Nicotinamide and its effects on endothelial dysfunction and secretion of antiangiogenic factors by primary human placental cells and tissues.

Preeclampsia is a serious pregnancy complication associated with elevated antiangiogenic markers and endothelial dysfunction. Recently nicotinamide (vitamin B3) was shown to reduce high blood pressure and proteinuria in mice models of the disease. Using primary human pregnancy tissue we show nicotinamide did not change antiangiogenic factor secretion including soluble fms-like tyrosine kinase 1 or soluble endoglin from primary cytotrophoblasts and placental explants. Furthermore, it did not reverse markers of endothelial dysfunction. Therefore, we did not demonstrate an effect of nicotinamide on reducing markers of preeclampsia from primary human placental tissues and vascular cells.

Combining metformin and sulfasalazine additively reduces the secretion of antiangiogenic factors from the placenta: Implications for the treatment of preeclampsia.

INTRODUCTION: The antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG) are elevated in preeclampsia and have been implicated in its pathogenesis. We have previously demonstrated metformin and sulfasalazine independently reduce antiangiogenic factor secretion. Here we examined whether combining metformin and sulfasalazine may be more effective than either alone in reducing placental expression and secretion of antiangiogenic and angiogenic factors and the expression of markers of endothelial dysfunction. METHODS: We performed functional experiments using primary human placenta to explore the effect of metformin and sulfasalazine, at lower doses than previously explored, individually and in combination, on sFlt-1 and sENG secretion and placental growth factor (PlGF) and vascular endothelial growth factor (VEGFα) expression. Using primary endothelial cells we induced dysfunction using cytokine tumor necrosis factor-α (TNF-α) and assessed the effect of low dose combination treatment on the expression of vascular cell adhesion molecule-1 (VCAM-1) and Endothelin-1 (a potent vasoconstrictor). RESULTS: We demonstrated combination metformin and sulfasalazine was additive in reducing sFlt-1 secretion from cytotrophoblasts and placental explants. Combination treatment was also additive in reducing sENG secretion from placental explants. Furthermore, combination treatment increased cytotrophoblast VEGFα mRNA expression. Whilst combination treatment increased PlGF mRNA expression this was similar to treatment with sulfasalazine alone. Combination therapy reduced TNFα induced endothelin-1 mRNA expression however did not change VCAM expression. DISCUSSION: Low dose combination metformin and sulfasalazine reduced cytotrophoblast sFlt-1 and sENG secretion, increased VEGFα expression and reduced TNFα induced endothelin-1 expression in primary endothelial cells. Combination therapy has potential to treat preeclampsia.

Sulfasalazine decreases soluble fms-like tyrosine kinase-1 secretion potentially via inhibition of upstream placental epidermal growth factor receptor signalling.

OBJECTIVES: Preeclampsia is a hypertensive disorder of pregnancy with no available medical treatment. We recently reported sulfasalazine, an anti-inflammatory medication, to be a candidate therapeutic for preeclampsia. We showed sulfasalazine decreases placental secretion of soluble Fms-like tyrosine kinase-1 (sFlt-1), an anti-angiogenic factor strongly implicated in the pathogenesis of preeclampsia. However, the cellular mechanism(s) by which sulfasalazine reduces placental sFlt-1 are yet to be determined. Recently we also reported that both the mitochondria and the epidermal growth factor receptor (EGFR) signalling pathways regulate secretion of placental sFlt-1. In this study we sought to assess directly whether sulfasalazine's capacity to reduce sFlt-1 secretion may be mediated via EGFR or the mitochondria. METHODS AND RESULTS: Using primary cytotrophoblast cells, we confirmed sulfasalazine reduced sFlt-1 secretion. Interestingly, when we measured the mRNA expression of EGFR, we found a reduction in EGFR expression which closely mirrored the changes in sFlt-1 secretion. At the protein level, sulfasalazine significantly reduced phosphorylated and active EGFR (phosphorylated/total) expression. Additionally, sulfasalazine significantly reduced the protein expression of ERK1/2 and STAT3 which are key adaptor molecules downstream of EGFR. Next, we assessed mitochondrial respiration following sulfasalazine treatment and found no effect on basal respiration, ATP production, proton leak or maximal respiration. CONCLUSION: Sulfasalazine reduces EGFR and down-stream signalling molecule expression coincident with reduced sFlt-1 secretion. EGFR signalling is a potential mechanism by which sulfasalazine decreases placental secretion of sFlt-1. Further interrogation of the EGFR may identify new candidate treatments for preeclampsia.

Sulfasalazine reduces placental secretion of antiangiogenic factors, up-regulates the secretion of placental growth factor and rescues endothelial dysfunction.

BACKGROUND: Preeclampsia is a major complication of pregnancy with no medical treatment. It is associated with placental oxidative stress, hypoxia and inflammation leading to soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sENG) secretion and reduced placental growth factor (PlGF). This results in widespread endothelial dysfunction causing hypertension and multisystem organ injury. Sulfasalazine is an anti-inflammatory and antioxidant medication used to treat autoimmune disease. Importantly, it is safe in pregnancy. We examined the potential of sulfasalazine to quench antiangiogenic factors and endothelial dysfunction and increase angiogenic factor secretion. METHODS: We performed functional experiments using primary human pregnancy tissues to examine the effects of sulfasalazine on sFlt-1, sENG and PlGF secretion. Sulfasalazine is known to inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) and upregulate heme-oxygenase 1 (HO-1) thus we explored the effect of these transcription factors on sFlt-1 secretion from human cytotrophoblasts. We examined the ability of sulfasalazine to reduce key markers of endothelial dysfunction and dilate whole blood vessels. FINDINGS: We demonstrate sulfasalazine administration reduces sFlt-1 and sENG and upregulates PlGF secretion from human placental tissues. Furthermore sulfasalazine mitigates endothelial dysfunction in several in vitro/ex vivo assays. It enhanced endothelial cell migration and proliferation, promoted blood vessel dilation (vessels obtained from women at caesarean section) and angiogenic sprouting from whole blood vessel rings. The effect of sulfasalazine on the secretion of sFlt-1 was not mediated through either the NFkB or HO-1 pathways. INTERPRETATION: We conclude that sulfasalazine reduces sFlt-1 and sENG secretion and endothelial dysfunction and upregulates PlGF. Sulfasalazine has potential to treat or prevent preeclampsia and warrants investigation in clinical trials. FUNDING: This work was funded by The National Health and Medical Research Council of Australia (NHMRC; #1048707, #1046484. #1101871, #1064845), an Arthur Wilson RANZCOG scholarship and a Norman Beischer Medical Research Foundation grant. FB was supported by a NHMRC Early Career Fellowship (NHMRC #1142636). NJH was supported by a CR Roper Research Fellowship. The NHMRC provided salary support (#1136418 to ST #1062418 to TKL, #1064845 to SS). The funders had no role in study design, data collection, analysis, decision to publish or the preparation of the manuscript.

Activating Transcription Factor 3 Is Reduced in Preeclamptic Placentas and Negatively Regulates sFlt-1 (Soluble fms-Like Tyrosine Kinase 1), Soluble Endoglin, and Proinflammatory Cytokines in Placenta.

Preeclampsia is a major pregnancy complication associated with poor placental perfusion and placental hypoxia. Systemic and placental inflammation and elevated placental secretion of the antiangiogenic factors sFlt-1 (soluble fms-like tyrosine kinase 1) and sEng (soluble endoglin) are hallmarks of preeclampsia, causing endothelial dysfunction and multiorgan injury. A molecule that links placental hypoxia, inflammation, and antiangiogenic factor release has not been described. ATF3 (activating transcription factor 3) is highly expressed in placenta. We assessed whether placental ATF3 is dysregulated in preterm preeclampsia, is altered by hypoxia, and regulates proinflammatory cytokine and antiangiogenic factor production. ATF3 mRNA and protein expression was significantly reduced in preterm preeclamptic placentas compared with gestation-matched controls. Hypoxia reduced ATF3 expression in primary cytotrophoblast and placental explants. Silencing ATF3 in primary cytotrophoblast increased proinflammatory cytokine (IL-6 [interleukin 6], TNF-α [tumor necrosis factor α]) and NF-κB (nuclear factor κB) expression. In silico analysis identified an ATF3-binding site in the promoter of Flt-1 (the transcript from which sFlt-1 is produced). Silencing ATF3 increased sFlt-1 and sEng secretion from primary cytotrophoblast possibly by increasing Rab11a and Arf1, cargo proteins that facilitate exosomal release of sFlt-1. ATF3 knockout mice did not have a preeclampsia phenotype, suggesting that these pathways may be specific to humans (preeclampsia is a uniquely human condition). To conclude, we have shown that ATF3 is decreased in preeclamptic placentas and that this decrease is likely to occur after prolonged hypoxia. We show that ATF3 is a regulator of placental proinflammatory cytokines and antiangiogenic factors sFlt-1 and sEng. Therefore, reduced ATF3 may be centrally involved in the pathology of preeclampsia.

Key players of the necroptosis pathway RIPK1 and SIRT2 are altered in placenta from preeclampsia and fetal growth restriction.

INTRODUCTION: Preeclampsia (PE) and fetal growth restriction (FGR) are among the leading causes of perinatal morbidity and mortality. Placental insufficiency is central to these conditions. The mechanisms underlying placental insufficiency are poorly understood. Apoptosis has long been considered the only form of regulated cell death, recent research has identified an alternate process of programmed cell death known as necroptosis [1]. Necroptosis is distinct from apoptosis, relying on the deacetylase sirtuin-2 [2], receptor interacting kinases RIPK1 and 3, and the pseudokinase MLKL [3]. We aimed to determine whether these key necroptosis effector molecules were present in human placenta and whether they are differentially expressed in severe preterm (PT) PE and FGR. METHODS: PT placentas from severe early onset (<34 weeks) PE (n = 30), FGR (n = 12) and control (18) pregnancies were collected. SIRT2 and RIPK1 localization and quantitation was determined by immunohistochemistry and western blot. Immunocytochemistry was used to detect SIRT2 and RIPK1 in trophoblastic debris from first trimester, term control and PE pregnancies. Expression of SIRT2, RIPK1, RIPK3 and MLKL was examined by qPCR. RESULTS: SIRT2 and RIPK1 were localized to the syncytiotrophoblast, villous leukocytes and vasculature in all PT placentas. A significant reduction in SIRT2 protein expression in both PE and FGR placentas was identified. RIPK1 mRNA expression was significantly increased in PE placentas. Immunofluorescence identified both SIRT2 and RIPK1 in the cytotrophoblast cytoplasm. DISCUSSION: We have identified the presence of activators of necroptosis in human placenta. Interestingly, there is differential expression in major pregnancy complications. We conclude necroptosis may contribute to placental pathophysiology that underlies serious pregnancy complications.

Proton Pump Inhibitors Decrease Soluble fms-Like Tyrosine Kinase-1 and Soluble Endoglin Secretion, Decrease Hypertension, and Rescue Endothelial Dysfunction.

Preeclampsia is a severe complication of pregnancy. Antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin are secreted in excess from the placenta, causing hypertension, endothelial dysfunction, and multiorgan injury. Oxidative stress and vascular inflammation exacerbate the endothelial injury. A drug that can block these pathophysiological steps would be an attractive treatment option. Proton pump inhibitors (PPIs) are safe in pregnancy where they are prescribed for gastric reflux. We performed functional studies on primary human tissues and animal models to examine the effects of PPIs on sFlt-1 and soluble endoglin secretion, vessel dilatation, blood pressure, and endothelial dysfunction. PPIs decreased sFlt-1 and soluble endoglin secretion from trophoblast, placental explants from preeclamptic pregnancies, and endothelial cells. They also mitigated tumor necrosis factor-α-induced endothelial dysfunction: PPIs blocked endothelial vascular cell adhesion molecule-1 expression, leukocyte adhesion to endothelium, and disruption of endothelial tube formation. PPIs decreased endothelin-1 secretion and enhanced endothelial cell migration. Interestingly, the PPI esomeprazole vasodilated maternal blood vessels from normal pregnancies and cases of preterm preeclampsia, but its vasodilatory effects were lost when the vessels were denuded of their endothelium. Esomeprazole decreased blood pressure in a transgenic mouse model where human sFlt-1 was overexpressed in placenta. PPIs upregulated endogenous antioxidant defenses and decreased cytokine secretion from placental tissue and endothelial cells. We have found that PPIs decrease sFlt-1 and soluble endoglin secretion and endothelial dysfunction, dilate blood vessels, decrease blood pressure, and have antioxidant and anti-inflammatory properties. They have therapeutic potential for preeclampsia and other diseases where endothelial dysfunction is involved.

Maternal plasma concentrations of the placental specific sFLT-1 variant, sFLT-1 e15a, in fetal growth restriction and preeclampsia.

OBJECTIVE: sFLT-1 e15a is a recently described sFlt-1 variant that is placental and primate specific. As such, it may have potential as a biomarker. Using a newly developed ELISA, we measured maternal plasma sFLT-1 e15a levels in women with fetal growth restriction and pre-eclampsia. METHOD: We performed a nested case-control study where we measured total sFLT-1 and sFLT-1 e15a plasma protein concentrations. Samples, selected from a prospective cohort study, consisted of 87 healthy controls, 11 cases that developed term preeclampsia and 20 cases where there was fetal growth restriction. We also measured sFLT-1 and sFLT-1 e15a plasma concentrations in a separate cohort: 15 cases of preterm preeclampsia and 24 healthy controls. RESULTS: The prospective case-control cohort demonstrated significantly increased sFLT-1 e15a among cases with term fetal growth restriction (p < 0.05). We also observed that total sFLT-1 (this ELISA indiscriminately detects all variants) was significantly increased in term preeclampsia (p < 0.0001), but not fetal growth restriction. The separate cohort of early-onset preeclamptics showed significantly increased sFLT-1 e15a levels (p < 0.0001). CONCLUSION: Plasma sFLT-1 e15a is significantly increased in early-onset preeclampsia and term fetal growth restriction. Further assessment of the benefit for sFLT-1 e15a testing in prediction or diagnosis of these disease states is warranted.

Loss of Akt increases soluble endoglin release from endothelial cells but not placenta.

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.

Identifying late-onset fetal growth restriction by measuring circulating placental RNA in the maternal blood at 28 weeks' gestation.

BACKGROUND: Late-onset fetal growth restriction (FGR) is often undetected prior to birth, which puts the fetus at increased risk of adverse perinatal outcomes including stillbirth. OBJECTIVE: Measuring RNA circulating in the maternal blood may provide a noninvasive insight into placental function. We examined whether measuring RNA in the maternal blood at 26-30 weeks' gestation can identify pregnancies at risk of late-onset FGR. We focused on RNA highly expressed in placenta, which we termed "placental-specific genes." STUDY DESIGN: This was a case-control study nested within a prospective cohort of 600 women recruited at 26-30 weeks' gestation. The circulating placental transcriptome in maternal blood was compared between women with late-onset FGR (<5th centile at >36+6 weeks) and gestation-matched well-grown controls (20-95th centile) using microarray (n = 12). TaqMan low-density arrays, reverse transcription-polymerase chain reaction (PCR), and digital PCR were used to validate the microarray findings (FGR n = 40, controls n = 80). RESULTS: Forty women developed late-onset FGR (birthweight 2574 ± 338 g, 2nd centile) and were matched to 80 well-grown controls (birthweight 3415 ± 339 g, 53rd centile, P < .05). Operative delivery and neonatal admission were higher in the FGR cohort (45% vs 23%, P < .05). Messenger RNA coding 137 placental-specific genes was detected in the maternal blood and 37 were differentially expressed in late-onset FGR. Seven were significantly dysregulated with PCR validation (P < .05). Activating transcription factor-3 messenger RNA transcripts were the most promising single biomarker at 26-30 weeks: they were increased in fetuses destined to be born FGR at term (2.1-fold vs well grown at term, P < .001) and correlated with the severity of FGR. Combining biomarkers improved prediction of severe late-onset FGR (area under the curve, 0.88; 95% CI 0.80-0.97). A multimarker gene expression score had a sensitivity of 79%, a specificity of 88%, and a positive likelihood ratio of 6.2 for subsequent delivery of a baby <3rd centile at term. CONCLUSION: A unique placental transcriptome is detectable in maternal blood at 26-30 weeks' gestation in pregnancies destined to develop late-onset FGR. Circulating placental RNA may therefore be a promising noninvasive test to identify pregnancies at risk of developing FGR at term.

Jumonji Domain Containing Protein 6 Is Decreased in Human Preeclamptic Placentas and Regulates sFLT-1 Splice Variant Production.

The anti-angiogenic protein, soluble fms-like tyrosine kinase-1 (sFLT-1), plays a central role in preeclamptic pathophysiology. A splice variant of FLT-1 (VEGF receptor 1), sFLT-1 is released in excessive amounts from the preeclamptic placenta into the maternal circulation, where it causes endothelial dysfunction manifesting as end-organ disease. However, the mechanisms regulating its production within the placenta remain poorly understood. Recently it was shown in endothelial cells that Jumonji domain containing protein 6 (JMJD6) hydroxylates U2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65, a component of the splicesome). The hydroxylation by JMJD6 is oxygen dependent. Under hypoxia, JMJD6 is less able to hydroxylate U2AF65, and this unhydroxylated form of U2AF65 biases splicing of FLT-1 to sFLT-1. We assessed whether oxygen-sensing JMJD6 is differentially expressed in preeclamptic placenta and regulates sFLT-1 splicing in placenta via U2AF65. JMJD6 protein expression was significantly reduced in preterm preeclamptic placenta (P < 0.0001; n = 21) relative to preterm controls (n = 10). Exposing both placental and endothelial cells to hypoxia significantly reduced JMJD6 mRNA and increased sFLT-1 mRNA and protein expression. Silencing JMJD6 in primary endothelial and trophoblast cells significantly increased sFLT-1 secretion. Next, we examined whether these molecules may be directly interacting. We demonstrated that placental U2AF65 colocalized with JMJD6. In turn, we found JMJD6 directly interacts with U2AF65, which in turn produces sFLT-1 mRNA transcripts. Taken together, our findings provide evidence that JMJD6 may play a role in regulating the production of sFLT-1 in the preeclamptic placenta. Decreased placental JMJD6 expression may be an important component to the pathophysiology of preeclampsia.

Metformin as a prevention and treatment for preeclampsia: effects on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion and endothelial dysfunction.

BACKGROUND: Preeclampsia is associated with placental ischemia/hypoxia and secretion of soluble fms-like tyrosine kinase 1 and soluble endoglin into the maternal circulation. This causes widespread endothelial dysfunction that manifests clinically as hypertension and multisystem organ injury. Recently, small molecule inhibitors of hypoxic inducible factor 1α have been found to reduce soluble fms-like tyrosine kinase 1 and soluble endoglin secretion. However, their safety profile in pregnancy is unknown. Metformin is safe in pregnancy and is also reported to inhibit hypoxic inducible factor 1α by reducing mitochondrial electron transport chain activity. OBJECTIVE: The purposes of this study were to determine (1) the effects of metformin on placental soluble fms-like tyrosine kinase 1 and soluble endoglin secretion, (2) to investigate whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion are regulated through the mitochondrial electron transport chain, and (3) to examine its effects on endothelial dysfunction, maternal blood vessel vasodilation, and angiogenesis. STUDY DESIGN: We performed functional (in vitro and ex vivo) experiments using primary human tissues to examine the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from placenta, endothelial cells, and placental villous explants. We used succinate, mitochondrial complex II substrate, to examine whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion were mediated through the mitochondria. We also isolated mitochondria from preterm preeclamptic placentas and gestationally matched control subjects and measured mitochondrial electron transport chain activity using kinetic spectrophotometric assays. Endothelial cells or whole maternal vessels were incubated with metformin to determine whether it rescued endothelial dysfunction induced by either tumor necrosis factor-α (to endothelial cells) or placenta villous explant-conditioned media (to whole vessels). Finally, we examined the effects of metformin on angiogenesis on maternal omental vessel explants. RESULTS: Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary endothelial cells, villous cytotrophoblast cells, and preterm preeclamptic placental villous explants. The reduction in soluble fms-like tyrosine kinase 1 and soluble endoglin secretion was rescued by coadministration of succinate, which suggests that the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin were likely to be regulated at the level of the mitochondria. In addition, the mitochondrial electron transport chain inhibitors rotenone and antimycin reduced soluble fms-like tyrosine kinase 1 secretion, which further suggests that soluble fms-like tyrosine kinase 1 secretion is regulated through the mitochondria. Mitochondrial electron transport chain activity in preterm preeclamptic placentas was increased compared with gestation-matched control subjects. Metformin improved features of endothelial dysfunction relevant to preeclampsia. It reduced endothelial cell messenger RNA expression of vascular cell adhesion molecule 1 that was induced by tumor necrosis factor-α (vascular cell adhesion molecule 1 is an inflammatory adhesion molecule up-regulated with endothelial dysfunction and is increased in preeclampsia). Placental conditioned media impaired bradykinin-induced vasodilation; this effect was reversed by metformin. Metformin also improved whole blood vessel angiogenesis impaired by fms-like tyrosine kinase 1. CONCLUSION: Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary human tissues, possibly by inhibiting the mitochondrial electron transport chain. The activity of the mitochondrial electron transport chain was increased in preterm preeclamptic placenta. Metformin reduced endothelial dysfunction, enhanced vasodilation in omental arteries, and induced angiogenesis. Metformin has potential to prevent or treat preeclampsia.

A wash step at collection of placental biopsies from preeclamptic pregnancies does not adversely affect levels of sFlt-1 or endoglin.

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.

Heme Oxygenase-1 Is Not Decreased in Preeclamptic Placenta and Does Not Negatively Regulate Placental Soluble fms-Like Tyrosine Kinase-1 or Soluble Endoglin Secretion.

Elevated placental release of the antiangiogenic factors, soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG), is central to the pathophysiology of preeclampsia. It is widely accepted that heme oxygenase-1 (HO-1) is decreased in preeclamptic placenta and negatively regulates sFlt-1 and sENG production. We set out to verify these contentions. There was no difference in HO-1 mRNA or protein levels in preterm preeclamptic placentas (n=17) compared with gestationally matched controls (n=27). In silico analysis of microarray studies did not identify decreased placental HO-1 expression in preeclamptic placenta. Silencing HO-1 in primary trophoblasts did not affect sFlt-1 protein secretion after 24 or 48 hours. Silencing nuclear factor (erythroid-derived 2)-like 2 (transcription factor that upregulates HO-1) in trophoblasts also did not affect sFlt-1 secretion. Administering tin protoporphyrin IX dichloride (HO-1 inhibitor) or cobalt protoporphyrin (HO-1 inducer) into placental explants did not affect sFlt-1 or sENG secretion. Silencing HO-1 in 2 types of primary endothelial cells (human umbilical vein endothelial and uterine microvascular endothelial cells) significantly increased sFlt-1 secretion but not sENG secretion. However, HO-1 silencing selectively increased mRNA expression of sFlt-1 i13 (generically expressed sFlt-1 variant) but not of sFlt-1 e15a (sFlt-1 variant mainly expressed in placenta). Furthermore, adding tin protoporphyrin IX dichloride decreased sFlt-1, whereas adding HO-1 inducers (cobalt protoporphyrin, dimethyl fumarate, and rosiglitazone) either had no effect or increased sFlt-1 or sENG secretion (these trends are opposite to what is expected). We conclude that HO-1 expression is not decreased in preeclamptic placenta and HO-1 does not negatively regulate placental sFlt-1 and sENG secretion in placental or endothelial cells.

Placental-Specific sFLT-1 e15a Protein Is Increased in Preeclampsia, Antagonizes Vascular Endothelial Growth Factor Signaling, and Has Antiangiogenic Activity.

In preeclampsia, the antiangiogenic factor soluble fms-like tyrosine kinase-1 (sFLT-1) is released from placenta into the maternal circulation, causing endothelial dysfunction and organ injury. A recently described splice variant, sFLT-1 e15a, is primate specific and the most abundant placentally derived sFLT-1. Therefore, it may be the major sFLT-1 isoform contributing to the pathophysiology of preeclampsia. sFLT-1 e15a protein remains poorly characterized: its bioactivity has not been comprehensively examined, and serum levels in normal and preeclamptic pregnancy have not been reported. We generated and validated an sFLT-1 e15a-specific ELISA to further characterize serum levels during pregnancy, and in the presence of preeclampsia. Furthermore, we performed assays to examine the bioactivity and antiangiogenic properties of sFLT-1 e15a protein. sFLT-1 e15a was expressed in the syncytiotrophoblast, and serum levels rose across pregnancy. Strikingly, serum levels were increased 10-fold in preterm preeclampsia compared with normotensive controls. We confirmed sFLT-1 e15a is bioactive and is able to inhibit vascular endothelial growth factor signaling of vascular endothelial growth factor receptor 2 and block downstream Akt phosphorylation. Furthermore, sFLT-1 e15a has antiangiogenic properties. sFLT-1 e15a decreased endothelial cell migration, invasion, and inhibited endothelial cell tube formation. Administering sFLT-1 e15a blocked vascular endothelial growth factor induced sprouts from mouse aortic rings ex vivo. We have demonstrated that sFLT-1 e15a is increased in preeclampsia, antagonizes vascular endothelial growth factor signaling, and has antiangiogenic activity. Future development of diagnostics and therapeutics for preeclampsia should consider targeting placentally derived sFLT-1 e15a.

YC-1 reduces placental sFlt-1 and soluble endoglin production and decreases endothelial dysfunction: A possible therapeutic for preeclampsia.

Preeclampsia is a serious complication of pregnancy with no medical treatment. It is caused by intermittent placental hypoxia and release of sFlt-1 and soluble endoglin, leading to wide spread maternal endothelial dysfunction and multisystem organ injury. YC-1 is a guanylyl cyclase activator and HIF1α inhibitor developed for use in hypertension and atherosclerosis. We examined whether YC-1 reduces sFlt-1 and sENG secretion and reverses endothelial dysfunction in primary human tissues. YC-1 significantly reduced sFlt-1 and sENG secretion from human umbilical vein endothelial cells, purified primary trophoblast cells and placental explants taken from patients with preterm preeclampsia. This was concordant with reduced HIF1α expression. YC-1 also reversed TNFα induced endothelial dysfunction, including reduced vascular cell adhesion molecule 1 expression and monocyte adhesion to primary endothelial cells. We conclude YC-1 decreases placental production of sFlt-1 and sENG and decreases endothelial dysfunction. It is a novel therapeutic candidate for preeclampsia.

Sofalcone upregulates the nuclear factor (erythroid-derived 2)-like 2/heme oxygenase-1 pathway, reduces soluble fms-like tyrosine kinase-1, and quenches endothelial dysfunction: potential therapeutic for preeclampsia.

Preeclampsia is a severe complication of pregnancy, characterized by hypertension, oxidative stress, and severe endothelial dysfunction. Antiangiogenic factors, soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin, play key pathophysiological roles in preeclampsia. Heme oxygenase-1 (HO-1) is a cytoprotective, antioxidant enzyme reported to be downregulated in preeclampsia. Studies propose that inducing HO-1 may also decrease sFlt-1 production. Sofalcone, a gastric antiulcer agent in clinical use, is known to induce HO-1 in gastric epithelium. We aimed to investigate whether sofalcone induces HO-1 and reduces sFlt-1 release from primary human placental and endothelial cells and blocks endothelial dysfunction in vitro. We isolated human trophoblasts and endothelial cells (human umbilical vein endothelial cells) and also used uterine microvascular cells. We investigated the effects of sofalcone on (1) HO-1 production, (2) activation of the nuclear factor (erythroid-derived 2)-like 2 pathway, (3) sFlt-1 and soluble endoglin release, (4) tumor necrosis factor α-induced monocyte adhesion and vascular cell adhesion molecule upregulation, and (5) endothelial tubule formation. Sofalcone potently increased HO-1 mRNA and protein in both primary trophoblasts and human umbilical vein endothelial cells. Furthermore, sofalcone treatment caused nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 and transactivation of other nuclear factor (erythroid-derived 2)-like 2 responsive genes (NQO1, TXN, and GCLC). Importantly, sofalcone significantly decreased the secretion of sFlt-1 from primary human trophoblasts. Sofalcone potently suppressed endothelial dysfunction in 2 in vitro models, blocking tumor necrosis factor α-induced monocyte adhesion and vascular cell adhesion molecule 1 expression in human umbilical vein endothelial cells. These results indicate that in primary human tissues, sofalcone can potently activate antioxidant nuclear factor (erythroid-derived 2)-like 2/HO-1 pathway, decrease sFlt-1 production, and ameliorate endothelial dysfunction. We propose that sofalcone is a novel therapeutic candidate for preeclampsia.

Pax5 loss imposes a reversible differentiation block in B-progenitor acute lymphoblastic leukemia.

Loss-of-function mutations in hematopoietic transcription factors including PAX5 occur in most cases of B-progenitor acute lymphoblastic leukemia (B-ALL), a disease characterized by the accumulation of undifferentiated lymphoblasts. Although PAX5 mutation is a critical driver of B-ALL development in mice and humans, it remains unclear how its loss contributes to leukemogenesis and whether ongoing PAX5 deficiency is required for B-ALL maintenance. Here we used transgenic RNAi to reversibly suppress endogenous Pax5 expression in the hematopoietic compartment of mice, which cooperates with activated signal transducer and activator of transcription 5 (STAT5) to induce B-ALL. In this model, restoring endogenous Pax5 expression in established B-ALL triggers immunophenotypic maturation and durable disease remission by engaging a transcriptional program reminiscent of normal B-cell differentiation. Notably, even brief Pax5 restoration in B-ALL cells causes rapid cell cycle exit and disables their leukemia-initiating capacity. These and similar findings in human B-ALL cell lines establish that Pax5 hypomorphism promotes B-ALL self-renewal by impairing a differentiation program that can be re-engaged despite the presence of additional oncogenic lesions. Our results establish a causal relationship between the hallmark genetic and phenotypic features of B-ALL and suggest that engaging the latent differentiation potential of B-ALL cells may provide new therapeutic entry points.

PAPPA2 is increased in severe early onset pre-eclampsia and upregulated with hypoxia.

Severe early onset pre-eclampsia is a serious pregnancy complication, believed to arise as a result of persistent placental hypoxia due to impaired placentation. Pregnancy-associated plasma protein A2 (PAPPA2) is very highly expressed in the placenta relative to all other tissues. There is some evidence that PAPPA2 mRNA and protein are increased in association with pre-eclampsia. The aim of the present study was to characterise the mRNA and protein expression, as well as localisation, of PAPPA2 in an independent cohort of severe early onset pre-eclamptic placentas. We also examined whether exposing placental explants to hypoxia (1% oxygen) changed the expression of PAPPA2. Expression of PAPPA2 mRNA and protein was upregulated in severe early onset pre-eclamptic placentas compared with preterm controls and localised to the syncytiotrophoblast. Interestingly, protein localisation was markedly reduced in term placenta. Syncytialisation of BeWo cells did not change PAPPA2 expression. However, hypoxia upregulated PAPPA2 mRNA and protein expression in primary placental explants. Together, our data suggest that PAPPA2 may be upregulated in severe pre-eclampsia and, functionally, this may be mediated via increased placental hypoxia known to occur with this pregnancy disorder.

Characterization of protocols for primary trophoblast purification, optimized for functional investigation of sFlt-1 and soluble endoglin.

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.