC6 , revealed over-representation of genes implicated in preeclampsia and other pregnancy disorders. Additionally, to enable in vitro functional studies of early placentation, we generated and thoroughly characterized two highly pure first trimester telomerase (TERT) immortalized rhesus trophoblast cell lines (iRP-D26 and iRP-D28A) that retained crucial features of isolated primary trophoblasts.
Conclusions
Overall, our findings help elucidate the molecular translatability between human and rhesus placenta and reveal notable expression differences in several HPGs and genes implicated in pregnancy complications that should be considered when using the rhesus animal model to study normal and pathological human placentation.
... These cell lineages express different levels of galectin-1 overall demonstrating increasing levels of galectin-1 in extravillous cytotrophoblasts essential for invasion and migration activities towards the decidua [107][108][109]. Upregulation of galectin-1 in human and animal trophoblastic cells can be induced by a variety of cell differentiation stimuli including sodium butyrate [110] and 8-Br-cAMP [111]. Studies with human choriocarcinoma cell line BeWo revealed that extracellular galectin-1 inhibited trophoblast cell proliferation and induced syncytium formation, which was associated with modulating activity of MAPK signaling pathway, the receptor tyrosine kinases, Janus kinase 2 (JAK2), and vascular endothelial growth factor receptor 3 [82]. ...
Galectins are a family of soluble β-galactoside-binding proteins with diverse glycan-dependent and glycan-independent functions outside and inside the cell. Human cells express twelve out of sixteen recognized mammalian galectin genes and their expression profiles are very different between cell types and tissues. In this review, we summarize the current knowledge on the changes in the expression of individual galectins at mRNA and protein levels in different types of differentiating cells and the effects of recombinant galectins on cellular differentiation. A new model of galectin regulation is proposed considering the change in O-GlcNAc homeostasis between progenitor/stem cells and mature differentiated cells. The recognition of galectins as regulatory factors controlling cell differentiation and self-renewal is essential for developmental and cancer biology to develop innovative strategies for prevention and targeted treatment of proliferative diseases, tissue regeneration, and stem-cell therapy.
... Butyrate may also regulate the melatoninergic pathway via its conversion of ceramide to glucosyl-ceramide, thereby preventing ceramide's inhibition of 14-3-3 [101]. Data in primates shows butyrate to significantly modulate trophoblast and placenta development [102]. ...
There is a growing awareness that pregnancy can set the foundations for an array of diverse medical conditions in the offspring, including obesity. A wide assortment of factors, including genetic, epigenetic, lifestyle, and diet can influence foetal outcomes. This article reviews the role of melatonin in the prenatal modulation of offspring obesity. A growing number of studies show that many prenatal risk factors for poor foetal metabolic outcomes, including gestational diabetes and night-shift work, are associated with a decrease in pineal gland-derived melatonin and associated alterations in the circadian rhythm. An important aspect of circadian melatonin's effects is mediated via the circadian gene, BMAL1, including in the regulation of mitochondrial metabolism and the mitochondrial melatoninergic pathway. Alterations in the regulation of mitochondrial metabolic shifts between glycolysis and oxidative phosphorylation in immune and glia cells seem crucial to a host of human medical conditions, including in the development of obesity and the association of obesity with the risk of other medical conditions. The gut microbiome is another important hub in the pathoetiology and pathophysiology of many medical conditions, with negative consequences mediated by a decrease in the short-chain fatty acid, butyrate. The effects of butyrate are partly mediated via an increase in the melatoninergic pathway, indicating interactions of the gut microbiome with melatonin. Some of the effects of melatonin seem mediated via the alpha 7 nicotinic receptor, whilst both melatonin and butyrate may regulate obesity through the opioidergic system. Oxytocin, a recently recognized inhibitor of obesity, may also be acting via the opioidergic system. The early developmental regulation of these processes and factors by melatonin are crucial to the development of obesity and many diverse comorbidities.
... Butyrate may also regulate the melatonergic pathway via its conversion of ceramide to glucosyl-ceramide, thereby preventing ceramide's inhibition of 14-3-3 [101]. Data in primates shows butyrate to significantly modulate trophoblast and placenta development [102]. ...
There is a growing awareness that pregnancy can set the foundations for an array of diverse medical conditions in the offspring, including obesity. An wide assortment of factors, including genetic, epigenetic, lifestyle and dietary can influence foetal outcomes. This article reviews the role of melatonin in the prenatal modulation of offspring obesity. A growing number of studies show that many prenatal risk factors for poor fetal metabolic outcomes, including gestational diabetes and night-shift work, are associated with a decrease in pineal gland-derived melatonin and associated alterations in the circadian rhythm. An important aspect of circadian melatonin's effects are mediated via the circadian gene, Bmal1, including in the regulation of mitochondrial metabolism and the mitochondrial melatonergic pathway. Alterations in the regulation of mitochondrial metabolic shifts between glycolysis and oxidative phosphorylation in immune and glia cells seem crucial to a host of human medical conditions, including in the development of obesity and the association of obesity with the risk of other medical conditions. The gut microbiome is another important hub in the pathoetiology and pathophysiology of many medical conditions, with negative consequences mediated by a decrease in the short-chain fatty acid, butyrate. The effects of butyrate are partly mediated via an increase in the melatonergic pathway, indicating interactions of the gut microbiome with melatonin. Some of the effects of melatonin seem mediated via the alpha 7 nicotinic receptor, whilst both melatonin and butyrate may regulate obesity the opioidergic system. Oxytocin, a recently recognized inhibitor of obesity may also be acting via the opioidergic system. The early developmental regulation of these processes and factors by melatonin are crucial to the development of obesity and many diverse comorbidities.
... Although released in the gut lumen, butyrate, and the other SCFAs, are readily taken up by intestinal epithelial cells, crossing into the general circulation, where butyrate can have wide regulatory effects, including in the regulation of the diverse cells of the immune system [36]. Maternal butyrate synthesis can directly and indirectly, via gut barrier maintenance, modulate prenatal processes, with butyrate significantly increasing trophoblast invasion and differentiation [37,38]. Such data suggest that the maternal microbiome may modulate prenatal risk factors in the pathophysiology of ASD (See Fig. 2). ...
Background
A diverse array of data has been associated with autism spectrum disorder (ASD), reflecting the complexity of its pathophysiology as well as its heterogeneity. Two important hubs have emerged, the placenta/prenatal period and the postnatal gut, with alterations in mitochondria functioning crucial in both.
Methods
Factors acting to regulate mitochondria functioning in ASD across development are reviewed in this article.
Results
Decreased vitamin A, and its retinoic acid metabolites, lead to a decrease in CD38 and associated changes that underpin a wide array of data on the biological underpinnings of ASD, including decreased oxytocin, with relevance both prenatally and in the gut. Decreased sirtuins, poly-ADP ribose polymerase-driven decreases in nicotinamide adenine dinucleotide (NAD+), hyperserotonemia, decreased monoamine oxidase, alterations in 14-3-3 proteins, microRNA alterations, dysregulated aryl hydrocarbon receptor activity, suboptimal mitochondria functioning, and decreases in the melatonergic pathways are intimately linked to this. Many of the above processes may be modulating, or mediated by, alterations in mitochondria functioning. Other bodies of data associated with ASD may also be incorporated within these basic processes, including how ASD risk factors such as maternal obesity and preeclampsia as well as more general prenatal stressors modulate the likelihood of offspring ASD.
Conclusion
Such an integrated model of the pathophysiology of ASD has important preventative and treatment implications.
... Zhuang et al (33) reported that the staining intensity of β-catenin is decreased in placental tissues from women with Pe. in addition, oxidative stress-induced c/eBPβ inhibits the activities of Wnt/β-catenin signaling, which subsequently contributes to the pathogenesis of Pe (34). a study using different activators of Wnt/β-catenin signaling revealed that Wnt/β-catenin signaling is closely related to trophoblast cell differentiation (35). in addition, decreased expression of WnT2 in the villi of patients with unexplained recurrent spontaneous abortion may cause trophoblast cell dysfunction via suppressing Wnt/β-catenin signaling (36). on this basis, the present study further examined the effects of FaM99a on Wnt/β-catenin signaling activity. ...
Preeclampsia (PE) is a complication of pregnancy, and a leading cause of maternal mortality and morbidity worldwide. Recently, the dysregulation of long non‑coding RNAs (lncRNAs) has been reported to contribute to the pathogenesis and progression of PE. This study aimed to examine the alterations in the lncRNA family with sequence similarity 99 member A (FAM99A) in PE and its effects on trophoblasts. The results of reverse transcription‑quantitative PCR indicated that the expression levels of FAM99A were downregulated in placental tissues from women with severe PE compared with in those from controls. A Transwell invasion assay and wound healing assay revealed that overexpression of FAM99A promoted invasion and migration of HTR‑8/SVneo cells; conversely, knockdown of FAM99A suppressed the invasive and migratory abilities of HTR‑8/SVneo cells. Flow cytometry demonstrated that FAM99A overexpression induced a decrease in the apoptotic rate of cells, whereas knockdown of FAM99A increased the apoptotic rate of HTR‑8/SVneo cells. Western blot analysis revealed that overexpression of FAM99A decreased the protein expression levels of cleaved caspase‑3, cleaved caspase‑9 and Bax, and increased Bcl‑2 protein expression, whereas knockdown of FAM99A had the opposite effects on these protein levels. Overexpression of FAM99A also decreased caspase‑3 activity in HTR‑8/SVneo cells; however, knockdown of FAM99A increased caspase‑3 activity. In addition, overexpression of FAM99A enhanced Wnt/β‑catenin signaling activity, whereas FAM99A knockdown exerted an inhibitory effect on the Wnt/β‑catenin signaling activity in HTR‑8/SVneo cells. In conclusion, these results indicated that FAM99A may serve a role in modulating the functions of trophoblasts, partially via targeting Wnt/β‑catenin signaling.
... Treatment of CTB cells derived from human placenta with troglitazone, a PPARγ agonist that activates AMPK, also induced cell fusion and significantly increased syncytin-1 and hCG [98,99]. Stress-induced AMPK activation may also facilitate trophoblast differentiation in non-human primates, as butyrate, a histone deacetylase (HDAC) inhibitor that activates AMPK, induces STB formation in early gestation rhesus monkey trophoblasts [100,101]. ...
Transposable elements (TEs), also known as “jumping genes”, are DNA sequences first described by Nobel laureate Barbara McClintock that comprise nearly half of the human genome and are able to transpose or move from one genomic location to another. As McClintock also noted that a genome “shock” or stress may induce TE activation and transposition, accumulating evidence suggests that cellular stress (e.g. mediated by increases in intracellular reactive oxygen species [ROS] and calcium [Ca2+], etc.) induces TE mobilization in several model organisms and L1s (a member of the retrotransposon class of TEs) are active and capable of retrotransposition in human oocytes, human sperm, and in human neural progenitor cells. Cellular stress also plays a critical role in human placental development, with cytotrophoblast (CTB) differentiation leading to the formation of the syncytiotrophoblast (STB), a cellular layer that facilitates nutrient and gas exchange between the mother and the fetus. Syncytin-1, a protein that promotes fusion of CTB cells and is necessary for STB formation, and its receptor is found in human sperm and human oocytes, respectively, and increases in ROS and Ca2+ promote trophoblast differentiation and syncytin-1 expression. Cellular stress is also essential in promoting human oocyte maturation and activation which, similar to TE mobilization, can be induced by compounds that increase intracellular Ca2+ and ROS levels. AMPK is a master metabolic regulator activated by increases in ROS, Ca2+, and/or an AMP(ADP)/ATP ratio increase, etc. as well as compounds that induce L1 mobilization in human cells. AMPK knockdown inhibits trophoblast differentiation and AMPK-activating compounds that promote L1 mobility also enhance trophoblast differentiation. Cellular stressors that induce TE mobilization (e.g. heat shock) also promote oocyte maturation in an AMPK-dependent manner and the antibiotic ionomycin activates AMPK, promotes TE activation, and induces human oocyte activation, producing normal, healthy children. Metformin promotes AMPK-dependent telomerase activation (critical for telomere maintenance) and induces activation of the endonuclease RAG1 (promotes DNA cleavage and transposition) via AMPK. Both RAG1 and telomerase are derived from TEs. It is our hypothesis that cellular stress and AMPK links TE activation and transposition with placental development and oocyte activation, facilitating both human genome evolution and the creation of all human life. We also propose the novel observation that various cellular stress-inducing compounds (e.g. metformin, resveratrol, etc.) may facilitate beneficial TE activation and transposition and enhance fertilization and embryological development through a common mechanism of AMPK activation.
Preeclampsia (PE) is a life-threatening pregnancy complication associated with diminished trophoblast migration and invasion. Wnt signalling is one of the most important regulators of placentation. Secreted frizzled-related protein 5 (SFRP5) is an anti-inflammatory adipokine that may inhibit Wnt signalling. In this study, we aimed to investigate the relationship between SFRP5 and PE and its effect on trophoblast function, as well as the underlying signalling pathways. SFRP5 levels in the serum and placental tissues were detected using enzyme-linked immunosorbent assay and immunohistochemistry, respectively. To evaluate the effect of SFRP5 on Wnt signalling, the human trophoblast cell line HTR8/SVneo was treated with recombinant human SFRP5 and Dickkopf-related protein 1 (Dkk-1, canonical Wnt inhibitor) proteins and lithium chloride (LiCl, canonical Wnt agonist). The migration and invasion ability of HTR8/SVneo cells was evaluated using wound-healing and Matrigel Transwell assays. The activities of multiple matrix metalloproteinases (MMP)-2/9 were detected using gelatin zymography. Expression of glycogen synthase kinase-3 beta (GSK3β) and β-catenin proteins was investigated using western blotting. The serum SFRP5 levels were elevated in patients with PE, but SFRP5 expression was not detected in the placental tissues. Furthermore, SFRP5 inhibited the migration and invasion of HTR8/SVneo cells in vitro, increased GSK3β, and decreased β-catenin expression and MMP-2/9 activity in HTR8/SVneo cells. In conclusion, this study suggests that SFRP5 inhibits trophoblast migration and invasion potentially via the inhibition of Wnt/β-catenin signalling, which might be involved in the development of PE. However, the primary cause of the increased SFRP5 levels needs to be investigated.
