ABSTRACT
Dysregulated progenitor cell populations may contribute to poor placental development and placental insufficiency pathogenesis. Side-population cells possess progenitor properties. Recent human trophoblast side-population isolation identified enrichment of 8 specific genes (CXCL8, ELL2, GATA6, HK2, HLA-DPB1, INTS6, SERPINE3 and UPP1) (Gamage et al. 2020, Stem Cell Rev Rep). We characterised these trophoblast side-population markers in human placenta and in placental insufficiency disorders: preeclampsia and fetal growth restriction (FGR). Trophoblast side-population markers localised to mononuclear trophoblasts lining the placental villous basement membrane in preterm control, preeclamptic and FGR placental sections (n = 3, panel of 3 markers/serial section). Analysis of single-cell transcriptomics of an organoid human trophoblast stem cell (hTSC) to extravillous trophoblast (EVT) differentiation model (Shannon et al. 2022, Development) identified that all side-population genes were enriched in mononuclear trophoblast and trophoblasts committed to differentiation under hTSC culture conditions. In vitro validation via 96 h time course hTSC differentiation to EVTs or syncytiotrophoblasts (n = 5) demonstrated ELL2 and HK2 increased with differentiation (p < 0.0024, p < 0.0039 respectively). CXCL8 and HLA-DPB1 were downregulated (p < 0.030, p < 0.011 respectively). GATA6 and INTS6 increased with EVT differentiation only, and UPP1 reduced with syncytialisation. SERPINE3 was undetectable. Trophoblast side-population marker mRNA was measured in human placentas (< 34-weeks' gestation; n = 78 preeclampsia, n = 30 FGR, and n = 18 gestation-matched controls). ELL2, HK2 and CXCL8 were elevated in preeclamptic (p = 0.0006, p < 0.0001, p = 0.0335 respectively) and FGR placentas (p = 0.0065, p < 0.0001, p = 0.0001 respectively) versus controls. Placental GATA6 was reduced in pregnancies with preeclampsia and FGR (p = 0.0014, p = 0.0146 respectively). Placental INTS6 was reduced with FGR only (p < 0.0001). This study identified the localisation of a unique trophoblast subset enriched for side-population markers. Aberrant expression of some side-population markers may indicate disruptions to unique trophoblast subtypes in placental insufficiency.
ABSTRACT
BACKGROUND: Preeclampsia is a serious condition of pregnancy, complicated by aberrant maternal vascular dysfunction. CNP (C-type natriuretic peptide) contributes to vascular homeostasis, acting through NPR-B (natriuretic peptide receptor-B) and NPR-C (natriuretic peptide receptor-C). CNP mitigates vascular dysfunction of arteries in nonpregnant cohorts; this study investigates whether CNP can dilate maternal arteries in ex vivo preeclampsia models. METHODS: Human omental arteries were dissected from fat biopsies collected during cesarean section. CNP, NPR-B, and NPR-C mRNA expression was assessed in arteries collected from pregnancies complicated by preeclampsia (n=6) and normotensive controls (n=11). Using wire myography, we investigated the effects of CNP on dilation of arteries from normotensive pregnancies. Arteries were preconstricted with either serum from patients with preeclampsia (n=6) or recombinant ET-1 (endothelin-1; vasoconstrictor elevated in preeclampsia; n=6) to model vasoconstriction associated with preeclampsia. Preconstricted arteries were treated with recombinant CNP (0.001-100 µmol/L) or vehicle and vascular relaxation assessed. In further studies, arteries were preincubated with NPR-B (5 µmol/L) and NPR-C (10 µmol/L) antagonists before serum-induced constriction (n=4-5) to explore mechanistic signaling. RESULTS: CNP, NPR-B, and NPR-C mRNAs were not differentially expressed in omental arteries from preeclamptic pregnancies. CNP potently stimulated maternal artery vasorelaxation in our model of preeclampsia (using preeclamptic serum). Its vasodilatory actions were driven through the activation of NPR-B predominantly; antagonism of this receptor alone dampened CNP vasorelaxation. Interestingly, CNP did not reduce ET-1-driven omental artery constriction. CONCLUSIONS: Collectively, these data suggest that enhancing CNP signaling through NPR-B offers a potential therapeutic strategy to reduce systemic vascular constriction in preeclampsia.
Subject(s)
Natriuretic Peptide, C-Type , Pre-Eclampsia , Receptors, Atrial Natriuretic Factor , Vasodilation , Female , Pre-Eclampsia/physiopathology , Pre-Eclampsia/metabolism , Pre-Eclampsia/drug therapy , Humans , Pregnancy , Natriuretic Peptide, C-Type/pharmacology , Adult , Receptors, Atrial Natriuretic Factor/genetics , Receptors, Atrial Natriuretic Factor/metabolism , Vasodilation/drug effects , Vasodilation/physiology , Omentum/blood supply , Vasoconstriction/drug effects , Arteries/drug effects , Arteries/metabolism , Arteries/physiopathologyABSTRACT
Metabolomics is the study of small molecules (metabolites), within cells, tissues and biofluids. Maternal metabolites can provide important insight into the health and development of both mother and fetus throughout pregnancy. This study assessed metabolic profiles in the maternal circulation prior to and at the time of diagnosis of preeclampsia and fetal growth restriction. Maternal plasma samples were collected from two independent cohorts: (1) Established disease cohort: 50 participants diagnosed with early-onset preeclampsia (< 34 weeks' gestation), 14 with early-onset fetal growth restriction, and 25 gestation-matched controls. (2) Prospective cohort, collected at 36 weeks' gestation before diagnosis: 17 participants later developed preeclampsia, 49 delivered infants with fetal growth restriction (birthweight < 5th centile), and 72 randomly selected controls. Metabolic evaluation was performed by Metabolomics Australia on the Agilent 6545 QTOF Mass Spectrometer. In the established disease cohort, 77 metabolites were altered in circulation from participants with preeclampsia - increased L-cysteine (3.73-fold), L-cystine (3.28-fold), L-acetylcarnitine (2.57-fold), and carnitine (1.53-fold) (p < 0.05). There were 53 metabolites dysregulated in participants who delivered a fetal growth restriction infant-including increased levulinic acid, citric acid (1.93-fold), and creatine (1.14-fold) (p < 0.05). In the prospective cohort, 30 metabolites were altered in participants who later developed preeclampsia at term - reduced glutaric acid (0.85-fold), porphobilinogen (0.77-fold) and amininohippuric acid (0.82-fold) (p < 0.05) was observed. There were 5 metabolites altered in participants who later delivered a fetal growth restriction infant - including reduced 3-methoxybenzenepropanoic acid (p < 0.05). Downstream pathway analysis revealed aminoacyl-tRNA biosynthesis to be most significantly altered in the established cohort in preeclampsia (13/48 hits, p < 0.001) and fetal growth restriction (7/48 hits, p < 0.001). The predictive cohort showed no significant pathway alterations. This study observed altered metabolites in maternal plasma collected before and after diagnosis of a preeclampsia or fetal growth restriction. While a significant number of metabolites were altered with established disease, few changes were observed in the predictive cohort. Thus, metabolites measured in this study may not be useful as predictors of preeclampsia or fetal growth restriction.
Subject(s)
Fetal Growth Retardation , Metabolomics , Pre-Eclampsia , Humans , Female , Pregnancy , Pre-Eclampsia/blood , Pre-Eclampsia/diagnosis , Fetal Growth Retardation/blood , Fetal Growth Retardation/diagnosis , Adult , Metabolomics/methods , Prospective Studies , Metabolome , Biomarkers/blood , Case-Control StudiesABSTRACT
This review aims to encapsulate the current knowledge in extracellular vesicles extracted from amniotic fluid and amniotic fluid derived stem/stromal cells. Amniotic fluid (AF) bathes the developing fetus, providing nutrients and protection from biological and mechanical dangers. In addition to containing a myriad of proteins, immunoglobulins and growth factors, AF is a rich source of extracellular vesicles (EVs). These vesicles originate from cells in the fetoplacental unit. They are biological messengers carrying an active cargo enveloped within the lipid bilayer. EVs in reproduction are known to play key roles in all stages of pregnancy, starting from fertilisation through to parturition. The intriguing biology of AF-derived EVs (AF-EVs) in pregnancy and their untapped potential as biomarkers is currently gaining attention. EV studies in numerous animal and human disease models have raised expectations of their utility as therapeutics. Amniotic fluid stem cell and mesenchymal stromal cell-derived EVs (AFSC-EVs) provide an established supply of laboratory-made EVs. This cell-free mode of therapy is popular as an alternative to stem cell therapy, revealing similar, if not better therapeutic outcomes. Research has demonstrated the successful application of AF-EVs and AFSC-EVs in therapy, harnessing their anti-inflammatory, angiogenic and regenerative properties. This review provides an overview of such studies and discusses concerns in this emerging field of research.
Subject(s)
Extracellular Vesicles , Mesenchymal Stem Cells , Animals , Humans , Female , Pregnancy , Amniotic Fluid , KnowledgeABSTRACT
In brief: Preterm birth is the leading cause of perinatal morbidity and mortality, and new therapies that delay preterm birth and improve neonatal outcomes are urgently needed. This study investigates whether ticagrelor inhibits uterine contractility and inflammation in preclinical in vitro, ex vivo (human) and in vivo (mouse) studies, to explore the potential of repurposing ticagrelor for the prevention of preterm birth. Abstract: Preterm birth remains a significant global health challenge, affecting approximately 10% of pregnancies and resulting in one million deaths globally every year. Tocolytic agents, used to manage preterm labour, have considerable limitations including lack of efficacy, and adverse side effects, emphasising the urgent need for innovative solutions. Here, we explore repurposing an antiplatelet cardioprotective drug, ticagrelor, as a potential treatment to prevent preterm birth. Ticagrelor has demonstrated pleiotropic actions beyond platelet inhibition, including relaxant effects on smooth muscle cells and anti-inflammatory effects in models of diabetes and sepsis. As preterm birth is underscored by inflammatory processes triggering uterine contractions, these actions position ticagrelor as an attractive candidate for prevention or delay of preterm birth. Utilising primary human myometrial tissue, human myometrial cells, and a mouse model of preterm birth, we investigated ticagrelor's potential as a safe and effective therapy for preterm birth. We showed that ticagrelor did not reduce the frequency or strength of spontaneous muscle contractions of ex vivo myometrial tissue nor did it reduce in vitro inflammation-induced contractility in myometrial cells. Additionally, ticagrelor did not exhibit the anticipated anti-inflammatory effects in myometrial cell culture experiments. In our mouse model of preterm birth, ticagrelor neither improved the preterm birth rate or fetal survival outcomes. Gene expression of pro-inflammatory cytokines and contraction-associated proteins in postpartum mouse uteri were unaltered by ticagrelor. In conclusion, ticagrelor is not a strong candidate to continue investigations in clinical trial for the treatment of preterm labour and prevention of preterm birth.
Subject(s)
Obstetric Labor, Premature , Premature Birth , Pregnancy , Female , Infant, Newborn , Humans , Animals , Mice , Premature Birth/prevention & control , Premature Birth/metabolism , Ticagrelor/pharmacology , Ticagrelor/metabolism , Ticagrelor/therapeutic use , Obstetric Labor, Premature/prevention & control , Obstetric Labor, Premature/metabolism , Myometrium/metabolism , Inflammation/metabolism , Anti-Inflammatory Agents/pharmacologyABSTRACT
There is growing interest in the use of the enzyme, insulin regulated aminopeptidase (IRAP), as a biomarker for conditions such as cardio-metabolic diseases and ischemic stroke, with upregulation in its tissue expression in these conditions. However, quantification of circulating IRAP has been hampered by difficulties in detecting release of the truncated, soluble form of this enzyme into the blood stream. The current study aimed to develop a sandwich ELISA using novel antibodies directed towards the soluble portion of IRAP (sIRAP), to improve accuracy in detection and quantification of low levels of sIRAP in plasma. A series of novel anti-IRAP antibodies were developed and found to be highly specific for sIRAP in Western blots. A sandwich ELISA was then optimised using two distinct antibody combinations to detect sIRAP in the low nanogram range (16-500 ng/ml) with a sensitivity of 9 ng/ml and intra-assay variability < 10%. Importantly, the clinical validity of the ELISA was verified by the detection of significant increases in the levels of sIRAP throughout gestation in plasma samples from pregnant women. The specific and sensitive sandwich ELISA described in this study has the potential to advance the development of IRAP as a biomarker for certain diseases.
Subject(s)
Aminopeptidases , Insulin , Humans , Female , Pregnancy , Enzyme-Linked Immunosorbent Assay , Antibodies , Blotting, Western , Cystinyl Aminopeptidase/metabolismABSTRACT
Gestational diabetes mellitus (GDM) is a condition of pregnancy defined by new-onset hyperglycemia. GDM is associated with impaired maternal endothelial and vascular reactivity. Endothelin-1 (ET-1) is a potent vasoconstrictor that contributes to endothelial dysfunction, however, its abundance and actions in GDM are unclear. Maternal plasma was obtained from pregnancies complicated by GDM (n = 24) and gestation-matched controls (n = 42); circulating ET-1 levels were assessed by ELISA. Human omental arteries from healthy pregnancies and those complicated by GDM were dissected from omental fat biopsies and collected at cesarean section. mRNA expression of ET-1 and its receptors, ETA and ETB, in addition to vascular cell adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1) were assessed by qPCR (n = 28). Using wire myography, we investigated vascular constriction to ET-1 (10-11-10-4 M) in omental arteries from pregnancies complicated by GDM, compared to gestation-matched controls (n = 7). GDM cases were stratified by clinical management, diet intervention (n = 5), or insulin treatment (n = 6). Additionally, arteries from healthy pregnancies were treated with insulin (1 mU/mL (n = 7) and 10 mU/mL (n = 5)) or vehicle control. Vasoactive response to ET-1 was measured via wire myography. Circulating ET-1 levels and mRNA expression of the ET-1 system in omental arteries were not found to be significantly different between pregnancies complicated by GDM compared to healthy controls. However, we found insulin treatment during pregnancy and in ex vivo models reduced ET-1 vasoconstriction of maternal vasculature in GDM. These data suggest insulin may improve vascular function in GDM, however, further investigation is needed to define the role of ET-1 in pregnancy.
ABSTRACT
BACKGROUND: Mucins are a family of proteins that protect the epithelium. A particular type of mucin, MUC15 is highly expressed in the placenta. This study aimed to characterise MUC15 in preeclampsia and investigate its role in placental stem cell biology. METHODS: MUC15 mRNA and protein were measured in placentas from patients with early onset (<34 weeks' gestation) preeclampsia. Circulating serum MUC15 was measured via ELISA. MUC15 was localised in the placenta using in situ hybridisation. MUC15 mRNA expression was measured across differentiation of human trophoblast stem cells (hTSCs) to syncytiotrophoblast and extravillous trophoblasts. MUC15 was measured after syncytialised hTSCs were cultured in hypoxic (1% O2) and proinflammatory (TNF α, IL-6) conditions. MUC15 secretion was assessed when syncytialised hTSCs were treated with brefeldin A (impairs protein trafficking) and batimastat (inhibits matrix metalloproteinases). RESULTS: MUC15 protein was significantly increased in the placenta (P = 0.0003, n = 32 vs n = 20 controls) and serum (P = 0.016, n = 32 vs n = 22 controls) of patients with preeclampsia, whilst MUC15 mRNA remained unchanged (n = 61 vs n = 18 controls). MUC15 mRNA (P = 0.005) and protein secretion (P = 0.006) increased following differentiation to syncytiotrophoblast cells. In situ hybridisation confirmed MUC15 localised to the syncytiotrophoblast cell within the placenta. Neither hypoxic or inflammatory conditions changed MUC15 mRNA expression or secretion. Brefeldin A treated hTSCs did not alter MUC15 secretion, whilst batimastat reduced MUC15 secretion (P = 0.044). CONCLUSIONS: MUC15 is increased in early onset preeclampsia and is cleaved by matrix metalloproteinases. Increased MUC15 may reflect a protective mechanism associated with placental dysfunction. Further research will aid in confirming this.
Subject(s)
Placenta , Pre-Eclampsia , Pregnancy , Humans , Female , Placenta/metabolism , Mucins/metabolism , Pre-Eclampsia/metabolism , Brefeldin A/metabolism , Trophoblasts/metabolism , RNA, Messenger/metabolism , Matrix Metalloproteinases/metabolismABSTRACT
BACKGROUND: Preeclampsia is a severe complication of pregnancy which is attributed to placental dysfunction. The retrotransposon, Paternal Expressed Gene 10 (PEG10) harbours critical placental functions pertaining to placental trophoblast cells. Limited evidence exists on whether PEG10 is involved in preeclampsia pathogenesis. This study characterised the expression and regulation of PEG10 in placentas from patients with early-onset preeclampsia compared to gestation-matched controls. METHODS: PEG10 expression was measured in plasma and placentas collected from patients with early-onset preeclampsia (< 34 weeks') and gestation-matched controls using ELISA (protein) and RT-qPCR (mRNA). First-trimester human trophoblast stem cells (hTSCs) were used for in vitro studies. PEG10 expression was measured during hTSC differentiation and hTSC exposure to hypoxia (1% O2) and inflammatory cytokines (IL-6 and TNFα) using RT-qPCR. Functional studies used PEG10 siRNA to measure the effect of reduced PEG10 on canonical TGF-[Formula: see text] signalling and proliferation using luciferase and xCELLigence assays, respectively. RESULTS: PEG10 mRNA expression was significantly reduced in placentas from patients with early-onset preeclampsia (< 34 weeks' gestation) relative to controls (p = 0.04, n = 78 vs n = 18 controls). PEG10 protein expression was also reduced in preeclamptic placentas (p = 0.03, n = 5 vs n = 5 controls, blinded assessment of immunohistochemical staining), but neither PEG10 mRNA nor protein could be detected in maternal circulation. PEG10 was most highly expressed in hTSCs, and its expression was reduced as hTSCs differentiated into syncytiotrophoblasts (p < 0.0001) and extravillous trophoblasts (p < 0.001). Trophoblast differentiation was not altered when hTSCs were treated with PEG10 siRNA (n = 5 vs n = 5 controls). PEG10 was significantly reduced in hTSCs exposed to hypoxia (p < 0.01). PEG10 was also reduced in hTSCs treated with the inflammatory cytokine TNF [Formula: see text] (p < 0.01), but not IL-6. PEG10 knocked down (siRNA) in hTSCs showed reduced activation of the canonical TGF-ß signalling effector, the SMAD binding element (p < 0.05) relative to controls. PEG10 knockdown in hTSCs however was not associated with any significant alterations in proliferation. CONCLUSIONS: Placental PEG10 is reduced in patients with early-onset preeclampsia. In vitro studies suggest that hypoxia and inflammation may contribute to PEG10 downregulation. Reduced PEG10 alters canonical TGF-[Formula: see text] signalling, and thus may be involved in trophoblast dysfunction associated with this pathway.
Subject(s)
Placenta , Pre-Eclampsia , Pregnancy , Humans , Female , Placenta/metabolism , Pre-Eclampsia/diagnosis , Pre-Eclampsia/genetics , Trophoblasts/metabolism , Cytokines/genetics , Cytokines/metabolism , RNA, Small Interfering , RNA, Messenger/metabolism , Hypoxia , DNA-Binding Proteins/metabolism , RNA-Binding Proteins/metabolism , Apoptosis Regulatory Proteins/metabolismABSTRACT
Preeclampsia is a heterogeneous and multiorgan cardiovascular disorder of pregnancy. Here, we report the development of a novel strip-based lateral flow assay (LFA) using lanthanide-doped upconversion nanoparticles conjugated to antibodies targeting two different biomarkers for detection of preeclampsia. We first measured circulating plasma FKBPL and CD44 protein concentrations from individuals with early-onset preeclampsia (EOPE), using ELISA. We confirmed that the CD44/FKBPL ratio is reduced in EOPE with a good diagnostic potential. Using our rapid LFA prototypes, we achieved an improved lower limit of detection: 10â pg ml-1 for FKBPL and 15â pg ml-1 for CD44, which is more than one order lower than the standard ELISA method. Using clinical samples, a cut-off value of 1.24 for CD44/FKBPL ratio provided positive predictive value of 100 % and the negative predictive value of 91 %. Our LFA shows promise as a rapid and highly sensitive point-of-care test for preeclampsia.
Subject(s)
Pre-Eclampsia , Pregnancy , Female , Humans , Pre-Eclampsia/diagnosis , Sensitivity and Specificity , Point-of-Care Testing , Biomarkers/analysis , Tacrolimus Binding ProteinsABSTRACT
Spontaneous preterm birth is the leading cause of perinatal morbidity and mortality. Tocolytics are drugs used in cases of imminent preterm birth to inhibit uterine contractions. Nifedipine is a calcium channel blocking agent used to delay threatened spontaneous preterm birth, however, has limited efficacy and lacks preclinical data regarding mechanisms of action. It is unknown if nifedipine affects the pro-inflammatory environment associated with preterm labour pathophysiology and we hypothesise nifedipine only targets myometrial contraction rather than also mitigating inflammation. We assessed anti-inflammatory and anti-contractile effects of nifedipine on human myometrium using in vitro and ex vivo techniques, and a mouse model of preterm birth. We show that nifedipine treatment inhibited contractions in myometrial in vitro contraction assays (P = 0.004 vs. vehicle control) and potently blocked spontaneous and oxytocin-induced contractions in ex vivo myometrial tissue in muscle myography studies (P = 0.01 vs. baseline). Nifedipine treatment did not reduce gene expression or protein secretion of pro-inflammatory cytokines in either cultured myometrial cells or ex vivo tissues. Although nifedipine could delay preterm birth in some mice, this was not consistent in all dams and was overall not statistically significant. Our data suggests nifedipine does not modulate preterm birth via inflammatory pathways in the myometrium, and this may account for its limited clinical efficacy.
Subject(s)
Obstetric Labor, Premature , Premature Birth , Tocolytic Agents , Pregnancy , Female , Infant, Newborn , Mice , Humans , Animals , Tocolytic Agents/pharmacology , Tocolytic Agents/therapeutic use , Nifedipine/metabolism , Premature Birth/metabolism , Obstetric Labor, Premature/drug therapy , Obstetric Labor, Premature/metabolism , Uterine Contraction , Myometrium/metabolismABSTRACT
Preeclampsia is associated with an increased lifelong risk of cardiovascular disease (CVD). It is not clear whether this is induced by persistent systemic organ and vascular damage following preeclampsia or due to a predisposition to both conditions that share cardiovascular pathophysiology. Common to both CVD and preeclampsia is the dysregulation of corin and its proteolytic product, atrial natriuretic peptide (ANP). ANP, a hypotensive hormone converted from pro-ANP by corin, is involved in blood pressure homeostasis. While corin is predominantly a cardiac enzyme, both corin and pro-ANP are significantly upregulated in the gravid uterus and dysregulated in preeclampsia. Relatively little is known about ANP function in the endothelium during a pregnancy complicated by preeclampsia. Here, we investigated the effect of ANP on endothelial cell proliferation and migration, markers of endothelial dysfunction, and receptor expression in omental arteries exposed to circulating preeclamptic toxins. ANP receptor expression is significantly upregulated in preeclamptic vasculature but not because of exposure to preeclampsia toxins tumour necrosis factor α or soluble fms-like tyrosine kinase-1. The supplementation of endothelial cells with ANP did not promote proliferation or migration, nor did ANP improve markers of endothelial dysfunction. The role of ANP in preeclampsia is unlikely to be via endothelial pathways.
Subject(s)
Cardiovascular Diseases , Pre-Eclampsia , Pregnancy , Female , Humans , Atrial Natriuretic Factor/metabolism , Endothelial Cells/metabolism , Endothelium/metabolismABSTRACT
BACKGROUND: Preeclampsia is a severe complication of pregnancy. Chemerin is an adipokine secreted from adipose tissue and highly expressed in placenta. This study evaluated the biomarker potential of circulating chemerin to predict preeclampsia. METHODS: Maternal plasma and placenta were collected from women with early-onset preeclampsia (<34 weeks), with preeclampsia and eclampsia, or before preeclampsia diagnosis (36 weeks). Human trophoblast stem cells were differentiated into syncytiotrophoblast or extravillous trophoblasts across 96â hours. Cells were cultured in 1% O2 (hypoxia) or 5% O2 (normoxia). Chemerin was measured by enzyme-linked immunosorbent assay (ELISA) and RARRES2 (gene coding chemerin) by reverse transcription-quantitative polymerase chain reaction. RESULTS: Circulating chemerin was increased in 46 women with early-onset preeclampsia (<34 weeks) compared to 17 controls (P < .0006). Chemerin was increased in placenta from 43 women with early-onset preeclampsia compared to 24 controls (P < .0001). RARRES2 was reduced in placenta from 43 women with early-onset preeclampsia vs 24 controls (P < .0001). Chemerin was increased in plasma from 26 women with established preeclampsia (P = .006), vs 15 controls. Circulating chemerin was increased in 23 women who later developed preeclampsia vs 182 who did not (P = 3.23 × 10-6). RARRES2 was reduced in syncytiotrophoblast (P = .005) or extravillous trophoblasts (P < .0001). Hypoxia increased RARRES2 expression in syncytiotrophoblast (P = .01) but not cytotrophoblast cells. CONCLUSIONS: Circulating chemerin was elevated in women with early-onset preeclampsia, established preeclampsia, and preceding preeclampsia diagnosis of preeclampsia. RARRES2 was dysregulated in placenta complicated by preeclampsia and may be regulated through hypoxia. Chemerin may have potential as a biomarker for preeclampsia but would need to be combined with other biomarkers.
Subject(s)
Pre-Eclampsia , Female , Humans , Pregnancy , Biomarkers/metabolism , Hypoxia/metabolism , Placenta/metabolism , Pre-Eclampsia/diagnosis , Trophoblasts/metabolismABSTRACT
INTRODUCTION: Development of a therapeutic that targets the pathophysiological elements of preeclampsia would be a major advance for obstetrics, with potential to save the lives of countless mothers and babies. We recently identified anti-inflammatory drug sulfasalazine as a prospective candidate therapeutic for treatment of preeclampsia. In primary human cells and tissues in vitro, sulfasalazine potently decreased secretion of anti-angiogenic sFlt-1 and sENG, increased production of pro-angiogenic PlGF, mitigated endothelial dysfunction, and promoted whole vessel vasodilation. METHODS: Using nitric oxide synthase antagonist Nω-Nitro-l-arginine methyl ester hydrochloride, a preeclampsia-like phenotype was induced in pregnant mice, including high blood pressure, fetal growth restriction, and elevated circulating sFlt-1. Mice were treated with sulfasalazine or vehicle from gestational day (D)13.5, with blood pressure measurements across gestation, fetal measurements at D17.5, and wire myograph assessment of vasoactivity. RESULTS: Sulfasalazine had a modest effect on blood pressure, decreasing diastolic and mean blood pressure on D13.5, but not later in gestation, or systolic blood pressure. Sulfasalazine was not able to rescue fetal growth, in male or female fetuses. There was a suggestion of improved vasoactivity with sulfasalazine, but further clarification is required. DISCUSSION: In this mouse model of preeclampsia, sulfasalazine did not sustain reductions in blood pressure nor affect fetal parameters of size and weight, both desirable attributes of a viable preeclampsia therapeutic. While these data suggest sulfasalazine might improve vasoactivity, murine toxicity considerations limited the dose range of sulfasalazine that could be tested in the current study.
Subject(s)
Hypertension , Pre-Eclampsia , Pregnancy , Female , Male , Mice , Animals , Humans , Pre-Eclampsia/drug therapy , Sulfasalazine/pharmacology , Sulfasalazine/therapeutic use , Blood Pressure , Disease Models, Animal , Nitric Oxide Synthase/pharmacology , Vascular Endothelial Growth Factor Receptor-1 , Nitric Oxide/pharmacologyABSTRACT
In brief: Preterm birth is the leading cause of perinatal morbidity and mortality; however, current therapies offer limited efficacy to delay birth and improve neonatal outcomes. This review explores the potential of repurposing drugs with known safety profiles to quench uterine contractions and inflammation, identifying promising agents for clinical trials. Abstract: Preterm birth is the leading cause of neonatal morbidity and mortality globally. Despite extensive research into the underlying pathophysiology, rates of preterm birth have not significantly reduced. Currently, preterm labour management is based on optimising neonatal outcomes. Treatment involves administering drugs (tocolytics) to suppress uterine contractions to allow sufficient time for transfer to an appropriate facility and administration of antenatal corticosteroids for fetal lung maturation. Current tocolytics are limited as they are associated with adverse maternal and fetal effects and only delay delivery for a short period. There has been a serious lack of therapeutic development for preterm birth, and new approaches to protect against or delay preterm birth are urgently needed. Repurposing drugs for the prevention of preterm birth presents as a promising approach by reducing the time and costs associated with pharmaceutical drug development. In this review, we explore the evidence for the potential of therapies, specifically proton pump inhibitors, tumour necrosis factor inhibitors, prostaglandin receptor antagonists, aspirin, and statins, to be repurposed as preventatives and/or treatments for preterm birth. Importantly, many of these innovative approaches being explored have good safety profiles in pregnancy. We also review how delivery of these drugs can be enhanced, either through targeted delivery systems or via combination therapy approaches. We aim to present innovative strategies capable of targeting multiple aspects of the complex pathophysiology that underlie preterm birth. There is an urgent unmet need for preterm birth therapeutic development, and these strategies hold great promise for improving neonatal outcomes.
Subject(s)
Obstetric Labor, Premature , Premature Birth , Tocolytic Agents , Infant, Newborn , Female , Pregnancy , Humans , Premature Birth/prevention & control , Tocolytic Agents/therapeutic use , Pharmaceutical Preparations , Drug Repositioning , Obstetric Labor, Premature/drug therapy , Obstetric Labor, Premature/prevention & controlABSTRACT
Preeclampsia is a multi-system disease that can have severe, even fatal implications for the mother and fetus. Abnormal placentation can lead to ischaemic tissue injury and placental inflammation. In turn, the placenta releases anti-angiogenic factors into the maternal circulation. These systemically act to neutralise angiogenic factors causing endothelial dysfunction causing preeclampsia. Hydroxychloroquine is an immune modulating drug that is considered safe in pregnancy. There is epidemiological evidence suggesting it may reduce the risk of preeclampsia. Here, we examined the effects hydroxychloroquine on the production and secretion of sFlt-1, soluble endoglin (sENG), placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) in primary human placenta, cytotrophoblasts and umbilical vein endothelial cells (endothelial cell model). Hydroxychloroquine treatment decreased mRNA expression of two sFlt-1 isoforms and its protein secretion. sENG was not reduced. Hydroxychloroquine treatment increased secretion of pro-angiogenic factor PIGF from endothelial cells. It did not significantly reduce the expression of the endothelial cell inflammation marker, ET-1, and inflammation induced expression of the adhesion molecule, VCAM. Hydroxychloroquine could not overcome leukocyte adhesion to endothelial cells. Hydroxychloroquine mitigates features of preeclampsia, but it does not reduce key markers of endothelial dysfunction.
Subject(s)
Pre-Eclampsia , Vascular Diseases , Female , Humans , Pregnancy , Trophoblasts/metabolism , Placenta Growth Factor/metabolism , Pre-Eclampsia/drug therapy , Pre-Eclampsia/metabolism , Placenta/metabolism , Endothelial Cells/metabolism , Hydroxychloroquine/therapeutic use , Vascular Endothelial Growth Factor A/metabolism , Endoglin/metabolism , Biomarkers/metabolism , Vascular Diseases/metabolism , Inflammation/metabolismABSTRACT
BACKGROUND: Metformin, widely used to treat diabetes, is now considered a candidate therapeutic for treatment of cardiovascular disease. This study aimed to assess whether metformin's non-glycaemic effects could mitigate cardiovascular disease indices in female mice consuming a high fat diet (HFD). METHODS: Four-week old female Arc:Arc(S) mice were placed on a standard (std) chow diet or Western-style HFD (22% fat, 0.15% cholesterol). At â¼8 months, the mice were administered 150 mg/kg metformin or vehicle (control) via intraperitoneal injection for 11 days. Blood pressure was measured (tail cuff plethysmography) at Day 9 and 11 of treatment. On Day 11, mice were weighed and culled. The mesenteric arcade and kidneys were collected for assessment of vascular reactivity (wire myography), and assessment of expression of cardiometabolic markers (qPCR), respectively. RESULTS: The HFD fed female mice were significantly heavier than those receiving the std diet at 1-12 weeks on diet, and at cull. Mice on a std diet with metformin treatment were significantly heavier at cull than the mice on a std diet administered the control treatment. Metformin treatment did not alter the weight of the mice receiving the HFD. Neither the HFD (compared to the std diet), nor metformin treatment (compared to control treatment) altered blood pressure, vascular reactivity, or expression of cardiometabolic markers in the kidney. CONCLUSION: Consumption of a Western-style HFD (without high salt/sugar levels) did not alter the cardiovascular markers measured. Further studies are required to establish the non-glycaemic, cardio-protective effects of metformin in high-risk cohorts.
Subject(s)
Cardiovascular Diseases , Metformin , Sexually Transmitted Diseases , Mice , Female , Animals , Metformin/pharmacology , Metformin/therapeutic use , Diet, High-Fat , Cardiovascular Diseases/etiology , Cardiovascular Diseases/prevention & control , Biomarkers , Sexually Transmitted Diseases/drug therapy , Mice, Inbred C57BLABSTRACT
Placental dysfunction is the leading cause of both preeclampsia and fetal growth restriction. This study aimed to characterize endothelial protein C receptor (EPCR) in preterm preeclampsia, term preeclampsia, and fetal growth restriction (defined by delivery of a small for gestational age [SGA] infant [<10% birthweight centile]) and examine its regulation in primary syncytiotrophoblast. Placental EPCR mRNA and protein were significantly increased in patients with preterm preeclampsia (<34 weeks gestation) compared to gestation-matched controls (p < .0001). In the plasma, EPCR was also significantly elevated (p = .01) in established preterm preeclampsia while its substrate, protein C (PC) was significantly reduced (p = .0083). Placentas from preterm small for gestational age (SGA) cases, had elevated EPCR mRNA expression (p < .0001) relative to controls. At 36 weeks, no significant changes in plasma EPCR were detected in samples from patients destined to develop preeclampsia or deliver an SGA infant at term. In terms of syncytiotrophoblast, hypoxia significantly increased EPCR mRNA expression (p = .008), but Tumor Necrosis Factor Alpha (TNF-α) decreased EPCR mRNA. Interleukin-6 (IL-6) had no significant effect on EPCR mRNA expression. When isolated syncytiotrophoblast was treated with metformin under hypoxia (1% O2 ) or normoxia (8% O2 ), EPCR mRNA expression was significantly reduced (p = .008) relative to control. In conclusion, EPCR is markedly elevated in the placenta and the circulation of patients with established preterm preeclampsia and placental increases may be associated with hypoxia. Additionally, fetal growth-restricted pregnancies (as defined by the delivery of an SGA infant) also demonstrated elevated placental EPCR.
Subject(s)
Pre-Eclampsia , Infant, Newborn , Humans , Female , Pregnancy , Pre-Eclampsia/metabolism , Fetal Growth Retardation/metabolism , Endothelial Protein C Receptor/metabolism , Placenta/metabolism , Hypoxia/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Preeclampsia affects â¼2-8% 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.
Subject(s)
Cardiovascular Diseases , Pre-Eclampsia , Animals , Female , Mice , Pregnancy , C-Reactive Protein/metabolism , Cardiovascular Diseases/etiology , Cardiovascular Diseases/metabolism , Disease Models, Animal , Endothelin-1/genetics , Endothelin-1/metabolism , Matrix Metalloproteinase 9/metabolism , NG-Nitroarginine Methyl Ester/pharmacology , NG-Nitroarginine Methyl Ester/metabolism , Phenylephrine/metabolism , Placenta/metabolism , Pre-Eclampsia/metabolism , RNA, Messenger/metabolism , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vasoconstrictor Agents/metabolismABSTRACT
Preeclampsia is a disease of pregnancy responsible for significant maternal and neonatal mortality. Galectin-3 is a ß-Galactoside binding protein. This study aimed to characterise galectin-3 in women with preeclampsia and human trophoblast stem cells (hTSCs). Galectin-3 was measured in placental lysates and plasma collected from patients with early-onset preeclampsia (delivered <34 weeks' gestation) and gestation matched controls. Placental galectin-3 protein was significantly reduced in 43 women with early-onset preeclampsia compared to 21 controls. mRNA expression of LGALS3 (galectin-3 encoding gene) was reduced in 29 women with early-onset preeclampsia, compared to 18 controls (p = 0.009). There was no significant difference in plasma galectin-3 protein in 46 women with early-onset preeclampsia compared to 20 controls. In a separate cohort of samples collected at 36 weeks' gestation, circulating galectin-3 was not altered in 23 women who later developed preeclampsia, versus 182 who did not. In syncytialised hTSCs, hypoxia increased mRNA expression of LGALS3 (p = 0.01). Treatment with inflammatory cytokines (TNF-α and IL-6) had no effect on LGALS3 mRNA expression. However, TNF-α treatment caused an increase in mRNA expression of LGALS3BP (galectin-3 binding protein encoding gene) in hTSCs (p = 0.03). This study showed a reduction of galectin-3 in placenta from pregnancies complicated by early-onset preeclampsia. LGALS3 mRNA expression was dysregulated by hypoxia exposure in placental stem cells.