Cell surface associated protein mucin 15 (MUC15) is elevated in preeclampsia.

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.

Paternal Expressed Gene 10 (PEG10) is decreased in early-onset preeclampsia.

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.

Circulating Chemerin Is Elevated in Women With Preeclampsia.

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 96 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.

Endothelial protein C receptor is increased in preterm preeclampsia and fetal growth restriction.

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.

Placental galectin-3 is reduced in early-onset preeclampsia.

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.

PSG7 and 9 (Pregnancy-Specific ß-1 Glycoproteins 7 and 9): Novel Biomarkers for Preeclampsia.

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.

Circulating syndecan-1 is reduced in pregnancies with poor fetal growth and its secretion regulated by matrix metalloproteinases and the mitochondria.

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.

Placental growth factor is negatively regulated by epidermal growth factor receptor (EGFR) signaling.

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.

Elevated Circulating and Placental SPINT2 Is Associated with Placental Dysfunction.

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.