The Role of the Placental Enzyme Indoleamine 2,3-Dioxygenase in Normal and Abnormal Human Pregnancy.

The biologically significant phenomenon that the fetus can survive immune attacks from the mother has been demonstrated in mammals. The survival mechanism depends on the fetus and placenta actively defending themselves against attacks by maternal T cells, achieved through the localized depletion of the amino acid L-tryptophan by an enzyme called indoleamine 2,3-dioxygenase. These findings were entirely unexpected and pose important questions regarding diseases related to human pregnancy and their prevention during human pregnancy. Specifically, the role of this mechanism, as discovered in mice, in humans remains unknown, as does the extent to which impaired activation of this process contributes to major clinical diseases in humans. We have, thus, elucidated several key aspects of this enzyme expressed in the human placenta both in normal and abnormal human pregnancy. The questions addressed in this brief review are as follows: (1) localization and characteristics of human placental indoleamine 2,3-dioxygenas; (2) overall tryptophan catabolism in human pregnancy and a comparison of indoleamine 2,3-dioxygenase expression levels between normal and pre-eclamptic pregnancy; (3) controlling trophoblast invasion by indoleamine 2,3-dioxygenase and its relation to the pathogenesis of placenta accrete spectrum.

Upregulation of METTL14 contributes to trophoblast dysfunction by elevating FOXO3a expression in an m6A-dependent manner.

INTRODUCTION: Preeclampsia, a specific complication of pregnancy, is a leading cause of perinatal and maternal mortality worldwide. N6-methyladenosine (m6A) is a prevalent and reversible modification of mammalian mRNAs, and is known to play an important role in various physiological and pathological processes. However, little is known about its possible effects on trophoblasts in preeclampsia. METHODS: Colorimetric RNA m6A methylation quantification assay and dot blotting were used to assess the levels of global RNA m6A modification in placental tissues collected from females with normal pregnancy and preeclampsia, while the mRNA levels of major m6A methyltransferases/demethylases were investigated by quantitative real-time polymerase chain reaction. The effects of methyltransferase-like 14 (METTL14) on trophoblasts were evaluated using cell counting kit-8, transwell invasion assay, autophagic flux assay, and Annexin V/propidium iodide apoptosis assay. The molecular mechanism underlying the regulation of forkhead box O3a (FOXO3a) expression by METTL14 was determined using methylated RNA immunoprecipitation and transcription inhibition assays. RESULTS: Global RNA m6A methylation and METTL14 expression were significantly increased in placental tissues obtained from patients with preeclampsia. In vitro studies showed that overexpression of METTL14 in HTR-8/SVneo cells inhibited trophoblast proliferation and invasion, but induced trophoblast autophagy and apoptosis. We further demonstrated that METTL14 epigenetically elevated FOXO3a expression via an m6A-dependent mechanism. FOXO3a inhibition effectively prevented the impairment of trophoblast proliferation and invasion, and diminished the induction of trophoblast autophagy and apoptosis in METTL14-overexpressing HTR-8/SVneo cells. DISCUSSION: Increased METTL14-mediated m6A modification results in an adverse impact on trophoblast function by elevating FOXO3a expression.

S-Nitrosoglutathione Reductase Deficiency Causes Aberrant Placental S-Nitrosylation and Preeclampsia.

Background Preeclampsia, a leading cause of maternal and fetal mortality and morbidity, is characterized by an increase in S-nitrosylated proteins and reactive oxygen species, suggesting a pathophysiologic role for dysregulation in nitrosylation and nitrosative stress. Methods and Results Here, we show that mice lacking S-nitrosoglutathione reductase (GSNOR-/-), a denitrosylase regulating protein S-nitrosylation, exhibit a preeclampsia phenotype, including hypertension, proteinuria, renal pathology, cardiac concentric hypertrophy, decreased placental vascularization, and fetal growth retardation. Reactive oxygen species, NO, and peroxynitrite levels are elevated. Importantly, mass spectrometry reveals elevated placental S-nitrosylated amino acid residues in GSNOR-/- mice. Ascorbate reverses the phenotype except for fetal weight, reduces the difference in the S-nitrosoproteome, and identifies a unique set of S-nitrosylated proteins in GSNOR-/- mice. Importantly, human preeclamptic placentas exhibit decreased GSNOR activity and increased nitrosative stress. Conclusions Therefore, deficiency of GSNOR creates dysregulation of placental S-nitrosylation and preeclampsia in mice, which can be rescued by ascorbate. Coupled with similar findings in human placentas, these findings offer valuable insights and therapeutic implications for preeclampsia.

ACE2: a key modulator of the renin-angiotensin system and pregnancy.

Angiotensin-converting enzyme 2 (ACE2) is a membrane-bound protein containing 805 amino acids. ACE2 shows approximately 42% sequence similarity to somatic ACE but has different biochemical activities. The key role of ACE2 is to catalyze the vasoconstrictor peptide angiotensin (ANG) II to Ang-(1-7), thus regulating the two major counterbalancing pathways of the renin-angiotensin system (RAS). In this way, ACE2 plays a protective role in end-organ damage by protecting tissues from the proinflammatory actions of ANG II. The circulating RAS is activated in normal pregnancy and is essential for maintaining fluid and electrolyte homeostasis and blood pressure. Renin-angiotensin systems are also found in the conceptus. In this review, we summarize the current knowledge on the regulation and function of circulating and uteroplacental ACE2 in uncomplicated and complicated pregnancies, including those affected by preeclampsia and fetal growth restriction. Since ACE2 is the receptor for SARS-CoV-2, and COVID-19 in pregnancy is associated with more severe disease and increased risk of abnormal pregnancy outcomes, we also discuss the role of ACE2 in mediating some of these adverse consequences. We propose that dysregulation of ACE2 plays a critical role in the development of preeclampsia, fetal growth restriction, and COVID-19-associated pregnancy pathologies and suggest that human recombinant soluble ACE2 could be a novel therapeutic to treat and/or prevent these pregnancy complications.

Elevated placental histone H3K4 methylation via upregulated histone methyltransferases SETD1A and SMYD3 in preeclampsia and its possible involvement in hypoxia-induced pathophysiological process.

INTRODUCTION: Disturbance in placental epigenetic regulation contributes to the pathogenesis of preeclampsia (PE). Although aberrant placental DNA methylation status in PE has been thoroughly studied, the role of histone modifications, including histone methylation, in PE remains unclear. Moreover, no study has ever reported the association between PE and placental histone methylation status by focusing on histone methyltransferases. The present study aimed to investigate the possible involvement of placental epigenetic regulation by histone methylation via histone methyltransferases in the pathophysiology of PE. METHODS: Placental mRNA expression of histone methyltransferases was examined using quantitative RT-PCR. Protein expression of histone methyltransferases and histone methylation status in placentas and trophoblast cell lines were assessed by immunoblotting and immunohistochemistry. RESULTS: Expression profile of histone methyltransferases in the placentas using quantitative RT-PCR revealed that the mRNA expression levels of histone 3 lysine 4 (H3K4) methyltransferases, SETD1A and SMYD3, were significantly increased in placentas from PE patients. Immunoblotting and immunohistochemistry revealed that not only protein expression levels of SETD1A and SMYD3, but also H3K4 methylation status was increased in the trophoblasts from PE placentas. In vitro studies using HTR-8/SV-neo and BeWo cells showed that hypoxia induced the expression levels of SETD1A and SMYD3, and subsequently enhanced H3K4 methylation. Furthermore, the overexpression of SETD1A and SMYD3 in HTR-8/SV-neo cells enhanced H3K4 methylation in response to hypoxia. DISCUSSION: Our study results suggest that placental epigenetic alteration by enhanced histone H3K4 methylation through upregulated SETD1A and SMYD3 might play a role in the pathophysiological process of PE associated with hypoxia.

Laboratory testing for ADAMTS13: Utility for TTP diagnosis/exclusion and beyond.

The metalloproteinase ADAMTS13 (a disintegrin with a thrombospondin type 1 motif, member 13), also known as VWF (von Willebrand factor) protease, may be assessed in a vast array of clinical conditions. Notably, a severe deficiency of ADAMTS13 characterizes TTP (thrombotic thrombocytopenic purpura), a rare but potentially fatal disorder associated with thrombosis due to accumulation of prothrombotic ultra-large VWF multimers. Although prompt identification/exclusion of TTP can be facilitated by rapid ADAMTS13 testing, the most commonly utilized assays are based on ELISA (enzyme linked immunosorbent assay) and require long turnaround time and have relatively limited throughput. Nevertheless, several rapid ADAMTS13 assays are now available, at least in select geographies. The current mini-review discusses these issues, as well as the potential utility of ADAMTS13 testing in a range of other conditions, including coronavirus disease 2019 (COVID-19).

Impaction of factors associated with oxidative stress on the pathogenesis of gestational hypertension and preeclampsia: A Chinese patients based study.

ABSTRACT: We aimed to investigate the effect of Kelch-like ECH-associated protein 1/NF-E2 p45-related factor 2 (Keap1/Nrf2) pathway on the biological function of trophoblast cells in oxidative stress model at the cellular level, and analyzed the expression level and clinical significance of Keap1/Nrf2 pathway and related antioxidant factors in placental tissues of Preeclampsia (PE) patients at clinical level. In present study, we found that under hypoxia/reoxygenation conditions, the activity of oxidative stress-related enzymes (CAT, GSH-Px, SOD) in HTR8/SVneo cells was significantly lower than that before treatment (P < .01). The activities of CAT, GSH-Px and SOD in HTR8/SVneo cells in SiRNA+H/R group decreased significantly (P < .01), indicating the important defense effect of Keap1/Nrf2 signaling pathway in oxidative stress. As a control group of Nrf2 SiRNA+H/R group, Si-NC+H/R group had CAT, GSH-Px and SOD activities decreasing, which was similar to that in H/R group. Moreover, the activities of oxidative stress-related active enzymes in patients with PE were further confirmed by detecting and comparing the activities of CAT, GSH-Px and SOD in placental tissues. The results showed that the activity of SOD (P < .001), GSH-Px (P < .01) and CAT (P < .01) in placental tissues of patients with PE were significant different from those of normal placental tissues. The expression level of Keap1 in placenta of patients with PE was slightly lower than that of normal placenta. While the expression of Nrf2 in placenta of patients with PE was significantly higher than that of normal placenta. HO-1 expression in placenta of patients with PE was significantly higher than that of normal placenta. These results implicate the importance of Keap-1/Nrf2 pathway in PE.

Energy deficiency caused by CTPS downregulation in decidua may contribute to pre-eclampsia by impairing decidualization.

Pre-eclampsia (PE) is a pregnancy-related disorder that occurs after 20 weeks of gestation. It seriously affects the health of maternity and the fetus. However, the pathogenesis of PE is still unknown. Decidualization deficiency is considered a contributing factor to the development of PE. CTP synthetase (CTPS) which is the rate-limiting enzyme in the CTP de novo biosynthesis, is essential for nucleic acid synthesis and cellular energy metabolism, and often appears as cytoophidium in many cell types. Here, we found that the expression of CTPS was significantly downregulated in decidual tissues of patients with severe PE compared with healthy pregnant women. During in vitro decidualization, changes in CTPS were accompanied by opposite fluctuation of the AMPK signaling pathway. Moreover, the downregulation of CTPS by glutamine analogs or CTPS small interfering RNA inhibited the decidualization process and the AMPK signaling pathway. Investigating the underlying mechanism of action by co-immunoprecipitation coupled with mass spectrometry showed that CTPS interacted with ATP synthase (ATPS) and maintained the content of ATP on Day 3 of decidualization. However, when combined with mitochondrial stress protein STRESS-70 instead of ATPS, the concentration of ATP on Day 6 of induction was reduced. Corresponding to this, CTPS was mainly distributes in the cytoplasm on Day 3 of induction, while it appeared both in the cytoplasm and the nucleus on Day 6 in decidualized cells, which was similar to that in cells before induction. In summary, we believe that CTPS plays an important role in decidualization by participating in energy metabolism. Abnormal expression of CTPS in decidualization would lead to abnormal decidualization and consequently result in the occurrence of PE.

Nesfatin-1 Promotes Proliferation, Migration and Invasion of HTR-8/SVneo Trophoblast Cells and Inhibits Oxidative Stress via Activation of PI3K/AKT/mTOR and AKT/GSK3ß Pathway.

Preeclampsia (PE) is a leading cause of perinatal and maternal mortality. Considering that Nesfatin-1 was reported to be downregulated in serum of PE patients, we aimed to explore the functional role of Nesfatin-1 in trophoblast cells. Cell transfection was conducted to overexpress or inhibit Nesfatin-1, and its expression was measured by quantitative PCR. Cell proliferation, migration, and invasion abilities were determined employing CCK-8, flow cytometry, wound-healing, and transwell assays. Immunofluorescence assay was performed to detect E-cadherin and vimentin. ROS, MDA, and SOD levels were measured using their corresponding commercial kits. Western blot was used to identify the expression of vital kinases in PI3K/AKT/mTOR or GSK3ß pathway and invasion-related proteins in trophoblast cells. Nesfatin-1 knockdown significantly suppressed proliferation, migration, and invasion and increased cell arrest in G1 phase, as well as downregulated expressions of MMP2/9 in HTR-8/SVneo cells. Besides, Nesfatin-1 knockdown promoted the expression of E-cadherin and reduced the expression of vimentin. Additionally, the levels of ROS, MDA, and SOD were elevated upon Nesfatin-1 knockdown. On the contrary, Nesfatin-1 overexpression exerted the opposite effects. Nesfatin-1 promoted the activation of PI3K/AKT/mTOR or GSK3ß pathway, blocking of which reversed the promotive effects on trophoblast invasion and the inhibitory effects on oxidative stress of Nesfatin-1 in HTR-8/SVneo cells. In short, this study revealed that Nesfatin-1 promoted trophoblast cell proliferation, migration, invasion, and EMT and suppressed oxidative stress by activating PI3K/AKT/mTOR and AKT/GSK3ß signaling pathway, laying the foundation for the development of therapeutic strategy for PE by targeting Nesfatin-1.

HDAC4 Knockdown Induces Preeclampsia Cell Autophagy and Apoptosis by miR-29b.

Preeclampsia (PE) is one of the main causes of maternal death and perinatal morbidity and mortality. Considering that histone deacetylase 4 (HDAC4) activity could relate to trophoblast cell motility and be antagonized by miR-29b, the aim of the present study was to investigate the ability of HDAC4 to regulate placental trophoblast cells by miR-29b. We assessed the cytological changes of PE patients, and the expression and cellular localization of HDAC4 and LC3 by histological analysis, immunohistochemistry, western blot assay, and immunofluorescence staining assay. We observed the effect of hypoxia on HDAC4, the correction of HDAC4/miR-29b, and the effects of HDAC4/miR-29b on HTR8 cells by dual-luciferase, quantitative real-time PCR, western blot assay, and flow cytometry assay. Here, we first found that HDAC4 was lowly expressed in PE tissues, while LC3 was highly expressed. In addition, the expression of HDAC4 was inhibited by hypoxia in HTR8 cells. Furthermore, our data showed that HDAC4 activity could be antagonized by miR-29b. We highlighted that miR-29b specifically targeted HDAC4 in trophoblast cells and both molecules were involved in a functional loop. Altogether, our findings demonstrated that silencing of HDAC4 could trigger cell autophagy and apoptosis directly by miR-29b in placental trophoblast cells of PE.