ABSTRACT
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
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
Biomarkers for placental dysfunction are currently lacking. We recently identified SPINT1 as a novel biomarker; SPINT2 is a functionally related placental protease inhibitor. This study aimed to characterise SPINT2 expression in placental insufficiency. Circulating SPINT2 was assessed in three prospective cohorts, collected at the following: (1) term delivery (n = 227), (2) 36 weeks (n = 364), and (3) 24-34 weeks' (n = 294) gestation. SPINT2 was also measured in the plasma and placentas of women with established placental disease at preterm (<34 weeks) delivery. Using first-trimester human trophoblast stem cells, SPINT2 expression was assessed in hypoxia/normoxia (1% vs. 8% O2), and following inflammatory cytokine treatment (TNFα, IL-6). Placental SPINT2 mRNA was measured in a rat model of late-gestational foetal growth restriction. At 36 weeks, circulating SPINT2 was elevated in patients who later developed preeclampsia (p = 0.028; median = 2233 pg/mL vs. controls, median = 1644 pg/mL), or delivered a small-for-gestational-age infant (p = 0.002; median = 2109 pg/mL vs. controls, median = 1614 pg/mL). SPINT2 was elevated in the placentas of patients who required delivery for preterm preeclampsia (p = 0.025). Though inflammatory cytokines had no effect, hypoxia increased SPINT2 in cytotrophoblast stem cells, and its expression was elevated in the placental labyrinth of growth-restricted rats. These findings suggest elevated SPINT2 is associated with placental insufficiency.
Subject(s)
Biomarkers/metabolism , Fetal Growth Retardation/diagnosis , Membrane Glycoproteins/metabolism , Placenta Diseases/diagnosis , Placenta/pathology , Pre-Eclampsia/diagnosis , Trophoblasts/pathology , Adolescent , Female , Fetal Growth Retardation/metabolism , Humans , Infant, Newborn , Infant, Small for Gestational Age , Longitudinal Studies , Placenta/metabolism , Placenta Diseases/metabolism , Pre-Eclampsia/metabolism , Pregnancy , Prospective Studies , Trophoblasts/metabolismABSTRACT
The circulating life span of blood platelets is regulated by the prosurvival protein BCL-XL It restrains the activity of BAK and BAX, the essential prodeath mediators of intrinsic apoptosis. Disabling the platelet intrinsic apoptotic pathway in mice by deleting BAK and BAX results in a doubling of platelet life span and concomitant thrombocytosis. Apoptotic platelets expose phosphatidylserine (PS) via a mechanism that is distinct from that driven by classical agonists. Whether there is any role for apoptotic PS in platelet function in vivo, however, is unclear. Apoptosis has also been associated with the platelet storage lesion (PSL), the constellation of biochemical deteriorations that occur during blood bank storage. In this study, we investigated the role of BAK/BAX-mediated apoptosis in hemostasis and thrombosis and in the development of the PSL. We show that although intrinsic apoptosis is rapidly induced during storage at 37°C, it is not detected when platelets are kept at the standard storage temperature of 22°C. Remarkably, loss of BAK and BAX did not prevent the development of the PSL at either temperature. BAK/BAX-deficient mice exhibited increased bleeding times and unstable thrombus formation. This phenotype was not caused by impaired PS exposure, but was associated with a defect in granule release from aged platelets. Strikingly, rejuvenation of BAK/BAX-deficient platelets in vivo completely rescued the observed hemostatic defects. Thus, apoptotic culling of old platelets from the bloodstream is essential to maintain a functional, hemostatically reactive platelet population. Inhibiting intrinsic apoptosis in blood banked platelets is unlikely to yield significant benefit.
Subject(s)
Apoptosis , Blood Platelets/metabolism , Disease Susceptibility , Animals , Apoptosis/genetics , Biomarkers , Bleeding Time , Blood Cell Count , Blood Coagulation , Caspases/metabolism , Cell Survival/genetics , Female , Genotype , Male , Mice , Mice, Knockout , Mitochondria/metabolism , Signal Transduction , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism , bcl-X Protein/genetics , bcl-X Protein/metabolismABSTRACT
BACKGROUND: Fetal growth restriction is a disorder of placental dysfunction with three to four-fold increased risk of stillbirth. Fetal growth restriction has pathophysiological features in common with preeclampsia. We hypothesised that angiogenesis-related factors in maternal plasma, known to predict preeclampsia, may also detect fetal growth restriction at 36 weeks' gestation. We therefore set out to determine the diagnostic performance of soluble fms-like tyrosine kinase 1 (sFlt-1), placental growth factor (PlGF), and the sFlt-1:PlGF ratio, measured at 36 weeks' gestation, in identifying women who subsequently give birth to small-for-gestational-age (SGA; birthweight <10th centile) infants. We also aimed to validate the predictive performance of the analytes for late-onset preeclampsia in a large independent, prospective cohort. METHODS: A nested 1:2 case-control study was performed including 102 cases of SGA infants and a matched group of 207 controls; and 39 cases of preeclampsia. We determined the diagnostic performance of each angiogenesis-related factor, and of their ratio, to detect SGA infants or preeclampsia, for a predetermined 10% false positive rate. RESULTS: Median plasma levels of PlGF at 36 weeks' gestation were significantly lower in women who subsequently had SGA newborns (178.5 pg/ml) compared to normal birthweight controls (326.7 pg/ml, p < 0.0001). sFlt-1 was also higher among SGA cases, but this was not significant after women with concurrent preeclampsia were excluded. The sensitivity of PlGF to predict SGA infants was 28.8% for a 10% false positive rate. The sFlt-1:PlGF ratio demonstrated better sensitivity for preeclampsia than either analyte alone, detecting 69.2% of cases for a 10% false positive rate. CONCLUSIONS: Plasma PlGF at 36 weeks' gestation is significantly lower in women who subsequently deliver a SGA infant. While the sensitivity and specificity of PlGF currently limit clinical translation, our findings support a blood-based biomarker approach to detect late-onset fetal growth restriction. Thirty-six week sFlt-1:PlGF ratio predicts 69.2% of preeclampsia cases, and could be a useful screening test to triage antenatal surveillance.
Subject(s)
Pregnancy Trimester, Third/blood , Vascular Endothelial Growth Factor Receptor-1/blood , Biomarkers/blood , Case-Control Studies , Female , Humans , Infant, Newborn , Infant, Small for Gestational Age/blood , Placenta Growth Factor , Pregnancy , Prospective StudiesABSTRACT
The anti-angiogenic protein, soluble fms-like tyrosine kinase-1 (sFLT-1), plays a central role in preeclamptic pathophysiology. A splice variant of FLT-1 (VEGF receptor 1), sFLT-1 is released in excessive amounts from the preeclamptic placenta into the maternal circulation, where it causes endothelial dysfunction manifesting as end-organ disease. However, the mechanisms regulating its production within the placenta remain poorly understood. Recently it was shown in endothelial cells that Jumonji domain containing protein 6 (JMJD6) hydroxylates U2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65, a component of the splicesome). The hydroxylation by JMJD6 is oxygen dependent. Under hypoxia, JMJD6 is less able to hydroxylate U2AF65, and this unhydroxylated form of U2AF65 biases splicing of FLT-1 to sFLT-1. We assessed whether oxygen-sensing JMJD6 is differentially expressed in preeclamptic placenta and regulates sFLT-1 splicing in placenta via U2AF65. JMJD6 protein expression was significantly reduced in preterm preeclamptic placenta (P < 0.0001; n = 21) relative to preterm controls (n = 10). Exposing both placental and endothelial cells to hypoxia significantly reduced JMJD6 mRNA and increased sFLT-1 mRNA and protein expression. Silencing JMJD6 in primary endothelial and trophoblast cells significantly increased sFLT-1 secretion. Next, we examined whether these molecules may be directly interacting. We demonstrated that placental U2AF65 colocalized with JMJD6. In turn, we found JMJD6 directly interacts with U2AF65, which in turn produces sFLT-1 mRNA transcripts. Taken together, our findings provide evidence that JMJD6 may play a role in regulating the production of sFLT-1 in the preeclamptic placenta. Decreased placental JMJD6 expression may be an important component to the pathophysiology of preeclampsia.
Subject(s)
Jumonji Domain-Containing Histone Demethylases/metabolism , Placenta/metabolism , Pre-Eclampsia/metabolism , Protein Isoforms/metabolism , Vascular Endothelial Growth Factor Receptor-1/metabolism , Down-Regulation , Female , Gene Expression Regulation/physiology , Humans , Jumonji Domain-Containing Histone Demethylases/genetics , Oxygen/metabolism , Pregnancy , Protein Isoforms/genetics , Splicing Factor U2AF/genetics , Splicing Factor U2AF/metabolism , Up-Regulation , Vascular Endothelial Growth Factor Receptor-1/geneticsABSTRACT
BACKGROUND: Preeclampsia is associated with placental ischemia/hypoxia and secretion of soluble fms-like tyrosine kinase 1 and soluble endoglin into the maternal circulation. This causes widespread endothelial dysfunction that manifests clinically as hypertension and multisystem organ injury. Recently, small molecule inhibitors of hypoxic inducible factor 1α have been found to reduce soluble fms-like tyrosine kinase 1 and soluble endoglin secretion. However, their safety profile in pregnancy is unknown. Metformin is safe in pregnancy and is also reported to inhibit hypoxic inducible factor 1α by reducing mitochondrial electron transport chain activity. OBJECTIVE: The purposes of this study were to determine (1) the effects of metformin on placental soluble fms-like tyrosine kinase 1 and soluble endoglin secretion, (2) to investigate whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion are regulated through the mitochondrial electron transport chain, and (3) to examine its effects on endothelial dysfunction, maternal blood vessel vasodilation, and angiogenesis. STUDY DESIGN: We performed functional (in vitro and ex vivo) experiments using primary human tissues to examine the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from placenta, endothelial cells, and placental villous explants. We used succinate, mitochondrial complex II substrate, to examine whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion were mediated through the mitochondria. We also isolated mitochondria from preterm preeclamptic placentas and gestationally matched control subjects and measured mitochondrial electron transport chain activity using kinetic spectrophotometric assays. Endothelial cells or whole maternal vessels were incubated with metformin to determine whether it rescued endothelial dysfunction induced by either tumor necrosis factor-α (to endothelial cells) or placenta villous explant-conditioned media (to whole vessels). Finally, we examined the effects of metformin on angiogenesis on maternal omental vessel explants. RESULTS: Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary endothelial cells, villous cytotrophoblast cells, and preterm preeclamptic placental villous explants. The reduction in soluble fms-like tyrosine kinase 1 and soluble endoglin secretion was rescued by coadministration of succinate, which suggests that the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin were likely to be regulated at the level of the mitochondria. In addition, the mitochondrial electron transport chain inhibitors rotenone and antimycin reduced soluble fms-like tyrosine kinase 1 secretion, which further suggests that soluble fms-like tyrosine kinase 1 secretion is regulated through the mitochondria. Mitochondrial electron transport chain activity in preterm preeclamptic placentas was increased compared with gestation-matched control subjects. Metformin improved features of endothelial dysfunction relevant to preeclampsia. It reduced endothelial cell messenger RNA expression of vascular cell adhesion molecule 1 that was induced by tumor necrosis factor-α (vascular cell adhesion molecule 1 is an inflammatory adhesion molecule up-regulated with endothelial dysfunction and is increased in preeclampsia). Placental conditioned media impaired bradykinin-induced vasodilation; this effect was reversed by metformin. Metformin also improved whole blood vessel angiogenesis impaired by fms-like tyrosine kinase 1. CONCLUSION: Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary human tissues, possibly by inhibiting the mitochondrial electron transport chain. The activity of the mitochondrial electron transport chain was increased in preterm preeclamptic placenta. Metformin reduced endothelial dysfunction, enhanced vasodilation in omental arteries, and induced angiogenesis. Metformin has potential to prevent or treat preeclampsia.
Subject(s)
Antigens, CD/metabolism , Cardiovascular Agents/therapeutic use , Endothelium, Vascular/drug effects , Metformin/therapeutic use , Pre-Eclampsia/drug therapy , Pre-Eclampsia/prevention & control , Receptors, Cell Surface/metabolism , Vascular Endothelial Growth Factor Receptor-1/metabolism , Biomarkers/metabolism , Cardiovascular Agents/pharmacology , Electron Transport Chain Complex Proteins/metabolism , Endoglin , Endothelium, Vascular/metabolism , Endothelium, Vascular/physiopathology , Female , Humans , In Vitro Techniques , Metformin/pharmacology , Placenta/drug effects , Placenta/metabolism , Pre-Eclampsia/metabolism , Pre-Eclampsia/physiopathology , Pregnancy , Treatment Outcome , Vasodilation/drug effectsABSTRACT
BACKGROUND: Preeclampsia is associated with the placental release of soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sENG). These anti-angiogenic factors cause hypertension and multi-organ injury. Pravastatin decreases placental secretion of sFlt-1 in vitro and is currently being examined in clinical trials as a potential treatment for preeclampsia. However, it is possible that different classes of statins may be more potent at decreasing sFlt-1 secretion. We compared the relative potency of three different generations of statins on sFlt-1 and sENG secretion from human endothelial cells, trophoblast cells, and placenta explants. METHODS: We performed functional experiments using primary human umbilical vein endothelial cells, trophoblast cells and preterm preeclamptic placental explants to assess the affect of simvastatin, rosuvastatin and pravastatin on sFlt-1 and sENG secretion and compared the relative potency of each statin at reducing these factors (Inhibitory Concentration 50). Furthermore we assessed the effect of each statin on the antioxidant and cytoprotective enzyme, heme-oxygenase 1. RESULTS: All statins reduced sFlt-1 secretion from endothelial cells, trophoblasts and preterm preeclamptic placental explants. Simvastatin was the most potent inhibitor of sFlt-1 secretion from endothelial cells (IC 50 3.2 µM), trophoblast cells (IC 50 61.4 µM) and placental explants. Simvastatin was 28 times and 3 times more potent at reducing sFlt-1 secretion from endothelial cells and 85 times and 33 times more potent at reducing sFlt-1 secretion from trophoblast cells than pravastatin or rosuvastatin respectively. All statins increased sENG secretion from endothelial cells however did not change secretion from placental explants. While all statins up-regulated heme-oxygenase 1 in endothelial cells, only simvastatin up-regulated its expression in placenta from patients with preterm preeclampsia. CONCLUSION: Simvastatin may be a more potent inhibitor of sFlt-1 secretion from endothelial cells, trophoblast cells and placenta from women with preterm preeclampsia than either pravastatin or rosuvastatin.
Subject(s)
Human Umbilical Vein Endothelial Cells/drug effects , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Placenta/drug effects , Pregnancy Proteins/drug effects , Trophoblasts/drug effects , Endoglin/drug effects , Female , Heme Oxygenase-1/drug effects , Humans , Placenta/metabolism , Pravastatin/pharmacology , Pre-Eclampsia/drug therapy , Pregnancy , Pregnancy Proteins/metabolism , Rosuvastatin Calcium/pharmacology , Simvastatin/pharmacology , Trophoblasts/cytology , Up-Regulation , Vascular Endothelial Growth Factor Receptor-1/drug effects , Vascular Endothelial Growth Factor Receptor-1/metabolismABSTRACT
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
To investigate the role of Aire in thymic selection, we examined the cellular requirements for generation of ovalbumin (OVA)-specific CD4 and CD8 T cells in mice expressing OVA under the control of the rat insulin promoter. Aire deficiency reduced the number of mature single-positive OVA-specific CD4(+) or CD8(+) T cells in the thymus, independent of OVA expression. Importantly, it also contributed in 2 ways to OVA-dependent negative selection depending on the T-cell type. Aire-dependent negative selection of OVA-specific CD8 T cells correlated with Aire-regulated expression of OVA. By contrast, for OVA-specific CD4 T cells, Aire affected tolerance induction by a mechanism that operated independent of the level of OVA expression, controlling access of antigen presenting cells to medullary thymic epithelial cell (mTEC)-expressed OVA. This study supports the view that one mechanism by which Aire controls thymic negative selection is by regulating the indirect presentation of mTEC-derived antigens by thymic dendritic cells. It also indicates that mTECs can mediate tolerance by direct presentation of Aire-regulated antigens to both CD4 and CD8 T cells.
Subject(s)
Antigen Presentation , Antigens/metabolism , Clonal Deletion/immunology , Dendritic Cells/immunology , Epithelial Cells/immunology , Immune Tolerance/immunology , Thymus Gland/immunology , Transcription Factors/immunology , Animals , Antigens/immunology , Bone Marrow Transplantation , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Crosses, Genetic , Cytokines/metabolism , Gene Expression Regulation/immunology , Insulin/genetics , Mice , Mice, Transgenic , Ovalbumin/genetics , Ovalbumin/immunology , Ovalbumin/metabolism , Promoter Regions, Genetic , Radiation Chimera , Recombinant Fusion Proteins/physiology , Thymus Gland/cytology , Transcription Factors/deficiency , Transcription Factors/genetics , AIRE ProteinABSTRACT
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
Platelets have been shown to enhance the survival of lymphoma cell lines. However, it remains unclear whether they play a role in lymphoma. Here, we investigated the potential role of platelets and/or megakaryocytes in the progression of Eµ-myc lymphoma. Eµ-myc tumor cells were transplanted into recipient wild-type (WT) control, Mpl-/-, or TpoTg mice, which exhibited normal, low, and high platelet and megakaryocyte counts, respectively. TpoTg mice that underwent transplantation exhibited enhanced lymphoma progression with increased white blood cell (WBC) counts, spleen and lymph node weights, and enhanced liver infiltration when compared with WT mice. Conversely, tumor-bearing Mpl-/- mice had reduced WBC counts, lymph node weights, and less liver infiltration than WT mice. Using an Mpl-deficient thrombocytopenic immunocompromised mouse model, our results were confirmed using the human non-Hodgkin lymphoma GRANTA cell line. Although we found that platelets and platelet-released molecules supported Eµ-myc tumor cell survival in vitro, pharmacological inhibition of platelet function or anticoagulation in WT mice transplanted with Eµ-myc did not improve disease outcome. Furthermore, transient platelet depletion or sustained Bcl-xL-dependent thrombocytopenia did not alter lymphoma progression. Cytokine analysis of the bone marrow fluid microenvironment revealed increased levels of the proinflammatory molecule interleukin 1 in TpoTg mice, whereas these levels were lower in Mpl-/- mice. Moreover, RNA sequencing of blood-resident Eµ-myc lymphoma cells from TpoTg and WT mice after tumor transplantation revealed the upregulation of hallmark gene sets associated with an inflammatory response in TpoTg mice. We propose that the proinflammatory microenvironment in TpoTg mice promotes lymphoma progression.
Subject(s)
Lymphoma , Thrombocytopenia , Mice , Animals , Humans , Megakaryocytes/metabolism , Receptors, Thrombopoietin , Blood Platelets/metabolism , Thrombocytopenia/genetics , Lymphoma/genetics , Tumor MicroenvironmentABSTRACT
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
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.
Subject(s)
Eclampsia , Placenta Growth Factor , Pre-Eclampsia , Vascular Endothelial Growth Factor Receptor-1 , Biomarkers , Eclampsia/diagnosis , Female , Humans , Infant, Newborn , Placenta Growth Factor/blood , Pre-Eclampsia/diagnosis , Pregnancy , Severity of Illness Index , Vascular Endothelial Growth Factor Receptor-1/bloodABSTRACT
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.
Subject(s)
Activins/blood , Placenta/metabolism , Pre-Eclampsia/blood , Adult , Biomarkers/blood , Case-Control Studies , Female , Humans , Pre-Eclampsia/diagnosis , Pregnancy , Pregnancy Trimester, Third , Prospective StudiesABSTRACT
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.
Subject(s)
ADAM12 Protein/blood , Pre-Eclampsia/blood , Biomarkers/blood , Female , Humans , Infant, Newborn , Infant, Small for Gestational Age , Pregnancy , Prospective StudiesABSTRACT
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.
ABSTRACT
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 ß-1 glycoprotein 7) and PSG9 (pregnancy-specific ß-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.
Subject(s)
Pre-Eclampsia , Pregnancy-Specific beta 1-Glycoproteins , Australia/epidemiology , Biomarkers/blood , Endothelial Cells/metabolism , Female , Glycoproteins , Humans , Placenta/metabolism , Placenta Growth Factor , Pre-Eclampsia/diagnosis , Pregnancy , Pregnancy-Specific beta 1-Glycoproteins/analysisABSTRACT
Fetal growth restriction (FGR), when undetected antenatally, is the biggest risk factor for preventable stillbirth. Maternal circulating SPINT1 is reduced in pregnancies, which ultimately deliver small for gestational age (SGA) infants at term (birthweight < 10th centile), compared to appropriate for gestational age (AGA) infants (birthweight ≥ 10th centile). SPINT1 is also reduced in FGR diagnosed before 34 weeks' gestation. We hypothesised that circulating SPINT1 would be decreased in co-existing preterm preeclampsia and FGR. Plasma SPINT1 was measured in samples obtained from two double-blind, randomised therapeutic trials. In the Preeclampsia Intervention with Esomeprazole trial, circulating SPINT1 was decreased in women with preeclampsia who delivered SGA infants (n = 75, median = 18,857 pg/mL, IQR 10,782-29,890 pg/mL, p < 0.0001), relative to those delivering AGA (n = 22, median = 40,168 pg/mL, IQR 22,342-75,172 pg/mL). This was confirmed in the Preeclampsia Intervention 2 with metformin trial where levels of SPINT1 in maternal circulation were reduced in SGA pregnancies (n = 95, median = 57,764 pg/mL, IQR 42,212-91,356 pg/mL, p < 0.0001) compared to AGA controls (n = 40, median = 107,062 pg/mL, IQR 70,183-176,532 pg/mL). Placental Growth Factor (PlGF) and sFlt-1 were also measured. PlGF was significantly reduced in the SGA pregnancies, while ratios of sFlt-1/SPINT1 and sFlt1/PlGF were significantly increased. This is the first study to demonstrate significantly reduced SPINT1 in co-existing FGR and preeclamptic pregnancies.
ABSTRACT
Plasmodium falciparum is predicted to transport over 300 proteins to the cytosol of its chosen host cell, the mature human erythrocyte, including 19 members of the Hsp40 family. Here, we have generated transfectant lines expressing GFP- or HA-Strep-tagged versions of these proteins, and used these to investigate both localization and other properties of these Hsp40 co-chaperones. These fusion proteins labelled punctate structures within the infected erythrocyte, initially suggestive of a Maurer's clefts localization. Further experiments demonstrated that these structures were distinct from the Maurer's clefts in protein composition. Transmission electron microscopy verifies a non-cleft localization for HA-Strep-tagged versions of these proteins. We were not able to label these structures with BODIPY-ceramide, suggesting a lower size and/or different lipid composition compared with the Maurer's clefts. Solubility studies revealed that the Hsp40-GFP fusion proteins appear to be tightly associated with membranes, but could be released from the bilayer under conditions affecting membrane cholesterol content or organization, suggesting interaction with a binding partner localized to cholesterol-rich domains. These novel structures are highly mobile in the infected erythrocyte, but based on velocity calculations, can be distinguished from the 'highly mobile vesicles' previously described. Our study identifies a further extra-parasitic structure in the P. falciparum-infected erythrocyte, which we name 'J-dots' (as their defining characteristic so far is the content of J-proteins). We suggest that these J-dots are involved in trafficking of parasite-encoded proteins through the cytosol of the infected erythrocyte.