Preeclampsia Mice Research Articles

Roles and action mechanisms of NRIP1 in pre-eclampsia.

Pre-eclampsia (PE) is characterized by the onset of hypertension and proteinuria during pregnancy. Here, we aimed to explore the functions of nuclear receptor-interacting protein 1 (NRIP1) in PE mice and human placental JEG-3 cells. We evaluated its effects on JEG-3 cell proliferation, apoptosis, invasion, and inflammatory response and regulation of Wnt/β-catenin pathway. NRIP1 levels in human serum and placental tissues, JEG-3 cells, and mouse models were assessed via quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. JEG-3 cell growth, apoptosis, migration, and invasion were evaluated via 5-ethynyl-2'-deoxyuridine, flow cytometry, and transwell assays. Levels of the inflammatory factors, matrix metalloproteinase (MMP)-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-6, were determined via enzyme-linked immunosorbent assay. Wnt/β-catenin pathway was assessed via western blotting and qRT-PCR. Systolic blood pressure and proteinuria were measured using the non-invasive tail cuff method and Coomassie brilliant blue assay, respectively. TdT-mediated dUTP nick-end labeling assay was used to assess cell apoptosis in the placental tissues of PE mice. NRIP1 levels were upregulated in the serum and placental tissues of patients with PE. In vitro experiments revealed that NRIP1-small interfering RNA (siRNA) increased the cell viability, migration, and invasion and reduced the cell apoptosis compared to the control siRNA. Moreover, NRIP1-siRNA activated the Wnt/β-catenin signaling pathway, as indicated by the increased Wnt3a, β-catenin, p-glycogen synthase kinase-3β, c-Myc, and cyclin D1 levels. Levels of the inflammatory factors, IL-6, TNF-α, and MMP-2, were decreased in the NRIP1-siRNA-treated group. Notably, NRIP1 downregulation improved the PE-like symptoms, inhibited the inflammatory responses, and reduced apoptosis in PE mice. This study revealed the crucial roles of NRIP1 in PE. Our findings revealed that NRIP1 downregulation relieved PE symptoms by inhibiting cell proliferation, migration, and invasion via the Wnt/β-catenin pathway, thus providing a novel candidate for PE treatment.

Down-regulation of CORO1C mediated by lncMALAT1/miR-133a-3p axis contributes to trophoblast dysfunction and preeclampsia

IntroductionPlacental trophoblast dysfunction has been proved to be closely related to the pathogenesis of preeclampsia. Coronaryxin-like actin-binding protein 1C (CORO1C) plays an important role in cell proliferation, apoptosis, invasion, and signal transduction, but its involvement in trophoblast dysfunction and preeclampsia remains uncertain. MethodsThe expression of CORO1C in placental tissues of preeclampsia (PE) pregnant women and pregnant mice PE model were detected by real-time quantitative polymerase chain reaction (RT-qPCR), western blotting (WB) and immunohistochemical (IHC) staining. Next, the proliferation, invasion, migration and apoptosis were performed to explore the functions of CORO1C in HTR8/SVneo cell. Furthermore, the expression of CORO1C were detected in lncMALAT1 knockdown and overexpression HTR-8/SVneo cell. And then we investigated the possible regulatory mechanism of lncMALAT1 on CORO1C through bioinformatics analysis, FISH assays, RIP assays, RNA pull down and dual luciferase reporter assays. Finally, we further validated that lncMALAT1 regulate the function of placental trophoblast cells through CORO1C. ResultsThe expression of CORO1C was significantly decreased in the placenta of PE patients and mice model, and positively associated with neonatal birth weight. And we found that CORO1C inhibited trophoblast proliferation, migration and invasion. Furthermore, reduced expression of lncMALAT1 impaired CORO1C level, thereby resulting in trophoblast dysfunction. Mechanistically, the dysregulation of lncMALAT1 promoted the expression of miR-133a-3p, strongly enhancing its binding to the 3′UTR region of CORO1C mRNA for degradation. DiscussionThis study demonstrated that the dysregulation of CORO1C via lncMALAT1/miR-133a-3p axis impairs trophoblast function and contributes to preeclampsia pathogenesis, providing novel insights in PE therapy through modulating CORO1C level.

Abstract 70: Cellular senescence as a driver of preeclampsia

Background: Preeclampsia (PE) is a severe hypertensive disorder of pregnancy of unknown pathophysiology. We previously have reported the presence of senescence in kidney and fat tissue of PE women, as well as accelerated epigenetic aging, which prompted us to postulate that cellular senescence may drive development of PE. Methods: We prospectively collected sera from pregnant women throughout pregnancy, and using multiplex Luminex analysis, measured 50 markers of the senescence-associated secretory phenotype (SASP). Comparisons were made between women who developed PE (n=10) vs. normotensive pregnancies (n=10). We then injected human PE serum into the IL-10KO mouse to induce PE and assess whether PE is associated with development of senescence. We measured plasma p16, a circulating indicator of cellular senescence, before and after inducing PE. We also compared plasma p16 and expressions of senescence-associated genes in the hearts and aortas of regular pregnant (PG, no PE serum injection) and PE mice (n=4). Results: There was a significant increase in several SASP markers in sera of women who developed PE, beginning in the first trimester, through delivery. Notably, eotaxin, MCP1, GROb, MIF, MMP2, TNFRII, IGFBP2 were significantly increased in PE women compared to their normotensive peers throughout all trimesters. Mice injected with PE serum exhibited all features of PE, as reported earlier. When measuring plasma p16 before and after PE serum injection, animals had higher values upon inducing PE. Plasma samples of PE mice demonstrated higher p16 concentrations compared to the PG group. The aortas of PE mice exhibited increased expressions of senescence associated genes p16, il6, and mcp1, whereas in the hearts, p16, Il1a and il8 were upregulated. Conclusion: The SASP is present very early in human pregnancy, even before PE has developed. Injection of SASP-enriched PE serum into IL-10KO mice induces cellular senescence in different tissues resulting in a PE phenotype. These results suggest that senescence may be an underlying mechanism of PE.

Abstract Tu064: Abnormal Calcium Regulation Leads to Pathological Cardiac Hypertrophy During Pregnancy in the GSNOR-Deficient Mouse Model of Preeclampsia

Introduction: Preeclampsia (PE), a leading cause of maternal mortality, is linked to cardiac hypertrophy and impaired relaxation, predisposing the mother to an increased risk of cardiovascular (CV) injury during pregnancy. Mice lacking S-nitrosoglutathione reductase (GSNOR –/– mice) exhibit all the clinical features of PE. Calcium regulation plays an important role in cardiac relaxation and hypertrophy, but whether alterations in calcium handling contribute to the impairment in these PE mice is unknown. Hypothesis: Cardiac hypertrophy and impaired relaxation in pregnant GSNOR –/– mice may be due in part to altered Ca 2+ handling mechanisms. Methods: Pregnant control (WT [C57B6]) and GSNOR –/– mice were examined at non-pregnant (NP) and late pregnancy (17.5 dpc). Cardiomyocytes (CMs) were isolated (N=3-4 mice/group; n=8-24 CMs/heart) and assessed for intracellular Ca 2+ and sarcomere shortening using IonOptix. Ca 2+ decay and sarcomere relaxation rates were recorded. Ventricular tissue homogenates were assessed for Ca 2+ handling and sarcomere-associated protein expression and post-translational modifications by Western Blot. Results: At late pregnancy, the CMs of GSNOR –/– mothers were significantly wider and systolic and diastolic Ca 2+ levels were elevated as compared to WT. At 17.5 dpc, expression of Ca 2+ handling proteins, RyR2, L-TCC and NCX1 were increased, and phosphorylation of RyR2 and L-TCC were increased in the GSNOR –/– mothers as compared to WT. The Ca 2+ decay rate improved in CMs from pregnant versus NP WT CMs but not in GSNOR –/– CMs. PLB phosphorylation at Thr-17 was augmented in pregnant WT hearts, while phosphorylation at Ser-16 was reduced in pregnant GSNOR –/– . Sarcomere relaxation parameters and sarcomere resting length were improved in CMs from pregnant WT mice but were impaired in pregnant versus NP GSNOR –/– mice in association with increased expression of MyBPC in pregnant GSNOR –/– . Conclusion: The PE-related cardiac hypertrophy and impaired relaxation at late pregnancy in GSNOR –/– mice were associated with elevated cytosolic Ca 2+ levels, altered expression and post-translational modifications of Ca 2+ handling and sarcomeric proteins, which may predispose these mice to higher CV injury during pregnancy.

Eplerenone Reduces Blood Pressure and Improves Vascular Function in a Mouse Model of Leptin-induced Preeclampsia

Preeclampsia is a hypertensive pregnancy disease that increases the risks for adverse outcomes of pregnancy for both mother and baby, notably by reducing fetal growth in utero. We have shown that mid-late gestation leptin infusion induces preeclampsia characteristics in pregnant mice in inducing hypertension, endothelial dysfunction, and fetal growth restriction, which is ablated in mice with endothelial mineralocorticoid receptor deletion (ECMR). However, there is a lack of preclinical data and rationale for MR antagonism for use in preeclampsia in the existing literature. Therefore, we hypothesized that leptin increases ECMR expression and that MR antagonism decreases hypertension, endothelial dysfunction, and fetal growth restriction in leptin-induced preeclamptic mice. We infused timed-pregnant Balb/C mice with saline (sham) or leptin (LEP, 0.9 mg/kg/day) via s.c. osmotic minipump and administered vehicle or eplerenone (EPL, 200mg/kg/day) in drinking water from gestation day (GD)11-18 (n=7-12). We measured mean arterial blood pressure (BP) via radiotelemetry, vascular function in 2nd order mesenteric arteries by wire myography, and pup/placental weights on GD18. We isolated endothelial cells from placentas of our mice via magnetic cell sorting. Pairwise comparisons were by student’s t test and myography curves analyzed by 2-W ANOVA w/RM. Leptin increased ECMR mRNA expression (3.1±0.32 WT LEP vs 1.1±0.48 WT sham, fold change, *p<0.05) compared to WT sham mice in placenta indicating a direct role for leptin to upregulate ECMR in pregnancy. EPL ablated leptin-induced increases in BP (98±4 LEP/EPL vs 113±3 lep/veh mmHg, *p<0.05), and when treated with EPL leptin did not induce endothelial dysfunction, as assessed as a lack of decrease in vascular relaxation to acetylcholine (p>0.05 LEP/EPL vs sham/EPL). Interestingly, EPL treatment did not prevent leptin from decreasing placental effciency in pregnant mice (7.42±0.17 LEP/veh vs 6.71±0.15 LEP/EPL, pup/placenta weight ratio, p>0.05). Collectively, these data show that leptin infusion from GD11-18 in pregnant mice increases ECMR activation by increasing expression. Furthermore, MR antagonism by eplerenone ablates the maternal hypertension and endothelial dysfunction in leptin-induced preeclampsia mice, however, fetal growth is not restored by eplerenone. Therefore, eplerenone may be a promising therapeutic for the maternal cardiovascular syndrome of preeclampsia patients, however, MR antagonism therapies may need additional agents to improve fetal outcomes. 1R01HL169576, 4R00HL146948. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Extracellular Vesicles Derived from Human Umbilical Cord MSC Improve Vascular Endothelial Function in In Vitro and In Vivo Models of Preeclampsia through Activating Arginine Metabolism.

Endothelial cell damage is an important feature of preeclampsia (PE). Human umbilical mesenchymal stem-cell-derived extracellular vesicles (HUMSCs-derived EVs) have been shown to have therapeutic effects on a variety of diseases and tissue damage. However, the therapeutic effect of HUMSCs-derived EVs on endothelial injury in PE remains unclear. This study explored the possible mechanism of HUMSCs-derived EVs in the treatment of endothelial cell injury. Tumor necrosis factor α- and lipopolysaccharide-induced endothelial dysfunction models were used to evaluate the therapeutic effect of HUMSCs-derived EVs on endothelial injury. We further constructed PE mouse models to explore the function of HUMSCs-derived EVs in vivo. The changes of metabolites in endothelial cells after HUMSCs-derived EVs treatment were analyzed by metabolomics analysis and further validated by cell experiments. HUMSCs-derived EVs treatment can alleviate endothelial cell injury in PE, involving cell proliferation, migration, angiogenesis, and anti-inflammatory. Importantly, administration of HUMSCs-derived EVs improves hypertension and proteinuria in PE mice, alleviates kidney damage, and promotes vascularization in the placenta. Furthermore, metabolomics analysis found that the arginine metabolic pathway is activated after HUMSCs-derived EVs treatment. We also observed increased arginine level, nitric oxide content, and nitric oxide synthase activity, and further experiments proved that activating the arginine metabolic pathway could alleviate endothelial dysfunction. Our results reveal that HUMSCs-derived EVs could ameliorate PE endothelial dysfunction by activating the arginine metabolic pathway and may serve as a therapeutic method for treating PE.

Abstract 065: Reduction Of Endoplasmic Reticulum Stress By Treatment With Nifedipine In A Mouse Model Of Pre-eclampsia And In Human Placental Endothelial Cells

Introduction: Pre-eclampsia (PE) is characterized by high blood pressure (BP) and metabolic disorders during pregnancy, being the leading cause of maternal-neonatal mortality worldwide. The causes of PE are still unclear. We observed enhanced endoplasmic reticulum (ER) stress and increased cytoplasmic mobility of calreticulin, a Ca2+ ion binding chaperone protein, in placental endothelial cells (pECs) of mice with PE, indicating changes in Ca2+ mobilization. We aimed to describe the effect of nifedipine, a Ca2+ channel blocker, on ER stress in placentas of mice with PE and in human pECs exposed to hypoxia. Methods: We used heterozygous mice overexpressing human renin and angiotensinogen, developing high BP from gestational day (GD) 13.5, and controls (C57BL/6). Nifedipine (1 mg/kg in 0.5 ml saline, s.c.) or saline was administered at GD14.5 and placentas removed at GD18.5 (n=8-12/group). Protein expressions of IREα, ATF6 and PERK were evaluated by western blot. ECs were extracted from human placentas of healthy pregnancies by magnetic sorting and exposed to hypoxia (0.5% O2) or control conditions (8% O2) for 24h while treated with nifedipine (0.1 μM) or DMSO. Angiogenesis was assessed by tube formation assay on matrigel. Results were compared by 2-way ANOVA (mean±SD). Results: IREα expression was higher in placentas of PE mice treated with saline versus control (+306±64%, p=0.020) and significantly reduced with nifedipine versus saline in PE mice (-235±66%, p=0.009). Results were similar with PERK, which was increased in placentas of saline-treated PE mice versus saline controls (116±40%, p=0.037) and significantly reduced with nifedipine versus saline in PE mice (-141±38%, p=0.006). ATF6 was not different between groups, but was significantly reduced by nifedipine versus saline in PE mice (-76±24%, p=0.022). In pECs, nifedipine increased 17x the numbers of tubes formed on matrigel of cells exposed to hypoxia in comparison to DMSO control (p<0.001). Conclusion: The results describe an enhanced ER stress in placentas of PE mice and a positive effect of nifedipine on this stress. The pro-angiogenic effect of nifedipine was shown in pECs exposed to hypoxia. Further investigations are needed to confirm a potential angiogenic effect of nifedipine in PE.

Abstract 023: Prior Sflt1 Induced Preeclampsia Exacerbates Post-partum Hypertension-mediated Aortic Stiffness And Hypercholesterolemia-induced Atherosclerotic Inflammation

Preeclampsia (PE) is new high blood pressure (BP) and organ damage occurring late in pregnancy in association with increased soluble VEGF receptor (sFlt1). PE survivors have increased risk of future hypertension and myocardial infarction/stroke. We hypothesized that exposure to high sFlt1 during pregnancy exacerbates hypertension and atherosclerosis post-partum. PE was induced by transient adenoviral expression of sFlt1 compared to control virus in pregnant mice. PE mice had elevated sFlt1, BP and kidney damage during pregnancy which resolve post partum. Two months post-partum, prior PE mice had significantly increased BP response to high salt diet. After high BP stimuli, we studied large and small artery function. Prior PE mice had diminished nitric oxide-mediated vasodilation and stiffer aortas with more fibrosis as measured by pulse wave velocity (Con:3.7 vs sFlt:5.9 mm/ms, p<0.0001) and trichrome staining (Con:13.7% vs sFlt:33.9%, p=0.0009). Atherosclerosis studies exposed LDLR knockout mice to PE followed by high fat diet feeding for 8 weeks. Body weight, fasting glucose and cholesterol were not altered by prior PE. Aortic roots were histologically analyzed for plaque size and composition. Aortic root plaque size (Con:73.3 vs sFlt:78.0 AU, p=0.60) and necrotic core were not different between groups. Aortic arch plaque inflammation was quantified via flow cytometry showing significantly increased plaque CD45+ leukocytes (sFlt:1.6 fold, p=0.036) and T cells (sFlt:1.8 fold, p=0.031) from mice with prior PE. Atheroprone mice with prior PE had significantly increased aortic expression of CCL22 (sFlt:1.5 fold, p=0.047), providing a potential mechanism for increased T cell infiltration into atherosclerotic plaques. Experimental PE produces a state of enhanced sensitivity to hypertensive stimuli and increased vascular inflammation in atherosclerotic plaques supporting the concept that PE directly exacerbates hypertension, vascular stiffness, and atherosclerotic inflammation independent of pre-existing risk factors.

Extracellular vesicles derived from Akkermansia muciniphila promote placentation and mitigate preeclampsia in a mouse model.

Preeclampsia (PE) is a multisystem disorder with high maternal morbidity and mortality rates. Currently, no practical therapeutic approach is available to prevent PE progression, except for early delivery. Gut dysbiosis is associated with PE development. Previous data showed that the abundance of Akkermansia muciniphila (Am) was lower in patients with PE than in normotensive pregnant women. Here, in this study, decreased abundance of Am was observed in a PE mouse model. Also, we found that administration with Am could significantly attenuate systolic blood pressure, promote foetal growth and improve the placental pathology in mice with PE. Moreover, Am-derived extracellular vesicles (AmEVs) were transferred from the gastrointestinal (GI) tract to the placenta and mitigated pre-eclamptic symptoms in PE mice. These beneficial effects of AmEVs were mediated by enhanced trophoblast invasion of the spiral artery (SpA) and SpA remodelling through activation of the epidermal growth factor receptor (EGFR)-phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT) signalling pathway. Collectively, our findings revealed the potential benefit of using AmEVs for PE treatment and highlighted important host-microbiota interactions.

Limosilactobacillus reuteri ameliorates preeclampsia in mice via improving gut dysbiosis and endothelial dysfunction

BackgroundAs a leading cause of maternal and neonatal morbidity and mortality, preeclampsia (PE) remains enigmatic. We confirmed that PE is associated with gut dysbiosis. However, whether probiotics could improve PE via regulating gut microbiota remains unclear. Exploring specific probiotic that could alleviate PE by modulating gut microbiota and elucidating detailed mechanisms will be essential for treating PE. MethodsPE model was induced by nitric oxide (NO) inhibition with L-NAME in mice, and treated with Limosilactobacillus reuteri (L. reuteri). 16 S rDNA analysis was conducted on feces from mice. In vitro and in vivo experiments were performed to explore the roles and mechanisms of L. reuteri in PE. ResultsNO inhibition by L-NAME induced PE and gut dysbiosis in mice, characterized by differential gut microbiome, reduced alpha diversity, and markedly decreased abundance of Lactobacillales and L. reuteri. Importantly, L. reuteri could improve PE induced by L-NAME in mice, and ameliorate the gut dysbiosis of PE mice, including restorative gut microbiota composition, increased alpha diversity, and upregulated L. reuteri content. Moreover, L. reuteri could improve NO synthesis, angiogenesis, inflammation and oxidative stress of PE mice. Consistently, L. reuteri could ameliorate NO synthesis, endothelial dysfunction and inflammation mediated by L-NAME in vitro. ConclusionsOur results reveal that L. reuteri could ameliorate PE induced by NO inhibition in mice via improving gut dysbiosis and endothelial dysfunction, supporting gut microbiota serving as therapeutic target for treating PE originated from endothelial dysfunction and L. reuteri protecting patients from PE via modulating gut microbiota and endothelial function.

Smooth Muscle Mineralocorticoid Receptor Promotes Hypertension After Preeclampsia.

Preeclampsia is a syndrome of high blood pressure (BP) with end organ damage in late pregnancy that is associated with high circulating soluble VEGF receptor (sFlt1 [soluble Fms-like tyrosine kinase 1]). Women exposed to preeclampsia have a substantially increased risk of hypertension after pregnancy, but the mechanism remains unknown, leaving a missed interventional opportunity. After preeclampsia, women have enhanced sensitivity to hypertensive stress. Since smooth muscle cell mineralocorticoid receptors (SMC-MR) are activated by hypertensive stimuli, we hypothesized that high sFlt1 exposure in pregnancy induces a postpartum state of enhanced SMC-MR responsiveness. Postpartum BP response to high salt intake was studied in women with prior preeclampsia. MR transcriptional activity was assessed in vitro in sFlt1-treated SMC by reporter assays and PCR. Preeclampsia was modeled by transient sFlt1 expression in pregnant mice. Two months post-partum, mice were exposed to high salt and then to AngII (angiotensin II) and BP and vasoconstriction were measured. Women exposed to preeclampsia had significantly enhanced salt sensitivity of BP verses those with a normotensive pregnancy. sFlt1 overexpression during pregnancy in mice induced elevated BP and glomerular endotheliosis, which resolved post-partum. The sFlt1 exposed post-partum mice had significantly increased BP response to 4% salt diet and to AngII infusion. In vitro, SMC-MR transcriptional activity in response to aldosterone or AngII was significantly increased after transient exposure to sFlt1 as was aldosterone-induced expression of AngII type 1 receptor. Post-partum, SMC-MR-KO mice were protected from the enhanced response to hypertensive stimuli after preeclampsia. Mechanistically, preeclampsia mice exposed to postpartum hypertensive stimuli develop enhanced aortic stiffness, microvascular myogenic tone, AngII constriction, and AngII type 1 receptor expression, all of which were prevented in SMC-MR-KO littermates. These data support that sFlt1-induced vascular injury during preeclampsia produces a persistent state of enhanced sensitivity of SMC-MR to activation. This contributes to postpartum hypertension in response to common stresses and supports testing of MR antagonism to mitigate the increased cardiovascular risk in women after PE.

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.

Paeonol alleviates placental inflammation and apoptosis in preeclampsia by inhibiting the JAK2/STAT3 signaling pathway.

Preeclampsia (PE) is a multisystemic and placental inflammatory disease that causes maternal and infant health issues. As one of the active components in peony root extract, paeonol (Pae) exerts anti-apoptosis and anti-inflammatory effects. Nonetheless, the protective role of Pae in PE has not yet been characterized. A mouse model of PE was constructed through tail vein injection of 1mg/d phosphatidylserine/dioleoyl-phosphatidycholine suspension. The levels of inflammatory cytokines in the placenta were examined via enzyme-linked immunosorbent assay (ELISA). The mRNA levels of inflammatory cytokines (TNF-α, IL-6, IFN-γ, and IL-4) and apoptosis markers (Bax, Bcl-2, and caspase-3) were tested using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Western blot analysis was performed to detect the protein levels of apoptosis markers and Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway-related molecules. Here, Pae repressed the inflammatory response in the placenta of PE-like mouse models, as demonstrated by the decreased concentrations and mRNA levels of TNF-α, IL-6, and IFN-γ and the increased concentrations and mRNA levels of IL-4. Apoptosis in the placentas of PE-like mouse models was attenuated by Pae, as manifested by the downregulated mRNA and protein levels of Bax and cleaved-caspase-3 and the upregulated Bcl-2. Administration of Pae inhibited the phosphorylation of JAK2 and STAT3 in the placental tissues of PE mice. The JAK2/STAT3 pathway agonist (SC-39100) reversed Pae treatment-mediated suppression of placental inflammation and apoptosis in PE mice. Overall, Pae inhibits the JAK2/STAT3 signaling pathway to attenuate placental inflammation and apoptosis in PE mice.

Empagliflozin Ameliorates Preeclampsia and Reduces Postpartum Susceptibility to Adriamycin in a Mouse Model Induced by Angiotensin Receptor Agonistic Autoantibodies.

Preeclampsia (PE) is the leading cause of maternal and perinatal morbidity and mortality and also is a risk factor for cardiovascular and kidney disease later in life. PE is associated with oversecretion of autoantibodies against angiotensin II type 1 receptor (AT1-AA) by the placenta into the maternal circulation. Here, we sought to determine the therapeutic value of the sodium-glucose co-transporter 2 (SGLT2) inhibitor empagliflozin (EMPA) in mice with AT1-AA-induced preeclampsia. Pregnant mice were injected with AT1-AA at gestation day (GD) 13 and treated daily with EMPA until GD 19, at which point some of the maternal mice were sacrificed and assessed. The other maternal mice were labored on time and challenged with adriamycin (ADR) at 12 weeks postpartum; their offspring were assessed for fetal outcomes. We showed that EMPA treatment significantly relieved high systolic blood pressure and proteinuria and ameliorated kidney injury in PE mice without affecting fetal outcomes. EMPA also ameliorated podocyte injury and oxidative stress, reduced the expression of SGLT2 and activated the AMPK/SIRT1 signaling pathway in vivo and in vitro. Remarkably, EMPA treatment during pregnancy reduced ADR-induced kidney and podocyte injury postpartum. These findings suggest that EMPA could be a potential pharmacological agent for PE.

LncRNA small nucleolar RNA host gene 5 inhibits trophoblast autophagy in preeclampsia by targeting microRNA-31-5p and promoting the transcription of secreted protein acidic and rich in cysteine

ABSTRACT Preeclampsia (PE) is a pregnancy-related complication. Dysregulation of long non-coding RNAs (lncRNAs) contributes to the pathogenesis of PE. The current study sought to investigate the effect of lncRNA small nucleolar RNA host gene 5 (SNHG5) on trophoblast autophagy in PE. A PE mouse model was established, followed by detection of parameters such as blood pressure, proteinuria, triglycerides, total cholesterol, low-density lipoprotein, and high-density lipoprotein, observation of alterations of mouse placenta and kidney, and detection of B-cell chronic lymphocytic leukemia/lymphoma-2, Bcl-2-associated X protein, and SNHG5 expression patterns. The expressions of LC3, Beclin-1, and p62 in the placenta of PE mice were detected. Moreover, the SNHG5 expression was downregulated in the established HTR-8/SVneo trophoblast model, followed by evaluation of cell proliferation, apoptosis, and autophagy. After combination treatment with 3-MA (an autophagy inhibitor) and si-SNHG5, the behaviors of HTR-8/SVneo cells were observed. The binding relations between SNHG5 and miR-31-5p, and miR-31-5p and SPARC were verified. The expressions of miR-31-5p and SPARC in the placenta of mice and trophoblasts were determined. Our results demonstrated a poor expression of lncRNA SNHG5 in PE mice. SNHG5 overexpression reduced the PE phenotype and tissue damage in mice. SNHG5 silencing reduced the proliferation, migration, and invasion of trophoblasts, but elevated apoptosis and autophagy. SNHG5 sponged miR-31-5p to promote SPARC transcription. Additionally, miR-31-5p knockdown or 3-MA treatment reverted the stimulative effect of SNHG5 silencing on trophoblast autophagy. Collectively, our study demonstrated that lncRNA SNHG5 alleviated the PE phenotype and inhibited trophoblast autophagy by sponging miR-31-5p and promoting SPARC transcription.

Effect of Placenta-Derived Mesenchymal Stromal Cells Conditioned Media on an LPS-Induced Mouse Model of Preeclampsia.

We tested the pro-angiogenic and anti-inflammatory effects of human placenta-derived mesenchymal stromal cells (hPDMSCs)-derived conditioned media (CM) on a mouse model of preeclampsia (PE), a severe human pregnancy-related syndrome characterized by maternal hypertension, proteinuria, endothelial damage, inflammation, often associated with fetal growth restriction (FGR). At d11 of pregnancy, PE was induced in pregnant C57BL/6N mice by bacterial lipopolysaccharide (LPS) intravenous injection. At d12, 300 μL of unconditioned media (control group) or 300 μL PDMSCs-CM (CM group) were injected. Maternal systolic blood pressure was measured from 9 to 18 days of pregnancy. Urine protein content were analyzed at days 12, 13, and 17 of pregnancy. At d19, mice were sacrificed. Number of fetuses, FGR, fetal reabsorption, and placental weight were evaluated. Placentae were analyzed for sFlt-1, IL-6, and TNF-α gene and protein expressions. No FGR and/or reabsorbed fetuses were delivered by PDMSCs-CM-treated PE mice, while five FGR fetuses were found in the control group accompanied by a lower placental weight. PDMSCs-CM injection significantly decreased maternal systolic blood pressure, proteinuria, sFlt-1, IL-6, and TNF-α levels in PE mice. Our data indicate that hPDMSCs-CM can reverse PE-like features during pregnancy, suggesting a therapeutic role for hPDMSCs for the treatment of preeclampsia.

Increased FUN14 domain containing 1 (FUNDC1) ubiquitination level inhibits mitophagy and alleviates the injury in hypoxia-induced trophoblast cells

ABSTRACT Preeclampsia (PE) is a pregnancy disorder characterized by excessive trophoblast cell death. This study aims to explore the exact mechanism of the ubiquitination level of FUN14 domain containing 1 (FUNDC1) in mitophagy and injury in hypoxic trophoblast cells. In this study, HTR-8/SVneo trophoblast cells were cultured under normoxic and hypoxic conditions and PE mouse model was established. We found low ubiquitination level of FUNDC1 in hypoxic trophoblast cells and placenta of pregnant women with PE. Proteasome inhibitor MG-132 and protease activator MF-094 were added into HTR-8/SVneo trophoblast cells. Proteasome inhibitor MG-132 decreased FUNDC1 ubiquitination level while protease activator MF-094 increased FUNDC1 ubiquitination level. Inhibition of FUNDC1 ubiquitination promoted mitophagy and mitochondrial membrane potential (Δψm) in normoxic trophoblast cells, increased levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and decreased levels of glutathione (GSH) and superoxide dismutase (SOD). In addition, FUNDC1 ubiquitination alleviated cell injury in PE mice in vivo. In conclusion, increased FUNDC1 ubiquitination level inhibited mitophagy and Δψm changes in hypoxic trophoblast cells, and thus alleviated oxidative injury.

Esomeprazole inhibits hypoxia/endothelial dysfunction\u2013induced autophagy in preeclampsia

Preeclampsia (PE) affects 3 to 5% of pregnant women worldwide and is associated with fetal and maternal morbidity and mortality. Although a complete understanding of PE remains elusive, it has been widely accepted that a dysfunction of the placenta plays a key role in the pathogenesis of PE. In this study, we investigated the role of excessive placental autophagy during PE pathogenesis and explored whether esomeprazole ameliorates PE by inhibiting the autophagy in the placenta. The PE cellular model was established by treating the cells’ L-NAME and hypoxia. The PE mice model was established by L-NAME administration and was confirmed by the increased systolic blood pressure (SBP) and urinary protein detected. The autophagy and key proteins were detected in human placental tissue, in cells, and in the mice model by Western blot and immunofluorescence staining. Results showed that excessive autophagy could be detected in human PE placental tissue, in the PE cellular model, and in the PE mice model. Hypoxia induces autophagy by activating AMPKα and inhibiting mTOR in vivo and in vitro. Esomeprazole inhibits L‐NAME-induced autophagy in mice by inhibiting AMPKα and activating mTOR. In conclusion, this study demonstrates that the excessive autophagy induced by the SIRT1/AMPKα-mTOR pathway plays a significant role in the pathogenesis of PE. However, esomeprazole treatment inhibits AMPKα but activates mTOR, resulting in the inhibition of autophagy in the placenta and, therefore, mitigates PE symptoms.

Angiotensin 1-7 and its analogue decrease blood pressure but aggravate renal damage in preeclamptic mice.

Preeclampsia (PE) is a multisystem disease that affects the health of both the pregnant women and the fetus during pregnancy. Agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA) play a significant role in the pathogenesis of PE. This study aimed to determine the effects of Angiotensin 1-7 (Ang 1-7) and its analogue AVE0991 on AT1-AA-induced PE model. Pregnant mice were divided into five groups: the normal pregnant group, AT1-AA-induced preeclampsia group, and AT1-AA-induced preeclampsia group treated with Losartan, Ang 1-7, and AVE0991, respectively. AT1-AA-induced PE model was established on gestational day 13 by tail intravenous injection of purified AT1-AA polyclonal antibody from serum of guinea pigs. Blood urea nitrogen (BUN), urine albumin and urinary creatinine were measured on day 18 of pregnancy. The systolic blood pressure (SBP) was measured from gestational day 13 to day 18. Renal structure changes were observed via light and electron microscopy. Compared with the normal pregnant group (NP group), AT1-AA-induced preeclampsia group (PE group) exhibited elevated blood pressure and proteinuria, consistent with the characteristics of PE. Ang 1-7 or AVE0991 treatment decreased blood pressure without showing renoprotective effects. The findings indicated that Ang 1-7 and its analogue reduced blood pressure but aggravated renal damage in AT1-AA-induced PE mice.

Effect of IL-15-Mediating IFN-γ on HTR-8/SVneo Cells and a Preeclampsia Mouse Model Induced by Lipopolysaccharides

Objective: Determine the effect of interleukin (IL)-15 on HTR-8/SVneo cells and a preeclampsia (PE) mouse model induced by LPS. Methods: Transwell and Annexin-V-FITC/PI assays were performed in HTR-8/SVneo cells transfected with IL-15 activation plasmid/siRNA prior to LPS treatment. Additionally, pregnant mice were injected with LPS and IL-15 siRNA followed by measurement of systolic blood pressure (SBP), urine protein, and serum NO. HE staining was used to observe the morphological changes of the placenta and kidney. Glycogen accumulation was detected using Best’s carmine. qRT-PCR, Western blotting, and ELISA were performed to detect mRNA and protein expression. Results: LPS increased IL-15 and IFN-γ expression in HTR-8/SVneo cells, and IL-15 positively regulated IFN-γ expression in LPS-induced HTR-8/SVneo cells. Moreover, LPS promoted apoptosis and reduced the invasion and migration of HTR-8/SVneo cells, which was, further, promoted by IL-15 overexpression but attenuated by IL-15 inhibition. Furthermore, LPS increased SBP and urine protein but decreased serum NO in mice, and these factors were reversed by IL-15 siRNA. Downregulation of IL-15 also mitigated kidney injury and improved pregnancy outcomes in LPS-induced PE mice. A significantly thicker junctional zone (JZ) and thinner labyrinth layer were found in placentas of PE mice treated with IL-15 siRNA, along with increased glycogen trophoblast cells in the JZ. Moreover, decreased IFN-γ and NKp46 were found in placentas of PE mice treated with IL-15 siRNA. Conclusion: IL-15 inhibition reduced cell apoptosis and increased the invasive and migratory abilities of LPS-induced HTR-8/SVneo cells, thereby alleviating the PE-like phenotype and improving pregnancy outcome.