Eucommia granules activate Wnt/ß-catenin pathway, and improve oxidative stress, inflammation, and endothelial injury in preeclampsia rats.

PURPOSE: Pre-eclampsia (PE) is a pregnancy-related complication. Eucommia is effective in the treatment of hypertensive disorders in pregnancy, but the specific effects and possible mechanisms of Eucommia granules (EG) in PE remain unknown. The aim of this study was to investigate the effects and possible mechanisms of EG in PE rats. METHODS: Pregnant Sprague Dawley rats were divided into five groups (n = 6): the control group, the model group, the low-dose group, the medium-dose group, and the high-dose group of EG. The PE model was established by subcutaneous injection of levonitroarginine methyl ester. Saline was given to the blank and model groups, and the Eucommia granules were given by gavage to the remaining groups. Blood pressure and urinary protein were detected. The body length and weight of the pups and the weight of the placenta were recorded. Superoxide dismutase (SOD) activity and levels of malondialdehyde (MDA), placental growth factor (PIGF), and soluble vascular endothelial growth factor receptor-1 (sFIt-1) were measured in the placenta. Pathological changes were observed by hematoxylin-eosin staining. Wnt/ß-catenin pathway-related protein expression was detected using Western blot. RESULTS: Compared with the model group, the PE rats treated with EG had lower blood pressure and urinary protein. The length and weight of the pups and placental weight were increased. Inflammation and necrosis in the placental tissue was improved. SOD level increased, MDA content and sFIt-1/PIGF ratio decreased, and Wnt/ß-catenin pathway-related protein expression level increased. Moreover, the results of EG on PE rats increased with higher doses of EG. CONCLUSIONS: EG may activate the Wnt/ß-catenin pathway and inhibit oxidative stress, inflammation, and vascular endothelial injury in PE rats, thereby improving the perinatal prognosis of preeclamptic rats. EG may inhibit oxidative stress, inflammation, and vascular endothelial injury through activation of the Wnt/ß-catenin pathway in preeclampsia rats, thereby improving perinatal outcomes in PE rats.

IL-33 supplementation improves uterine artery resistance and maternal hypertension in response to placental ischemia.

Preeclampsia (PE), a leading cause of maternal/fetal morbidity and mortality, is a hypertensive pregnancy disorder with end-organ damage that manifests after 20 wk of gestation. PE is characterized by chronic immune activation and endothelial dysfunction. Clinical studies report reduced IL-33 signaling in PE. We use the Reduced Uterine Perfusion Pressure (RUPP) rat model, which mimics many PE characteristics including reduced IL-33, to identify mechanisms mediating PE pathophysiology. We hypothesized that IL-33 supplementation would improve blood pressure (BP), inflammation, and oxidative stress (ROS) during placental ischemia. We implanted intraperitoneal mini-osmotic pumps infusing recombinant rat IL-33 (1 µg/kg/day) into normal pregnant (NP) and RUPP rats from gestation day 14 to 19. We found that IL-33 supplementation in RUPP rats reduces maternal blood pressure and improves the uterine artery resistance index (UARI). In addition to physiological improvements, we found decreased circulating and placental cytolytic Natural Killer cells (cNKs) and decreased circulating, placental, and renal TH17s in IL-33-treated RUPP rats. cNK cell cytotoxic activity also decreased in IL-33-supplemented RUPP rats. Furthermore, renal ROS and placental preproendothelin-1 (PPET-1) decreased in RUPP rats treated with IL-33. These findings demonstrate a role for IL-33 in controlling vascular function and maternal BP during pregnancy by decreasing inflammation, renal ROS, and PPET-1 expression. These data suggest that IL-33 may have therapeutic potential in managing PE.NEW & NOTEWORTHY Though decreased IL-33 signaling has been clinically associated with PE, the mechanisms linking this signaling pathway to overall disease pathophysiology are not well understood. This study provides compelling evidence that mechanistically links reduced IL-33 with the inflammatory response and vascular dysfunction observed in response to placental ischemia, such as in PE. Data presented in this study submit the IL-33 signaling pathway as a possible therapeutic target for the treatment of PE.

High-Dose Vitamin D Supplementation Significantly Affects the Placental Transcriptome.

Vitamin D deficiency is a highly prevalent obstetrical concern associated with an increased risk of complications like pre-eclampsia, gestational diabetes, and growth retardation. Vitamin D status in pregnancy is also linked to long-term offspring health, e.g., the risk of obesity, metabolic disease, and neurodevelopmental problems. Despite the suspected role of vitamin D in placental diseases and fetal development, there is limited knowledge on the effect of vitamin D on placental function. Thus, we performed next-generation RNA sequencing, comparing the placental transcriptome from uncomplicated term pregnancies receiving the often-recommended dose of 10 µg vitamin D/day (n = 36) with pregnancies receiving 90 µg/day (n = 34) from late first trimester to delivery. Maternal vitamin D status in the first trimester was also considered. We found that signaling pathways related to cell adhesion, immune function, and neurodevelopment were affected, supporting that increased vitamin D supplementation benefits placental function in established pregnancies without severe vitamin D deficiency, also underlining the importance of vitamin D in brain development. Specific effects of the first trimester vitamin D status and offspring sex were also identified. Further studies are warranted, addressing the optimal vitamin status during pregnancy with a focus on organ-specific vitamin D needs in individual pregnancies.

Erythrocyte fatty acids and desaturase indices in early pregnancy are associated with risk of preeclampsia.

Preeclampsia (PE) is a pregnancy disorder that may be associated with inadequate maternal nutrition. Fatty acids are vital for placental and fetal growth. Fatty acid desaturases, key enzymes influencing the metabolism of polyunsaturated fatty acids, are reported to be associated with cardiometabolic risk. Any imbalance in the levels of omega-3 and omega-6 fatty acids can result in increased inflammatory response. The current study reports the levels of erythrocyte fatty acids and desaturase index across gestation in women who develop PE (n = 108) and compares them with non-PE women (n = 216). Maternal erythrocyte fatty acids were measured at 4 time points during pregnancy (i.e., 11-14, 18-22, 26-28 weeks and at delivery) using gas chromatography. Maternal total erythrocyte saturated fatty acids and omega-6/omega-3 fatty acid ratio was higher in the PE group as compared to the non-PE group at 11-14 weeks and 18-22 weeks respectively. Maternal Δ5 desaturase index was lower while Δ6 desaturase index was higher in the PE group at 11-14 and 18-22 weeks. Maternal stearoyl CoA desaturase-18 (SCD-18) index was lower at 11-14 weeks and at delivery. These changes were mainly observed in the early onset PE (EOP) group. Δ6 desaturase index at 11-14 weeks predicted the risk of EOP. Imbalance in fatty acid levels and desaturase indices predate the clinical diagnosis of PE, indicating their role in its pathophysiology. Measurement of fatty acids and desaturase indices in early pregnancy merits evaluation as predictors of risk of PE.

Pathophysiology of Preeclampsia and L-Arginine/L-Citrulline Supplementation as a Potential Strategy to Improve Birth Outcomes.

In preeclampsia, the shallow invasion of cytotrophoblast cells to uterine spiral arteries, leading to a reduction in placental blood flow, is associated with an imbalance of proangiogenic/antiangiogenic factors to impaired nitric oxide (NO) production. Proangiogenic factors, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), require NO to induce angiogenesis through antioxidant regulation mechanisms. At the same time, there are increases in antiangiogenic factors in preeclampsia, such as soluble fms-like tyrosine kinase type 1 receptor (sFIt1) and toll-like receptor 9 (TLR9), which are mechanism derivates in the reduction of NO bioavailability and oxidative stress in placenta.Different strategies have been proposed to prevent or alleviate the detrimental effects of preeclampsia. However, the only intervention to avoid the severe consequences of the disease is the interruption of pregnancy. In this scenario, different approaches have been analysed to treat preeclamptic pregnant women safely. The supplementation with amino acids is one of them, especially those associated with NO synthesis. In this review, we discuss emerging concepts in the pathogenesis of preeclampsia to highlight L-arginine and L-citrulline supplementation as potential strategies to improve birth outcomes. Clinical and experimental data concerning L-arginine and L-citrulline supplementation have shown benefits in improving NO availability in the placenta and uterine-placental circulation, prolonging pregnancy in patients with gestational hypertension and decreasing maternal blood pressure.

Association between NET-1 Level in Placenta from Preeclampsia Pregnancies and Trophoblast Proliferation and Apoptosis: A Correlation Analysis.

Objective: This study investigates the expression pattern of neuroepithelial cell transforming 1 (NET-1) in placental tissues from pregnancies with preeclampsia (PE) and explores its role in mediating proliferative and apoptotic capacities of trophoblasts. Methods: The relative mRNA levels of NET-1 in placental tissues obtained from preeclampsia (PE) pregnancies (n = 60) and healthy pregnancies (n = 60) were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Pearson correlation test was conducted to assess the correlation between NET-1 level and systolic (Sp) and diastolic pressure (Dp) in PE pregnancies. After the knockdown of NET-1 in HTR-8/SVneo cells, changes in proliferation and apoptosis were examined using cell counting kit-8 (CCK-8) and flow cytometry, respectively. Results: NET-1 was highly expressed in placental tissues from PE pregnancies. PE patients with a high level of NET-1 had higher Sp and Dp, and NET-1 level was positively correlated with both Sp and Dp. Knockdown of NET-1 in HTR-8/SVneo cells decreased the proliferative rate but increased the apoptotic rate. Conclusions: NET-1 stimulates the development of PE by triggering trophoblast proliferation and inhibiting apoptosis. Therefore, NET-1 could be a potential therapeutic target for treating PE and other related hypertensive disorders during pregnancy.

Vitamin D regulates microflora and ameliorates LPS-induced placental inflammation in rats.

Preeclampsia is a pregnancy-specific disease, which has become an essential cause of perinatal and neonatal death. Gut microflora becomes the regulator of host immunity through the metabolic pathway. Epidemiological studies provide convincing evidence that vitamin D supplementation can prevent the onset of preeclampsia. However, research on the microbial mechanisms and effective treatment strategies for placental inflammation induced by lipopolysaccharide is lacking. In this study, pregnant rats were induced by LPS to establish a rat model of preeclampsia. Sixteen-sequence analysis was used to determine the composition of microflora in feces. In addition, the protective effect of vitamin D supplementation on LPS-preeclampsia rats was evaluated. The results showed that the blood pressure and creatinine of pregnant rats in the LPS group were significantly higher than those in the control group. In addition, LPS disturbed the intestinal microbial community and reduced microbial diversity. Vitamin D supplementation improves the symptoms of preeclampsia, increases the abundance of intestinal beneficial flora, normalizes the level of inflammatory factors LPS-induced by inhibiting the TLR4/MYD88/NF-κB pathway, and effectively resists the disturbance of uterine spiral artery remodeling induced by LPS. This study established that vitamin D-mediated microbial mechanisms and their inhibition are potential therapeutic targets for the treatment of preeclampsia.

Dietary supplements and vascular function in hypertensive disorders of pregnancy.

Hypertensive disorders of pregnancy are complications that can lead to maternal and infant mortality and morbidity. Hypertensive disorders of pregnancy are generally defined as hypertension and may be accompanied by other end organ damages including proteinuria, maternal organ disturbances including renal insufficiency, neurological complications, thrombocytopenia, impaired liver function, or uteroplacental dysfunction such as fetal growth restriction and stillbirth. Although the causes of these hypertensive disorders of pregnancy are multifactorial and elusive, they seem to share some common vascular-related mechanisms, including diseased spiral arteries, placental ischemia, and endothelial dysfunction. Recently, preeclampsia is being considered as a vascular disorder. Unfortunately, due to the complex etiology of preeclampsia and safety concerns on drug usage during pregnancy, there is still no effective pharmacological treatments available for preeclampsia yet. An emerging area of interest in this research field is the potential beneficial effects of dietary intervention on reducing the risk of preeclampsia. Recent studies have been focused on the association between deficiencies or excesses of some nutrients and complications during pregnancy, fetal growth and development, and later risk of cardiovascular and metabolic diseases in the offspring. In this review, we discuss the involvement of placental vascular dysfunction in preeclampsia. We summarize the current understanding of the association between abnormal placentation and preeclampsia in a vascular perspective. Finally, we evaluate several studied dietary supplementations to prevent and reduce the risk of preeclampsia, targeting placental vascular development and function, leading to improved pregnancy and postnatal outcomes.

Alterations in Antioxidant Micronutrient Concentrations in Placental Tissue, Maternal Blood and Urine and the Fetal Circulation in Pre-eclampsia.

Trace elements such as selenium and zinc are vital components of many enzymes, including endogenous antioxidants, and can interact with each other. Women with pre-eclampsia, the hypertensive disease of pregnancy, have been reported as having changes in some individual antioxidant trace elements during pregnancy, which are related to maternal and fetal mortality and morbidity. We hypothesised that examination of the three compartments of (a) maternal plasma and urine, (b) placental tissue and (c) fetal plasma in normotensive and hypertensive pregnant women would allow identification of biologically significant changes and interactions in selenium, zinc, manganese and copper. Furthermore, these would be related to changes in the angiogenic markers, placental growth factor (PlGF) and Soluble Fms-Like Tyrosine Kinase-1 (sFlt-1) concentrations. Venous plasma and urine were collected from healthy non-pregnant women (n = 30), normotensive pregnant controls (n = 60) and women with pre-eclampsia (n = 50) in the third trimester. Where possible, matched placental tissue samples and umbilical venous (fetal) plasma were also collected. Antioxidant micronutrient concentrations were measured by inductively coupled plasma mass-spectrometry. Urinary levels were normalised to creatinine concentration. Plasma active PlGF and sFlt-1 concentrations were measured by ELISA. Maternal plasma selenium, zinc and manganese were all lower in women with pre-eclampsia (p < 0.05), as were fetal plasma selenium and manganese (p < 0.05 for all); maternal urinary concentrations were lower for selenium and zinc (p < 0.05). Conversely, maternal and fetal plasma and urinary copper concentrations were higher in women with pre-eclampsia (p < 0.05). Differences in placental concentrations varied, with lower overall levels of selenium and zinc (p < 0.05) in women with pre-eclampsia. Maternal and fetal PlGF were lower and sFlt-1 higher in women with pre-eclampsia; maternal plasma zinc was positively correlated with maternal plasma sFlt-1 (p < 0.05). Because of perceptions that early- and late-onset pre-eclampsia have differing aetiologies, we subdivided maternal and fetal data accordingly. No major differences were observed, but fetal sample sizes were small following early-onset. Disruption in these antioxidant micronutrients may be responsible for some of the manifestations of pre-eclampsia, including contributing to an antiangiogenic state. The potential benefits of mineral supplementation, in women with deficient intakes, during pregnancy to reduce pre-eclampsia remain an important area for experimental and clinical research.

Mechanism of astragalus injection to relieve symptoms of preeclampsia rat model by inhibiting MMP-9/sFlt-1/TNF-α.

Objective: The aim of this study was to observe the effect of astragalus injection on rats with preeclampsia. Methods: A total of 30 pregnant Sprague Dawley (SD) rats were randomly assigned to the model group (MG), the astragalus group (AG) or the control group (CG), with 10 rats in each group. The rat model of preeclampsia was established by subcutaneous injection of 50 mg/(kg∙d) of N-nitro-L-arginine methyl ester (L-NAME), and 0.024 ml/(g∙d) astragalus injection was administered intraperitoneally. The arterial pressure, urinary protein, placental mass, fetal weight, inflammatory factors in peripheral blood of pregnant rats, protein and mRNA levels of nuclear factor- κB (NF-κB), matrix metalloproteinase-9 (MMP-9), nuclear transcription factor 5 (NFAT-5), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and reactive oxygen species (ROS) activity, malondialdehyde (MDA) and nitric oxide (NO) levels in placental tissues were compared in the 3 groups. Results: After treatment, the arterial pressure and urinary protein levels in pregnant rats in the MG group were significantly higher than in the CG and AG groups (P < .05). The placental mass in the MG group was lower than in the CG and AG groups (P < .05). The messenger RNA (mRNA) and protein levels of sFlt-1, NFAT-5 and NF-κB, as well as ROS activity, MDA, inerleukin (IL)-6, tumor necrosis factor alpha (TNF-α) and interferon gamma (INF-γ) in the AG group were significantly lower than in the MG group, and mRNA and protein expression of MMP-9 and PlGF, as well as the NO level in the AG group, were significantly higher than in the MG (P < .05). Conclusions: Astragalus injection can effectively inhibit the expression of sFlt-1, NFAT-5, NF-κB and enhance the expression of PlGF and MMP-9 in the placental tissue of rats with preeclampsia, which may be the mechanism of preeclampsia treatment.

LC-MS/MS based untargeted lipidomics uncovers lipid signatures of late-onset preeclampsia.

The mechanisms underlying late-onset preeclampsia (LOPE) remain unknown. Metabolic disturbances have been implicated as a primary factor in LOPE development. Lipids have been shown to have great clinical value in recent years. This study aimed to use lipidomics to provide evidence for the etiology and potential therapeutic approaches for LOPE. Twenty patients with LOPE and 20 healthy controls were enrolled in this study. Placental lipidomic data were acquired using liquid chromatographymass spectrometry (LC-MS/MS), and the data were analyzed by weighted gene correlation network analysis (WGCNA) and statistical methods. Of 1508 identified lipids, 226 were differentially expressed between the LOPE and control groups. In the LOPE group, the abundance of most unsaturated triglycerides (TG) increased, whereas that of other lipids, including phosphatidylcholine (PC), sphingomyelin, and phosphatidylserine (PS) increased. The WGCNA implied that the correlation network module of lipids was highly related to clinical traits. Pathway analysis revealed that these dysregulated lipids are closely related to glycerophospholipid metabolism. Lipidomics may help identify the pathogenesis underlying placental dysfunction in LOPE patients and provide potential therapeutic targets in the future.

Puerariae lobatae Radix Alleviates Pre-Eclampsia by Remodeling Gut Microbiota and Protecting the Gut and Placental Barriers.

Pre-eclampsia (PE) is a serious pregnancy complication, and gut dysbiosis is an important cause of it. Puerariae lobatae Radix (PLR) is a medicine and food homologous species; however, its effect on PE is unclear. This study aimed to investigate the efficacy of PLR in alleviating PE and its mechanisms. We used an NG-nitro-L-arginine methyl ester (L-NAME)-induced PE mouse model to examine the efficacy of preventive and therapeutic PLR supplementation. The results showed that both PLR interventions alleviated hypertension and proteinuria, increased fetal and placental weights, and elevated the levels of VEGF and PlGF. Moreover, PLR protected the placenta from oxidative stress via activating the Nrf2/HO-1/NQO1 pathway and mitigated placental damage by increasing intestinal barrier markers (ZO-1, Occludin, and Claudin-1) expression and reducing lipopolysaccharide leakage. Notably, preventive PLR administration corrected gut dysbiosis in PE mice, as evidenced by the increased abundance and positive interactions of beneficial bacteria including Bifidobacterium, Blautia, and Turicibacter. Fecal microbiota transplantation confirmed that the gut microbiota partially mediated the beneficial effects of PLR on PE. Our findings revealed that modulating the gut microbiota is an effective strategy for the treatment of PE and highlighted that PLR might be used as an intestinal nutrient supplement in PE patients.

Attenuated effect of zinc gluconate on oxidative stress, inflammation, and angiogenic imbalance in pre-eclampsia rats.

AIMS: Pre-eclampsia (PE) is a common obstetric disease associated with oxidative stress, systemic inflammation, and angiogenic imbalance, whereas zinc (Zn) presents anti-oxidative and anti-inflammatory effects. This study is to investigate whether zinc gluconate (ZG) supplementation may ameliorate the early signs, adverse pregnancy outcomes, and pathogenic processes of PE in an animal model. MAIN METHODS: Forty pregnant Wistar rats were randomly divided into four groups: blank control (treated with normal saline, NS), Zn control (treated with ZG and followed by NS), PE model (treated with NS and followed by nitro-L-arginine methyl ester, L-NAME), and PE intervention (treated with ZG and followed by L-NAME). ZG (5 mg/kg/day) or NS was administered by gavage from day 0 to 19 of gestation, and L-NAME (80 mg/kg/day) or NS was subcutaneously injected from day 4 to 19 of gestation. The blood pressure, urinary protein, and pregnancy outcomes were recorded. Oxidative stress, inflammation, and angiogenic homeostasis were evaluated. KEY FINDINGS: PE rats exhibited oxidative stress (reduced SOD, CAT, and GSH, and increased MDA and 3-NT), inflammation (increased IL-6 and TNF-α), and angiogenic imbalance (reduced VEGF and PlGF, and increased sFlt-1). After intervention with ZG, the blood pressure and urinary protein levels were reverted, and the pregnancy outcomes were improved. The oxidative stress, inflammation, and angiogenic imbalance were effectively restored in accompany by increased Zn and MT levels. SIGNIFICANCE: ZG can ameliorate the early signs and pathological processes of PE in the animal model, indicating the value of zinc supplementation during pregnancy for PE prevention.

Cadmium-induced preeclampsia-like phenotype in the rat is related to decreased progesterone synthesis in the placenta.

Cadmium (Cd) is a toxic element, which is associated with preeclampsia (PE).We treated pregnant rats with cadmium chloride from gestational days (GDs) 9-12 to introduce the PE-like animal model. Maternal systolic blood pressures (SBPs) and body weights were measured on GDs 0, 5, 10, 15, and 20. Foetuses were delivered by Caesarean section on GD20. Then, the dams were sacrificed and the specimens were obtained. The morphological analysis of the placentae was carried out by haematoxylin and eosin staining examination and immunohistochemistry assay.Our study showed that Cd-treated rats developed PE-like phenotypes, such as hypertension, albuminuria, and foetal growth restriction. Moreover, Cd-injected rats displayed abnormal placental angiogenesis and lower progesterone (P4) levels. We further demonstrated that Cd also inhibited the mRNA and protein expressions of steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A1), and 3ß-hydroxysteroid dehydrogenase 1 (3ß-HSD1), which are involved in P4 synthesis in the rat placenta.Our study demonstrates that maternal Cd exposure disrupts the local synthesis of P4 in the placenta, which contributes to the onset of PE in pregnant rats. Supplementing P4 at the early gestational stage may be a promising therapeutic strategy to prevent PE, which requires further investigation.

Inflammation in Preeclampsia: Genetic Biomarkers, Mechanisms, and Therapeutic Strategies.

Objective: Preeclampsia is a common and serious complication of pregnancy, posing a threat to maternal and fetal safety due to the lack of effective biomarkers and treatment strategies. This study aimed to identify potential biomarkers that can be used to predict preeclampsia and identify the molecular mechanisms of preeclampsia pathogenesis and drug prediction at the transcriptome level. Methods: We analyzed differential expression genes (DEGs) in preeclampsia and non-preeclampsia groups in the GSE75010 dataset, cross-linking with extracted inflammatory response-related genes to obtain differentially expressed inflammation-related genes (DINRGs). Enrichment analysis and protein-protein interaction (PPI) networks were constructed to understand the functions and enrichment pathways. Machine learning models were used to identify key genes associated with preeclampsia and build a nomogram in the training set, which was validated in the validation set. The R package RcisTarget was used to predict transcription factors, and Cytoscape was used to construct miRNA-mRNA pathways, which could identify the molecular mechanisms. Then, we conducted molecular docking of the obtained key genes INHBA (inhibin subunit beta A), OPRK1 (opioid receptor kappa 1), and TPBG (trophoblast glycoprotein), as well as predicted transcription factors with drug molecules. Additionally, the CIBERSORT method explored the differences in immune cell infiltration between preeclampsia and non-preeclampsia samples based on the GSE75010 dataset. Results: A total of 69 DINRGs associated with preeclampsia patients were screened. INHBA, OPRK1, and TPBG were the key genes based on machine learning models. A nomogram for prediction was further constructed, and the receiver operating curves (ROCs) showed good performance. Based on the transcriptome level of key genes, we proposed that RELA-miR-548K/miR-1206-TPBG may be a potential RNA regulatory pathway regulating the progression of early preeclampsia. Molecular docking suggested the effectiveness of curcumin in the treatment of preeclampsia. Additionally, regulatory T cells (Tregs) and resting mast cells were significantly different between the two groups. Conclusion: In summary, we identified three key inflammation-associated genes, namely INHBA, OPRK1, and TPBG, which can be used as potential genetic biomarkers for preeclampsia prediction and treatment, and established a nomogram as a predictive model. Additionally, we provided insights into the mechanisms of preeclampsia development at the transcriptome level and performed corresponding drug predictions.

Vitamin D Supplementation Improves Mitochondrial Function and Reduces Inflammation in Placentae of Obese Women.

Background: About 30% of women entering pregnancy in the US are obese. We have previously reported mitochondrial dysregulation and increased inflammation in the placentae of obese women. Vitamin D (VitD) is a major player in calcium uptake and was shown to modulate mitochondrial respiration and the immune/inflammation system. Studies show decreased VitD levels in obese individuals; however, the effect of maternal obesity on VitD metabolism and its association with placental function remains understudied. Methods: Maternal and cord blood plasma and placental samples were collected upon C-section from normal-weight (NW, body mass index [BMI]<25) and obese (OB, BMI>30) women with uncomplicated pregnancies at term. We measured 25(OH)D3 (calcidiol) levels in maternal and cord blood plasma using ELISA. We assessed the expression of CYP27B1, an activator of calcidiol, and Vitamin D receptor (VDR) in placentae from NW and OB, and women with gestational diabetes and preeclampsia. In addition, we examined the effects of VitD supplementation on mitochondrial function and inflammation in trophoblasts from NW and OB, using the Seahorse Bioanalyzer and Western blot, respectively. Results: Vitamin D levels in blood from OB but not NW women and in cord blood from babies born to NW and OB women showed a significant inverse correlation with maternal pre-pregnancy BMI (r=-0.50, p<0.1 and r=-0.55, p=0.004 respectively). Cord plasma VitD levels showed a positive correlation with placental efficiency, i.e., the ratio between fetal and placental weight, as well as with maternal blood VitD levels (r=0.69 and 0.83 respectively, p<0.00). While we found no changes in CYP27B1 in OB vs. NW women, VDR expression were decreased by 50% (p<0.03) independent of fetal sex. No changes in VDR expression relative to BMI-matched controls were observed in the placentae of women with gestational diabetes or preeclampsia. Cytotrophoblasts isolated from placentae of OB women showed a dose-dependent increase in VDR expression after 24-hour treatment with calcitriol (10 nM and 100 nM), an active form of VitD. Trophoblasts isolated from OB women and treated with calcitriol improved mitochondrial respiration (p<0.05). We also found a two-fold increase in expression of the NLRP3 inflammasome and the pro-inflammatory cytokine IL-18 in trophoblasts isolated from placentae of OB women (p<0.05), with IL-18 expression being reversed by calcitriol treatment (100 nM). Conclusions: We show that VitD deficiency is at least partially responsible for mitochondrial dysfunction and increased inflammation in the placentae of obese women. Vitamin D supplementation could be beneficial in improving placental dysfunction seen in obese women.

Mast cell-derived exosomal miR-181a-5p modulated trophoblast cell viability, migration, and invasion via YY1/MMP-9 axis.

BACKGROUND: Mast cells regulate the process of preeclampsia (PE). Since we previously identified mast cells specifically expressing miR-181a-5p in the placenta of PE patients, it is plausible to examine the effect and mechanism of mast cell-derived exosomal miR-181a-5p on trophoblast cells. METHODS: The miR-181a-5p and YY1 levels were determined by quantitative real-time reverse transcription-polymerase chain reaction. Exosomes were identified by transmission electron microscopy, Western blot, and PKH-26 labeling. Mast cells or trophoblast cell malignant phenotype were detected using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, wound healing, and Transwell assays. Quantification of YY1 and metastasis-related proteins was performed using Western blot. TargetScan, JASPAR, dual-luciferase reporter genes, and chromatin immunoprecipitation were exploited to verify the relationship between miR-181a-5p, YY1, and MMP-9. RESULTS: MiR-181a-5p was overexpressed in mast cells of PE patients. Overexpressed miR-181a-5p restrained mast cell viability. Mast cell exosomes were successfully isolated, containing high expressions of CD63 and HSP70 and low expression of Calnexin and could be transported to the cytoplasm of trophoblast cells. Mast cell exosomes attenuated the viability, migration, and invasion of HTR-8/SVneo cells, inhibited YY1, N-cadherin, Vimentin, and MMP-9 protein expressions, and promoted E-cadherin protein expression. The effect of exosomes was enhanced by miR-181a-5p mimic but was reversed by miR-181a-5p inhibitor. MiR-181a-5p targeted YY1 which bound to the MMP-9 promoter. Overexpressed YY1 in HTR-8/SVneo cells accelerated the malignant phenotype of the cells and reversed the regulatory effects of exosomal miR-181a-5p. CONCLUSION: Mast cell-derived exosomal miR-181a-5p modulates HTR-8/SVneo cell viability, migration, and invasion via YY1/MMP-9.

Red Light Mitigates the Deteriorating Placental Extracellular Matrix in Late Onset of Preeclampsia and Improves the Trophoblast Behavior.

Preeclampsia is a serious pregnancy disorder which in extreme cases may lead to maternal and fetal injury or death. Preexisting conditions which increase oxidative stress, e.g., hypertension and diabetes, increase the mother's risk to develop preeclampsia. Previously, we established that when the extracellular matrix is exposed to oxidative stress, trophoblast function is impaired, and this may lead to improper placentation. We investigated how the oxidative ECM present in preeclampsia alters the behavior of first trimester extravillous trophoblasts. We demonstrate elevated levels of advanced glycation end products (AGE) and lipid oxidation end product 4-hydroxynonenal in preeclamptic ECM (28%, and 32% increase vs control, respectively) accompanied with 35% and 82% more 3-chlorotyrosine and 3-nitrotyrosine vs control, respectively. Furthermore, we hypothesized that 670 nm phototherapy, which has antioxidant properties, reverses the observed trophoblast dysfunction as depicted in the improved migration and reduction in apoptosis. Since NO is critical for placentation, we examined eNOS activity in preeclamptic placentas compared to healthy ones and found no differences; however, 670 nm light treatment triggered enhanced NO availability presumably by using alternative NO sources. Light exposure decreased apoptosis and restored trophoblast migration to levels in trophoblasts cultured on preeclamptic ECM. Moreover, 670 nm irradiation restored expression of Transforming Growth Factor (TGFß) and Placental Growth Factor (PLGF) to levels observed in trophoblasts cultured on healthy placental ECM. We conclude the application of 670 nm light can successfully mitigate the damaged placental microenvironment of late onset preeclampsia as depicted by the restored trophoblast behavior.

Evidence From Human Placenta, Endoplasmic Reticulum-Stressed Trophoblasts, and Transgenic Mice Links Transthyretin Proteinopathy to Preeclampsia.

BACKGROUND: We have demonstrated that protein aggregation plays a pivotal role in the pathophysiology of preeclampsia and identified several aggregated proteins in the circulation of preeclampsia patients, the most prominent of which is the serum protein TTR (transthyretin). However, the mechanisms that underlie protein aggregation remain poorly addressed. METHODS: We examined TTR aggregates in hypoxia/reoxygenation-exposed primary human trophoblasts (PHTs) and the preeclampsia placenta using complementary approaches, including a novel protein aggregate detection assay. Mechanistic analysis was performed in hypoxia/reoxygenation-exposed PHTs and Ttr transgenic mice overexpressing transgene-encoded wild-type human TTR or Ttr-/- mice. High-resolution ultrasound analysis was used to measure placental blood flow in pregnant mice. RESULTS: TTR aggregation was inducible in PHTs and the TCL-1 trophoblast cell line by endoplasmic reticulum stress inducers or autophagy-lysosomal disruptors. PHTs exposed to hypoxia/reoxygenation showed increased intracellular BiP (binding immunoglobulin protein), phosphorylated IRE1α (inositol-requiring enzyme-1α), PDI (protein disulfide isomerase), and Ero-1, all markers of the unfolded protein response, and the apoptosis mediator caspase-3. Blockade of IRE1α inhibited hypoxia/reoxygenation-induced upregulation of Ero-1 in PHTs. Excessive unfolded protein response activation was observed in the early-onset preeclampsia placenta. Importantly, pregnant human TTR mice displayed aggregated TTR in the junctional zone of the placenta and severe preeclampsia-like features. High-resolution ultrasound analysis revealed low blood flow in uterine and umbilical arteries in human TTR mice compared with control mice. However, Ttr-/- mice did not show any pregnancy-associated abnormalities. CONCLUSIONS: These observations in the preeclampsia placenta, cultured trophoblasts, and Ttr transgenic mice indicate that TTR aggregation is an important causal contributor to preeclampsia pathophysiology.

Differential effects of cyclo-oxygenase 1 and 2 inhibition on angiogenesis inhibitor-induced hypertension and kidney damage.

Vascular endothelial growth factor antagonism with angiogenesis inhibitors in cancer patients induces a 'preeclampsia-like' syndrome including hypertension, proteinuria and elevated endothelin (ET)-1. Cyclo-oxygenase (COX) inhibition with aspirin is known to prevent the onset of preeclampsia in high-risk patients. In the present study, we hypothesised that treatment with aspirin would prevent the development of angiogenesis inhibitor-induced hypertension and kidney damage. Our aims were to compare the effects of low-dose (COX-1 inhibition) and high-dose (dual COX-1 and COX-2 inhibition) aspirin on blood pressure, vascular function, oxidative stress, ET-1 and prostanoid levels and kidney damage during angiogenesis-inhibitor therapy in rodents. To this end, Wistar Kyoto rats were treated with vehicle, angiogenesis inhibitor (sunitinib) alone or in combination with low- or high-dose aspirin for 8 days (n=5-7/group). Our results demonstrated that prostacyclin (PGI2) and ET-1 were increased during angiogenesis-inhibitor therapy, while thromboxane (TXA2) was unchanged. Both low- and high-dose aspirin blunted angiogenesis inhibitor-induced hypertension and vascular superoxide production to a similar extent, whereas only high-dose aspirin prevented albuminuria. While circulating TXA2 and prostaglandin F2α levels were reduced by both low- and high-dose aspirin, circulating and urinary levels PGI2 were only reduced by high-dose aspirin. Lastly, treatment with aspirin did not significantly affect ET-1 or vascular function. Collectively our findings suggest that prostanoids contribute to the development of angiogenesis inhibitor-induced hypertension and renal damage and that targeting the prostanoid pathway could be an effective strategy to mitigate the unwanted cardiovascular and renal toxicities associated with angiogenesis inhibitors.