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Previously, we identified elevated transcripts for the gene Oleoyl-ACP Hydrolase ( OLAH ) in the maternal circulation of pregnancies complicated by preterm fetal growth restriction. As placental dysfunction is central to the pathogenesis of both fetal growth restriction and preeclampsia, we aimed to investigate OLAH levels and function in the human placenta. We assessed OLAH mRNA expression (qPCR) throughout pregnancy, finding placental expression increased as gestation progressed. OLAH mRNA and protein levels (Western blot) were elevated in placental tissue from cases of preterm preeclampsia, while OLAH protein levels in placenta from growth-restricted pregnancies were comparatively reduced in the preeclamptic cohort. OLAH expression was also elevated in placental explant tissue, but not isolated primary cytotrophoblast cultured under hypoxic conditions (as models of placental dysfunction). Further, we discovered that silencing cytotrophoblast OLAH reduced the expression of pro- and anti-apoptosis genes, BAX and BCL2 , placental growth gene, IGF2 , and oxidative stress gene, NOX4 . Collectively, these findings suggest OLAH could play a role in placental dysfunction and may be a therapeutic target for mitigating diseases associated with this vital organ. Further research is required to establish the role of OLAH in the placenta, and whether these changes may be a maternal adaptation or consequence of disease.

Background The angiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) are postulated to be pathogenic disease drivers of preeclampsia. If true, then circulating levels should become more deranged with increasing disease severity. Methods and Results We investigated the association between circulating sFlt-1 and PlGF levels and severe adverse maternal outcomes among 348 women with preeclampsia. Compared with 125 women with preeclampsia without severe features, 25 women with preeclampsia and any of hemolysis, elevated liver enzymes, low platelet count syndrome, disseminated intravascular coagulation, or severe renal involvement had sFlt-1 levels that were 2.63-fold higher (95% CI, 1.81-3.82), sFlt-1/PlGF levels that were 10.07-fold higher (95% CI, 5.36-18.91) and PlGF levels that were 74% lower (adjusted fold change, 0.26 [95% CI, 0.18-0.39]). Compared with 125 women with preeclampsia without severe features, 37 with eclampsia had sFlt-1 levels that were 2-fold higher (2.02 [95% CI, 1.32-3.09]), sFlt-1/PIGF levels that were 4.71-fold higher (95% CI, 2.30-9.66) and PIGF levels that were 63% lower (0.43-fold change [95% CI, 0.27-0.68]). Compared with those without severe features, preeclampsia with severe hypertension (n=146) was also associated with altered angiogenic levels (sFlt-1, 1.71-fold change [95% CI, 1.39-2.11]; sFlt/PlGF, 2.91 [95% CI, 2.04-4.15]; PlGF, 0.59 [95%CI 0.47-0.74]). We also found that sFlt-1 and PlGF levels were altered by the number of maternal complications experienced. Conclusions Further angiogenic imbalance among women with preeclampsia is likely a pathogenic disease driver responsible for the life-threatening maternal complications.

Preeclampsia affects ∼2-8% of pregnancies worldwide. It is associated with increased long-term maternal cardiovascular disease risk. This study assesses the effect of the vasoconstrictor N(ω)-nitro-L-arginine methyl ester (L-NAME) in modelling preeclampsia in mice, and its long-term effects on maternal cardiovascular health. In this study, we found that L-NAME administration mimicked key characteristics of preeclampsia, including elevated blood pressure, impaired fetal and placental growth, and increased circulating endothelin-1 (vasoconstrictor), soluble fms-like tyrosine kinase-1 (anti-angiogenic factor), and C-reactive protein (inflammatory marker). Post-delivery, mice that received L-NAME in pregnancy recovered, with no discernible changes in measured cardiovascular indices at 1-, 2-, and 4-wk post-delivery, compared with matched controls. At 10-wk post-delivery, arteries collected from the L-NAME mice constricted significantly more to phenylephrine than controls. In addition, these mice had increased kidney Mmp9:Timp1 and heart Tnf mRNA expression, indicating increased inflammation. These findings suggest that though administration of L-NAME in mice certainly models key characteristics of preeclampsia during pregnancy, it does not appear to model the adverse increase in cardiovascular disease risk seen in individuals after preeclampsia., (© 2022 de Alwis et al.)

Background Preeclampsia is pregnancy specific, involving significant maternal endothelial dysfunction. Predictive biomarkers are lacking. We evaluated the biomarker potential, expression, and function of PSG7 (pregnancy-specific β-1 glycoprotein 7) and PSG9 (pregnancy-specific β-1 glycoprotein 9) in preeclampsia. Methods and Results At 36 weeks gestation preceding term preeclampsia diagnosis, PSG7 and PSG9 (in Australian cohorts of n=918 and n=979, respectively) were significantly increased before the onset of term preeclampsia (PSG7, P =0.013; PSG9, P =0.0011). In samples collected at 28 to 32 weeks from those with preexisting cardiovascular disease and at high risk of preeclampsia (Manchester Antenatal Vascular Service, UK cohort, n=235), both PSG7 and PSG9 were also significantly increased preceding preeclampsia onset (PSG7, P <0.0001; PSG9, P =0.0003) relative to controls. These changes were validated in the plasma and placentas of patients with established preeclampsia who delivered at <34 weeks gestation (PSG7, P =0.0008; PSG9, P <0.0001). To examine whether PSG7 and PSG9 are associated with increasing disease severity, we measured them in a cohort from South Africa stratified for this outcome, the PROVE (Preeclampsia Obstetric Adverse Events) cohort (n=72). PSG7 ( P =0.0027) and PSG9 ( P =0.0028) were elevated among patients who were preeclamptic with severe features (PROVE cohort), but not significantly changed in those without severe features or with eclampsia. In syncytialized first trimester cytotrophoblast stem cells, exposure to TNFα (tumor necrosis factor α) or IL-6 (interleukin 6) significantly increased the expression and secretion of PSG7 and PSG9. In contrast, when we treated primary endothelial cells with recombinant PSG7 and PSG9, we only observed modest changes in Flt-1 (FMS-like tyrosine kinase-1) expression and Plgf (placental growth factor) expression, and no other effects on proangiogenic/antiangiogenic or endothelial dysfunction markers were observed. Conclusions Circulating PSG7 and PSG9 are increased before preeclampsia onset and among those with established disease with their production and release potentially driven by placental inflammation.

Activin A is aberrantly expressed by the preeclamptic placenta and circulating levels have been investigated as a potential biomarker for the disease. In a nested case-control study we measured Activin A levels in maternal plasma at 28- and 36-weeks' gestation preceding term preeclampsia diagnosis. At 28 weeks Activin A was not significantly altered (n = 73 destined to develop preeclampsia vs n = 191 controls). At 36 weeks' gestation Activin A was significantly increased in 40 women destined to develop preeclampsia relative to 201 controls (p < 0.0001). These findings provide further validation of Activin A as a potential biomarker for subsequent term preeclampsia., (Copyright © 2021 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

There has been increasing research momentum to identify new therapeutic agents for the prevention or treatment of preeclampsia, drugs that can affect the underlying disease pathophysiology. Molecular targets of candidate treatments include oxidative stress, antiangiogenic factors, and the angiotensin, nitric oxide, and proinflammatory pathways. The proposed treatments undergoing preclinical and clinical trial evaluation are thought to act on placental or endothelial disease or both. Most have adopted the pragmatic strategy of repurposing drugs. Of all the therapeutic agents proposed, pravastatin has received the most interest. There are preclinical studies showing that it has pleiotropic actions that favorably impact on multiple molecular targets and can resolve a preeclampsia phenotype in many animal models. An early phase clinical trial suggests that it may have therapeutic activity. Several large prevention trials are planned or ongoing and, when completed, could definitively address whether pravastatin can prevent preeclampsia. Proton-pump inhibitors, metformin, and sulfasalazine are other drugs with preclinical evidence of multiple molecular actions that could resolve the pathophysiology of preeclampsia. These agents are also currently being evaluated in clinical trials. There have been many recent preclinical studies identifying the potential of numerous natural compounds to treat preeclampsia, such as plant extracts and micronutrients that have potent anti-inflammatory or antioxidant activity. Recent preclinical studies have also proposed novel molecular-targeted strategies, such as monoclonal antibodies targeting tumor necrosis factor alpha, placental growth factor, and short interfering RNA technology, to silence the gene expression of soluble fms-like tyrosine kinase-1 or angiotensinogen. Other treatment approaches that have transitioned to human trials (ranging from single-arm to phase III trials that have been completed or are ongoing) include folic acid, nitric oxide donors (such as L-arginine), recombinant antithrombin III, digoxin immune antigen-binding fragment, and melatonin. There have been case series showing the removal of circulating soluble fms-like tyrosine kinase-1 may help stabilize the disease and prolong pregnancy. Interestingly, there are case reports suggesting that monoclonal antibody eculizumab (complement inhibitor) may have therapeutic potential. If new agents are discovered that are proven to be effective in preventing or treating preeclampsia, the potential to improve global maternal and perinatal health will be significant., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

First trimester circulating ADAM12 is reduced in fetal growth restriction (FGR) and preeclampsia. We measured plasma ADAM12 at 36 weeks' gestation preceding diagnosis of term preeclampsia or delivery of a small for gestational age (SGA; birthweight <10th centile) infant in two independent cohorts (Cohort 1 90 SGA, 41 preeclampsia, 862 controls; Cohort 2121 SGA 23 preeclampsia; 190 controls). ADAM12 was reduced with SGA in both cohorts (p = 0.0015 and 0.011 respectively), and further reduced with birthweight <5th centile (p = 0.0013 and 0.0058 respectively). This validates ADAM12 as an SGA biomarker near term. Circulating ADAM12 preceding preeclampsia was not consistently altered., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Preeclampsia is pregnancy-specific, and significantly contributes to maternal, and perinatal morbidity and mortality worldwide. An effective predictive test for preeclampsia would facilitate early diagnosis, targeted surveillance and timely delivery; however limited options currently exist. A first-trimester screening algorithm has been developed and validated to predict preterm preeclampsia, with poor utility for term disease, where the greatest burden lies. Biomarkers such as sFlt-1 and placental growth factor are also now being used clinically in cases of suspected preterm preeclampsia; their high negative predictive value enables confident exclusion of disease in women with normal results, but sensitivity is modest. There has been a concerted effort to identify potential novel biomarkers that might improve prediction. These largely originate from organs involved in preeclampsia's pathogenesis, including placental, cardiovascular and urinary biomarkers. This review outlines the clinical imperative for an effective test and those already in use and summarises current preeclampsia biomarker research., Competing Interests: Declaration of Competing Interest TKL, TM, SW and ST hold a provisional patent (PCT/AU2019/050516) relating to the use of SPINT1 and syndecan-1 as diagnostic markers for placental insufficiency. The remaining authors have no conflicts of interest to declare., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Introduction: Preeclampsia is associated with reduced pro-angiogenic Placental Growth Factor (PlGF) and increased levels of anti-angiogenic soluble FMS like tyrosine kinase-1 (sFlt-1). We have previously shown that sFlt-1 secretion is positively regulated via the Epidermal Growth Factor Receptor (EGFR) and mitochondrial respiration pathways. We assessed whether these pathways also regulate endothelial and placental secretion of PlGF., Methods: Primary cytotrophoblast cells and primary human umbilical vein endothelial cells (HUVECs) were treated with EGFR inhibitor gefitinib, or small molecules that inhibit down-stream pathways of the receptor: U0126, PD98059 (ERK/MEK pathway inhibitors), ZM336372 (JAK/STAT inhibitor) or AG490 (JAK inhibitor). We inhibited mitochondrial respiration in primary cytotrophoblasts using mitochondrial complex inhibitors rotenone (complex I), antimycin (complex III) or oligomycin (complex IV). We then measured PlGF secretion in the condition media., Results: Three inhibitors of the EGFR pathway significantly increased PlGF secretion: gefitinib (p = 0.03), AG490 (p < 0.0001) and U0126 (p = 0.03) in primary cytotrophoblasts, while PD98059 reduced PlGF secretion (p = 0.002). In the same cells, neither gefitinib or UO126 altered PlGF mRNA expression, but AG490 significantly increased its expression (p = 0.02). Primary endothelial cell PlGF secretion was significantly reduced when treated with PD98059 and U0126 while ZM336372 had no effect. Rotenone significantly reduced cytotrophoblast PlGF secretion (p = 0.0005). Neither antimycin (p = 0.9) or oligomycin (p = 0.9) had an effect., Discussion: We have shown that PlGF secretion from primary cytotrophoblast and HUVECs is altered by inhibiting EGFR signaling and potentially mitochondrial respiration, coincident with reduced sFlt-1 secretion. This suggests that common pathways are regulating both pro and anti-angiogenic molecules that are changed in association with preeclampsia and provides insight into the pathogenesis of this serious disease., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction: The human placenta has a defined lifespan and placental aging is a key feature as pregnancy progresses. Placental aging and mitochondrial dysfunction are known to play a key role in pregnancy pathophysiology. Premature aging of the placenta has also been linked with placental dysfunction resulting in poor fetal development and premature birth., Methods: The expression of key mitochondrial-related genes were analysed in a series of publicly available databases then expression changes were validated in placental samples collected from term, pre-term, post-term pregnancies and pregnancies complicated by fetal growth restriction (FGR). Gene and protein expression levels of MFN1, MFN2, TFAM, TOMM20, OPA3 and SIRT4 were measured in placental tissues via qPCR and western blotting., Results: Initial analysis found that key mitochondrial transcripts related to biogenesis, bioenergetics and mitophagy clustered by pregnancy trimester. A refined list of 13 mitochondrial-related genes were investigated in additional external datasets of pregnancy complications. In the new cohort, protein expression of MFN1 was decreased in FGR and MFN2 is decreased in post-term placenta. Analysis of placental tissues revealed that TOMM20 gene and protein expression was altered in FGR and post-term placenta., Discussion: MFN1 and MFN2 play a major role in mitochondrial dynamics, and alterations in these markers have been highlighted in early unexplained miscarriage. TOMM20 is an importer protein that plays a major role in mitophagy and changes have also been identified in age-related diseases. Significant changes in MFN1, MFN2 and TOMM20 indicate that mitochondrial regulators play a critical role in placental aging and placental pathophysiology., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Background We investigated the biomarker potential of growth differentiation factor 15 (GDF-15), a stress response protein highly expressed in placenta, to predict preeclampsia. Methods and Results In 2 prospective cohorts (cohort 1: 960 controls, 39 women who developed preeclampsia; cohort 2: 950 controls, 41 developed preeclampsia), plasma concentrations of GDF-15 at 36 weeks' gestation were significantly increased among those who developed preeclampsia ( P <0.001), area under the receiver operating characteristic curves (AUC) of 0.66 and 0.71, respectively. In cohort 2 a ratio of sFlt-1/PlGF (a clinical biomarker for preeclampsia) had a sensitivity of 61.0% at 83.2% specificity to predict those who will develop preeclampsia (AUC of 0.79). A ratio of GDF-15×sFlt-1/PlGF yielded a sensitivity of 68.3% at 83.2% specificity (AUC of 0.82). GDF-15 was consistently elevated across a number of international cohorts: levels were higher in placenta and blood from women delivering <34 weeks' gestation due to preterm preeclampsia in Melbourne, Australia; and in the blood at 26 to 32 weeks' gestation among 57 women attending the Manchester Antenatal Vascular Service (MAViS, UK) who developed preeclampsia ( P =0.0002), compared with 176 controls. In the Preeclampsia Obstetric adVerse Events biobank (PROVE, South Africa), plasma GDF-15 was significantly increased in women with preeclampsia with severe features ( P =0.02; n=14) compared to controls (n=14). Conclusions We conclude circulating GDF-15 is elevated among women more likely to develop preeclampsia or diagnosed with the condition. It may have value as a clinical biomarker, including the potential to improve the sensitivity of sFlt-1/PlGF ratio.

Fetal growth restriction is a leading cause of stillbirth that often remains undetected during pregnancy. Identifying novel biomarkers may improve detection of pregnancies at risk. This study aimed to assess syndecan-1 as a biomarker for small for gestational age (SGA) or fetal growth restricted (FGR) pregnancies and determine its molecular regulation. Circulating maternal syndecan-1 was measured in several cohorts; a large prospective cohort collected around 36 weeks' gestation (n = 1206), a case control study from the Manchester Antenatal Vascular service (285 women sampled at 24-34 weeks' gestation); two prospective cohorts collected on the day of delivery (36 + 3-41 + 3 weeks' gestation, n = 562 and n = 405 respectively) and a cohort who delivered for preterm FGR (< 34 weeks). Circulating syndecan-1 was consistently reduced in women destined to deliver growth restricted infants and those delivering for preterm disease. Syndecan-1 secretion was reduced by hypoxia, and its loss impaired proliferation. Matrix metalloproteinases and mitochondrial electron transport chain inhibitors significantly reduced syndecan-1 secretion, an effect that was rescued by coadministration of succinate, a mitochondrial electron transport chain activator. In conclusion, circulating syndecan-1 is reduced among cases of term and preterm growth restriction and has potential for inclusion in multi-marker algorithms to improve detection of poorly grown fetuses., (© 2021. The Author(s).)

Objectives: The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is upregulated in the maternal vasculature in preeclampsia, and contributes to oxidative stress and endothelial dysfunction. However, its function in the placenta is unclear. This paper investigated LOX-1 expression in models of placental dysfunction and preeclampsia, and whether candidate therapeutics for preeclampsia could alter its expression., Study Design: Placentas were collected from preterm pregnancies and cases of preterm preeclampsia and fetal growth restriction. Blood was collected from participants whose pregnancies were complicated by preterm fetal growth restriction and/or preeclampsia. Primary cytotrophoblast and placental explant tissue were cultured under hypoxic (1% O 2 ) or normoxic (8% O 2 ) conditions. Cytotrophoblast were exposed to 10% preeclamptic or control serum. Cytotrophoblast and preeclamptic explant tissue were treated with 100 µM esomeprazole, lansoprazole or rabeprazole., Main Outcome Measures: LOX-1 expression was assessed in all samples via qPCR., Results: LOX-1 expression was reduced in placentas from cases of preterm preeclampsia, but not fetal growth restriction, compared to controls. LOX-1 expression was reduced in cytotrophoblast under hypoxia, but not in explant tissue. Treatment with preeclamptic serum in vitro did not alter cytotrophoblast LOX-1 expression. Circulating LOX-1 mRNA was unaltered in patients with fetal growth restriction, preeclampsia, and fetal hypoxia, compared to controls. Treatment with esomeprazole or lansoprazole in vitro increased placental LOX-1 expression., Conclusions: LOX-1 expression is reduced in preeclamptic placentas and hypoxic cytotrophoblast. Esomeprazole and lansoprazole increase placental LOX-1 expression. These findings demonstrate a role for LOX-1 in the placenta, and improve our understanding of maternal adaptations in pregnancy complications., (Copyright © 2021 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Preeclampsia is a pregnancy complication associated with angiogenic dysbalance, maternal endothelial dysfunction and end-organ injury. A predictive test to identify those who will develop preeclampsia could substantially decrease morbidity and mortality. MicroRNAs (miRs) are small RNA molecules involved in post-transcriptional gene regulation. We screened for circulating miRs differentially expressed at 36 weeks' gestation in pregnancies before the development of preeclampsia. We used a case-control group (198 controls, 34 pre-preeclampsia diagnosis) selected from a prospective cohort (n = 2015) and performed a PCR-based microarray to measure the expression of 41 miRs. We found six circulating miRs (miRs 363, 149, 18a, 1283, 16, 424) at 36 weeks' had significantly reduced expression (p < 0.0001-0.04). miR363 was significantly downregulated at 28 weeks' gestation, 10-12 weeks before the onset of clinical disease. In the circulation of another cohort of 34 participants with established preterm preeclampsia (vs 23 controls), we found miRs363, 18a, 149 and 16 were significantly down regulated (p < 0.0001-0.04). Combined expression of miRs149 and 363 in the circulation at 36 weeks' gestation provides a test with 45% sensitivity (at a specificity of 90%) which suggests measuring both miRs may have promise as part of a multi-marker test to predict preeclampsia.

Development and repurposing of therapies that show promise in the prevention or treatment of preeclampsia would be a major advance for the obstetrics field. We recently identified esomeprazole and sulfasalazine as potential candidates for the treatment of preeclampsia. Both reduce placental and endothelial secretion of sFlt-1 and sENG and mitigate endothelial dysfunction in vitro. Here we assessed whether esomeprazole and sulfasalazine in combination would additively attenuate the elevated release of anti-angiogenic factors and markers of endothelial dysfunction, key characteristics of preeclampsia. Primary placental tissue and cells, and primary endothelial cells were treated with esomeprazole and sulfasalazine alone and in combination. We assessed secretion of sFlt-1 and sENG and performed in vitro assays of endothelial dysfunction. Combining esomeprazole and sulfasalazine in lower concentrations caused an additive reduction in sFlt-1 secretion in primary cytotrophoblasts, placental explants and endothelial cells. No additive reduction was observed in sENG secretion when esomeprazole and sulfasalazine were combined. Together, esomeprazole and sulfasalazine additively reduced TNF-α-induced VCAM and ET-1 mRNA expression, and monocyte adhesion to endothelial cells. In conclusion, combining esomeprazole and sulfasalazine additively reduced secretion of sFlt-1 and markers of endothelial dysfunction. Combined administration of esomeprazole and sulfasalazine may provide a more effective treatment or prevention for preeclampsia compared to either as single agents., (Copyright © 2020 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Objectives: There is avid interest in pravastatin as a therapeutic intervention for pre-eclampsia, however little is known on statin action on endothelial dysfunction. This study aimed to evaluate the ability of pravastatin, simvastatin and rosuvastatin to reduce pre-eclampsia-associated markers of endothelial dysfunction in human endothelial cells., Study Design: Primary human umbilical vein endothelial cells (HUVECs) and uterine microvascular cells (UtMVs) were isolated and treated with 0.2, 2, 20 and 200 µM pravastatin, simvastatin and rosuvastatin for 24 h, either with or without pre-treatment with TNF-α to induce endothelial dysfunction., Main Outcome Measures: Cell viability (MTS) assays were performed and cells were visually inspected. Expression of endothelial dysfunction markers, endothelin-1 (ET-1) and vascular cell adhesion molecule-1 (VCAM-1) were assessed by qPCR (n=3). Intracellular VCAM-1 protein was examined by Western Blotting (n=5). ET-1 and soluble fms-like tyrosine kinase-1 (sFLT-1) protein secretion was assessed by ELISA in HUVEC conditioned media (n=3)., Results: High doses of simvastatin and rosuvastatin significantly compromised HUVEC survival. 200 µM simvastatin significantly reduced UtMV survival. Abnormal cell structure was observed with these doses and thus were excluded from further analysis. The statins did not mitigate TNF-α induced ET-1 or VCAM-1 expression in either HUVECs or UtMVs, nor VCAM-1 protein expression in HUVECs. 0.2 µM pravastatin and simvastatin significantly reduced ET-1 and sFLT-1 protein secretion., Conclusions: Pravastatin significantly reduced secretion of both ET-1 and sFLT-1, key mediators of endothelial dysfunction. Importantly, pravastatin had no toxic effects, in contrast to rosuvastatin and simvastatin. This further supports selection of pravastatin for clinical applications to combat pre-eclampsia., (Copyright © 2020 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Preeclampsia is a pregnancy complication associated with elevated placental secretion of anti-angiogenic factors, maternal endothelial dysfunction and end-organ injury. Adrenomedullin (ADM) is a pro-angiogenic peptide hormone which regulates blood pressure and vascular integrity. It is highly expressed in both the placenta and vascular endothelial cells. We performed a nested case-control study, selected from a large prospective cohort of over 2000 participants. Circulating ADM mRNA was reduced at both 28 (n = 39 vs 248 controls, p = 0.005) and 36 weeks' of pregnancy (n = 39 vs 205 controls, p < 0.0001) in those destined to develop term preeclampsia. It was also significantly reduced in the circulation of women with established early-onset preeclampsia (n = 34 vs 21 controls, p = 0.01). ADM mRNA (n = 34 vs 12 controls) and protein (n = 53 vs 17 controls) were significantly decreased in placental tissue from women with early-onset preeclampsia (p = 0.02, p = 0.0002 respectively), suggesting the placenta is a possible source of the reduced circulating mRNA. Functional studies in primary endothelial cells revealed significantly reduced ADM mRNA expression when cells were exposed to cytotrophoblast conditioned media (derived from normotensive pregnancies, p < 0.0001) or TNFα (p < 0.0001), suggesting another possible source of reduced circulating ADM mRNA is the endothelium. Circulating ADM mRNA, but not protein, is reduced 10-12 weeks before the diagnosis of term preeclampsia. It may be of endothelial or placental origin. Whole blood mRNA is a rich source of potential biomarker discovery in the prediction of preeclampsia., (Copyright © 2019 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Preeclampsia is a hypertensive disorder of pregnancy characterized by maternal endothelial dysfunction and end-organ damage. The antiangiogenic factor, sFlt-1 (soluble FMS-like tyrosine kinase-1) has been strongly implicated in the pathogenesis of preeclampsia. sFlt-1 is secreted into the maternal circulation where it antagonizes VEGF (vascular endothelial growth factor) and ultimately disrupts vascular homeostasis. However, the upstream mechanisms regulating release of sFlt-1 are poorly characterized. We investigated the roles of key prosurvival pathways, EGFR (epidermal growth factor receptor) signaling, and the mitochondria, in regulating sFlt-1 production. We initially found that the mRNA and protein of EGFR and downstream adaptor molecules were significantly increased in preeclamptic placental tissue relative to normotensive controls. Inhibiting the EGFR signaling cascade using siRNA (small interfering ribonucleic acid) or small molecule inhibitors significantly reduced sFlt-1 release from primary cytotrophoblast (placental cells). Additionally, inhibiting the mitochondrial electron transport chain or activating downstream energy-sensing molecules (AMPK [AMP-activated kinase], SIRT1 [sirtuin 1 ], and PGC1α [PPAR-γ co-activator 1]) also significantly reduced sFlt-1 secretion from cytotrophoblast cells, without affecting EGFR signaling. In vivo, treating pregnant mice with gefitinib (an EGFR inhibitor) or resveratrol (perturbs mitochondrial function) significantly reduced circulating murine sFlt-1 compared with vehicle control. Furthermore, treating primary cytotrophoblasts with therapeutics which have been previously found to reduce sFlt-1 secretion, either reduced EGFR signaling (esomeprazole and statins) or perturbed mitochondrial function (esomeprazole, resveratrol, and metformin). Additionally, targeting both pathways simultaneously produced additive reductions in sFlt-1 secretion. Thus, we have identified 2 key survival pathways that seem to be overactive in preeclampsia and involved in the regulation of placental sFlt-1 secretion.

Introduction: Preeclampsia is a severe complication of pregnancy, and likely arises from abnormal placental development in early pregnancy. Persistent placental hypoxia is thought to trigger the release of anti-angiogenic factors into the maternal circulation leading to widespread endothelial dysfunction. Epidermal growth factor-like domain 7 (EGFL7) is a secreted angiogenic factor that may play a key role in the disrupted angiogenesis seen in response to placental hypoxia that characterizes preeclampsia., Methods: Primary trophoblasts were isolated and cultured in both normoxic and hypoxic conditions. Under hypoxia HIF1α was silenced and EGFL7 mRNA expression was assessed. EGFL7 mRNA expression was measured in placentas obtained from women with early (<34 weeks) and late onset preeclampsia; and in peripheral whole blood maternal samples from women with preeclampsia and gestation matched controls. EGFL7 plasma levels were assessed in plasma from women with preeclampsia, compared to gestation-matched controls., Results: EGFL7 expression was significantly upregulated in primary human trophoblasts cultured in hypoxia (>2-fold, p < 0.0001), however this was not regulated via a HIF1α dependent manner. EGFL7 mRNA expression was not altered in placenta from women with early or late onset preeclampsia. Circulating EGFL7 protein levels were not different in women with severe preeclampsia. In contrast, EGFL7 mRNA expression was increased in maternal blood in women with early onset preeclampsia (∼1.6-fold, p < 0.05)., Discussion: EGFL7 mRNA expression is increased with hypoxia in human trophoblast and is increased in the maternal circulation in women with preeclampsia. Further studies aimed at understanding the role and regulation of EGLF7 in the pathophysiology of preeclampsia are required., (Copyright © 2018. Published by Elsevier B.V.)

Preeclampsia is associated with intermittent placental hypoxia, inflammation and the release of antiangiogenic factors, namely sFLT-1 and sEng. These factors cause maternal endothelial dysfunction and the manifestation of clinical disease. Disulfiram is a dehydrogenase inhibitor used to treat alcoholism and has been suggested as a proteasome inhibitor. Inhibiting the proteasome has been previously shown to reduce FLT-1 gene expression. Thus, we aim to investigate whether disulfiram alters the secretion of sFLT-1 and sEng and reduces endothelial dysfunction. METHODS AND RESULTS: We assessed the effects of disulfiram on primary cytotrophoblast and human umbilical vein endothelial cells (HUVECs). Disulfiram significantly reduced mRNA expression of membrane bound FLT-1 and sFLT-1 variants in primary cytotrophoblasts, which translated into a significant reduction in the protein secretion of sFLT-1. Additionally, sFLT-1 was reduced in primary HUVECs treated with disulfiram, whilst sEng was only reduced in primary cytotrophoblasts. Next, we investigated the effect of disulfiram on endothelial dysfunction using primary HUVECs treated with 5% preeclamptic serum ± disulfiram. Serum from preeclamptic women induced endothelial dysfunction evidenced by increased mRNA expression of vascular cell adhesion molecule-1 (VCAM-1) and adhesion of peripheral blood mononuclear cells (PBMCs) to HUVECs. The addition of disulfiram reduced VCAM-1 mRNA expression, however did not affect the adhesion of PBMCs to endothelial cells. Lastly, we assessed the effects of disulfiram on the 20S subunit of the proteasome and found disulfiram did not inhibit this subunit in either primary cytotrophoblast or HUVECs. CONCLUSIONS: Disulfiram quenches sFLT-1 and sEng via mechanisms independent of the 20S subunit of the proteasome. Understanding of the mechanisms by which disulfiram inhibits antiangiogenic secretion may reveal insights into the pathogenesis and potential therapeutic targets for preeclampsia., (Copyright © 2018 International Society for the Study of Hypertension in Pregnancy. All rights reserved.)

Objectives: To examine the effect of sildenafil on level of antiangiogenic proteins of preeclampsia. Firstly to examine the effect of sildenafil on serum biomarkers in a patient with preterm preeclampsia. Secondly, to examine the effect of sildenafil on sFlt-1 and soluble endoglin secretion from primary trophoblasts and placental explants., Study Design: The clinical team administered 50 mg tds sildenafil to a 26-year-old primigravid woman with severe preeclampsia at the threshold of viability (24 3/7 weeks gestation) and we collected bloods to examine the effect of slidenafil on antiangiogenic factors sFlt-1 and soluble endoglin (sENG), pro-angiogenic factor PlGF and vascular cell adhesion molecule 1 (VCAM-1) and endothelin-1 (ET1). We administered sildenafil to human primary trophoblasts and placental explants and explored its effect on sFlt-1 and sENG secretion., Main Outcome Measures: We examined serum anti-angiogenic factors sFlt-1 and sENG, pro-angiogenic factor PlGF, the potent vasoconstrictor ET1 and VCAM-1 by ELISA. We explored the effect of sildenafil on sFlt-1 secretion from primary trophoblasts and sFlt-1 and sENG secretion from placental explants., Results: We found a reduction in serum sFlt-1, stabilisation in sENG and PlGF in a patient with peri-viable preterm preeclampsia administered oral sildenafil 50 mg three times daily (tds). Furthermore there was an initial stabilisation in serum VCAM-1 and a decline in ET1 with sildenafil administration. This was concordant with stabilisation of clinical and biochemical features of preeclampsia. Interestingly, treating placental cells and tissues in vitro with sildenafil did not appear to change sFlt-1 or sENG secretion., Conclusion: Sildenafil administration was associated with a reduction in serum sFlt-1 and sENG secretion and increase in PlGF secretion in a patient with preterm preeclampsia, potentially via increasing placental perfusion rather than acting directly on the placenta., (Copyright © 2018 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Introduction: The discovery of new treatments that prevent or treat preeclampsia would be a major advance. Antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG) are secreted in excess from the placenta, causing hypertension, endothelial dysfunction, and multiorgan injury. We recently identified metformin and esomeprazole as potential treatments for preeclampsia. Both reduce placental and endothelial secretion of sFlt-1 and soluble endoglin, and reduce endothelial dysfunction., Objectives: We set out to assess whether combining metformin and esomeprazole would additively reduce sFlt-1 and soluble endoglin secretion and reduce endothelial dysfunction (verses drug alone). Metformin and esomeprazole were added to primary placental cells and tissues, and endothelial cells and their effects on sFlt-1 and soluble endoglin secretion were assessed in vitro. Tumor necrosis factor-α (TNF-α) was added to endothelial cells to induce dysfunction in vitro. We examined the ability of metformin + esomeprazole to rescue TNF-α induced vascular cell adhesion molecule-1 (VCAM-1) and Endothelin-1 (ET-1) expression, leukocyte adhesion (markers of endothelial dysfunction)., Results: Combining metformin and esomeprazole was additive at reducing sFlt-1 secretion and expression of sFlt-1 e15a mRNA isoform in primary cytotrophoblast, placental explants and endothelial cells. In contrast, no additive reduction in sENG was observed with combined metformin and esomeprazole. The low-dose combination of metformin + esomeprazole additively reduced TNF-α-induced VCAM-1 mRNA, but not VCAM-1 protein expression. There was no additive reduction when combining metformin and esomeprazole on TNF-α induced PBMC adhesion to endothelial cells. However, combining metformin and esomeprazole additively reduced ET-1 mRNA expression., Conclusions: In conclusion combining metformin and esomeprazole additively reduced secretion of sFlt-1, and markers of endothelial dysfunction. The combination of metformin and esomeprazole may provide a more effective treatment or prevention for preeclampsia compared to either as single agents.

Preeclampsia is a severe complication of pregnancy. Antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin are secreted in excess from the placenta, causing hypertension, endothelial dysfunction, and multiorgan injury. Oxidative stress and vascular inflammation exacerbate the endothelial injury. A drug that can block these pathophysiological steps would be an attractive treatment option. Proton pump inhibitors (PPIs) are safe in pregnancy where they are prescribed for gastric reflux. We performed functional studies on primary human tissues and animal models to examine the effects of PPIs on sFlt-1 and soluble endoglin secretion, vessel dilatation, blood pressure, and endothelial dysfunction. PPIs decreased sFlt-1 and soluble endoglin secretion from trophoblast, placental explants from preeclamptic pregnancies, and endothelial cells. They also mitigated tumor necrosis factor-α-induced endothelial dysfunction: PPIs blocked endothelial vascular cell adhesion molecule-1 expression, leukocyte adhesion to endothelium, and disruption of endothelial tube formation. PPIs decreased endothelin-1 secretion and enhanced endothelial cell migration. Interestingly, the PPI esomeprazole vasodilated maternal blood vessels from normal pregnancies and cases of preterm preeclampsia, but its vasodilatory effects were lost when the vessels were denuded of their endothelium. Esomeprazole decreased blood pressure in a transgenic mouse model where human sFlt-1 was overexpressed in placenta. PPIs upregulated endogenous antioxidant defenses and decreased cytokine secretion from placental tissue and endothelial cells. We have found that PPIs decrease sFlt-1 and soluble endoglin secretion and endothelial dysfunction, dilate blood vessels, decrease blood pressure, and have antioxidant and anti-inflammatory properties. They have therapeutic potential for preeclampsia and other diseases where endothelial dysfunction is involved., (© 2017 American Heart Association, Inc.)

Introduction: Preeclampsia is a serious pregnancy complication for which there are no medical treatments. Soluble endoglin is an anti-angiogenic factor implicated in the pathogenesis of the disease, however little is known about its molecular regulation. The PI3K/Akt pathway is down regulated in preeclamptic placentas and decreased PI3K/Akt signaling has been linked to increased soluble endoglin release from endothelial cells. MMP14 is a key protease that functions to release soluble endoglin from the placental surface., Objective: This study aimed to determine whether reduced placental PI3K/Akt causes elevated release of soluble endoglin via MMP14., Study Design: Akt mRNA and protein expression were assessed in early onset preeclamptic and preterm control placentas (delivered <34weeks gestation). PI3K/Akt inhibition was achieved by administering PI3K inhibitor wortmannin, a specific Akt inhibitor or Akt siRNA to primary human umbilical vein endothelial cells, primary trophoblast and placental explants. The effect of PI3K/Akt inhibition on soluble endoglin release, MMP14, endoglin and TIMP-3 mRNA expression was determined., Results: We identified significantly reduced pAkt and total Akt in preeclamptic placentas relative to preterm control. Inhibition of PI3K/Akt resulted in significantly elevated soluble endoglin release from HUVECs, had no effect on MMP14 mRNA expression but resulted in significantly reduced TIMP3. In contrast inhibiting PI3K/Akt in placental explants or primary trophoblast did not change soluble endoglin release., Conclusion: This study confirms that the PI3K/Akt cell protection pathway is down regulated in preeclampsia, but demonstrates that this dysregulation is unlikely to be responsible for the excessive placental soluble endoglin release characteristic of preeclampsia., (Copyright © 2016 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

There is recent interest for uniform placental collection protocols so laboratories can share samples. However, concerns have been raised a wash step at collection causes significant loss of sFlt-1 and soluble endoglin, among the most studied proteins in placentology. We measured Flt-1 and endoglin mRNA and protein in 10 preeclamptic placentas that were washed, or left unwashed. Reassuringly, there was no significant change in the Flt-1, sFlt-1-e15a or sFlt-1-i13 mRNA or Flt-1 or sFlt-1 protein expression or localization. There was also no change in endoglin mRNA expression or protein localization. Washing preeclamptic placental samples does not alter anti-angiogenic factor expression., (Copyright © 2015 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Objectives: Soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) are the most studied molecules in preeclampsia. However, most trophoblast cell lines do not secrete both these factors. Thus, we set out to characterize protocols to functionally investigate sFlt-1 and sEng from primary trophoblast., Study Design: Primary trophoblasts were isolated from term placenta by percoll gradient, then negative selection using a CD9 antibody. Purity was assessed by cytokeratin 7 immunostaining. We first examined the effects of CD9 negative selection on sFlt-1, sEng and hCG secretion and the ability of forskolin to enhance syncytialization. We then examined the effects of hypoxia on sFlt-1 production and assessed gene knockdown using siRNA., Results: CD9 negative selection produced a pure population of primary trophoblasts. Secretion of sEng was 5-fold lower when CD9-positive cells were removed, sFlt1 was unchanged, and hCG was significantly increased. hCG analysis of the purified population indicated spontaneous syncytialization, which was not enhanced by forskolin. Forskolin similarly did not alter sFlt-1 secretion. Hypoxia significantly increased sFlt-1 secretion as expected. Importantly, high gene silencing efficiencies were readily achieved., Conclusion: In conclusion, we present a protocol that yields primary trophoblasts of high purity that produce abundant sFlt-1 and low but detectable levels of sEng. Furthermore, these cells are readily amenable to gene silencing by siRNAs and hence suitable for functional studies., (Copyright © 2014 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Background: We have recently shown first trimester Macrophage inhibitory cytokine-1 (MIC-1) and Pregnancy Associated Plasma Protein-A (PAPP-A) serum concentrations are depressed among asymptomatic women destined to miscarry. Here we examined whether plasma levels of MIC-1 and PAPP-A are depressed among women presenting to an Early Pregnancy Assessment Unit (EPAU), noted to have a confirmed viable fetus, but subsequently miscarry., Methods: We performed a prospective cohort study, recruiting 462 women in the first trimester presenting to EPAU and had fetal viability confirmed by ultrasound. We obtained plasma samples on the same day and measured MIC-1, PAPP-A and human chorionic gonadotrophin (hCG), grouping the cohort according to whether they later miscarried or not. To correct for changes in analyte levels across gestation, we expressed the data as Multiples of the normal Median (MoMs)., Results: We recruited 462 participants presenting to EPAU at 5-12 weeks gestation. Most (80%) presented with symptoms of threatened miscarriage (e.g. abdominal pain, vaginal bleeding). 34 (7.4%) subsequently miscarried. Median plasma MIC-1 levels among those who miscarried were 50% of those with ongoing pregnancies (Miscarriage cohort MoM 0.50 (25(th)-75(th) centiles: 0.29-1.33) vs ongoing pregnancies MoM 1.00 (0.65-1.38); p=0.0025). Median plasma PAPP-A MoMs among those who miscarried was 0.57 (0.00-1.12), significantly lower than those with ongoing pregnancies (MoMs 1.00 (0.59-1.59); p=0.036). Plasma hCG levels were also significantly depressed among those who miscarried compared to those with ongoing pregnancies. However, the performance of MIC-1 as a diagnostic marker to predict miscarriage in this cohort was modest, and not improved with the addition of hCG., Conclusion: MIC-1 and PAPP-A levels are significantly depressed in women presenting to EPAU with ultrasound evidence of fetal viability, but later miscarry. While they are unlikely to be useful as predictive biomarkers in this clinical setting, they probably play important roles in the maintenance of early pregnancy.

Abnormal trophoblast growth can cause life-threatening disorders such as ectopic pregnancy, choriocarcinoma, and placenta accreta. EnGeneIC Delivery Vehicles (EDVs) are nanocells that can promote tissue-specific delivery of drugs and may be useful to medically treat such disorders. The objective of this study was to determine whether EDVs loaded with the chemotherapeutic doxorubicin and targeting the epidermal growth factor receptor (EGFR, very highly expressed on the placental surface) can regress placental cells in vitro, ex vivo, and in vivo. In female SCID mice, EGFR-targeted EDVs induced greater inhibition of JEG-3 (choriocarcinoma cells) tumor xenografts, compared with EDVs targeting an irrelevant antigen (nontargeted EDVs) or naked doxorubicin. EGFR-targeted EDVs were more readily taken up by human placental explants ex vivo and induced increased apoptosis (M30 antibody) compared with nontargeted EDVs. In vitro, EGFR-targeted EDVs administered to JEG-3 cells resulted in a dose-dependent inhibition of cell viability, proliferation, and increased apoptosis, a finding confirmed by continuous monitoring by xCELLigence. In conclusion, EGFR-targeted EDVs loaded with doxorubicin significantly inhibited trophoblastic tumor cell growth in vivo and in vitro and induced significant cell death ex vivo, potentially mediated by increasing apoptosis and decreasing proliferation. EDVs may be a novel nanoparticle treatment for ectopic pregnancy and other disorders of trophoblast growth.

A shift from type 1 to type 2 immune bias is essential for successful implantation and pregnancy. ST2 is a receptor present on type 2 immune cells and IL-33 was recently discovered to be its ligand. We measured serum levels of soluble ST2 (sST2) and IL-33 across the mid-first trimester and examined whether levels were dysregulated among women with a viable fetus but destined to miscarry. While there was no significant change in sST2 or IL-3 across the first trimester, we found that women with live pregnancies destined to fail had dysregulated levels of serum IL-33, and potentially sST2 at six weeks' gestation., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Abnormal uterine bleeding can severely affect the quality of life for women. After menstruation, the endometrium must adequately repair to limit and stop bleeding. Abnormal uterine bleeding may result from incorrect or inadequate endometrial repair after menstruation. Previous studies have shown an important contribution of activin to skin wound healing, with severely delayed wound repair observed in animals transgenically induced to overexpress activin's natural inhibitor, follistatin. Activin subunits have also been identified within human endometrium; however, their role in endometrial repair is unknown. We assessed the contribution of activin to endometrial repair after menses using a human in vitro cell wounding method and our well-characterized mouse model of endometrial breakdown and repair applied to mice overexpressing follistatin. Endometrial repair after menses is initiated with reepithelialization of the uterine surface. To mimic this repair, we utilized a human endometrial epithelial cell line (ECC-1) and demonstrated significant stimulation of wound closure after activin A administration, and attenuation of this response by addition of follistatin. Immunolocalization of activin subunits, betaA and betaB, in control endometrium from the mouse model demonstrated specific epithelial and stromal localization and some leukocyte staining (betaA) around sites of endometrial repair, suggestive of a role for activin in this process. Follistatin-overexpressing animals had significantly higher circulating follistatin levels than wild-type littermates. There was a significant delay in endometrial repair after breakdown in follistatin transgenic animals compared with control animals. This study demonstrates for the first time a functional role for activin in endometrial repair after menses.

The current dogma surrounding endometrial regeneration after menses includes a critical need for estrogen-primed proliferation. Although some evidence suggests that estrogen may not be required for the initial reepithelialization of the uterine surface, it is widely believed that it is essential for successful stromal renewal. This study aimed to identify proliferating cell types during endometrial repair and to examine whether estrogen is required for successful repair using a previously developed mouse model. In the model, decidualization is artificially induced, and progesterone support withdrawn; the endometrial tissue progressively breaks down by 24 h after progesterone withdrawal and by 48 h has usually undergone complete repair. Although the mice are ovariectomized, restoration of both the stromal and epithelial components proceeds rapidly after breakdown and results in what appears to be a normal endometrium. However, potential estrogenic influences from extraovarian sources (particularly the diet and fat) remain. In this study, complete removal of extraovarian estrogen was achieved by maintenance of animals on a soy-free diet and administration of aromatase inhibitor letrozole. No significant differences in uterine weight or estrogen-responsive genes lactoferrin and progesterone receptor were observed compared with control ovariectomized but otherwise untreated mice, whereas significantly higher measurements were obtained from an estrogen-added group. Importantly, no significant difference in the rate of endometrial repair was observed in the complete absence of estrogen, demonstrating that estrogen is not essential for complete endometrial restoration in this model.