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Oxidative stress and endothelial dysfunction contribute substantially to the pathogenesis of preeclampsia (PE). Decidual mesenchymal stem/stromal cells (DMSC), reportedly reduce endothelial cell dysfunction and alleviate PE-like symptoms in a murine model. However, as a therapeutic strategy, the use of whole DMSC presents significant technical limitations, which may be overcome by employing DMSC-secreted extracellular vesicles (DMSC_EV). DMSC_EV restoration of endothelial dysfunction through a paracrine effect may alleviate the clinical features of PE.Objective: To determine whether DMSC-secreted, extracellular vesicles (DMSC_EV) restore endothelial cell function and reduce oxidative stress.Methods: DMSC were isolated from the placentae of uncomplicated term pregnancies and DMSC_EV prepared by ultracentrifugation. Human umbilical vein endothelial cells (HUVEC) were treated with bacterial lipopolysaccharide (LPS), or with serum from PE patients, to model the effects of PE. DMSC_EV were then added to treated HUVEC and their growth profiles, inflammatory state, and oxidative stress levels measured.Results: DMSC_EV displayed characteristic features of extracellular vesicles. In both LPS- and PE serum-treatment models, addition of DMSC_EV significantly increased HUVEC cell attachment and proliferation, and significantly reduced production of pro-inflammatory cytokine IL-6. The addition of DMSC_EV to LPS-treated HUVEC had no significant effect on total antioxidant capacity, superoxide dismutase levels or on lipid peroxidation levels. In contrast, the addition of DMSC_EV to PE serum-treated HUVEC resulted in a significant reduction in levels of lipid peroxidation.Conclusion: Addition of DMSC_EV had beneficial effects in both LPS- and PE serum- treated HUVEC but the two treatment models to induce endothelial cell dysfunction showed differences. The LPS treatment of HUVEC model may not accurately model the endothelial cell dysfunction characteristic of PE. Human cell culture models of PE show that DMSC_EV improve endothelial cell dysfunction in PE, but testing in in vivo models of PE is required. [ABSTRACT FROM AUTHOR]

Introduction: The maternal endothelial dysfunction characteristic of preeclampsia arises, in part, from excessive placental production of anti-angiogenic factors, including soluble Flt-1, soluble endoglin and activin A, inducing oxidative stress. We assessed whether the antioxidant and NRF2-activator sulforaphane could mitigate endothelial and trophoblast dysfunction in vitro.Methods: We induced dysfunction in human umbilical vein endothelial cells (HUVECs) with TNF-α, assessing endothelial activation and dysfunction (endothelin-1, vascular cell adhesion molecule; VCAM1, intracellular adhesion molecule; ICAM1, e-selectin and endothelial permeability) in the presence or absence of sulforaphane. We also assessed the effects of sulforaphane in mitigating hypoxic and hyperoxic injury in term placental explants by measuring secretion of anti-angiogenic factors. To assess the role of NRF2 we silenced NRF2 in HUVECs and primary trophoblast cells.Results: Sulforaphane reduced TNF-α mediated HUVEC secretion of endothelin-1, VCAM1, ICAM1 and E-selectin, and prevented increased endothelial permeability. In placental explants, sulforaphane reduced the secretion of soluble Flt-1, soluble endoglin and activin A. Sulforaphane induced activation and nuclear translocation of NRF2 in HUVECs, inducing heme oxygenase 1. NRF2 silencing blocked some but not all of sulforaphane's effects in HUVECs. NRF2 silencing did not prevent sulforaphane's inhibition of trophobast secretion of soluble Flt-1 or activin A.Conclusion: In reducing placental and endothelial oxidative stress, sulforaphane may offer a new adjuvant therapeutic approach for the treatment of preeclampsia. [ABSTRACT FROM AUTHOR]

Decidual stromal cells form the largest proportion of maternal cells at the maternal-fetal interface. Our aim was to investigate the role of the pre-eclampsia associated decidual activin receptor, ACVR2A, in regulating trophoblast functions at this interface. St-T1b and HTR-8/SVneo cell lines were used to model decidual stromal and trophoblast cells respectively. St-T1b conditioned medium inhibited HTR-8/SVneo adhesion, proliferation, migration and invasion; all effects that were attenuated by decidual ACVR2A siRNA transfection. These findings suggest that altered decidual ACVR2A expression perturbs the maternal-fetal crosstalk involved in regulating trophoblast function at the interface, which may affect placentation and lead to pre-eclampsia. [ABSTRACT FROM AUTHOR]

Hydroxychloroquine is an anti-malarial drug which, due to its anti-inflammatory and immunomodulatory effects, is widely used for the treatment of autoimmune diseases. In a model of systemic lupus erythematosus hydroxychloroquine has been shown to exert protective endothelial effects. In this study, we aimed to investigate whether hydroxychloroquine was endothelial protective in an in vitro model of TNF-α and preeclamptic serum induced dysfunction. We showed that hydroxychloroquine significantly reduced the production of TNF-α and preeclamptic serum induced endothelin-1 (ET-1). Hydroxychloroquine also significantly mitigated TNF-α induced impairment of angiogenesis. These findings support the further assessment of hydroxychloroquine as an adjuvant therapy in preeclampsia. [ABSTRACT FROM AUTHOR]

Objectives: Activin A, a TGFβ family member, circulates in the maternal blood at increasing concentrations throughout gestation during a healthy pregnancy. The circulating concentration of activin A is further increased in pre-eclampsia (PE), a hypertensive disorder of pregnancy that is marked by systemic maternal vascular endothelial cell dysfunction. The effect of increasing activin A concentrations on the maternal vascular endothelium is unknown. The study aim was to investigate the effect of physiological and pathological activin A concentrations observed in normotensive and PE pregnancies respectively, on vascular endothelial cell function.Methods and Results: Immunostaining demonstrated the presence of the activin A receptor, ACVR2A, in SGHEC-7 cells used to model the vascular endothelium. SGHEC-7 cells were treated with activin A concentrations representative of concentrations throughout gestation in normotensive (0-10ng/ml) and PE (50ng/ml) pregnancies. xCELLigence functional assays revealed that normotensive activin A concentrations increased SGHEC-7 proliferation and migration, which was inhibited by PE concentrations. Additionally, fluorescence based assays showed that PE concentrations increased endothelial permeability. None of the tested activin A concentrations affected cell apoptosis. PE concentrations also resulted in an imbalance of the vasoactive factors eNOS, PTGIS and EDN1, as determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assays.Conclusion: Compared with normotensive activin A concentrations, the higher PE activin A concentrations resulted in abnormal endothelial functions, which may contribute to the systemic maternal vascular endothelial cell dysfunction observed in the disorder. [ABSTRACT FROM AUTHOR]

Objectives Preeclampsia (PE) is a common and serious pregnancy disorder with a strong genetic component. Numerous genetic studies performed, including our own, have yielded many susceptibility genes from distinct functional groups. Additionally, transcriptome profiling at the maternal-fetal interface has likewise yielded many differentially expressed genes. Often there is little overlap between the two approaches, although both are significantly informative with respect to PE. We have thus taken a novel approach by analysing pathways common to the susceptibility genes and the PE transcriptome. Methods Using Illumina Human Ht12v4 and Wg6v3 BeadChips, transcriptome profiling was conducted on n = 65 normotensive and n = 60 PE decidua basalis tissues collected at delivery. R software was used to pre-process transcript data for pathway analysis. Pathways were analysed and constructed using Ariadne Pathway Studio 9. We examined the following susceptibility genes: ACVR1, ACVR1C, ACVR2A, COL4A1, COL4A2, ERAP1, ERAP2, INHA, INHBB and LNPEP (Yong et al., 2014). Significance was set at p < 0.05. Results Major common targets/regulators of these susceptibility genes were AGT, IFNG, IL6, INHBA, SERPINE1, TGFB1 and VEGFA (Fig. 1). The top downstream pathway categories of susceptibility genes that were significantly altered in the PE decidual transcriptome were apoptosis and cell signalling. Conclusions Susceptibility genes from distinct functional groups have similar downstream pathways through common targets/regulators. Several of these pathways matched those altered in PE. This study contributes to a better understanding on how susceptibility genes may interact in the development of PE. With this knowledge, more targeted functional analyses of PE susceptibility genes in these key pathways can be performed to examine their contributions to the pathogenesis and severity of PE. Disclosures H.E. Yong: None. P.E. Melton: None. M.P. Johnson: None. K.A. Freed: None. B. Kalionis: None. P. Murthi: None. S.P. Brennecke: None. R.J. Keogh: None. E.K. Moses: None. [ABSTRACT FROM AUTHOR]

Introduction Circulating activin A concentrations are significantly higher in pre-eclampsia (PE) compared with those in a normotensive pregnancy. We and others have previously identified ACVR2A, which codes for the activin A binding receptor, as a maternal PE susceptibility gene with reduced expression in PE. The PE-associated rs1424954 genetic polymorphism also results in decreased receptor expression. Objective Given that PE is characterised by systemic vascular endothelial dysfunction, we used an in vitro cell-line model to investigate how reduced ACVR2A expression modifies the response of maternal vascular endothelial cells to normotensive and PE activin A concentrations. Methods The SGHEC-7 cell line was used as a model of vascular endothelial cells. ACVR2A expression was silenced using siRNA transfection and confirmed with real-time PCR and immunoblotting. A non-targeting siRNA was used as a negative control (NC). Following a 72 h transfection, SGHEC-7s were treated for 24 h with activin A concentrations representative of normotensive (0, 10 ng/ml) and PE (50 ng/ml) pregnancies. SGHEC-7 proliferation was determined using the electrical impedance-based xCELLigence system, while permeability was measured with a Millipore fluorescence-based assay. Student’s t test and one-way ANOVA with Bonferroni’s post-hoc test where appropriate were used for statistical evaluations. Each experiment was performed at least n = 3 times. A p -value of < 0.05 was considered statistically significant. Results ACVR2A siRNA markedly reduced ACVR2A mRNA and protein levels by 78% ( p < 0.05) and 54% ( p < 0.01) respectively. Activin A at 10 ng/ml significantly increased proliferation of both NC and ACVR2A siRNA transfected cells by at least 43% ( p < 0.01) above that of 0 ng/ml. These increases were inhibited by at least 23% ( p < 0.01) by the PE 50 ng/ml concentration. Nevertheless, at all tested concentrations of activin A, proliferation of ACVR2A siRNA transfected cells was significantly reduced by at least 32% ( p < 0.01) compared with that of NC cells. The PE concentration of 50 ng/ml also significantly increased permeability of NC cells by 39% ( p < 0.01) compared with that of the 0 ng/ml baseline. In contrast, permeability of ACVR2A siRNA transfected cells was significantly increased by 36% ( p < 0.01) even at 0 ng/ml, with no further increase observed at 50 ng/ml, when compared with that of NC cells. Conclusions This study demonstrates that decreased expression of the ACVR2A receptor, in addition to activin A, can also result in dysfunctional vascular endothelial proliferation and permeability. Therefore, the altered expression of the ACVR2A receptor, resulting from a genetic predisposition, may modify the threshold required for activin A to impact vascular endothelial function, thereby exacerbating systemic vascular endothelial dysfunction in PE. [ABSTRACT FROM AUTHOR]

Introduction Preeclampsia (PE) is a multi-system disorder typically involving pregnancy-induced maternal hypertension and proteinuria presenting in the second half of pregnancy. The placenta is the key organ responsible for the pathogenesis of PE, with altered release of placenta-derived anti- (eg sFlt-1) and pro- (eg PlGF) angiogenic proteins implicated in maternal PE pathophysiological events. Epidemiological data indicate that low-dose aspirin (LDA) commenced before 16 weeks gestation is clinically effective in preventing PE, but its mechanism of action is unclear. We hypothesized that the prophylactic efficacy of LDA treatment may be associated with a corrective effect of LDA on the altered release of placenta-derived angiogenic proteins seen in PE pregnancies. Objective To test this hypothesis, we examined the effects of pregnancy sera from normotensive and PE cases with and without LDA on the in vitro production and release of sFlt-1 and PlGF from a trophoblast-derived cell line (BeWo). Methods The effects of LDA (5 μg/mL) following treatment of BeWo cells in vitro with either PE or normotensive serum on the expression of angiogenic proteins sFlt-1 and PlGF were determined using real-time PCR. The secretion of these angiogenic proteins in the conditioned media was screened with a commercially designed protein array. Candidate protein validation was then performed using an ELISA. All experiments were performed in triplicate at least six independent occasions. A p -value <0.05 was considered statistically significant. Results Angiogenic profiling at the mRNA level identified significantly increased expression of Flt-1 [Normotensive: 1.04 ± 0.03 vs PE: 2.68 ± 0.07, p < 0.05] and significantly decreased expression of PlGF [Normotensive: 0.99 ± 0.05 vs PE: 0.39 ± 0.01, p < 0.05] in sera treated BeWo cells. Following LDA treatment of cells in the presence of PE sera, a significant decrease in Flt -1 (2 fold) and a significant increase in PlGF (1.7 fold) mRNA were observed compared with PE sera alone treated cells. Screening for angiogenic proteins in the pooled conditioned media revealed a 2-fold increase in sFlt-1 and a 1.5-fold decrease in PlGF in PE sera treated cells compared with normotensive sera treated cells. However, treatment of BeWo cells with LDA inhibited the PE sera-induced sFlt-1 increase and the PE-sera induced decrease in PlGF to levels observed in cells treated with normotensive sera. Further validation by ELISA confirmed that both the PE serum-induced increase in sFlt-1 protein (PE: 1068 ± 72 vs PE+LDA: 541 ± 102, p < 0.01) and the PE serum-induced decrease in PlGF protein (PE:311 ± 43 vs PE+LDA: 890 ± 34, p < 0.01) were significantly attenuated by LDA treatment. Conclusion Increased placental release of the anti-angiogenic protein sFlt-1 and reduced placental release of the pro-angiogenic protein PlGF into the maternal circulation are key contributors to the maternal vascular endothelial cell dysfunction that is central to the development of the maternal syndrome of PE. The ability of LDA to rebalance these key angiogenic proteins in an in vitro trophoblast cell-line model may offer an important mechanistic insight at a molecular level regarding the clinical utility of LDA in preventing PE. [ABSTRACT FROM AUTHOR]

Objectives Arresten is an endogenous anti-angiogenic peptide derived from the non-collagenous domain of collagen IV α -chain COL4A1. We previously identified COL4A1 as a maternal preeclampsia (PE) susceptibility gene and also shown increased mRNA expression of COL4A1 in PE decidual tissue (Johnson et al., 2007; Yong et al., 2014). This increased expression also significantly correlated with an earlier onset of PE, a marker of clinical severity. The study aims were to investigate the circulating levels of arresten in plasma from normotensive and PE women throughout pregnancy and correlate these levels with the clinical severity of PE. Methods Plasma samples were collected from n = 44 normotensive and n = 39 PE women from 16 to 40 weeks’ gestation with informed consent. Arresten levels were determined by semi-quantitative Western immunoblotting of plasma samples and expressed as densitometric units ±SEM (DU). Arresten levels were then correlated with several parameters of clinical severity in PE. Statistical tests used were Student’s t test with Welch’s correction and Pearson’s correlation. Significance was set at p < 0.05. Results Arresten levels were significantly increased in PE compared with normotensive plasma by 2.3-fold (PE: 1148 ± 163DU, n = 14 vs normotensive: 505 ± 46DU, n = 30) in the 2nd trimester. This increase was maintained during the 3rd trimester with an increase of 1.4-fold (PE: 776 ± 75DU, n = 25 vs normotensive: 568 ± 40DU, n = 14). Increased 3rd trimester levels were significantly associated with PE women requiring MgSO 4 treatment ( r = 0.46), with an increase of 1.7-fold observed (MgSO 4 required: 907 ± 101DU, n = 16 vs MgSO 4 Not required: 543 ± 55DU, n = 9). Conclusions Arresten levels were significantly increased in PE plasma from 16 weeks’ gestation, many weeks before the onset of clinical disease. Higher 3rd trimester levels were also associated with greater disease severity as indicated by a requirement for MgSO 4 treatment. Given its significant increase before the onset of clinical disease and its association with clinical severity, arresten may be useful in predicting PE and monitoring its severity. Disclosures H.E. Yong: None. P. Murthi: None. M.H. Wong: None. B. Kalionis: None. S.P. Brennecke: None. R.J. Keogh: None. [ABSTRACT FROM AUTHOR]

Objectives Pre-eclampsia (PE) is a serious pregnancy complication which affects 3–5% of the pregnant population. To date there is no ‘cure’ for PE. However, the placenta seems to play an important role in the pathogenesis of this disease. Human placenta is an abundant source of proteoglycans to which glycosaminoglycan (GAG) side chains are attached. Heparan sulphate proteoglycans (HSPGs) have been implicated in many biological processes including, anticoagulation, angiogenesis and inflammation. However, their role in human placentae is largely unknown. The aim of this study was to determine the mRNA expression and protein abundance of HSPGs as well as the structure and relative abundance of GAGs in PE-affected placentae compared to gestation-matched controls. Methods The mRNA expression of HSPGs was determined using real-time PCR. Proteoglycans (PGs) were isolated from placental tissue by anion-exchange chromatography. PG enriched samples were investigated using ELISA with antibodies against the protein core of a range of PGs, as well as GAG chains, and Heparan Sulphate (HS) linkage regions following 0.01 U/mL heparinase III digestion. Results The mRNA expression of HSPGS, syndecan 1 & 2, glypican 1 & 3 and perlecan was significantly reduced in PE-affected placentae compared to controls ( p < 0.05, n = 40 each, Mann–Whitney U test). Syndecans1-4, glypicans1 & 3, biglycan, decorin and perlecan were identified in placental tissues by ELISA following anion exchange chromatography. HS abundance was further investigated using ELISA following digestion with heparinase III to reveal the HS linkage region common to all HS and heparin chains. In PE-affected placentae there was significantly less HS linkage regions compared to controls (1.53 ± 0.0.19 vs 1.03 ± 0.11, n = 20 each, p = 0.0391, Mann–Whitney U test). This correlated with the reduction in HSPG mRNA expression. Conclusions It is plausible that the reduction in PG expression and HS abundance may contribute to the pathogenesis of PE by altering thrombin management or angiogenic and inflammatory processes within the placenta. Disclosures T. Gunatillake: None. A. Chui: None. M. Lord: None. J. Whitelock: None. P. Murthi: None. V. Ignatovic: None. P. Monagle: None. S. Brennecke: None. J. Said: None. [ABSTRACT FROM AUTHOR]