sEng Release Research Articles

Pressure Overload-Mediated Sustained PKR2 (Prokineticin-2 Receptor) Signaling in Cardiomyocytes Contributes to Cardiac Hypertrophy and Endotheliopathies.

[Figure: see text].

Effects of modified lipoproteins on human trophoblast cells: a role in pre-eclampsia in pregnancies complicated by diabetes

IntroductionPre-eclampsia (PE) is increased ~4-fold by maternal diabetes. Elevated plasma antiangiogenic factors, soluble fms-like tyrosine kinase (sFLT-1) and soluble endoglin (sENG), precede PE onset. We investigated whether diabetes-related stresses, modified...

Hydroxychloroquine Mitigates the Production of 8-Isoprostane and Improves Vascular Dysfunction: Implications for Treating Preeclampsia.

In preeclampsia, widespread maternal endothelial dysfunction is often secondary to excessive generation of placental-derived anti-angiogenic factors, including soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), along with proinflammatory cytokines such as tumour necrosis factor-α (TNF-α) and activin A, understanding of which offers potential opportunities for the development of novel therapies. The antimalarial hydroxychloroquine is an anti-inflammatory drug improving endothelial homeostasis in lupus. It has not been explored as to whether it can improve placental and endothelial function in preeclampsia. In this in vitro study, term placental explants were used to assess the effects of hydroxychloroquine on placental production of sFlt-1, sEng, TNF-α, activin A, and 8-isoprostane after exposure to hypoxic injury or oxidative stress. Similarly, human umbilical vein endothelial cells (HUVECs) were used to assess the effects of hydroxychloroquine on in vitro markers of endothelial dysfunction. Hydroxychloroquine had no effect on the release of sFlt-1, sEng, TNF-α, activin A, or 8-isoprostane from placental explants exposed to hypoxic injury or oxidative stress. However, hydroxychloroquine mitigated TNF-α-induced HUVEC production of 8-isoprostane and Nicotinanamide adenine dinucleotide phosphate (NADPH) oxidase expression. Hydroxychloroquine also mitigated TNF-α and preeclamptic serum-induced HUVEC monolayer permeability and rescued the loss of zona occludens protein zona occludens 1 (ZO-1). Although hydroxychloroquine had no apparent effects on trophoblast function, it may be a useful endothelial protectant in women presenting with preeclampsia.

Association between endoglin/transforming growth factor beta receptors 1, 2 gene polymorphisms and the level of soluble endoglin with preeclampsia in Egyptian women

IntroductionPreeclampsia (PE) is a pregnancy-specific hypertensive disease whose etiopathogenesis remains unclear. ObjectivesThis study was designed to assess association between PE and 3 single nucleotide polymorphisms (SNPs): ENG(G/A) rs11792480, TGFβR1(A/C) rs10739778 and TGFβR2(G/A) rs6550005, beside circulating soluble endoglin (sENG), oxidative stress biomarkers and nitric oxide (NO) in Egyptian women. MethodsThe study included 75 preeclamptic women stratified into 4 clinical subgroups and 50 normotensive pregnant women. Genotyping was performed by real time polymerase chain reaction-Taqman allelic discrimination. ResultsPreeclamptic women showed significantly increased sENG and malondialdehyde (MDA), decreased total antioxidant capacity (TAC), endothelial nitric oxide synthase (eNOS) and NO, without change in transforming growth factor beta 1 (TGFβ1) versus controls. Moreover, sENG was significantly higher in severe and early than mild and late PE. Higher MDA and lower TAC and NO were observed in severe than mild PE. ENG(G/A) and TGFβR2(G/A) showed no association with PE. However, CC genotype of TGFβR1(A/C) was more frequent in controls than either PE, early-onset or severe revealing a reduced PE risk in CC genotype versus AA or AA + AC. Importantly, patients carrying AA genotype had higher SBP and MDA with lower TAC, gestational age at delivery (GA) and birth weight than those carrying CC genotype. ConclusionsExcessive sENG release with decreased eNOS/NO may be involved in PE pathogenesis. Women who carry C allele or CC genotype of TGFβR1(A/C) may be less prone to develop PE.

Disulfiram inhibits placental soluble FMS-like tyrosine kinase-1 and soluble endoglin secretion independent of the proteasome.

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

Effects of Modified Lipoproteins on Human Trophoblast Cells-A Role in Preeclampsia in Pregnancies Complicated by Diabetes

Preeclampsia (PE) complicates 2-8% of pregnancies worldwide. In women with diabetes, the risk for PE is substantially increased by approximately 4 fold. Evidence indicates that an increased release of two anti-angiogenic factors, soluble fms-like tyrosine kinase (sFlt-1) and soluble endoglin (sEng) from placental trophoblasts into the maternal circulation promotes endothelial dysfunction associated with PE. In this study, we investigated sFlt-1 and sEng release from the human placental trophoblast cell line, HTR8/SVneo, in response to modified lipoproteins and/or elevated glucose. HTR8/SVneo cells were exposed to native low-density lipoprotein (N-LDL) vs. ’highly-oxidized glycated’ LDL (HOG-LDL) (50 µg protein/ml) for 24h, with and without pre-treatment with 30mM glucose for 72h. The mRNA expressions of sFlt-1 isoforms (sFlt-1-i13, sFlt-1-e15a), endoglin and MMP-14 were measured by RT-PCR, and levels of sFlt-1 and sEng in cell supernatant were determined by ELISA. Compared with N-LDL, HOG-LDL increased the mRNA expression of sFlt-1-i13 (2.0 fold, p<0.05), sFlt-1-e15a (1.9 fold, p<0.01), endoglin (1.5 fold, p<0.05) and MMP-14 (1.8 fold, p<0.05). HOG-LDL increased the levels of sFlt-1 and sEng proteins in the supernatant (2.9 fold, p<0.and 3.6 fold, p<0.01, respectively). High glucose alone had no significant effect on sFlt-1 and sEng expressions; however, pre-treatment of high glucose potentiated the effects of HOG-LDL: the supernatant levels of sFlt-1 and sEng were increased by 1.5 fold and 1.8 fold, respectively, compared to HOG-LDL treatment alone (p<0.for both). Exposure of trophoblasts to modified lipoproteins (which accumulate in the tissues of patients with diabetes due to vascular leakage) may contribute to the development of PE in diabetes, which is amplified by the presence of high glucose. These findings may explain, in part, the high risk of PE in women with diabetes. Disclosure R.H. McLeese: None. J. Zhao: None. J. Yu: None. D. Brazil: None. T. Lyons: None.

MMP-14 aggravates onset of severe preeclampsia by mediating soluble endoglin release.

Gestational hypertension is a pregnancy complication that serious damages the maternal and child health. Early onset severe preeclampsia accounts for about 0.9% of the gestational hypertension disease. Conservative treatment is proposed in recent years to early onset severe preeclampsia through delay delivery. Therefore, it is particularly important to explore the pathogenesis of severe preeclampsia. Soluble endoglin (sEng) has been identified as a central factor to induce endothelium dysfunction of preeclampsia, while its specific mechanism is unclear. Matrix metallopeptidase 14 (MMP-14) and endoglin expressions and tissue localization in the placenta of preeclampsia and premature were detected by Western blot and immunohistochemistry. Endoglin level, mean arterial blood pressure (MABP), and urinary protein/creatinine ratio were analyzed for correlation to investigate their relationship and the influence of endoglin on eclampsia severity. MMP specific or broad spectrum inhibitor combining MMP-14 siRNA were used in JAR cell line BeWo to explore the regulatory role of MMP-14 on endoglin. MMP-14, endoglin, and sEng expression levels significantly increased in the placenta of severe preeclampsia patients. MMP-14 and endoglin exhibited expression co-localization. Endoglin expression was positively correlated with the severity of eclampsia. MMP-14 directly mediated the release of sEng. MMP-14 aggravated the onset of severe preeclampsia by mediating sEng release. MMP-14 was proposed as the effective target for the treatment of severe preeclampsia. Blocking the interaction between MMP-14 and endothelial protein may be an important treatment method.

LIFR increases the release of soluble endoglin via the upregulation of MMP14 expression in preeclampsia.

Preeclampsia (PE) is a pregnancy-specific disorder that is the main cause of maternal and perinatal morbidity and mortality worldwide. Inadequate trophoblastic invasion and endothelial dysfunction in the placenta are considered the foundation of the pathogenesis of preeclampsia in which soluble endoglin (sENG) plays an antiangiogenic role in the development of PE. The leukemia inhibitory factor receptor (LIFR) has been widely studied and is highly involved in arterial injury in vivo and in the migration of cancer cells in vitro Here, we tested the hypothesis that LIFR may be correlated with preeclampsia through its regulation of the release of sENG. Our data showed that LIFR protein, the expression of which significantly decreased with the progression of pregnancy, was located in the syncytiotrophoblast and cytotrophoblast. The LIFR protein level was increased in pregnancies with preeclampsia compared with normotensive full-term pregnancies. After the overexpression of LIFR in HTR8/SVneo cells, the release of sENG as well as the migration and invasion were significantly enhanced. Moreover, we also observed that LIFR induced the expression of matrix metalloproteinase14 (MMP14) and that the knockdown or inhibition of MMP14 decreased the release of sENG, as well as increased the LIFR-induced migration and invasion of HTR8/SVneo cells. These studies demonstrated that LIFR promoted the release of sENG through MMP14 in vitro, which indicates that LIFR may be involved in the development of preeclampsia.

Chronic Venous Insufficiency: Transforming Growth Factor-β Isoforms and Soluble Endoglin Concentration in Different States of Wound Healing.

Venous leg ulcer (VLU) is a huge healthcare problem with poorly understood pathophysiology. Transforming growth factor-β (TGF-β) and endoglin (Eng), are inflammatory and wound healing mediators. Eng, co-receptor for TGF-β type-II receptors, may be cleaved forming soluble Eng (sEng), antagonizing TGF-β signaling, a crucial process in vascular pathologies. We evaluated the accumulation in wound fluid (WF) of TGF-β isoforms and sEng in healing stages, showing the effects of sulodexide treatments, a glycosaminoglycan with clinical efficacy in VLU healing. Patients with inflammatory (Infl) and granulating (Gran) VLU were recruited. WFs and THP-1 monocytes exposed to Infl and Gran WF (treated/untreated with sulodexide) were analyzed for TGF-β isoforms and sEng by multiplex immunoassay. In both Infl and Gran WF, TGF-β1 and β2 were similar; TGF-β3 was significantly increased in Infl compared to Gran WFs (p = 0.033). sEng was significantly elevated in Gran compared to Infl WFs (p = 0.002). In THP-1 monocytes there was a significant increase in sEng after co-treatment of WF and sulodexide. The increase in TGF-β3 found in Infl WF highlights its negative effect on wound healing, while the increased levels of sEng in Gran WF affects the leukocyte adhesion/transmigration through the endothelium, reducing the inflammatory response and favoring the wound healing. Glycosaminoglycan sulodexide potentiates the effects of sEng release from monocyte, representing an important therapeutic option for wound healing.

Evidence-Based Revised View of the Pathophysiology of Preeclampsia.

Preeclampsia is a life-threatening vascular disorder of pregnancy due to a failing stressed placenta. Millions of women risk death to give birth each year and globally each year, almost 300,000 lose their life in this process and over 500,000 babies die as a consequence of preeclampsia. Despite decades of research, we lack pharmacological agents to treat it. Maternal endothelial oxidative stress is a central phenomenon responsible for the preeclampsia phenotype of high maternal blood pressure and proteinuria. In 1997, it was proposed that preeclampsia arises due to the loss of VEGF activity, possibly due to elevation in anti-angiogenic factor, soluble Flt-1 (sFlt-1). Researchers showed that high sFlt-1 and soluble endoglin (sEng) elicit the severe preeclampsia phenotype in pregnant rodents. We demonstrated that heme oxygenase-1 (HO-1)/carbon monoxide (CO) pathway prevents placental stress and suppresses sFlt-1 and sEng release. Likewise, hydrogen sulphide (H2S)/cystathionine-γ-lyase (Cth) systems limit sFlt-1 and sEng and protect against the preeclampsia phenotype in mice. Importantly, H2S restores placental vasculature, and in doing so improves lagging fetal growth. These molecules act as the inhibitor systems in pregnancy and when they fail, preeclampsia is triggered. In this review, we discuss what are the hypotheses and models for the pathophysiology of preeclampsia on the basis of Bradford Hill causation criteria for disease causation and how further in vivo experimentation is needed to establish 'proof of principle'. Hypotheses that fail to meet the Bradford Hill causation criteria include abnormal spiral artery remodelling and inflammation and should be considered associated or consequential to the disorder. In contrast, the protection against cellular stress hypothesis that states that the protective pathways mitigate cellular stress by limiting elevation of anti-angiogenic factors or oxidative stress and the subsequent clinical signs of preeclampsia appear to fulfil most of Bradford Hill causation criteria. Identifying the candidates on the roadmap to this pathway is essential in developing diagnostics and therapeutics to target the pathogenesis of preeclampsia.

IFI16 mediates soluble Flt-1 and endoglin production by trophoblast cells.

Preeclampsia is a serious pregnancy-specific hypertensive syndrome that is characterized by widespread maternal endothelial dysfunction. Previous studies have shown that increased levels of circulating cell-free fetal DNA in women with preeclampsia correspond to the degree of disease severity; however, it is unknown whether this DNA is a key signal that contributes to the development of preeclampsia. The detection of DNA is critical to appropriate innate immune responses. The interferon-inducible protein 16 (IFI16) - a member of the HIN-200 family - is an innate immune receptor for intracellular DNA, which is implicated in the control of cell growth, apoptosis, angiogenesis, and immunomodulation; however, its role in preeclampsia remains unresolved. Here, we tested the hypothesis that this DNA can activate IFI16 in the placentas of women with preeclampsia and is sufficient to induce soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng) production. We characterized IFI16 in severe preeclamptic placentas and assessed whether DNA increased the release of sFlt-1 and sEng from trophoblast cells and placental explants. Furthermore, we determined whether IFI16 was involved in DNA-induced sFlt-1 and sEng production. Placental immunoreactivity and protein levels of IFI16 were significantly increased in women with preeclampsia compared to matched control women. Treatment of human trophoblasts with the IFI16 agonist poly(dA:dT) significantly increased IFI16 levels. Furthermore, poly(dA:dT) induced sFlt-1 and sEng production by human trophoblasts in an IFI16-dependent manner. We conclude that trophoblast cells respond to cell-free fetal DNA through the IFI16 receptor, resulting in the production of the preeclampsia-related antiangiogenic factors sFlt-1 and sEng.

6-OR

Objectives Key pathophysiological steps in preeclampsia (PE) are (1) release of anti-angiogenic factors sFlt-1 and soluble endoglin (sEng), (2) oxidative stress and (3) maternal endothelial dysfunction. We performed functional studies in primary human tissues to examine whether proton pump inhibitors (PPIs) can counter these pathophysiological effects. Methods Functional studies were performed on: (1) primary trophoblast, (2) primary human umbilical vein endothelial cells (HUVEC), (3) uterine microvascular cells and (4) placental explants, obtained from women with severe PE. Five PPIs (including esomeprazole, rabeprazole, lansoprazole) were added at increasing doses (0-100μM), and the following outputs examined: (1) sFlt-1/sEng production, (2) heme-oxygenase-1 (HO-1) expression and Nrf2 translocation and (3) endothelial dysfunction (modeled by adding TNFα or PE patient serum to endothelial cells). Results PPIs caused a dose-dependent reduction in mRNA expression of sFlt-1 variants (e15a and i13) and reduced secretion of sFlt1 and sEng in all cell types. The decrease was potent: for example the top dose of esomeprazole decreased sFlt-1 release from trophoblast by 50%, and sEng release by 70%. In contrast, pravastatin at the same dose did not decrease sFlt-1/sEng production. All PPIs induced a highly significant dose dependent increase in HO-1 mRNA and protein in all tissues. PPIs also induced nuclear translocation of the master antioxidant transcription factor, Nrf2, and upregulated Nrf2 regulated genes. Importantly, PPIs markedly reduced sFlt-1 production (and increased HO-1) when added to placental explants from women with severe PE. PPIs rescued both TNFα and PE serum induced endothelial dysfunction (quenching leukocyte adhesion and attenuating VCAM1 and endothelin-1 expression). Furthermore, PPIs blocked TNFα-induced disruption of endothelial tube formation (both in HUVECs and uterine microvascular cells). Conclusions PPIs potently decrease sFlt-1/sEng release, switch on anti-oxidant defenses and quench endothelial dysfunction. Widely prescribed for gastric reflux during pregnancy, they represent an exciting novel candidate therapeutic to treat severe preeclampsia. Disclosures K. Onda: None. N. Hannan: None. S. Beard: None. N. Binder: None. F. Brownfoot: None. T. Kaitu’u-Lino: None. L. Tuohey: None. R. Hastie: None. S. Tong: None.

273-POS

Objectives Sulfasalazine is an anti-inflammatory and immune modulating drug. Its mode of action has remained elusive, however recent evidence suggests it induces potent antioxidant enzyme heme-oxygenase1 (HO1) via nuclear erythroid-2-related factor 2 (Nrf2). Given its anti-oxidant capacity and that it is safe in pregnancy, we examined the potential for sulfasalazine as a novel therapeutic for preeclampsia. In particular, we examined its ability to quench anti-angiogenic factors sFlt1 and soluble endoglin (sEng) and reverse endothelial dysfunction in vitro. Methods Increasing doses of sulfasalazine were administered to primary human umbilical vein endothelial cells (HUVECs) and sFlt1 and sEng release assessed. To induce endothelial dysfunction, HUVECs were treated with TNF α and the effect of sulfasalazine on monocyte adhesion determined. Finally the effect of sulfasalazine on HUVEC migration and proliferation was assessed. Results Excitingly, we observed a significant dose dependent reduction in both sFlt1 and sEng release. Importantly, mRNA expression of newly described human and placental specific variant sFlt1-e15a mRNA was significantly decreased, as was MMP14 mRNA expression (MMP14 is the protease that produces sEng). As expected, sulfasalazine also significantly increased mRNA expression of HO-1. Treatment of HUVECs with TNF α induced significant upregulation of VCAM which was potently reversed by Sulfasalazine, indicating its ability to quench endothelial dysfunction. Furthermore, sulfalsazine significantly reduced monocyte adhesion to HUVECs treated with TNF α , again supporting its anti-inflammatory properties. Finally we demonstrated that sulfasalazine could reverse the negative effects of sFlt1 on VEGF-stimulated HUVEC migration, and enhance HUVEC proliferation. Conclusions Sulfasalazine is a novel agent, safe in pregnancy that significantly quenches sFlt1 and sEng release from human endothelial cells. In addition, it significantly reduces endothelial dysfunction and enhances endothelial cell migration and proliferation. This provides strong evidence to suggest that sulfasalazine may be able to quench the endothelial dysfunction of preeclampsia and could be an effective treatment for preeclampsia. Disclosures F.C. Brownfoot: None. S. Tong: None. N. Hannan: None. R. Hastie: None. P. Cannon: None. T.J. Kaitu’u-Lino: None.

26-OR

Objectives The soluble endoglin (sEng) is an antiangiogenic protein inhibiting TGF- β signaling and eNOS activation. It is well known the levels of sEng increase in preeclamptic patients. However, the factors increasing the sEng in preeclampsia remains poorly understood. In preeclampsia, many autoantibodies such as antiphospholipid, angiotensin II type IA receptor autoantibody (AT1-AA) have been reported. To investigate factors increasing sEng in preeclampsia, we studied the effect of IgG from preeclampsia sera on the production of sEng from human trophoblast cells. Methods Serum samples were from women with normal pregnancies and those with preeclampsia. Villous tissues were from healthy pregnant women having artificial abortion. IgGs isolated from normal pregnancy and preeclampsia sera were eluted by Protein G affinity chromatography. To investigate the possibility of AT1-AA, the production and mRNA expression of sEng were evaluated by AT1-receptor antagonist (losaltan). We also analyzed the various kinds of cytokine productions with or without losartan to find the relationship between sEng, AT1-AA and cytokine productions. Results The addition of preeclampsia IgG into primary trophoblast led to increased release of sEng and increased expression of Eng mRNA. The increased production and mRNA expression of sEng were blocked by the treatment of losartan. The production of TGF- β was increased under the presence of losartan. We considered this effect comes from the decreased production of sEng with losaltan. Conclusions We suggest that AT1-AA in IgG fraction from preeclampsia may play an important role of high level of serum sEng in preeclampsia. Disclosures Y. Koabayshi: None. T. Yamamoto: None. F. Chishima: None. T. Murase: None. H. Azuma: None. A. Nakamura: None. H. Takahashi: None.

44-OR

Objectives Preclinical studies suggest pravastatin is a candidate therapeutic for preeclampsia, leading to clinical trials. Surprisingly, the amount of functional interrogation of pravastatin (indeed all statins) on primary gestational tissues published thus far is very limited. The objective of this study was to determine the effect of statin administration to primary gestational tissues in vitro. Methods Pravastatin, simvastatin and rosuvastatin (HMG Co-A reductase inhibitors) were administered to primary endothelial cells (HUVECs), primary trophoblasts and preterm preeclamptic placental explants and the effect on sFlt1 and sEng secretion assessed. To identify the mechanism involved, farnesyl pyrophosphate, a HMG Co-A reductase pathway activator was added and sFlt1 and sENG was measured. Endothelial dysfunction was induced by addition of TNF α or trophoblast conditioned media to HUVECs, and the effect of statins on VCAM and monocyte adhesion determined. Finally HUVEC migration in the presence of sFlt1 ± statins was measured using the xCELLigence system (measures in real time). Results Statins significantly reduced sFlt1 release, reducing mRNA expression of the human placental specific variant, sFlt1-e15a and up-regulating heme-oxygenase-1. Pravastatin also significantly reduced sFlt1 release from preeclamptic placental explants. Concerning however, all three statins significantly increased secretion of sEng in a dose dependent manner. Addition of farnesyl pyrophosphate blocked statin-induced sFlt-1 reduction and sEng increase from HUVEC cells, indicating these changes are mediated via inhibition of HMG Co-A reductase. Primary trophoblast conditioned media and TNF α significantly upregulated markers of endothelial dysfunction, VCAM and Endothelin-1, which were quenched by adding statins. Statins also decreased monocyte adhesion to primary HUVECs, enhanced HUVEC tube forming and restored sFlt1 impaired HUVEC migration towards VEGF. Conclusions We characterized the effect of three statins on primary human endothelial cells and trophoblast cells. Our data confirms statins reduce sFlt1 and improve endothelial dysfunction. However, of potential concern is that statins increase sEng release. Disclosures F.C. Brownfoot: None. T.J. Kaitu’u-Lino: None. N. Hannan: None. R. Hastie: None. P. Cannon: None. K. Onda: None. S. Tong: None.

Autoantibodies isolated from patients with preeclampsia induce soluble endoglin production from trophoblast cells via interactions with angiotensin II type 1 receptor.

This study investigated whether angiotensin II type 1 receptor agonistic autoantibodies (AT1 -AAs) mediate the increased release of soluble endoglin (sEng) in women with preeclampsia. Serum samples were obtained from women with normal pregnancies or with preeclampsia. Human first-trimester trophoblast cells were cultured with purified IgG derived from these sera, and the sEng protein and mRNA expression levels were measured in the supernatants. We also determined the effects of the AT1 -AAs on these cells following treatment with an AT1 receptor antagonist (losartan). Compared with the IgG isolated from the women with normal pregnancies, treatments of the preeclamptic patients markedly increased sEng production and mRNA expression in trophoblast cells. Co-treatment with losartan significantly attenuated the release of sEng and sEng mRNA expression in the trophoblast cells. AT1 -AAs may be related to the increased release of sEng observed during preeclampsia and may play important roles in the pathology of this disorder.

Peptides do not prevent cleavage of endoglin to produce soluble endoglin.

MMP14 cleaves membrane bound endoglin to produce soluble endoglin (sEng), an anti-angiogenic factor that causes endothelial dysfunction in preeclampsia. A recent publication proposed peptides with an amino acid sequence straddling the MMP14 cleavage site on endoglin decreases sEng release. This may be an exciting therapeutic approach and requires validation. We administered peptides to JAR cells, and primary placental explants and endothelial cells. The peptides had no effect on sEng production, and did not block sEng production in HEK293 with MMP14 and endoglin overexpressed. Peptides with an amino acid sequence encompassing the cleavage site do not prevent sEng production in vitro.

Soluble endoglin production is upregulated by oxysterols but not quenched by pravastatin in primary placental and endothelial cells

IntroductionPreeclampsia is a serious pregnancy complication. Soluble endoglin (sEng) is released from the placenta and contributes to the maternal endothelial dysfunction seen in preeclampsia. Recently oxysterols, which activate the Liver X Receptor (LXR), have been implicated in producing sEng, by upregulating matrix metalloproteinase-14 (MMP14; cleaves endoglin to produce sEng) and down-regulating tissue inhibitor of metalloproteinase-3 (TIMP-3; inhibitor of MMP14). The functional experiments in that study were performed on JAR cells (human choriocarcinoma cell line) and placental explants. MethodsWe characterized LXR in severe preeclamptic placentas, and assessed whether oxysterols increase release of sEng from primary human umbilical vein endothelial cells (HUVECs), primary trophoblasts and placental explants. Given pravastatin is thought to block oxysterol production and inhibit the LXR, we examined whether pravastatin reduces sEng release. ResultsLXRα and β were localized to the syncytiotrophoblast and villous tips and were significantly up-regulated in preeclamptic placenta. Oxysterols upregulated sEng production in HUVECs and placental explants although the increases were far more modest than that recently reported. Oxysterols did not upregulate sEng in primary trophoblasts. Furthermore, mRNA expression of MMP14 and TIMP-3 were not altered by oxysterols in any tissue. Surprisingly, pravastatin did not decrease oxysterol-induced upregulation of sEng. DiscussionLXR is up-regulated in preeclamptic placenta. Oxysterols upregulate sEng production from human tissues, but the increase is modest, suggesting this may not be the main mechanism for the very significant elevations in sEng seen in preeclampsia. Pravastatin does not decrease sEng production. ConclusionOxysterols modestly up-regulate sEng production which is not quenched by pravastatin.

Molecular mechanisms and therapeutic implications of the carbon monoxide/hmox1 and the hydrogen sulfide/CSE pathways in the prevention of pre-eclampsia and fetal growth restriction

The exact aetiology of preeclampsia is unknown, but there is a good association with an imbalance in angiogenic growth factors and abnormal placentation (Ahmad and Ahmed, 2004). The incidence of preeclampsia is reduced by a third in smokers, but not in snuff users. Soluble Flt-1 (sFlt-1) and soluble endoglin (sEng) are increased prior to the clinical onset of preeclampsia. Animals exposed to high circulating levels of sFlt-1 and sEng elicit severe preeclampsia-like symptoms. Smokers have reduced circulating sFlt-1 and cigarette smoke extract decreases sFlt-1 release from placental villous explants. An anti-inflammatory enzyme, heme oxygenase-1 (HO-1) and its metabolite carbon monoxide (CO), inhibit sFlt-1 and sEng release. Women with preeclampsia exhale less CO than women with normal pregnancies and HO expression decreases as the severity of preeclampsia increases. In contrast, sFlt-1 levels increase with increasing severity. More importantly, chorionic villous sampling from women at eleven weeks gestation shows that HO-1 mRNA expression is decreased in women who go on to develop preeclampsia. Collectively, these facts provide compelling evidence to support the proposition that the pathogenesis of preeclampsia is largely due to loss of HO activity. This results in an increase in inflammation and excessive elevation of the two key anti-angiogenic factors responsible for the clinical signs of preeclampsia. The identification of a protective role for HO-1 in pregnancy, offers HO/CO pathway as a target for the treatment of preeclampsia. The cardiovascular drugs, statins, stimulate HO-1 expression and inhibit sFlt-1 release in vivo and in vitro, suggesting statins have the potential to ameliorate preeclampsia. The StAmP trial is underway to address this and if positive, its outcome will lead to the very first therapeutic intervention to prolong affected pregnancies. Hydrogen sulphide (H2S), a gaseous messenger produced mainly by cystathionineY-lyase (CSE), is pro-angiogenic vasodilator (Yang et al., 2008; Papapetropoulos et al., 2009). We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Plasma levels of H2S were significantly decreased in preeclamptic women (p<-0.01), which was associated with reduced CSE message and protein expression in human placenta as determined by real - time PCR and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine (PAG) in first trimester (8-12weeks gestation) human placental explants had reduced placenta growth factor (PIGF) production as assessed by ELISA and inhibited trophoblast invasion in vitro. Endothelial CSE knockdown by siRNA transfection increased the endogenous release of soluble fms-Like tyrosine kinase-1 (sFlt-1) and soluble endoglin, (sEng) from human umbilical vein endothelial cells while adenoviral-mediated CSE overexpression inhibited their release. Administration of PAG to pregnant mice induced hypertension, liver damage, and promoted abnormal labyrinth vascularisation in the placenta and decreased fetal growth. Finally, a slow releasing, H2S-generating compound, GYY4137, inhibited circulating sFlt-1 and sEng levels and restored fetal growth that was compromised by PAG-treatment demonstrating that the effect of CSE inhibitor was due to inhibition of H2S production. These results imply that endogenous H2S is required for healthy placental vasculature and a decrease in of CSE/ H2S activity may contribute to the pathogenesis of preeclampsia. This work was supported by grants from the Medical Research Council (G0601295 and G0700288) and Aston University StrategicFunds.

Therapeutic potential of statins and the induction of heme oxygenase-1 in preeclampsia

Heme oxygenase (Hmox) is an endogenous system that offers protection against placental cytotoxic damage associated with preeclampsia. The Hmox1/carbon monoxide (CO) pathway inhibits soluble Flt-1 (sFlt-1) and soluble Endoglin (sEng). More importantly, statins induce Hmox1 and suppress the release of sFlt-1 and sEng; thus, statins and Hmox1 activators are potential novel therapeutic agents for treating preeclampsia. The contribution of the Hmox system to the pathogenesis of preeclampsia has been further indicated by the incidence of preeclampsia being reduced by a third in smokers, who had reduced levels of circulating sFlt-1. Interestingly, preeclamptic women exhale less CO compared with women with healthy pregnancies. Hmox1 is reduced prior to the increase in sFlt-1 as Hmox1 mRNA expression in the trophoblast is decreased in the first trimester in women who go on to develop preeclampsia. Induction of Hmox1 or exposure to CO or bilirubin has been shown to inhibit the release of sFlt-1 and sEng in animal models of preeclampsia. The functional benefit of statins and Hmox1 induction in women with preeclampsia is valid not only because they inhibit sFlt-1 release, but also because statins and Hmox1 are associated with anti-apoptotic, anti-inflammatory, and anti-oxidant properties. The StAmP trial is the first randomized control trial (RCT) evaluating the use of pravastatin to ameliorate severe preeclampsia. This proof-of-concept study will pave the way for future global RCT, the success of which will greatly contribute to achieving the United Nations Millennium Development Goals (MDG4 and MDG5) and offering an affordable and easily accessible therapy for preeclampsia.