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Soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG) are increased in the circulation of women with preeclampsia. We examined whether they differ in concentration between plasma and serum samples. Maternal blood was collected and processed for serum and plasma, from 28 women with preterm preeclampsia and 18 gestation matched controls (<34weeks). Significantly higher sFlt-1 was detected in preeclamptic serum compared to plasma. Significantly higher levels of sENG were identified in serum compared to plasma from control pregnancies. Thus these data show the sample type (serum or plasma) needs to be consistent within comparisons for both sFlt-1 and sENG. [ABSTRACT FROM AUTHOR]

Introduction: Preeclampsia is a serious pregnancy complication for which there are no medical treatments. Soluble endoglin is an anti-angiogenic factor implicated in the pathogenesis of the disease, however little is known about its molecular regulation. The PI3K/Akt pathway is down regulated in preeclamptic placentas and decreased PI3K/Akt signaling has been linked to increased soluble endoglin release from endothelial cells. MMP14 is a key protease that functions to release soluble endoglin from the placental surface.Objective: This study aimed to determine whether reduced placental PI3K/Akt causes elevated release of soluble endoglin via MMP14.Study Design: Akt mRNA and protein expression were assessed in early onset preeclamptic and preterm control placentas (delivered <34weeks gestation). PI3K/Akt inhibition was achieved by administering PI3K inhibitor wortmannin, a specific Akt inhibitor or Akt siRNA to primary human umbilical vein endothelial cells, primary trophoblast and placental explants. The effect of PI3K/Akt inhibition on soluble endoglin release, MMP14, endoglin and TIMP-3 mRNA expression was determined.Results: We identified significantly reduced pAkt and total Akt in preeclamptic placentas relative to preterm control. Inhibition of PI3K/Akt resulted in significantly elevated soluble endoglin release from HUVECs, had no effect on MMP14 mRNA expression but resulted in significantly reduced TIMP3. In contrast inhibiting PI3K/Akt in placental explants or primary trophoblast did not change soluble endoglin release.Conclusion: This study confirms that the PI3K/Akt cell protection pathway is down regulated in preeclampsia, but demonstrates that this dysregulation is unlikely to be responsible for the excessive placental soluble endoglin release characteristic of preeclampsia. [ABSTRACT FROM AUTHOR]

There is recent interest for uniform placental collection protocols so laboratories can share samples. However, concerns have been raised a wash step at collection causes significant loss of sFlt-1 and soluble endoglin, among the most studied proteins in placentology. We measured Flt-1 and endoglin mRNA and protein in 10 preeclamptic placentas that were washed, or left unwashed. Reassuringly, there was no significant change in the Flt-1, sFlt-1-e15a or sFlt-1-i13 mRNA or Flt-1 or sFlt-1 protein expression or localization. There was also no change in endoglin mRNA expression or protein localization. Washing preeclamptic placental samples does not alter anti-angiogenic factor expression. [ABSTRACT FROM AUTHOR]

Objectives Soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) are the most studied molecules in preeclampsia. However, most trophoblast cell lines do not secrete both these factors. Thus, we set out to characterize protocols to functionally investigate sFlt-1 and sEng from primary trophoblast. Study design Primary trophoblasts were isolated from term placenta by percoll gradient, then negative selection using a CD9 antibody. Purity was assessed by cytokeratin 7 immunostaining. We first examined the effects of CD9 negative selection on sFlt-1, sEng and hCG secretion and the ability of forskolin to enhance syncytialization. We then examined the effects of hypoxia on sFlt-1 production and assessed gene knockdown using siRNA. Results CD9 negative selection produced a pure population of primary trophoblasts. Secretion of sEng was 5-fold lower when CD9-positive cells were removed, sFlt1 was unchanged, and hCG was significantly increased. hCG analysis of the purified population indicated spontaneous syncytialization, which was not enhanced by forskolin. Forskolin similarly did not alter sFlt-1 secretion. Hypoxia significantly increased sFlt-1 secretion as expected. Importantly, high gene silencing efficiencies were readily achieved. Conclusion In conclusion, we present a protocol that yields primary trophoblasts of high purity that produce abundant sFlt-1 and low but detectable levels of sEng. Furthermore, these cells are readily amenable to gene silencing by siRNAs and hence suitable for functional studies. [ABSTRACT FROM AUTHOR]

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 . [ABSTRACT FROM AUTHOR]

Introduction Although discovered more than a decade ago, the molecular pathways regulating sFLT-1 release in preeclampsia are still poorly understood. We have discovered the EGFR superhighway and the mitochondria (either via inhibiting the electron transport chain (ETC) or increasing molecules associated with mitochondrial biogenesis) independently regulate sFLT-1 secretion from placenta. The aim of this study was to assess whether targeting both pathways simultaneously additively reduced sFLT-1. Methods Isolated primary trophoblast were used for all studies. Metformin is a mitochondrial ETC inhibitor and resveratrol stimulates expression of mitochondrial biogenesis molecules SIRT1, AMPK and PGC1a. We initially assessed their effects on EGFR expression and activation using western blot. Gefitinib is an EGFR inhibitor, PD98059 a MEK-1 inhibitor and AG490 a STAT3 inhibitor (pathways downstream of the EGFR). We assessed their effects on mitochondrial respiration using a seahorse flux analyser. Having identified molecules that were either "EGFR-specific" or "Mitochondrial specific" we subsequently treated primary trophoblast simultaneously with drugs targeting both pathways and measured the effect on sFLT-1 secretion. Results Treatment of primary trophoblast with metformin or resveratrol significantly reduced sFLT-1 secretion and increased down-stream mitochondrial biogenesis molecules but had no significant effect on EGFR, pEGFR or downstream adaptor molecules ERK, pERK, STAT-3 or pSTAT-3. Although inhibitors targeting the EGFR super-highway also significantly reduced sFLT-1 secretion, none significantly altered mitochondrial respiration, or ATP production. When we targeted the two pathways simultaneously (combining gefitinib and metformin, gefitinib and resveratrol, metformin and PD980 or metformin and AG490) we found the reduction in sFLT-1 was additive compared to targeting either pathway alone. Conclusions Our study identifies EGFR signalling and the mitochondria as two parallel pathways that both regulate sFLT-1 secretion and which, when targeted simultaneously, can additively reduce sFLT-1. These pathways provide new therapeutic targets to reduce excess sFLT-1 secretion in pathological conditions and improve vascular homeostasis. [ABSTRACT FROM AUTHOR]

Introduction Preeclampsia is associated with elevated placental secretion of the anti-angiogenic factor sFlt1. Surprisingly, little is understood about its regulation, characterizing this may identify therapeutic leads. Placenta has by far the highest Epidermal Growth Factor Receptor (EGFR) expression of non-malignant tissue. It is localized to the placental surface and elevated in preeclamptic placenta. Objectives Our objective was to characterize EGFR pathway expression in preeclamptic placenta and to assess whether EGFR signaling regulates sFlt1 secretion in primary human placenta. Methods RNA and protein were extracted from a cohort of severe early onset preeclamptic ( n = 25) placentas as well as gestational matched normotensive controls ( n = 25). EGFR mRNA expression was assessed as well as protein levels of phosphorylated EGFR and the key downstream signaling molecules Erk1/2, Akt and STAT3. We next isolated primary trophoblast from normotensive term placenta and (1) inhibited EGFR signaling with gefitinib (orally available EGFR inhibitor), or (2) inhibited the downstream EGFR signaling cascades with seven different small molecule inhibitors. mRNA expression of sFlt1 variants as well as protein secretion was assessed. EGFR expression was also assessed in primary trophoblasts treated with esomeprazole, pravastatin and sulfasalazine. Results mRNA expression of EGFR was significantly upregulated in preeclamptic placentas. Active EGFR, Erk1/2, Akt and STAT3 were all also significantly increased in preeclamptic placenta. Inhibiting EGFR in primary trophoblasts with gefitinib significantly reduced sFlt1-e15a mRNA and protein expression and total sFlt1 secretion by 30–70% from baseline (Fig. 1). Inhibiting the downstream adaptor molecules MEK1/2 (U0126), Erk/12 (PD98059), c-RAF (ZM336372) and JAK2 (AG490) also induced a significant dose dependent reduction in both sFlt1-e15a and total sFlt1 mRNA and protein expression. Additionally, treating primary trophoblasts with esomeprazole, pravastatin and sulfasalazine all significantly down regulated EGFR mRNA and protein expression coincident with significantly reduced sFlt1 secretion. Conclusions We have identified a new level of molecular regulation for sFlt1. EGFR signaling positively regulate sFlt1 secretion. The implications are that identifying drugs that block EGFR signaling may be a novel therapeutic strategy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction Preeclampsia is a serious complication of pregnancy. Responsible for thousands of maternal and fetal deaths worldwide, there is no cure. Key pathophysiological steps in preeclampsia include 1) placental damage and oxidative stress, 2) elevated sFlt1 and soluble endoglin (sEng) and 3) endothelial dysfunction. While the antiplatelet drug aspirin is used clinically to prevent preeclampsia, its effectiveness, if any, remains limited. New generation antiplatelet therapies (Clopidogrel, Prasugrel and Ticagrelor) have health benefits beyond anti-thrombus formation (they reduce oxidative damage, inflammation and endothelial dysfunction) and thus may be more effective to prevent preeclampsia. We examined whether new generation antiplatelet agents can counter the pathophysiological steps in human preeclampsia models and compared their efficacy directly to aspirin. Methods Primary human trophoblast, placental explants and endothelial cells (human umbilical vein endothelial cells (HUVECs) and uterine microvascular cells (UtMVs)) were treated with increasing doses of Aspirin, Clopidogrel, Prasugrel or Ticagrelor (0–100 μ M). Endothelial dysfunction was induced by addition of the pro-inflammatory cytokine Tumor Necrosis Factor (TNF)- α (10 μ g/mL) stimulation of endothelial cells (antiplatelet agents were titrated in). Peripheral blood monocytes were isolated and applied to stimulated HUVECs; their adhesion was assessed flurometrically. Media and cell lysates were collected to assess 1) antioxidant response element signaling pathways, 2) anti-angiogenic factors (sFlt1/sEng) and the pro-angiogenic factor PlGF and 3) endothelial dysfunction (vascular cell adhesion molecule-1 (VCAM1) and endothelin-1 (ET-1)). Results New generation antiplatelet agents induced nuclear Nrf2 translocation (antioxidant transcription factor) and increased antioxidant gene expression: HO-1, NQO1 and GCLC. In contrast, aspirin did not affect antioxidant pathways. Furthermore Clopidogrel, Prasugrel and Ticagrelor potently reduced sFlt1 secretion from both placental and endothelial cells; and sEng secretion from endothelial cells. In contrast, aspirin did not affect sFlt or sEng secretion. PlGF mRNA expression was significantly enhanced with new generation anti-platelet treatment. Clopidogrel and Prasugrel rescued endothelial dysfunction, mitigating TNF α induced monocyte-endothelial adhesion, as well as VCAM1 and ET-1 mRNA and protein expression. Again aspirin had no effect. Conclusions New generation antiplatelet therapies potently upregulate anti-oxidant defences, decrease sFlt1/sEng secretion and counter endothelial dysfunction in human models of preeclampsia. Given they are classified as category B/C drugs, they represent exciting candidate therapies to prevent preeclampsia. [ABSTRACT FROM AUTHOR]

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 TNF α 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 TNF α and PE serum induced endothelial dysfunction (quenching leukocyte adhesion and attenuating VCAM1 and endothelin-1 expression). Furthermore, PPIs blocked TNF α -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. [ABSTRACT FROM AUTHOR]

Objectives It is now widely accepted that pre-eclampsia is associated with decreased placental heme oxygenase-1 (HO-1; antioxidant enzyme) expression [1,2] . Furthermore, it also appears widely accepted HO-1 directly regulates the release of soluble fms-like tyrosine kinase-1 (sFlt1). HO-1 is proposed to inhibit sFlt1 production. These observations have prompted the investigation of drugs, such as pravastatin, that induce HO-1 as an approach to treat PE. However, the links between HO-1 and pre-eclampsia and/or sFlt1 production have not been independently validated. Methods Placental tissue was obtained from pregnancies complicated by severe PE (<34 weeks gestation; n = 18) and gestationally-matched normotensive controls ( n = 27), and HO-1 measured. We also examined data on HO-1 expression in pre-eclamptic placentas from publically available microarray repositories (5 independent studies identified). Functionally, we induced (using small molecules) and silenced HO-1 (using siRNAs) in primary human trophoblast and human umbilical vein endothelial cells (HUVEC), and measured sFlt-1 and sEng secretion. Results Surprisingly, there were no significant differences in HO-1 mRNA or protein expression between PE and control placentas (mRNA measured using both conventional qPCR and digital PCR [the latter is more precise]). Public microarray datasets did not show HO-1 to be differentially expressed in PE placenta compared to controls. Silencing either HO-1 or Nrf2 (nuclear factor erythroid-2-related factor 2, transcription factor upstream of HO-1) had no effect on sFlt1 or sEng secretion, in either primary trophoblast or HUVECs. We next induced HO-1 using BG-12. While this known HO-1 inducer upregulates HO-1 expression approximately 200-fold, there was no change in sFlt-1 production. Conclusions HO-1 expression is not reduced in severe PE placenta. Furthermore, it does not appear that HO-1 regulates sFlt1 or sEng regulation in primary human gestational tissues. Disclosures T. Kaitu’u-Lino: None. S. Tong: None. K. Onda: None. F. Brownfoot: None. S. Beard: None. C. Whitehead: None. C. Cluver: None. N.J. Hannan: None. [ABSTRACT FROM AUTHOR]

Objectives sFlt1 is central to the pathophysiology of pre-eclampsia. Excitingly, in 2008-09 three groups reported the predominant placental-derived sFlt-1 isoform is a newly identified splice variant named sFlt1-e15a. Given this is the main sFlt-1 isoform released from placenta, it may be the key pathophysiological factor of pre-eclampsia. To date, the protein expression of sFlt1-e15a has not been described and there have been no functional studies of sFlt1-e15a. We aimed to characterize sFlt1-e15a in preterm and preeclamptic placenta and examine whether sFlt1-e15a protein is biologically active. Methods To investigate expression of sFlt1-e15a we developed polyclonal antibodies targeting a unique peptide sequence in the C-terminal region of sFlt1-e15a. To test whether sFlt1-e15a neutralizes VEGF activity, we used a novel bioassay; BAF3 cells engineered so that they require VEGF to survive. We next assessed whether sFlt1-e15a blocks VEGF induced migration and invasion using the xCELLigence system (monitors experiments in real time), and finally assessed whether sFlt-1-e15a blocked VEGF induced signaling by Western Blot. Results The specificity of our antibodies was established by western analysis. Immunofluorescence confirmed expression of sFlt1-e15a principally within the syncytiotrophoblast layer of placenta, with expression appearing increased in pre-eclampsia. Recombinant sFlt1-e15a dose dependently reduced BAF3 cell viability in the presence of VEGF, suggesting it directly antagonizes VEGF and is biologically active. In the presence of VEGF, sFlt1-e15a also potently blocked primary HUVEC migration and invasion. We next assessed whether sFlt1-e15a blocked VEGF induced signaling of Akt by exposing primary HUVECs to VEGF ± sFlt1-e15a. VEGF induced phosphorylation of Akt (normalized to total Akt) was potently blocked by sFlt1-e15a. Conclusions The novel primate and placental specific sFlt1-e15a protein is present in pre-eclamptic placenta, biologically active and neutralizes VEGF. Being the main sFlt1 splice variant expressed in preeclampsia, sFlt1-e15a may be the main sFlt-1 isoform responsible for the pathophysiology of preeclampsia. Disclosures T. Kaitu’u-Lino: None. K. Palmer: None. R. Hastie: None. P. Cannon: None. N.J. Hannan: None. S. Tong: None. [ABSTRACT FROM AUTHOR]

Introduction: Preeclampsia is a serious pregnancy complication for which there are no medical treatments. Soluble endoglin is an anti-angiogenic factor implicated in the pathogenesis of the disease, however little is known about its molecular regulation. The PI3K/Akt pathway is down regulated in preeclamptic placentas and decreased PI3K/Akt signaling has been linked to increased soluble endoglin release from endothelial cells. MMP14 is a key protease that functions to release soluble endoglin from the placental surface., Objective: This study aimed to determine whether reduced placental PI3K/Akt causes elevated release of soluble endoglin via MMP14., Study Design: Akt mRNA and protein expression were assessed in early onset preeclamptic and preterm control placentas (delivered <34weeks gestation). PI3K/Akt inhibition was achieved by administering PI3K inhibitor wortmannin, a specific Akt inhibitor or Akt siRNA to primary human umbilical vein endothelial cells, primary trophoblast and placental explants. The effect of PI3K/Akt inhibition on soluble endoglin release, MMP14, endoglin and TIMP-3 mRNA expression was determined., Results: We identified significantly reduced pAkt and total Akt in preeclamptic placentas relative to preterm control. Inhibition of PI3K/Akt resulted in significantly elevated soluble endoglin release from HUVECs, had no effect on MMP14 mRNA expression but resulted in significantly reduced TIMP3. In contrast inhibiting PI3K/Akt in placental explants or primary trophoblast did not change soluble endoglin release., Conclusion: This study confirms that the PI3K/Akt cell protection pathway is down regulated in preeclampsia, but demonstrates that this dysregulation is unlikely to be responsible for the excessive placental soluble endoglin release characteristic of preeclampsia., (Copyright © 2016 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

There is recent interest for uniform placental collection protocols so laboratories can share samples. However, concerns have been raised a wash step at collection causes significant loss of sFlt-1 and soluble endoglin, among the most studied proteins in placentology. We measured Flt-1 and endoglin mRNA and protein in 10 preeclamptic placentas that were washed, or left unwashed. Reassuringly, there was no significant change in the Flt-1, sFlt-1-e15a or sFlt-1-i13 mRNA or Flt-1 or sFlt-1 protein expression or localization. There was also no change in endoglin mRNA expression or protein localization. Washing preeclamptic placental samples does not alter anti-angiogenic factor expression., (Copyright © 2015 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Objectives: Soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) are the most studied molecules in preeclampsia. However, most trophoblast cell lines do not secrete both these factors. Thus, we set out to characterize protocols to functionally investigate sFlt-1 and sEng from primary trophoblast., Study Design: Primary trophoblasts were isolated from term placenta by percoll gradient, then negative selection using a CD9 antibody. Purity was assessed by cytokeratin 7 immunostaining. We first examined the effects of CD9 negative selection on sFlt-1, sEng and hCG secretion and the ability of forskolin to enhance syncytialization. We then examined the effects of hypoxia on sFlt-1 production and assessed gene knockdown using siRNA., Results: CD9 negative selection produced a pure population of primary trophoblasts. Secretion of sEng was 5-fold lower when CD9-positive cells were removed, sFlt1 was unchanged, and hCG was significantly increased. hCG analysis of the purified population indicated spontaneous syncytialization, which was not enhanced by forskolin. Forskolin similarly did not alter sFlt-1 secretion. Hypoxia significantly increased sFlt-1 secretion as expected. Importantly, high gene silencing efficiencies were readily achieved., Conclusion: In conclusion, we present a protocol that yields primary trophoblasts of high purity that produce abundant sFlt-1 and low but detectable levels of sEng. Furthermore, these cells are readily amenable to gene silencing by siRNAs and hence suitable for functional studies., (Copyright © 2014 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)