sFlt-1 Release Research Articles

Angiogenic and vasoactive proteins in the maternal-fetal interface in healthy pregnancies and preeclampsia

Preeclampsia is characterized by maternal endothelial activation and placental dysfunction. Imbalance in maternal angiogenic and vasoactive factors has been linked to the pathophysiology. The contribution of the placenta as a source to these factors remains unclear. Furthermore, little is known about fetal angiogenic and vasoactive proteins and the relation between maternal and fetal levels. We describe Placental growth factor (PGF), soluble Fms-like tyrosine kinase 1 (sFLT1), soluble Endoglin (sENG), as well as Endothelin 1, 2 and 3 (EDN1, EDN2 and EDN3) in five vessels in healthy pregnancies, early onset (EOPE) and late onset (LOPE) preeclampsia. Specifically, we aimed to 1) compare protein abundance in vessels at the maternal-fetal interface between EOPE, LOPE and healthy pregnancies, 2) describe placental uptake and release of proteins, and 3) describe protein abundance in the maternal vs fetal circulations. Samples were collected from the maternal radial artery, uterine vein and antecubital vein as well as the fetal umbilical vein and artery in 75 healthy and 37 preeclamptic mother-fetus pairs (including 19 EOPE and 18 LOPE), during scheduled cesarean delivery. This method allows estimation of placental release and uptake of proteins by calculation of venoarterial differences on each side of the placenta. The microarray based SomaScan assay quantified the proteins. Abundance of sFLT1 and EDN1 was higher in the maternal vessels in preeclampsia as compared to healthy pregnancies, with highest abundance in EOPE. PGF was lower in the maternal vessels in EOPE as compared to both healthy and LOPE. Maternal EDN2 was higher in preeclampsia, with LOPE having the highest abundance. Our model confirmed placental release of PGF and sFLT1 to the maternal circulation in all groups. The placenta released sFLT1 into the fetal circulation in healthy and LOPE pregnancies. Fetal EDN1 and sFLT1 were higher in early onset preeclampsia, whereas sENG and EDN3 were lower in both preeclampsia groups, compared to healthy. Across groups, abundances of PGF, sFLT1 and EDN3 were higher in the maternal artery as compared to the fetal umbilical vein, whereas EDN2 was lower. Increasing abundance of maternal sFLT1 and EDN1 across the groups healthy, LOPE and EOPE combined with a positive correlation may suggest that these proteins are associated with the pathophysiology and severity of the disease. Elevated EDN1 in the fetal circulation in EOPE represents a novel finding. Long term effects of altered protein abundance in preeclampsia on fetal development and health remain unknown. Further investigation of these proteins' involvement in the pathophysiology and as treatment targets is warranted.

An Examination of the Effect of Aspirin and Salicylic Acid on Soluble Fms-like Tyrosine Kinase-1 Release from Human Placental Trophoblasts.

Low-dose aspirin (LDA) is efficacious in preventing preeclampsia, but its mechanism of action is unclear. Conflicting evidence suggests that it may inhibit placental trophoblast release of soluble fms-like tyrosine kinase-1 (sFlt1), a key mediator of preeclampsia. We examined whether, and at what concentrations, aspirin and its principal metabolite, salicylic acid, modulate sFlt1 release and/or expression in trophoblasts. Human trophoblast lines BeWo and HTR-8/SVneo were cultured; BeWo cells were also treated with 1% oxygen vs. normoxia to mimic hypoxia in preeclamptic placentas. Cells were treated with aspirin or salicylic acid vs. vehicle for 24 h at concentrations relevant to LDA and at higher concentrations. Protein concentrations (ELISA) and mRNA expression (RT-PCR) of sFlt1 were determined. Under normoxia, LDA-relevant concentrations of aspirin (10-50 µmol/L) or salicylic acid (20-100 µmol/L) had no significant effect on sFlt1 protein release or mRNA expression in BeWo cells. However, inhibition was observed at higher concentrations (1 mmol/L for aspirin and ≥200 μmol/L for salicylic acid). Hypoxia enhanced sFlt1 protein release and mRNA expression in BeWo cells, but these responses were not significantly affected by either aspirin or salicylic acid at LDA concentrations. Similarly, neither drug altered sFlt1 protein secretion or mRNA expression in normoxic HTR-8/SVneo cells at LDA concentrations. We suggest that direct modulation of trophoblast release or expression of sFlt1 is unlikely to be a mechanism underlying the clinical efficacy of LDA in preeclampsia.

A defined clathrin-mediated trafficking pathway regulates sFLT1/VEGFR1 secretion from endothelial cells.

FLT1/VEGFR1 negatively regulates VEGF-A signaling and is required for proper vessel morphogenesis during vascular development and vessel homeostasis. Although a soluble isoform, sFLT1, is often mis-regulated in disease and aging, how sFLT1 is trafficked and secreted from endothelial cells is not well understood. Here we define requirements for constitutive sFLT1 trafficking and secretion in endothelial cells from the Golgi to the plasma membrane, and we show that sFLT1 secretion requires clathrin at or near the Golgi. Perturbations that affect sFLT1 trafficking blunted endothelial cell secretion and promoted intracellular mis-localization in cells and zebrafish embryos. siRNA-mediated depletion of specific trafficking components revealed requirements for RAB27A, VAMP3, and STX3 for post-Golgi vesicle trafficking and sFLT1 secretion, while STX6, ARF1, and AP1 were required at the Golgi. Live-imaging of temporally controlled sFLT1 release from the endoplasmic reticulum showed clathrin-dependent sFLT1 trafficking at the Golgi into secretory vesicles that then trafficked to the plasma membrane. Depletion of STX6 altered vessel sprouting in 3D, suggesting that endothelial cell sFLT1 secretion influences proper vessel sprouting. Thus, specific trafficking components provide a secretory path from the Golgi to the plasma membrane for sFLT1 in endothelial cells that utilizes a specialized clathrin-dependent intermediate, suggesting novel therapeutic targets.

Bioflavonoid luteolin prevents sFlt-1 release via HIF-1α inhibition in cultured human placenta.

Preeclampsia (PE) is a serious hypertensive complication of pregnancy and is a leading cause of maternal death and major contributor to maternal and perinatal morbidity, including establishment of long-term complications. The continued prevalence of PE stresses the need for identification of novel treatments which can target prohypertensive factors implicated in the disease pathophysiology, such as soluble fms-like tyrosine kinase 1 (sFlt-1). We set out to identify novel compounds to reduce placental sFlt-1 and determine whether this occurs via hypoxia-inducible factor (HIF)-1α inhibition. We utilized a commercially available library of natural compounds to assess their ability to reduce sFlt-1 release from primary human placental cytotrophoblast cells (CTBs). Human placental explants from normotensive (NT) and preeclamptic (PE) pregnancies were treated with varying concentrations of luteolin. Protein and mRNA expression of sFlt-1 and upstream mediators were evaluated using ELISA, western blot, and real-time PCR. Of the natural compounds examined, luteolin showed the most potent inhibition of sFlt-1 release, with >95% reduction compared to vehicle-treated. Luteolin significantly inhibited sFlt-1 in cultured placental explants compared to vehicle-treated in a dose- and time-dependent manner. Additionally, significant decreases in HIF-1α expression were observed in luteolin-treated explants, suggesting a mechanism for sFlt-1 downregulation. The ability of luteolin to inhibit HIF-1α may be mediated through the Akt pathway, as inhibitors to Akt and its upstream regulator phosphatidylinositol-3 kinase (PI3K) resulted in significant HIF-1α reduction. Luteolin reduces anti-angiogenic sFlt-1 through inhibition of HIF-1α, making it a novel candidate for the treatment of PE.

Abstract 037: Serum Chemerin As A Novel Biomarker For Preeclampsia Complications: Effects Of Statins On Its Release From The Placenta

Background: The adipokine chemerin is increased in preeclampsia (PE), possibly because it is released from the placenta. We aimed to evaluate the potential of chemerin as novel biomarker to predict PE or PE-related complications. Methods: Making use of a secondary analysis of a prospective cohort study that evaluated the usefulness of the sFlt-1 (soluble Fms-like tyrosine kinase-1)/PlGF (placental growth factor), we measured chemerin in 371 women with suspected or confirmed PE. In addition, we obtained placentas from 25 women (15 healthy controls, 10 PE) to determine whether statins are capable of reducing chemerin release from isolated perfused placentas and placental explants. For this purpose, chemerin, sFlt-1 and PlGF were measured in the perfusate before and after the addition of 1000 ng/mL pravastatin or 6000 ng/mL fluvastatin to the maternal side, and in explant cultured medium before and after exposure to pravastatin (10, 100 or 1000 μmol/L) or fluvastatin (10 or 100 μmol/L) for 72 hours at 37°C with 5% O 2 and 5% CO 2 . Results: At time of biomarker measurement, women had a median gestational age of 35.6 weeks (range 31.7-38.0). Serum chemerin levels were elevated (P<0.001) in women with confirmed PE (n=85) and gestational hypertension (n=68) in comparison to those without hypertensive disease of pregnancy (n=218; 243±13 and 205±19 vs. 185±6 ng/mL). Serum chemerin correlated positively with the sFlt-1/PlGF ratio (r=0.27, P<0.001), the protein/creatinine ratio (r=0.27, P<0.001), and mean arterial blood pressure (r=0.20, P<0.001), and negatively with birth weight (r=-0.24, P<0.001). Levels of chemerin were significantly higher in maternal perfusate of preeclamptic placentas in comparison to healthy placentas. Pravastatin, but not fluvastatin, suppressed chemerin and sFlt-1 release from healthy placentas, and upregulated PlGF release. Studies in placental explants confirmed this observation. Conclusions: Chemerin is increased in both serum and placental perfusate of women with PE, and should therefore be investigated as a potential biomarker for PE or PE-related complications. Moreover, pravastatin lowered placental chemerin release in parallel with sFlt-1, suggesting that the 2 might be causally linked.

Increased Placental sFLT1 (Soluble fms-Like Tyrosine Kinase Receptor-1) Drives the Antiangiogenic Profile of Maternal Serum Preceding Preeclampsia but Not Fetal Growth Restriction.

Preeclampsia and fetal growth restriction (FGR) are both associated with an increased ratio of sFLT1 (soluble fms-like tyrosine kinase-1) to PlGF (placenta growth factor) in maternal serum. In preeclampsia, it is assumed that increased placental release of sFLT1 results in PlGF being bound and inactivated. However, direct evidence for this model is incomplete, and it is unclear whether the same applies in FGR. We conducted a prospective cohort study where we followed 4212 women having first pregnancies from their dating ultrasound, obtained blood samples serially through the pregnancy, and performed systematic sampling of the placenta after delivery. The aim of the present study was to determine the relationship between protein levels of sFLT1 and PlGF in maternal serum measured at ≈36 weeks and placental tissue lysates obtained after term delivery in 82 women with preeclampsia, 50 women with FGR, and 132 controls. The sFLT1:PlGF ratio was increased in both preeclampsia and FGR in both the placenta and maternal serum. However, in preeclampsia, the maternal serum ratio of sFLT1:PlGF was strongly associated with placental sFLT1 level (r=0.45; P<0.0001) but not placental PlGF level (r=-0.17; P=0.16). In contrast, in FGR, the maternal serum ratio of sFLT1:PlGF was strongly associated with placental PlGF level (r=-0.35; P=0.02) but not sFLT1 level (r=0.04; P=0.81). We conclude that the elevated sFLT1:PlGF ratio is primarily driven by increased placental sFLT1 in preeclampsia, whereas in FGR, it is primarily driven by decreased placental PlGF.

Omeprazole Administration in Preterm Preeclampsia: a Randomized Controlled Trial to Study Its Effect on sFlt-1 (Soluble Fms-Like Tyrosine Kinase-1), PlGF (Placental Growth Factor), and ET-1 (Endothelin-1).

Background:Low sFlt-1 (soluble Fms-like tyrosine kinase-1) and ET-1 (endothelin-1) levels have been reported in preeclamptic women using proton pump inhibitors.Methods:Here, we examined whether the proton pump inhibitor omeprazole could acutely reduce sFlt-1 and ET-1 (measured as CT-proET-1 [C-terminal pro-endothelin-1]), or increase free PlGF (placental growth factor) in 20 women with confirmed preeclampsia. Primary outcome was specified as the difference in sFlt-1, PlGF, or CT-proET-1 after 4 days of omeprazole versus 20 preeclamptic women not receiving omeprazole.Results:Mean maternal age was 30 years, and median gestational age was 30+3 weeks. Baseline sFlt-1 levels were identical in both groups, and the same was true for PlGF or CT-proET-1. After 4 days, sFlt-1 levels remained similar in women not receiving omeprazole compared with women receiving omeprazole, while the levels of PlGF and CT-proET-1 also did not differ between groups. Women receiving omeprazole had a similar prolongation of pregnancy after inclusion compared with those in the nonomeprazole group (median 15 versus 14 days). Except for a higher neonatal intubation rate in the nonomeprazole group (31% versus 4%, P=0.02), there were no differences in maternal/perinatal complications. Finally, making use of the placenta perfusion model, we established that both omeprazole and its S-isomer, esomeprazole, when maternally applied, reached the fetal compartment (fetal-to-maternal ratio’s 0.43–0.59), while only esomeprazole inhibited placental sFlt-1 release.Conclusions:Administration of omeprazole to women with confirmed preeclampsia does not alter their circulating levels of sFlt-1, PlGF, or ET-1, arguing against a role of this drug as a treatment for this syndrome.

Induction of the PPARγ (Peroxisome Proliferator-Activated Receptor γ)-GCM1 (Glial Cell Missing 1) Syncytialization Axis Reduces sFLT1 (Soluble fms-Like Tyrosine Kinase 1) in the Preeclamptic Placenta.

[Figure: see text].

MODULATION OF ANGIOGENIC BALANCE DURING PREGNANCY BY EXOGENOUS HEPARIN

Objective: Preeclampsia is a pregnancy-specific hypertensive disorder, commonly believed to result from placental dysfunction. Increased soluble vascular endothelial growth factor receptor-1 (sFlt-1) is consistently observed, and is believed to be pathogenic. sFlt-1 contains a binding site for the extracellular matrix (ECM) component heparan sulfate and, therefore, the anticoagulant heparin. Here we hypothesized that infusion of unfractionated heparin during pregnancy in rodents would displace and solubilize ECM-bound sFlt-1, cause maternal angiogenic imbalance, and maternal hypertension. Design and method: To determine the effects of heparin on sFlt-1 release in vitro. BeWo cytotrophoblasts and rodent placental explants were cultured at normoxic (8%) or hypoxic (1%) oxygen with or without exogenous heparin (10U/ml). Media sFlt-1 was measured by commercial ELISA, membrane-bound sFlt-1 was determined by quantitative immunofluorescence, and mRNA levels of Flt-1, sFlt-1, and heparanase were monitored by qRT-PCR. Pregnant rats were treated from GD14–19 with i.p. unfractionated heparin (300U/kg/day) or saline. MAP was monitored by carotid catheter on GD19, when tissues were collected and morphometrics noted. Placental and plasma sFlt-1 and VEGF were determined by commercial ELISA. Results: In BeWo cells, heparin increased media sFlt-1 in both normoxic (p = 0.0002) and hypoxic samples (p = 0.0066) compared to controls. Total Flt-1 and sFlt-1 was significantly decreased in heparin treated cells compared to controls (p = 0.0063) by immunofluorescence. In placental explants, 10U/mL heparin for 30 minutes significantly increased media sFlt-1 levels (p = 0.0005). Flt-1 mRNA was significantly decreased (p = 0.003) in heparin treated samples, while sFlt-1 and HPSE showed trends towards reduction. Infusion of unfractionated heparin increased MAP in pregnant rats compared to controls (110 ± 2.6 mmHg vs. 99.6 ± 1.3 mmHg, respectively; p = 0.0038). Placental mass (p = 0.0116) and pup weight (p = 0.0075) decreased with heparin treatment. Placental sFlt-1 levels were reduced with heparin (761.8 ± 48.4 pg/mg vs. 913.8 ± 33 pg/mg; p = 0.0178), while placental VEGF levels trended toward reduction. Heparin treatment reduced plasma bioavailable VEGF levels compared to controls (1174 ± 38.5 pg/mL vs. 1492 ± 62.2 pg/mL, respectively; p = 0.0006). Conclusions: Heparin infusion released ECM-bound sFlt-1, altered the angiogenic balance in the placenta and circulation of pregnant rats, and elevated their mean arterial pressure.

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...

Bioenergetic effects of hydrogen sulfide suppress soluble Flt-1 and soluble endoglin in cystathionine gamma-lyase compromised endothelial cells

Endothelial dysfunction is a hallmark of preeclampsia, a life-threatening complication of pregnancy characterised by hypertension and elevated soluble Fms-Like Tyrosine Kinase-1 (sFlt-1). Dysregulation of hydrogen sulfide (H2S) by inhibition of cystathionine γ-lyase (CSE) increases sFlt-1 and soluble endoglin (sEng) release. We explored whether compromise in CSE/H2S pathway is linked to dysregulation of the mitochondrial bioenergetics and oxidative status. We investigated whether these effects were linked to CSE-induced sFlt-1 and sEng production in endothelial cells. Here, we demonstrate that CSE/H2S pathway sustain endothelial mitochondrial bioenergetics and loss of CSE increases the production of mitochondrial-specific superoxide. As a compensatory effect, low CSE environment enhances the reliance on glycolysis. The mitochondrial-targeted H2S donor, AP39, suppressed the antiangiogenic response and restored the mitochondrial bioenergetics in endothelial cells. AP39 revealed that upregulation of sFlt-1, but not sEng, is independent of the mitochondrial H2S metabolising enzyme, SQR. These data provide new insights into the molecular mechanisms for antiangiogenic upregulation in a mitochondrial-driven environment. Targeting H2S to the mitochondria may be of therapeutic benefit in the prevention of endothelial dysfunction associated with preeclampsia.

Contribution of placental 11β-HSD2 to the pathogenesis of preeclampsia.

Preeclampsia, a major human pregnancy-specific disorder, leads to maternal and fetal morbidity and mortality. Here we reported that 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), an enzyme that degrades active glucocorticoids, is one of the key factors that contributes to preeclampsia development. In the pregnant rat model, we firstly confirmed that administration of 11β-HSD2 inhibitor carbenoxolone (CBX) subcutaneously or by placenta-targeted delivery system could lead to a decrease in placental 11β-HSD2 expression and activity and an increase in corticosterone level in placenta and maternal circulation. Then, we showed that subcutaneous administration and placenta-targeted delivery of CBX resulted in the hallmark of preeclampsia-like features including hypertension, proteinuria, renal damages as well as elevated circulatory soluble fms-like tyrosine kinase 1 (sFlt1) and increased sFlt1/placental growth factor (PlGF) ratio in pregnant rats. These animals displayed decreased trophoblast invasion in uterus, impaired spiral artery remodeling, and reduced placental blood flow. Preeclampsia-like features could also be induced by administration of dexamethasone in pregnant rats. In the cultured human trophoblast models, we found that cortisol only inhibited migration and invasion of the extravillous trophoblasts with 11β-HSD2 knockdown, and promoted sFlt1 release in the cultured syncytiotrophoblasts with 11β-HSD2 knockdown. Furthermore, we elucidated that cortisol stimulated a disintegrin and metalloprotease (ADAM)17 expression in placentas, thereby promoting sFlt1 release in placenta. Collectively, our study provided the evidence that placental 11β-HSD2 dysfunction plays a key role in the development of preeclampsia and immediately identified innovative target to counteract preeclampsia.

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.

Conditional Deletion of Phd2 in Spongiotrophoblasts Mimics Early Onset Preeclampsia

Preeclampsia is a common pregnancy disorder which is characterized by new onset hypertension and endothelial dysfunction. Despite efforts to determine the causal factors of this disease, little progress has been made in discerning the etiology. The hypoxic and ischemic placenta, however, is generally accepted as the source for secreted factors in the maternal circulation, such as sFLT-1, which drive the maternal syndrome.Using BeWo placental trophoblast cells, we measured the role of hypoxia on sFLT-1 mRNA as well as protein production. We also exposed the cells to treatment with heparin and heparanase inhibitor OGT-2115.We found that under hypoxic conditions mRNA levels of sFLT-1 were unchanged compared to normoxic controls. Although the message level did not differ under hypoxic conditions, the sFLT-1 release into the media was significantly greater in hypoxia. Additionally, we found that sFLT-1 is able to bind heparan strands in the extracellular matrix with its heparin binding site. These heparan strands can be cleaved by the extracellular enzyme heparanase. We found that heparanase expression was significantly increased in hypoxia, and inhibiting the actions of heparanase attenuated the release of sFLT-1 into the media.While the placenta remains a source of sFLT-1, the mechanism of increased circulating sFLT-1 may differ than simple upregulation of the protein. These data demonstrate the potential importance of the role heparanase may play in releasing previously made sFLT-1 into the maternal circulation.

Sexual Dimorphism in Regulation of Trophoblast Respiration and Metabolic Reprogramming with Obesity and Gestational Diabetes

Preeclampsia is a common pregnancy disorder which is characterized by new onset hypertension and endothelial dysfunction. Despite efforts to determine the causal factors of this disease, little progress has been made in discerning the etiology. The hypoxic and ischemic placenta, however, is generally accepted as the source for secreted factors in the maternal circulation, such as sFLT-1, which drive the maternal syndrome.Using BeWo placental trophoblast cells, we measured the role of hypoxia on sFLT-1 mRNA as well as protein production. We also exposed the cells to treatment with heparin and heparanase inhibitor OGT-2115.We found that under hypoxic conditions mRNA levels of sFLT-1 were unchanged compared to normoxic controls. Although the message level did not differ under hypoxic conditions, the sFLT-1 release into the media was significantly greater in hypoxia. Additionally, we found that sFLT-1 is able to bind heparan strands in the extracellular matrix with its heparin binding site. These heparan strands can be cleaved by the extracellular enzyme heparanase. We found that heparanase expression was significantly increased in hypoxia, and inhibiting the actions of heparanase attenuated the release of sFLT-1 into the media.While the placenta remains a source of sFLT-1, the mechanism of increased circulating sFLT-1 may differ than simple upregulation of the protein. These data demonstrate the potential importance of the role heparanase may play in releasing previously made sFLT-1 into the maternal circulation.

Heparanase regulation of sFLT-1 release in trophoblasts in vitro

IntroductionPreeclampsia is a common pregnancy disorder which is characterized by new onset hypertension and endothelial dysfunction. Despite efforts to determine the causal factors of this disease, little progress has been made in discerning the etiology. The hypoxic and ischemic placenta, however, is generally accepted as the source for secreted factors in the maternal circulation, such as sFLT-1, which drive the maternal syndrome. MethodsUsing BeWo placental trophoblast cells, we measured the role of hypoxia on sFLT-1 mRNA as well as protein production. We also exposed the cells to treatment with heparin and heparanase inhibitor OGT-2115. ResultsWe found that under hypoxic conditions mRNA levels of sFLT-1 were unchanged compared to normoxic controls. Although the message level did not differ under hypoxic conditions, the sFLT-1 release into the media was significantly greater in hypoxia. Additionally, we found that sFLT-1 is able to bind heparan strands in the extracellular matrix with its heparin binding site. These heparan strands can be cleaved by the extracellular enzyme heparanase. We found that heparanase expression was significantly increased in hypoxia, and inhibiting the actions of heparanase attenuated the release of sFLT-1 into the media. DiscussionWhile the placenta remains a source of sFLT-1, the mechanism of increased circulating sFLT-1 may differ than simple upregulation of the protein. These data demonstrate the potential importance of the role heparanase may play in releasing previously made sFLT-1 into the maternal circulation.

Hypertension Editors' Picks Preeclampsia.

Hypertension Editors' Picks Preeclampsia.

1392-P: The Potential Role of Placental α2-Adrenergic Receptors in the High Risk of Preeclampsia in Diabetes

Preeclampsia is a life-threatening complication of pregnancy that occurs four times more frequently in the presence of maternal diabetes. The anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt1), released from the placenta, plays a crucial role in PE development. Alpha 2-adrenoceptor (A2AR) dysfunction has been reported in diabetes, and is implicated in the development of vascular complications. Furthermore, A2AR agonist antihypertensive agents are frequently used to treat patients with preeclampsia, whether they have a disease-modifying role is unclear. We hypothesized that A2AR dysfunction could underlie up-regulation of placental sFlt1 expression, thus ‘driving’ preeclampsia in diabetes. We treated cultured human trophoblasts (HTR8/SVneo) with a stressor often present in the diabetic vasculature, ‘heavily oxidized, glycated’ low-density lipoproteins (HOG-LDL), vs. native LDL (at 100 µg protein/ml) with or without the A2AR agonist, clonidine, and antagonist, yohimbine (24h; both drugs at 10 μM and 100 μM). We determined α2A-, α2B- and α2C-AR mRNA expression (RT-PCR) and immunostaining; sFlt1 mRNA expression and protein release (ELISA); and cell viability (Cell Counting Kit-8 assay). Experiments were repeated three times independently. We found that all three A2AR subtypes were expressed in human trophoblasts. HOG- vs. N-LDL increased sFlt1 mRNA expression (2.0-fold, p&amp;lt;0.001) and protein release (2.5-fold, p&amp;lt;0.01), but neither clonidine nor yohimbine had any effect at either dose. Neither HOG- nor N-LDL altered A2AR expression or cell viability. Overall, the data confirm that modified lipoproteins may promote PE development in women with diabetes via upregulation and release of sFlt1, but do not provide clear evidence that A2ARs are implicated in this process. Further work will investigate whether A2ARs may be modified by other stressors of diabetes, and whether they may affect non-sFlt1 mechanisms for PE. Disclosure R.P. Chow: None. J. Zhao: None. T. Curtis: None. T. Lyons: None. J. Yu: None.

Low-dose aspirin reduces hypoxia-induced sFlt1 release via the JNK/AP-1 pathway in human trophoblast and endothelial cells.

Pre-eclampsia (PE) is a serious hypertensive disorder of pregnancy that remains a leading cause of perinatal and maternal morbidity and mortality worldwide. Placental ischemia/hypoxia and the secretion of soluble fms-like tyrosine kinase 1 (sFlt1) into maternal circulation are involved in the pathogenesis of PE. Although low-dose aspirin (LDA) has beneficial effects on the prevention of PE, the exact mechanisms of action of LDA, particularly on placental dysfunction, and sFlt1 release, have not been well investigated. This study aimed to determine whether LDA exists the protective effects on placental trophoblast and endothelial functions and prevents PE-associated sFlt1 release. First, we observed that LDA mitigated hypoxia-induced trophoblast apoptosis, showed positive effects on trophoblast cells migration and invasion activity, and increased the tube-forming activity of human umbilical vein endothelial cells (HUVECs). In addition, LDA decreased hypoxia-induced sFlt1 production, and the c-Jun NH2 -terminal kinase/activator protein-1 (JNK/AP-1) pathway was shown to mediate the induction of sFlt1. Moreover, the transcription factor AP-1 was confirmed to regulate the Flt1 gene expression by directly binding to the Flt1 promoter in luciferase assays. The result of chromatin immunoprecipitation assays further demonstrated that LDA could directly decrease the expression of the transcription factor AP-1, and thus decrease sFlt1 production. Finally, the effects of LDA on sFlt1 production were proved in human placental explants. Taken together, our data show the protective effects of LDA against trophoblast and endothelial cell dysfunction and reveal that the LDA-mediated inhibition of sFlt1 via the JNK/AP-1 pathway may be a potential cellular/molecular mechanism for the prevention of PE.

SFlt‐1 Splicing Regulation by U2AF65 and JMJD6 in Endothelial Cells

The obstetric disorder preeclampsia (PE) is a condition presenting with new onset hypertension and other co‐morbidities such as proteinuria and endothelial dysfunction. Women with preeclamptic pregnancies have significantly increased circulating levels of the soluble vascular endothelial growth factor (VEGF) receptor Flt‐1 (sFlt‐1). Acting as a VEGF antagonist, which is crucial for the growth and proliferation of endothelial cells, sFlt‐1 causes decreased bioavailability of VEGF. The mechanism regulating the alternative splicing of full length Flt‐1 to the soluble variant are not understood, though U2AF65 and JMJD6 are two proteins which have been implicated in splicing event. Previous research has suggested that U2AF65 promotes the splicing of sFlt‐1, while JMJD6 antagonizes splicing by marking U2AF65 for degradation. Using human umbilical vein endothelial cells (HUVECs), we tested the hypothesis that U2AF65 and JMJD6 were both necessary for alternative splicing in endothelial cells. siRNA was utilized to knock down both U2AF65 (1± 0.012 Fold vs 0.136± 0.017 Fold; p&lt;0.001) and JMJD6 (0.87±0.17 Fold vs 0.077 ± 0.019 Fold; p&lt;0.001) when normalized to β‐actin. Surprisingly, when we measured sFlt‐1 variants 2 and 3, the two most common variants in humans, we found no difference in sFlt‐1 expression. Additionally, we measured expression of the full length Flt‐1, and similarly saw no changes. Next, we overexpressed both U2AF65 and JMJD6 and assessed levels by qRT‐PCR. We observed a trend for ~2‐fold increase in U2AF65 200% (0.85 ± 0.20 Fold vs 1.899 ± 0.39 Fold; p=0.22) though this did not reach significance. Interestingly, in addition to significantly overexpressing JMJD6 (1.27 ± 0.27 Fold vs 2.40 ± 0.31 Fold; p&lt;0.01), cells overexpressing JMJD6 also had significantly higher expression of U2AF65 (0.85 ± 0.20 Fold vs 2.63 ± 0.30 Fold; p&lt;0.05). We measured expression of the FLT1 gene, with no changes seen in the expression of Flt‐1 or sFlt‐1 version 2. The sFlt‐1 variant 3, however, was significantly higher in cells overexpressing JMJD6 (1.008 ± 0.09 Fold vs 1.56 ± 0.14 Fold; p&lt;0.05). Despite the message levels remaining unchanged, sFlt‐1 protein was affected by U2AF65 and JMJD6, with strong trends toward decreased sFlt‐1 in with knock down of U2AF65 (5066 ± 644.3 pg/mL vs 3231 ± 193.5 pg/mL; p=0.067) and JMJD6 (5066 ± 644.3 pg/mL vs 3447 ± 605 pg/mL; p=0.09). Cells which had expression of both U2AF65 and JMJD6 knocked down exhibited an additive effect of sFlt‐1 protein decrease (5066 ± 644.3 pg/mL vs 2166 ± 63.6 pg/mL; p&lt;0.01). Similar, though non‐significant, trends of a positive correlation were seen in cells overexpressing U2AF65 and JMJD6 having slightly increased sFlt‐1 in the media. Because the message levels did not differ greatly among the transformed cells, we looked at heparanase (HPSE) expression as a modulator of sFlt‐1 release into the media. Though there was no change in HPSE expression in knockdown cells, significantly higher HPSE expression was seen in cells overexpressing both U2AF65 and JMJD6 (1.0 ± 0.03 Fold vs 1.97 ± 0.15 Fold; p&lt;0.001). These data suggest that U2AF65 and JMJD6 play an important role in the release of sFlt‐1, but may not be part of the alternative splicing machinery. Future experiments will look at other potential modulators regulating both sFlt‐1 and heparanase, such as MMP‐9.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.