sFlt-1 mRNA Expression Research Articles

Edaravone inhibits hypoxia-induced trophoblast-soluble Fms-like tyrosine kinase 1 expression: A possible therapeutic approach to preeclampsia

ObjectiveTo investigate the effects of edaravone, a potent free radical scavenger used clinically, on hypoxia-induced trophoblast-soluble Fms-like tyrosine kinase 1 (sFlt-1) expression. MethodsA trophoblast cell line (HRT-8/SVneo) impaired by cobalt chloride (CoCl2) was used as the cell model under hypoxic conditions. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) was used to measure the viability of cells exposed to CoCl2 and edaravone. The levels of intracellular reactive oxygen species (ROS) were analyzed by flow cytometry. mRNA expression of sFlt-1, vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) in trophoblasts was measured by real-time polymerase chain reaction, and the secretion of sFlt-1, VEGF, and PlGF proteins was analyzed by enzyme-linked immunosorbent assays (ELISAs). A human umbilical vein endothelial cell (HUVEC) tube-formation assay was performed to identify the effects of CoCl2 and edaravone on vascular development. ResultsCoCl2 treatment caused the loss of trophoblast viability, the formation of ROS, and sFlt-1 mRNA and protein expression in a dose-dependent manner. Pretreatment with edaravone significantly inhibited hypoxia-induced oxidative stress formation and sFlt-1 expression in trophoblasts. Neither PlGF nor VEGF mRNA or protein expression was increased by CoCl2. In the in vitro tube formation assay, edaravone showed a protective role in vascular development under hypoxic conditions. ConclusionThis study demonstrated that hypoxia leading to increased sFlt-1 release in trophoblasts may contribute to the placental vascular formation abnormalities observed in preeclampsia and suggested that the free radical scavenger edaravone could be a candidate for the effective treatment of preeclampsia.

Mitochondrial Channel Opener Diazoxide Attenuates Hypoxia-Induced sFlt-1 Release in Human Choriocarcinoma Cells.

ObjectivesTo examine the effect of diazoxide on hypoxia-induced soluble fms-like tyrosin kinase-1 (sFlt-1) release in JEG-3 choriocarcinoma cells.MethodsCells were cultured under normoxia (20% O2) or hypoxia (1% O2), and expression of sFlt-1 mRNA and protein release was determined by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) assays and enzyme-linked immunosorbent assay (ELISA).ResultsTumor necrosis factor-alpha (TNF-α) as well as hypoxia stimulated sFlt-1 release and diazoxide inhibited both of them. The selective inhibitor of mitochondrial adenosine triphosphat (ATP)-sensitive K+ channel opener (KATP) 5-hydroxydecanoate (5-HD) completely reversed the diazoxide-induced inhibition of hypoxia-stimulated sFlt-1 release. qRT-PCR and Western blot analyses showed that diazoxide up-regulated the heme oxygenase-1 (HO-1) expression. In addition, the HO-1 inducer cobalt protoporphyrin (CoPP) and the metabolic product of HO-1 bilirubin mimicked diazoxide to inhibit sFlt-1 release and reactive oxygen species (ROS) production under hypoxia, whereas the HO-1 inhibitor zinc protoporphyrin IX (ZnPP IX) antagonized the effect of diazoxide. In cells transfected with the HO-1 siRNA, diazoxide did not exert any effect on sFlt-1 release and ROS production under hypoxia.ConclusionThese results, taken together, strongly suggest that up-regulation of the HO-1 expression is the crucial mechanism responsible for the diazoxide-induced inhibition of the sFlt-1 release and ROS production under hypoxia.

Sildenafil Inhibits Advanced Glycation End Products-induced sFlt-1 Release Through Upregulation of Heme Oxygenase-1.

ObjectivesWe examined the effect of sildenafil citrate on advanced glycation end products (AGEs)-induced soluble fms-like tyrosine kinase 1 (sFlt-1) release in JEG-3 choriocarcinoma cells.MethodsCells were incubated with control bovine serum albumin (BSA) or AGEs-BSA, and expression of sFlt-1 mRNA and protein release was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. AGEs-BSA increased sFlt-1 mRNA expression and protein release in a dose-dependent manner.ResultsSildenafil citrate suppressed sFlt-1 mRNA expression and protein release in cells treated with AGEs-BSA in a dose-dependent manner. Likewise, it inhibited the increase of reactive oxygen species (ROS) production and NF-κB activity in these cells. Cobalt protoporphyrin (CoPP) and bilirubin also inhibited sFlt-1 release and ROS production in cells treated with AGEs-BSA, whereas zinc protoporphyrin IX (ZnPP IX) antagonized the effect of sildenafil citrate. In cells transfected with the heme oxygenase-1 (HO-1) siRNA, sildenafil citrate failed to inhibit the sFlt-1 release and ROS production.ConclusionThese results strongly suggest that sildenafil citrate inhibits sFlt-1 release and ROS production in cells treated with AGEs-BSA through upregulation of the HO-1 expression in JEG-3 cells.

343: Placental sFLT1 mRNA expression in the selective reduced uterine perfusion pressure model

343: Placental sFLT1 mRNA expression in the selective reduced uterine perfusion pressure model

Advanced glycation end products as an upstream molecule triggers ROS-induced sFlt-1 production in extravillous trophoblasts: A novel bridge between oxidative stress and preeclampsia

Although abnormal soluble fms-like tyrosine kinase-1 (sFlt-1) production is thought to be an important factor in the pathogenesis of pre-eclampsia, the mechanisms that regulate the production of sFlt-1 during pre-eclampsia are unclear. Accumulation of advanced glycation end products (AGEs) is prevalent in obesity, advanced maternal age, diabetes mellitus, and polycystic ovary syndrome. Alterations in the regulation and signaling of angiogenic pathways have been considered as a link between these conditions and pre-eclampsia. The purpose of this study was to explore the possible effects of AGEs on sFlt-1 secretion in extravillous trophoblasts (EVT). A EVT cell line (HRT-8/SVneo) was treated with various concentrations of AGEs–BSA. The mRNA expression of sFlt-1, vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) in EVT were detected with real-time polymerase chain reaction. The secretion of sFlt-1, VEGF, and PlGF protein from EVT was measured with ELISA. The levels of intracellular reactive oxygen species (ROS) production were determined by DCFH-DA. Exposure of EVT to AGEs–BSA induced increased intracellular ROS generation and overexpression of sFlt-1 at mRNA and protein levels in a dose dependent manner. Anti-RAGE immunoglobulin G or apocynin (an inhibitors of NADPH oxidase) could decrease the intracellular ROS generation and subsequently suppressed the production of sFlt-1 at mRNA and protein levels. Our data suggested that AGEs may be a new class of important mediator in the regulation of angiogenic pathways of EVT. Accumulation of AGEs might contribute to the pathogenesis of preeclampsia by promoting sFlt-1 production through activation of RAGE/NADPH oxidase dependent pathway in EVT.

Screening models for early detection of late-onset preeclampsia with various markers in low-risk pregnancy population

Screening models for early detection of late-onset preeclampsia with various markers in low-risk pregnancy population

Static, but not cyclic tensional forces on the heart increase anti-angiogenic sFlt-1 expression in endothelial cells and inhibit VEGFR-2 activation

Purpose: The left ventricle (LV) is subject to increased mechanical forces in a variety of diseases that cause chronic increased systolic or diastolic pressures. These are often associated with endothelial dysfunction and/or reduced numbers of capillaries. Further mechanical forces are known to modulate endothelial gene expression. Therefore, we hypothesized, that increase in static tensional forces on capillary endothelial cells of the LV result in up-regulation of soluble VEGF receptor 1 (sFlt-1) and thus leading to an imbalance of proand anti-angiogenic stimuli in the heart. Methods: A rabbit Langendorff heart with retrograde constant pressure perfusion was used as model. As perfusate oxygenated Dulbecco’s modified Eagle’s medium was used to keep the heart viable as long as possible without deterioration of its function or sings of ischemia. For the tensional force 5g per g of heart weight was applied to the apex of the beating LV either as static load or every 30 seconds (cyclic). For a set of experiments cardiac contractions were completely arrested using butanedione monoxime (BDM). Quantitative RT-PCR, immunoblots and co-immunoprecipitation was used to quantify mRNA and proteins of sFlt-1, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2). Immunohistochemistry was performed for colocalized of proteins. Results: Increased static tensional force on the LV leads to significant increase in mRNA (3,8 fold, p < 0.05) and protein expression of sFlt-1 (1,4 fold, p < 0.05), which can be detected as early as after 30min and remains increased for the duration of the experiments (up to 6h). Cyclic tensional forces, however, do not affect sFlt-1 mRNA or protein levels. VEGF levels are unaffected by both static and cycle forces on the beating heart. Complete cardiac arrest without affecting the endothelium by BDM, an ATPase inhibitor of skeletal myosin 2, does not affect sFlt-1 or VEGF expression. Increased sFlt-1 due to increased static force on the LV binds more VEGF, shown by co-immunoprecipitation of sFlt-1 and VEGF, and reduced VEGFR-2 phosphorylation. Endothelial cells of the heart were colocalized by immunohistochemistry as source of sFlt-1 release. Conclusions: Static, but not cyclic tensional forces on the LV result in increased release of anti-angiogenic sFlt-1 from capillary endothelial cells. While expression levels of VEGF remain unchanged, sFlt-1 binds VEGF and reduces activation of the signalling Receptor VEGFR-2. Thus, chronic increased forces in the LV contribute to the anti-angiogenic setting and reduced capillary density as seen in many heart diseases.

Advanced oxidation protein products enhances soluble Fms-like tyrosine kinase 1 expression in trophoblasts: A possible link between oxidative stress and preeclampsia

Accumulation of advanced oxidation protein products (AOPPs) is prevalent in obesity, advanced maternal age, diabetes mellitus, and polycystic ovary syndrome. Alterations in the regulation and signaling of angiogenic pathways have been recognized as a link between these conditions and pre-eclampsia. To investigate the possible impact of AOPPs on soluble Fms-like tyrosine kinase 1 (sFlt-1) expression in trophoblasts. A trophoblast cell line (HRT-8/SVneo) was treated with various concentrations of AOPPs. The mRNA expression of sFlt-1, vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) in trophoblasts were measured with the use of real-time polymerase chain reaction; and the secretion of sFlt-1, VEGF, and PlGF protein from trophoblasts were detected with the use of ELISA. Exposure of HRT-8/SVneo cells to AOPPs induced overexpression of sFlt-1 at mRNA and protein levels in a dose dependent manner. These effects could be inhibited by apocynin, an inhibitors of NADPH oxidase. Our data identified AOPPs as a class of important mediator in the regulation and signaling of angiogenic pathways of trophoblasts. Accumulation of AOPPs might contributes to the pathogenesis of preeclampsia by promoting sFlt-1 production in trophoblasts.

Thrombin enhances soluble Fms-like tyrosine kinase 1 expression in trophoblasts; possible involvement in the pathogenesis of preeclampsia

To investigate the possible impact of thrombin on soluble Fms-like tyrosine kinase 1 (sFlt-1) expression in trophoblasts. Experimental. University hospital laboratory. A trophoblast cell line (HRT-8/SVneo) was treated with thrombin, protease-activated receptor 1 (PAR-1)-specific agonist SFLLERN, and thrombin antagonist PPACK. mRNA expression of sFlt-1, vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) in trophoblasts, with the use of real-time polymerase chain reaction; and the secretion of sFlt-1, VEGF, and PlGF protein from trophoblasts, with the use of ELISA. Administration of thrombin (10 U/mL) and PAR-1-specific agonist SFLLRN (300 μmol/L) increased sFlt-1 mRNA expression (4.24 ± 0.74- and 4.21 ± 0.79-fold, respectively) and protein secretion (5.08 ± 0.42- and 1.89 ± 0.16-fold, respectively) in HRT-8/SVneo. The induction of sFlt-1 protein secretion by thrombin was dose dependent. The effect of thrombin was completely reduced by thrombin inhibitor PPACK. Thrombin increased mRNA expression of VEGF but did not change VEGF secretion and PlGF mRNA expression and secretion. During placental development, thrombin, generated in the local hemorrhage of the uteroplacenta increases trophoblast expression of sFlt-1. Consequently, thrombin may contribute to the pathogenesis of preeclampsia.

PP121. Expression of PlGF, sFlt, MTF-1, HO-1 and HIF-1 alpha mRNAs in preeclampsia placenta and effect of preeclampsia sera on their expression of choriocarcinoma cells

Placenta growth factor (PlGF) is a growth factor originated from placenta. The sFlt-1 is soluble receptor for PlGF and suppresses PlGF function. It has been reported that in preeclampsia, serum level of PlGF decreased and sFlt-1 level increased and that preeclampsia placenta is in hypoxic condition. Metal-responsive transcription factor (MTF)-1, Hemoxigenase 1 (HO-1) and Hypoxia responsive factor -1 (HIF-1) may be induced in hypoxic condition. In order to investigate pathophysiology in preeclampsia, we studied the expression of PlGF, sFlt-1, MTF-1, HO-1 and HIF-1 alpha mRNAs in placenta taken from preeclampsia and the effect of preeclampsia sera on their expression of choriocarcinoma cells and analysed the effect of placental hypoxia and serum factor on the expression of PlGF and sFlt-1 mRNA. Placenta and serum samples were taken from preeclampsia and normal pregnancy with informed consent. The choriocarcinoma cells (JEG-3) were cultured in 24-well tissue culture plate. The cells were cultured with preeclampsia and normal pregnant sera. The RNAs were purified from these cells 24h after and placenta. The expressions of these mRNA were measured by using the real time PCR method (Applied Biosystems-7500). The expression of PlGF mRNA decreased and that of sFlt-1mRNA increased in preeclampsia placenta. The expression of MTF-1 and HO-1 mRNA decreased. The correlation was found between the expression of PlGF and MTF-1 mRNA, PlGF and HO-1 mRNA and sFlt-1 and HO-1mRNA. Moreover, expression of sFlt-1mRNA increased and HO-1mRNA decreased in JEG-3 cells after incubation of preeclampsia sera. The changes of PlGFmRNA in preeclampsia placenta may relate to the expression of MTF-1 and HO-1 mRNA. The changes of sFlt-1mRNA may relate to the expression of HO-1 mRNA and serum factor. Not only hypoxia but also serum factor may play a role of the levels of PlGF and sFlt-1 in preeclampsia placenta.

Morpholino-Mediated Increase in Soluble Flt-1 Expression Results in Decreased Ocular and Tumor Neovascularization

BackgroundAngiogenesis is a key process in several ocular disorders and cancers. Soluble Flt-1 is an alternatively spliced form of the Flt-1 gene that retains the ligand-binding domain, but lacks the membrane-spanning and intracellular kinase domains of the full-length membrane bound Flt-1 (mbFlt-1) protein. Thus, sFlt-1 is an endogenous inhibitor of VEGF-A mediated angiogenesis. Synthetic mopholino oligomers directed against splice site targets can modulate splice variant expression. We hypothesize that morpholino-induced upregulation of sFlt-1 will suppress angiogenesis in clinically relevant models of macular degeneration and breast cancer.Methods and Findings In vivo morpholino constructs were designed to target murine exon/intron 13 junction of the Flt-1 transcript denoted VEGFR1_MOe13; standard nonspecific morpholino was used as control. After nucleofection of endothelial and breast adenocarcinoma cell lines, total RNA was extracted and real-time RT-PCR performed for sFlt-1 and mbFlt-1. Intravitreal injections of VEGFR1_MOe13 or control were done in a model of laser-induced choroidal neovascularization and intratumoral injections were performed in MBA-MD-231 xenografts in nude mice. VEGFR1_MOe13 elevated sFlt-1 mRNA expression and suppressed mbFlt-1 mRNA expression in vitro in multiple cellular backgrounds (p<0.001). VEGFR1_MOe13 also elevated sFlt/mbFlt-1 ratio in vivo after laser choroidal injury 5.5 fold (p<0.001) and suppressed laser-induced CNV by 50% (p = 0.0179). This latter effect was reversed by RNAi of sFlt-1, confirming specificity of morpholino activity through up-regulation of sFlt-1. In the xenograft model, VEGFR1_MOe13 regressed tumor volume by 88.9%, increased sFlt-1 mRNA expression, and reduced vascular density by 50% relative to control morpholino treatment (p<0.05).ConclusionsMorpholino oligomers targeting the VEGFR1 mRNA exon/intron 13 junction promote production of soluble FLT-1 over membrane bound FLT-1, resulting in suppression of lesional volume in laser induced CNV and breast adenocarcinoma. Thus, morpholino manipulation of alternative splicing offers translational potential for therapy of angiogenic disorders.

Nitric oxide generation affects pro- and anti-angiogenic growth factor expression in primary human trophoblast

ObjectivesPreeclampsia is associated with reduced trophoblast placenta growth factor (PGF) expression, elevated soluble fms-like tyrosine kinase-1 (sFlt-1) and decreased bioactivity of nitric oxide (NO). Elevated sFlt-1 reduces bio-availability of PGF and vascular endothelial growth factor (VEGF) leading to maternal endothelial dysfunction. Although NO can regulate gene expression, its ability to regulate trophoblast expression of angiogenic growth factors is not known. Study designHuman primary term trophoblast and JEG-3 choriocarcinoma cells were cultured under 21%O2 or 1%O2 conditions in the presence or absence of NO donor (SNP) or inhibitor (L-NAME). Effects on PGF, VEGF and Flt-1 isoform mRNA expression were determined by quantitative real-time PCR. Changes in expression of soluble protein isoforms of FLT-1 was monitored by ELISA. ResultsHypoxia decreased PGF mRNA but increased VEGF, sFlt-1 and Flt-1 mRNA expression in trophoblast. Generation of NO in trophoblast under 1%O2 culture conditions significantly reversed sFlt-1 mRNA and protein expression, independent of mFlt-1. Conversely NO generation in hypoxic trophoblast increased VEGF and PGF mRNA expression. ConclusionsNO production in primary human trophoblast cultures had divergent effects on pro-angiogenic (PGF, VEGF) versus anti-angiogenic (sFlt-1) mRNA expression, resulting in an enhanced pro-angiogenic gene expression environment in vitro.

Tumor necrosis factor α induces a model of preeclampsia in pregnant baboons (Papio hamadryas)

Preeclampsia is a common disease of pregnancy characterised by maternal hypertension and proteinuria. Abnormal placentation in early pregnancy and abnormal cytokine and anti-angiogenic factor expression are thought to contribute to the clinical syndrome of endothelial dysfunction evident in the second half of gestation. The mechanisms underlying both the placental pathology and its translation to the maternal clinical syndrome are not fully understood. A model of preeclampsia manifest by clinically evident endothelial dysfunction (increased blood pressure and proteinuria) was induced by administration of low-dose TNF-α for 2weeks at mid-gestation in pregnant baboons (Papio hamadryas). Blood pressure was monitored continuously and remotely by intra-arterial radiotelemetry. Following TNF-α infusion, there was an increase in systolic and diastolic blood pressure and development of proteinuria in pregnant treated animals, but not in pregnant saline controls nor in non-pregnant TNF-α treated animals. The treated pregnant animals also developed elevated plasma soluble FMS-like tyrosine kinase-1 (sFLT-1) and increased placental mRNA expression of sFLT-1 and soluble endoglin (sEng). These results clearly demonstrate that the cytokine TNF-α can induce the clinical and biochemical features of human preeclampsia. The results identify a link between cytokines, placental dysfunction and endothelial dysfunction resulting in a loss of maternal blood pressure control.

Hypoxic trophoblast-derived sFlt-1 may contribute to endothelial dysfunction: An implication for the mechanism of trophoblast-endothelial dysfunction in preeclampsia

The maternal systemic disorder of widespread endothelial dysfunction is a primary focus in understanding the development of preeclampsia. sFlt-1 (soluble fms-like tyrosine kinase receptor 1), an endogenous inhibitor of VEGF (vascular endothelial growth factor), may play important roles in endothelial dysfunction. The present study aimed to determine whether hypoxic trophoblast-derived sFlt-1 could lead to endothelial dysfunction by establishing a cocultured model of anoxic TEV-1s (human first-trimester extravillous trophoblasts) and HUVECs (human umbilical vein endothelial cells). The results showed that the hypoxic treatment significantly promoted sFlt-1 mRNA and protein expression in TEV-1s in a time-dependent manner compared with the effect in HUVECs. When HUVECs were cocultured with anoxic TEV-1s, the endothelial function, which was characterized by NO (nitric oxide) synthesis and monolayer barrier function of HUVECs, were notably decreased, accompanied by increasing sFlt-1 and decreasing VEGF in cell-conditioned medium. Moreover, the observed endothelial dysfunction described above was consistent with the dysfunction observed in VEGF siRNA-treated cultures. The findings presented herein imply that chronically hypoxic trophoblasts may release sufficient sFlt-1 to cause endothelial dysfunction by depriving cells of VEGF activity.

Reduction of Circulating Soluble Fms-Like Tyrosine Kinase-1 Plays a Significant Role in Renal Dysfunction–Associated Aggravation of Atherosclerosis

Renal dysfunction is commonly accompanied by a worsening of atherosclerosis; however, the underlying molecular mechanism is not fully understood. We examined the role played by soluble fms-like tyrosine kinase-1 (sFlt-1), an endogenous antagonist of the proatherogenic cytokine placental growth factor (PlGF), in the worsening of atherosclerosis in patients with renal dysfunction and in an animal model of renal failure. In this study, 329 patients who received cardiac catheterization and 76 patients who underwent renal biopsy were enrolled. Both plasma sFlt-1 levels and renal sFlt-1 mRNA expression were positively correlated with estimated glomerular filtration rate (P<0.01). The PlGF/sFlt-1 ratio was negatively correlated with estimated glomerular filtration rate (P<0.01), whereas plasma PlGF levels were not affected by it. The PlGF/sFlt-1 ratio was significantly higher in patients with multivessel coronary artery disease than in patients with single-vessel or no coronary artery disease. The reduction of circulating sFlt-1 and renal sFlt-1 mRNA levels was confirmed in five-sixths (5/6)-nephrectomized apolipoprotein E-deficient mice that developed experimental renal dysfunction. Atherosclerotic plaque area and macrophage infiltration into the plaque were significantly higher in 5/6-nephrectomized apolipoprotein E-deficient mice than in control mice, but replacement therapy with recombinant sFlt-1 significantly reduced both plaque formation and macrophage infiltration. The present study demonstrates that a reduction in the circulating levels of sFlt-1 is associated with the worsening of atherosclerosis that accompanies renal dysfunction.

Abstract 5018: Soluble fms-like Tyrosine Kinase (sFLT1): A Biomarker of Endothelial Origin in Acute ST-segment Elevation Myocardial Infarction (STEMI)

In STEMI, early recognition improves clinical outcomes. Current biomarkers of myocardial necrosis are limited by poor sensitivity and specificity during the acute phase of coronary occlusion. sFLT1 is an endothelial-derived, hypoxia-inducible protein. We hypothesized that vascular occlusion induces early expression of sFLT1 in STEMI. Methods &amp; Results: In a prospective, observational study, serum sFLT1 levels were measured by ELISA in 30 patients with STEMI referred for emergent catheterization. Compared to 20 healthy, age-matched controls, sFLT1 levels were markedly increased at the time of presentation (11.7±2 vs 210.5±80 pg/mL, p&lt;0.0001), then substantially reduced within 24 hours of reperfusion (22.3±15 pg/mL, p&lt;0.0001). On admission, 100% of STEMI patients had elevated sFLT-1 levels (&gt;50pg/mL), whereas 73% and 64% had levels of CK-MB &gt;5ng/mL and Troponin-I &gt;1.0 ng/mL respectively. Two patients undergoing alcohol septal ablation were studied as positive controls. sFLT1 levels increased within 40 minutes of occlusion (27.8 vs 216.2 pg/mL, pre- vs post-ablation, p&lt;0.03). We also studied sFLT1 expression in a mouse model of myocardial infarction (MI). After 1 hour of left coronary ligation in 10-week old male wild-type mice (n=6/group), left ventricular sFLT1 mRNA levels were increased (p&lt;0.01 MI vs. sham), while aortic and lung mRNA expression were unchanged. Serum sFLT1 levels were also elevated in MI (170±71 vs 541±107pg/mL, sham vs. MI, p&lt;0.002). To explore the cellular origin of sFLT1, human coronary endothelial (HCE) and smooth muscle cells (HSMC) were rendered hypoxic. Within 1 hour, sFLT1 mRNA expression in HCE increased by 173±35% (p=0.03). By 12 hours, sFLT1 mRNA and protein expression in hypoxic HCE increased by 313±15% (p&lt;0.01) and 256±17% (p&lt;0.001) respectively. To simulate the effects of reperfusion on sFLT1 expression, HCE were exposed to 12 hours of hypoxia, then 6 hours of normoxia. After normoxic rescue, sFLT1 protein expression decreased by 36% (p&lt;0.01, vs hypoxic HCE). Hypoxia failed to induce sFLT1 mRNA or protein expression in HSMC. Conclusion: sFLT1 is a highly sensitive biomarker of vascular occlusion released by the endothelium during the acute phase of STEMI and may be an important new marker for acute ischemic syndromes. This research has received full or partial funding support from the American Heart Association, Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).

Toll-like Receptor-3 Stimulation Upregulates sFLT-1 Production by Trophoblast Cells

Background Preeclampsia is characterized by a systemic inflammatory response involving cytokines, chemokines, and anti-angiogenic factors such as sFLT-1. In many other inflammatory diseases related responses are triggered by toll-like receptor (TLR) stimulation. Therefore, we tested the hypothesis that TLR stimulation of a trophoblast cell line induces inflammatory mediator production and, in particular, production of the preeclampsia-related anti-angiogenic factor sFLT-1. Methods We stimulated human first trimester extravillous trophoblast cells (HTR-8/SV neo cell line) with a variety of TLR ligands and measured downstream NF-κB and IRF signaling, inflammatory mediator (RANTES), and sFLT-1 mRNA expression and protein production. Results Of all TLR ligands, we found that TLR3 ligation with polyI:C resulted in the biggest response with 5.6-fold increased signaling via NF-κB and 5.8-fold increased signaling via IRF. RANTES mRNA expression increased 2900 fold and protein production increased 1600 fold in response to TLR3 ligation. sFLT-1 mRNA expression increased 1.7-fold and protein production increased 3.1-fold in response to TLR3 ligation. Inhibitors of the NF-κB and IRF signaling pathway decreased TLR3 ligation-induced sFLT-1 protein production by 31.8% and 24.9%, respectively. Conclusion We conclude that trophoblast cells respond to TLR3 ligation by signaling through both NF-κB and IRF pathways resulting in expression of inflammatory mediators and, in particular, the preeclampsia-related anti-angiogenic factor sFLT-1.

71: Nicotine suppresses soluble FMS-like tyrosine kinase-1 (SFLT-1) expression in trophoblasts via alpha 7 nicotinic acetylcholine receptor but not in human umbilical vein endothelial cells (HUVEC)

EXPRESSION IN TROPHOBLASTS VIA ALPHA 7 NICOTINIC ACETYLCHOLINE RECEPTOR BUT NOT IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC) JA YOUNG KWON, YOUNG HAN KIM, MUONG HWA KANG, YONG SUN MAENG, YOUNG GUEN KWON, YONG WON PARK, Yonsei University College of Medicine, Department of Obstetrics and Gynecology, Seoul, South Korea, Yonsei University, Department of Biochemistry, Seoul, South Korea OBJECTIVE: Cigarette smoking is a unique factor known to reduce the risk of preeclampsia and serum sFlt-1 level is known to be decreased in smokers. Present study was to evaluate the effect of nicotine on production in trophoblasts and HUVEC STUDY DESIGN: Commercially available term placenta trophoblasts and HUVEC extracted from umbilical cord of normal pregnancy were treated with nicotine of 10-7 and 10-6 M. Gene expression of sFlt-1 mRNA expressions were evaluated by RT-PCR and protein level in supernatants were assayed by ELISA following 0, 3, 6, and 12 h of nicotine treatment. Subsequently, alphabungarotoxin (BGT), an a7 nicotinic acetylcholine receptor (nAchR) antagonist was pretreated to evaluate this receptor=s potential role in sFlt-1 production modulation by nicotine. The presence of a7 nAchR in both cell types were confirmed by immunostaining. RESULTS: After 3, 6, 12 h of incubation of trophoblasts and HUVECs with nicotine, sFlt-1 mRNA expression significantly decreased in trophoblasts but not in HUVECs. Concordantly, sFlt-1 protein level in the supernatants of trophoblast following 3, 6, and 12 h of nicotine treatment significantly decreased (19.0 0.6, 23.7 2.0, 32.1 1.9 pg/mL vs. 29.0 1.0, 41.5 3.1, 57.4 5.6 pg/mL, p 0.001). To evaluate whether this effect on trophoblast was mediated via a7 nAchR, aBGT was pretreated for 2 h prior to nicotine treatment and as a result, previous nicotinic effect of reduced sFlt-1 protein level was reversed in the aBGTtreated group (28.8 0.8, 40.7 1.3, 58.9 2.4 pg/mL vs. 19.0 0.6, 23.7 2.0, 32.1 1.9 pg/mL, p 0.05). CONCLUSION: Nicotine downregulated sFlt-1 production in trophoblast but not in HUVEC. And this effect was mediated by a7 nAchR. This mechanism may be involved in preeclampsia risk reduction in smokers and thus may provide a potential therapeutic target for preventing or treating preeclampsia.

Placental Expression of Soluble fms-Like Tyrosine Kinase 1 is Increased in Singletons and Twin Pregnancies with Intrauterine Growth Restriction

Intrauterine growth restriction (IUGR) is characterized by decreased placental perfusion. Low oxygen has been shown to increase soluble fms-like tyrosine kinase 1 (sFlt-1) expression in the human placenta. The objective of this study was to examine sFlt-1 expression in different types of IUGR pregnancies, including early-onset severe cases characterized by abnormal umbilical and uterine artery Doppler and discordant IUGR twins in which the normal cotwin represents the optimal control because both placentas share the same uterine environment. Placentas from four subgroups were collected: early severe IUGR with umbilical artery absent end diastolic flow (n = 19), small for gestational age with normal uterine and umbilical artery Doppler (n = 11), severely growth-restricted dichorionic and monochorionic twins with abnormal umbilical artery Doppler (n = 9), preeclamptic twins (n = 3), and age-matched normal singletons (n = 19) and twin controls (n = 8). Expression of sFlt-1 mRNA and protein was significantly increased in IUGR placentas compared with small for gestational age and normal control placentas. sFlt-1 expression levels were also significantly greater in the small IUGR twin placentas from discordant twin pregnancies compared with the normal cotwin. In preeclamptic twins, sFlt-1 expression was increased in only one of the two placentas. Our results demonstrate that sFlt-1 expression is increased in severe IUGR placentas with abnormal umbilical artery Doppler of singletons and also in discordant IUGR twins. Reduced placental perfusion may contribute to the increased expression of sFlt-1 in IUGR pregnancies. Our data are compatible with differential sFlt-1 expression in placentas from discordant twins.

Uteroplacental ischemia results in proteinuric hypertension and elevated sFLT-1

Preeclampsia is a complication of pregnancy with significant morbidity and mortality for the mother and the fetus. Presumptions are made that placental hypoxia has a causative role in the clinical syndrome. Furthermore, soluble fms-like tyrosine kinase 1 (sFLT-1) has been shown to have a role in the maternal syndrome of preeclampsia. We investigated the relationship between uteroplacental ischemia (UPI), the maternal clinical syndrome of preeclampsia and sFLT-1 in non-human primates. The induction of UPI in a pregnant non-human primate resulted in the development of a clinical entity analogous to human preeclampsia. This was illustrated by the increase in blood pressure, development of proteinuria, and renal histological changes identical to human preeclampsia. A significant elevation in the placental and peripheral blood mononuclear cell sFLT-1 mRNA expression was noted, translating to a significant elevation in circulating sFLT-1. Thus, this sequence suggests that a pathogenic reduction in placental perfusion results in the development of the maternal syndrome of preeclampsia and an increase in circulating sFLT-1, which is derived both from placental and extra-placental sources.