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.     

Maternal serum soluble fms-like tyrosine kinase 1 concentrations are not increased in early pregnancy and decrease more slowly postpartum in women who develop preeclampsia

OBJECTIVE: We measured maternal serum soluble fms-like tyrosine kinase 1 concentrations across pregnancy and immediately postpartum in women who developed preeclampsia and normal pregnant women. STUDY DESIGN: This was a nested case control study of 113 normal pregnant women and 55 women with preeclampsia. RESULTS: Serum soluble fms-like tyrosine kinase 1 concentrations increased similarly in early pregnancy in both groups. Mean serum soluble fms-like tyrosine kinase 1 concentrations were increased in women who developed preeclampsia, compared with normal pregnant women, and this increase was most pronounced in severe preeclampsia. However, many women with preeclampsia had soluble fms-like tyrosine kinase 1 concentrations similar to normal pregnant women. Lastly, soluble fms-like tyrosine kinase 1 decreased rapidly after delivery, but this decrease was significantly slower in women with severe preeclampsia. CONCLUSION: Increased soluble fms-like tyrosine kinase 1 is not an early-pregnancy event among women who later develop preeclampsia. Increased soluble fms-like tyrosine kinase 1 is more likely to be present in women with severe preeclampsia, but it is not present in all women with preeclampsia. Soluble fms-like tyrosine kinase 1 concentrations decrease more slowly after delivery in women with preeclampsia, consistent with a decreased rate of excretion or continued production.     

Increased plasma soluble fms-like tyrosine kinase 1 and endoglin levels in pregnancies complicated with preeclampsia

Background.?Increased maternal plasma levels of proinflammatory cytokines as well as the anti-angiogenic agents soluble fms-like tyrosine kinase 1 (sFlt-1) and endoglin (sEng) are associated with promoting vascular dysfunction leading to the maternal syndrome of preeclampsia.

Objective and method.?Nulliparous women complicated with preeclampsia (n = 29) and their corresponding controls (n = 29) delivering at the Enrique C. Sotomayor Obstetrics and Gynecology Hospital, Guayaquil-Ecuador were requested to participate in a study evaluating plasma levels of soluble anti-angiogenic factors (sFlt-1 and sEng) and pro-inflammatory cytokines: interleukin 6 (IL-6), interleukin 8 (IL-8), granulocyte colony stimulating factor (G-CSF), and tumor necrosis factor-alpha (TNF-α). Maternal and neonatal data were also assessed and compared among the study groups.

Results.?No significant differences in either maternal baseline or delivery characteristics were observed among the study groups. Compared with controls, preeclamptic women exhibited higher plasma levels of sFlt-1 (19.0 ± 15.1 vs. 12 ± 8.3 ng/mL) and of sEng (20.4 ± 9.9 vs.15.9 ± 9.4 ng/mL); respectively, p < 0.05. Women with severe disease displayed higher sFlt-1 and sEng levels when compared with mild ones (34.5 ± 11.6 vs. 9.5 ± 1.6 ng/mL, and 29.5 ± 9.0 vs. 14.8. ± 5.2 ng/mL, respectively; p < 0.001). In contrast, women with preeclampsia exhibited significant lower IL-8 and G-CSF levels compared with controls. No differences existed between either group in IL-6 levels or TNF-α.

Conclusion.?Consistent with previous reports, increased sFlt-1 and Eng levels in maternal plasma is consistent with vascular dysfunction found in gestations complicated with preeclampsia.     

Variable expression of soluble fms-like tyrosine kinase 1 in patients at high risk for preeclampsia

Objective.?To explore angiogenic factor differences in preeclamptic patients according to the absence or presence of underlying vascular disease.

Methods.?We prospectively compared serum soluble fms-like tyrosine kinase 1 (sFlt1), soluble endoglin, and placental growth factor (PlGF) from 41 normal-risk and 32 high-risk (preexisting conditions) subjects at serial gestational ages.

Results.?Median sFlt1 was lower at delivery in preeclamptic patients with underlying chronic hypertension and/or chronic proteinuria (5115?pg/ml) compared with normal risk preeclamptic patients (16375?pg/ml). PlGF was consistently low in patients who developed preeclampsia.

Conclusions.?Effects of sFlt1 may be contextual, varying according to the health or disease state of vascular endothelium.     

Serum heme oxygenase 1 (HO-1), soluble FMS like tyrosine kinase (sFlt-1) level,and neonatal outcome in early onset,late onset preeclampsia,and normal pregnancy

ABSTRACT

Objective: To compare the level of serum heme oxygenase 1 (HO-1), soluble FMS like tyrosine kinase (sFlt-1), and neonatal outcome in early onset preeclampsia (EO-PE), late onset preeclampsia (LO-PE), and normal pregnancy (NP).

Methods: In this prospective observational case control study, HO-1 and sFlt-1 levels were measured in blood samples within 24 h of hospital admission. Preeclampsia cases were divided into two groups based on gestational age at delivery: EO-PE (<34 weeks) and LO-PE (≥34 weeks). A total of 45 patients were involved in this study.

Result: Maternal serum level of sFlt-1 was higher in EO-PE than LO-PE and NP groups (mean ± SD; 14.50 ± 17.12 ng/ml vs 5.20 ± 6.69 ng/ml vs 2.72 ± 1.2 ng/ml [p = 0.020]. Maternal serum level of HO-1 was not different between EO-PE, LO-PE, and NP groups (p = 0.681). Birthweights were significantly lower in the EO-PE group compared with the LO-PE and NP groups (1580 ± 536 g vs 2635 ± 578 g vs 3010 ± 371 g [p = 0.000]). The rate of small for gestational age infant (26.7% vs 6.7% vs 0%; p = 0.046) and perinatal death (20% vs 0 vs 0; p = 0.037) was also significantly higher in EO-PE compared to LO-PE and NP. The maternal sFlt-1 level was negatively correlated with birthweight (p = 0.006; CC = ?0.445).

Conclusion: This study did not find a correlation between maternal HO-1 levels and sFlt-1 levels. Maternal serum sFLt-1 levels in preeclampsia were higher in EO-PE and were associated with a worse perinatal outcome.     

The preeclampsia biomarkers soluble fms-like tyrosine kinase-1 and placental growth factor: current knowledge, clinical implications and future application

Preeclampsia is a leading cause of maternal and fetal/neonatal morbidity and mortality worldwide. Currently there is no single reliable parameter for the diagnosis of preeclampsia and attention has turned towards identifying non-invasive testing methods, including Doppler sonography and blood-borne or urinary biomarkers in women who go on to develop preeclampsia. Biomarkers of angiogenesis are currently at the most advanced state of development for the diagnosis of preeclampsia. In this article we will highlight the diagnostic potential of the novel biomarkers soluble fms-like tyrosine kinase-1 and placental growth factor and discuss the opportunities and challenges involved in bringing these preeclampsia biomarkers from bench to bedside.     

可溶性血管内皮生长因子受体-1在子痫前期胎盘及在外周血的研究

目的研究可溶性血管内皮生长因子受体-1(sFlt-1) mRNA在正常妊娠和子痫前期胎盘中的表达差异及相应的母体外周血中sFlt-1水平的变化。方法选取15例子痫前期孕妇(子痫前期组)和14例孕周与之相匹配的血压正常妊娠孕妇(对照组),用ELISA方法测定其外周血中sFlt-1的水平;并应用实时荧光定量PCR测定两组胎盘组织中sFlt-1 mRNA水平。结果外周血中sFlt-1的水平子痫前期组明显高于对照组,分别为(16.9±8.32)μg/L、(5.48±2.09)μg/L,两者差异有显著性(P<0.05)。sFlt-1 mRNA在两组胎盘中均有表达,在子痫前期组的表达明显高于对照组的表达分别为(4.11±4.09)、(1.69±1.61),两者差异有显著性(P<0.05)。子痫前期组血清sFlt-1水平与24h尿蛋白定量呈明显正相关(r=0.741,P<0.05)。结论sFlt-1 mRNA在子痫前期组孕妇的外周血中及胎盘组织表达升高,可能与子痫前期的病因及病理生理有关。     

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

Objectives

Preeclampsia 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 design

Human 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.

Results

Hypoxia 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.

Conclusions

NO 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.     

Increased placental sFlt-1 but unchanged PlGF expression in late-onset preeclampsia

Objective: To investigate whether differences between late-onset preeclampsia (PE) and intrauterine growth restriction (IUGR) can be explained by differential placental expression patters of sFlt-1, Flt-1, and placental growth factor (PlGF).

Methods: Placental tissues and maternal blood samples from seven patients with PE, five IUGR, and seven age-matched controls were studied for mRNA and protein levels as well as protein localization and expression intensity.

Results: Placental PlGF mRNA and protein expression were not altered by placental dysfunction while placental villous trophoblast expression intensity of PlGF was increased.

Conclusion: High sFlt-1 concentrations may account for diminished maternal serum PlGF levels.     

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.     

Placental growth factor and soluble fms-like tyrosine kinase-1 are useful markers for the prediction of preeclampsia but not for small for gestational age neonates: a longitudinal study

Objective

To determine maternal serum concentrations of placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) longitudinally in normal pregnancies, pregnancies that developed preeclampsia and pregnancies that deliver a small for gestational age (SGA) infant, in order to evaluate them as markers for the prediction of preeclampsia.

Study design

In this case–control study we included 12 singleton pregnancies that developed preeclampsia and 104 randomly selected singleton normal pregnancies. Fourteen of the normal pregnancies gave birth to an SGA infant. Blood samples and ultrasonographic data were collected during the 1st, 2nd and 3rd trimesters of pregnancy.

Results

In preeclamptic pregnancies, PlGF (pg/mL) (median; inter-quartile range) was significantly lower in the 2nd (208; 84–339) (p = 0.035) and in the 3rd trimester (202; 109–284) (p = 0.002) while sFlt-1 was significantly higher only in the 3rd trimester (2521; 2101–3041) (p = 0.011) compared to normal pregnancies (PlGF 2nd: 311; 243–440, PlGF 3rd: 780; 472–1037, sFlt-1 3rd: 1616; 1186–2220). In pregnancies with SGA infants, PlGF and sFlt-1 did not differ significantly from normal pregnancies in any trimester. The sFlt-1 to PlGF ratio was significantly higher in preeclamptic pregnancies than in normal pregnancies, in both the 2nd and 3rd trimesters. The relative difference and the slope of PlGF concentration between 1st and 2nd trimester were significantly reduced in preeclampsia compared to normal pregnancies. A logistic regression model with predictors BMI, 2nd trimester Doppler PI and relative difference of PlGF from the 1st to the 2nd trimester gave 46% sensitivity and 99% specificity for the prediction of preeclampsia, with a very high negative predictive value of 98.3%.

Conclusions

Our study confirms that maternal serum PlGF concentration is significantly lower, at least after 20th week, while sFlt-1 concentration is significantly higher in 3rd trimester, in pregnancies destined to develop preeclampsia. Pregnancies that gave birth to SGA infants do not have altered angiogenic factor concentrations throughout pregnancy. The relative difference of PlGF from the 1st to the 2nd trimester, uterine artery Doppler PI in the 2nd trimester and BMI are the most powerful markers for the prediction of preeclampsia.     

A trio of risk factors for the onset of preeclampsia in the second and early third trimesters

ObjectiveWe evaluated the biological interaction among mean blood pressure (MBP), uterine artery Doppler (UAD), and the soluble fms-like tyrosine kinase 1 (sFlt-1)/placental growth factor (PlGF) ratio for preeclampsia (PE) risk.Study designA prospective cohort study.Main outcome measuresIn 1239 pregnant women, MBP and UAD were measured at 16–23 weeks of gestation, and plasma levels of the sFlt-1/PlGF ratio at 19–25 weeks and 26–31 weeks. A Cox proportional hazard model was used. Women with a low sFlt-1/PlGF ratio and either low BP or normal UAD were set as controls. The relative excess risk due to biological interaction (RERI) was calculated using the following equation: RERI = hazard ratio (HR) in women with high sFlt-1/PlGF and both high BP and abnormal UAD (group 3) – HR in women with both high BP and abnormal UAD alone (group 1) – HR in women with high sFlt-1/PlGF alone (group 2) + 1. RERI ⩾ 10 was considered to be strong.ResultsAt 19–25 weeks, the HR and 95% confidence intervals (CI) in group 1, group 2, and group 3 were 7.4 (3.1–17.4), 15.3 (4.5–52.2), and 107.0 (41.0–279), respectively, and the RERI for PE was 85.3. At 26–31 weeks, the HR and 95% CI in each group were 8.3 (2.9–23.2), 7.5 (0.97–57.8), and 69.0 (18.5–256), respectively; the RERI for PE was 54.2.ConclusionsWe found a trio of risk factors for the onset of PE in the second and early third trimesters: high BP, abnormal UAD, and high sFlt-1/PlGF ratio.     

Serum sFlt1:PlGF Ratio,PlGF, and Soluble Endoglin Levels in Gestational Proteinuria

Objective: It was recently reported that both a high soluble fms-like tyrosine kinase 1 (sFlt1): placental growth factor (PlGF) ratio (sFlt1:PlGF ratio) and high soluble endoglin (sEng) levels are related to the later occurrence of preeclampsia. We compared the serum sFlt1:PlGF ratio, PlGF and sEng levels in women with gestational proteinuria (GP) to those in women with preeclampsia. Methods: Seven women with GP and 34 women with preeclampsia were recruited in this study. The 95^th percentile values in the reference curves of sFlt1, sFlt1:PlGF ratio and sEng, and the 5^th percentile values in the reference curve of PlGF were respectively set as the cutoff values. Results: The incidence rates of a high sFlt1:PlGF ratio, low PlGF and high sEng in women with GP were 57%, 29% and 86%, respectively, whereas those in women with preeclampsia were 94%, 77%, and 88%, respectively (p?=?0.028, p?=?0.024, and p?=?1.000, respectively). The incidence rates of a both high sFlt1:PlGF ratio and high sEng in women with GP and preeclampsia were 57% and 88%, respectively (p?=?0.082). Conclusion: The majority of women with GP showed both increases of the sFlt1:PlGF ratio and sEng, thus suggesting some women with GP may represent subclinical preeclampsia. In addition, women with GP showed a significantly lower sFlt1:PlGF ratio and higher PlGF level than those with preeclampsia, suggesting that the PlGF level is a key regulator for developing hypertension in some pregnant women, even with increases of both sFlt1:PlGF ratio and sEng levels.