Antiangiogenic Factor Soluble Fms-like Tyrosine Research Articles

SFlt-1 impairs neurite growth and neuronal differentiation in SH-SY5Y cells and human neurons.

Pre-eclampsia (PE) is a hypertensive disorder of pregnancy which is associated with increased risk of neurodevelopmental disorders in exposed offspring. The pathophysiological mechanisms mediating this relationship are currently unknown, and one potential candidate is the anti-angiogenic factor soluble Fms-like tyrosine kinase 1 (sFlt-1), which is highly elevated in PE. While sFlt-1 can impair angiogenesis via inhibition of VEGFA signalling, it is unclear whether it can directly affect neuronal development independently of its effects on the vasculature. To test this hypothesis, the current study differentiated the human neural progenitor cell (NPC) line ReNcell® VM into a mixed culture of mature neurons and glia, and exposed them to sFlt-1 during development. Outcomes measured were neurite growth, cytotoxicity, mRNA expression of nestin, MBP, GFAP, and βIII-tubulin, and neurosphere differentiation. sFlt-1 induced a significant reduction in neurite growth and this effect was timing- and dose-dependent up to 100 ng/ml, with no effect on cytotoxicity. sFlt-1 (100 ng/ml) also reduced βIII-tubulin mRNA and neuronal differentiation of neurospheres. Undifferentiated NPCs and mature neurons/glia expressed VEGFA and VEGFR-2, required for endogenous autocrine and paracrine VEGFA signalling, while sFlt-1 treatment prevented the neurogenic effects of exogenous VEGFA. Overall, these data provide the first experimental evidence for a direct effect of sFlt-1 on neurite growth and neuronal differentiation in human neurons through inhibition of VEGFA signalling, clarifying our understanding of the potential role of sFlt-1 as a mechanism by which PE can affect neuronal development.

Pericardial Adipose Tissue Thrombospondin-1 Associates With Antiangiogenesis in Ischemic Heart Disease

Accumulation of ectopic pericardial adipose tissue has been associated with cardiovascular complications which, in part, may relate to adipose-derived factors that regulate vascular responses and angiogenesis. We sought to characterize adipose tissue microvascular angiogenic capacity in individuals undergoing elective cardiac surgeries including aortic, valvular, and coronary artery bypass grafting. Pericardial adipose tissue was collected intraoperatively and examined for angiogenic capacity. Capillary sprouting was significantly blunted (2-fold, p<0.001) in subjects with CAD (age 60±9 years, BMI 32±4 kg/m2, LDL-C 95±46 mg/dl, n=29) compared to age-, BMI-, and LDL-C matched individuals without angiographic obstructive CAD (age 59±10 years, BMI 35±9 kg/m2, LDL-C 101±40 mg/dl, n=12). For potential mechanistic insight, we performed mRNA expression analyses using quantitative RT- PCR, and observed no significant differences in pericardial fat gene expression of pro-angiogenic mediators vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), and angiopoietin-1 (angpt1), or anti-angiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and endostatin. In contrast, mRNA expression of anti-angiogenic thrombospondin-1 (TSP-1) was significantly upregulated (2-fold, p=0.008) in CAD compared to non-CAD subjects, which was confirmed by protein western-immunoblot analysis. TSP-1 gene knockdown using shRNA lentiviral delivery significantly improved angiogenic deficiency in CAD (p<0.05). In conclusion, pericardial fat in subjects with CAD may be associated with an anti-angiogenic profile linked to functional defects in vascularization capacity. Local paracrine actions of TSP-1 in adipose depots surrounding the heart may play a role in mechanisms of ischemic heart disease.

Nitroxide-HMP-Protects Human Trophoblast HTR-8/SVneo Cells from H2O2-Induced Oxidative Stress by Reducing the HIF1A Signaling Pathway.

Preeclampsia (PE) is a pregnancy-specific syndrome affecting 5-7% of patients. There is no effective treatment available. Early abnormal placental development is associated with oxidative stress (OS) and a release of reactive oxygen species (ROS) in the placenta. This phenomenon leads to downstream signaling, Hypoxia Inducible Factor 1A (HIF1A) stabilization and transcription of the anti-angiogenic factors soluble fms-like tyrosine kinase 1 (sFLT1) and soluble endoglin (sEng), which are known to cause endothelial and trophoblast dysfunction and cardinal features of PE: hypertension, proteinuria and, in severe cases, eclampsia. We tested whether 3-(Hydroxymethyl)-1-oxy-2,2,5,5-tetramethylpyrrolidine (HMP)-a nitroxide-type antioxidant molecule-can reduce placental OS and mitigate PE symptoms in vitro. We induced OS in human trophoblast (HTR-8/SVneo) cells with hydrogen peroxide (H2O2) and assessed whether modulating cell redox function with HMP reduces cell injury, mitochondrial stress and HIF1A and sFLT1 production. Pre-treatment with HMP reduced mitochondrial-derived ROS production, restored LC3B expression and reduced HIF1A and sFLT1 expression in H2O2-exposed HTR-8/SVneo trophoblast cells. HMP improved the mitochondrial electron chain enzyme activity, indicating that a reduction in OS alleviates mitochondrial stress and also reduces anti-angiogenic responses. In reducing placental trophoblast OS, HMP presents a potential novel therapeutic approach for the treatment of PE. Future investigation is warranted regarding the in vivo use of HMP.

Updated study of peripartum cardiomyopathy and preeclampsia

HIGHLIGHTS 1. Cardiovascular disease that occurs during pregnancy is the peripartum cardiomyopathy (PPCM).2. Literature on the association between preeclampsia (PE) and PPCM was reviewed.3. It was not certain whether PE is the independent risk factor of PPCM or an early predictor of PPCM development. ABSTRACT Objectives: This paper aims to review the literature related to peripartum cardiomyopathy (PPCM) and preeclampsia (PE) in order to know their frequency and relationship and the current knowledge on their pathophysiology and management. Materials and Methods: The articles reviewed in this study were primary clinical studies published around 2016 and 2021, retrieved using Google Scholar and PUBMED databases. After several evaluations, 14-full-text studies written in English were examined. Results: Overall prevalence of PE in PPCM cases varied, about 9.9% - 44% in the individual studies. The lactation hormone prolactin and placental-derived anti-angiogenic factor soluble Fms-like tyrosine kinase 1 (sFlt-1), which had been known to be able to cause cardiac dysfunction, were elevated in both PE and PPCM. This partly explained the pathophysiology that the incidence of concurrent PE in women diagnosed with PPCM was four times more than that in the general population. Conclusion: Epidemiologic studies showed significant overlap between PE and PPCM patients. However, there were not enough good quality data to fully draw conclusions about the relationship between PE and PPCM, whether PE as the independent risk factor of PPCM or an early predictor of PPCM development.

The Relationship between Angiogenic Factors and Energy Metabolism in Preeclampsia.

Antiangiogenic factors are currently used for the prediction of preeclampsia. The present study aimed to evaluate the relationship between antiangiogenic factors and lipid and carbohydrate metabolism in maternal plasma and placenta. We analyzed 56 pregnant women, 30 healthy and 26 with preeclampsia (including early and late onset). We compared antiangiogenic factors soluble Fms-like Tyrosine Kinase-1 (sfLt-1), placental growth factor (PlGF), and soluble endoglin (sEng)), lipid and carbohydrate metabolism in maternal plasma, and lipid metabolism in the placenta from assays of fatty acid oxidation, fatty acid esterification, and triglyceride levels in all groups. Antiangiogenic factors sFlt-1, sFlt-1/PlGF ratio, and sEng showed a positive correlation with triglyceride, free fatty acid, and C-peptide maternal serum levels. However, there was no relationship between angiogenic factors and placental lipid metabolism parameters. Free fatty acids were predictive of elevated sFlt-1 and sEng, while C-peptide was predictive of an elevated sFlt1/PlGF ratio. The findings in this study generate a model to predict elevated antiangiogenic factor values and the relationship between them with different products of lipid and carbohydrate metabolism in maternal serum and placenta in preeclampsia.

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

IntroductionThe 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. MethodsWe 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). ResultsWe 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. DiscussionLow 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.

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.

Cardiac function, exercise capacity and biomarkers after peripartum cardiomyopathy and preeclampsia: A Danish long-term follow-up study

Introduction: Peripartum cardiomyopathy (PPCM) is a rare but potentially life-threatening disease defined by idiopathic heart failure towards the end of pregnancy or up to six months after delivery. 6–12 months recovery rates are 47–72% in recent cohorts, but data on longer term prognosis are few. PPCM is often preceded by preeclampsia and the two conditions share pathophysiological mechanisms including disturbed levels of the antiangiogenic factor soluble Fms-like tyrosine kinase (sFlt-1) and placental growth factor (PlGF).

Prediction of imminent preeclampsia at 35–37 weeks gestation

In the weeks preceding the clinical onset of preeclampsia, the maternal serum level of the angiogenic placental growth factor is decreased and that of the antiangiogenic factor soluble fms-like tyrosine kinase-1 is increased. Women presenting at specialist clinics with signs or symptoms of hypertensive disorders have been stratified according to concentrations of placental growth factor or the ratio of concentrations of soluble fms-like tyrosine kinase-1 and placental growth factor to determine clinical management for the subsequent 1-4 weeks. An alternative approach for the prediction of preeclampsia is use of the competing risks model, a Bayes' theorem based method, to derive patient-specific risk for preeclampsia by various combinations of maternal characteristics and medical history with multiples of the median values of biomarkers. The purpose of this study was to compare the performance of screening for delivery with preeclampsia at ≤2 and ≤4 weeks after assessment at 35+0-36+6 weeks gestation between the use of percentile cut-offs in placental growth factor alone or the soluble fms-like tyrosine kinase-1/placental growth factor ratio and the competing risks model. This was a prospective observational study in women who attended a routine hospital visit at 35+0-36+6 weeks gestation in 2 maternity hospitals in England. The visits included the recording of maternal demographic characteristics and medical history and the measurement of serum placental growth factor and soluble fms-like tyrosine kinase-1 and mean arterial pressure. The areas under the receiver operating characteristics curves were used to compare the predictive performance for preeclampsia with delivery at ≤2 and ≤4 weeks from assessment of screening by placental growth factor alone and the soluble fms-like tyrosine kinase-1/placental growth factor ratio with that of a previously developed competing risks model with a combination of maternal factors, placental growth factor, soluble fms-like tyrosine kinase-1, and mean arterial pressure (triple test). First, the study population of 15,247 pregnancies included 326 pregnancies (2.1%) that subsequently experienced preeclampsia. Second, in the screening for delivery with preeclampsia at ≤2 and ≤4 weeks from assessment, the performance of the triple test was superior to that of placental growth factor alone or the soluble fms-like tyrosine kinase-1/placental growth factor ratio. The area under the receiver operating characteristics curves for preeclampsia at ≤2 weeks in screening by the triple test (0.975; 95% confidence interval, 0.964-0.985) was higher than that of placental growth factor alone (0.900; 95% confidence interval, 0.866-0.935; P<.0001) and the soluble fms-like tyrosine kinase-1/placental growth factor ratio (0.932; 95% confidence interval, 0.904-0.960; P=.0001). Similarly, the areas under the receiver operating characteristics curves for preeclampsia at ≤4 weeks in screening by the triple test (0.907; 95% confidence interval, 0.886-0.928) was higher than that of placental growth factor alone (0.827; 95% confidence interval, 0.800-0.854; P<.0001) or the soluble fms-like tyrosine kinase-1/placental growth factor ratio (0.857; 95% confidence interval, 0.830-0.883; P<.0001). Third, at most, screen-positive rates of 2-30% the detection rate of delivery with preeclampsia at ≤2 and ≤4 weeks that was achieved by the triple test was approximately 10% higher than that of the soluble fms-like tyrosine kinase-1/placental growth factor ratio and 20% higher than that of placental growth factor alone; the negative predictive value was similar for the 3 tests. At 35+0-36+6 weeks gestation, the performance of screening for imminent delivery with preeclampsia by the competing risks model is superior to that of placental growth factor alone or the soluble fms-like tyrosine kinase-1/placental growth factor ratio.

Molecular determinants of microvascular dysfunction in hypertensive pregnancy and preeclampsia.

Preeclampsia is a pregnancy-related disorder characterized by hypertension and often fetal intrauterine growth restriction, but the underlying mechanisms are unclear. Defective placentation and apoptosis of invasive cytotrophoblasts cause inadequate remodeling of spiral arteries, placental ischemia, and reduced uterine perfusion pressure (RUPP). RUPP causes imbalance between the anti-angiogenic factors soluble fms-like tyrosine kinase-1 and soluble endoglin and the pro-angiogenic vascular endothelial growth factor and placental growth factor, and stimulates the release of proinflammatory cytokines, hypoxia-inducible factor, reactive oxygen species, and angiotensin AT1 receptor agonistic autoantibodies. These circulating factors target the vascular endothelium, smooth muscle and various components of the extracellular matrix. Generalized endotheliosis in systemic, renal, cerebral, and hepatic vessels causes decreases in endothelium-derived vasodilators such as nitric oxide, prostacyclin and hyperpolarization factor, and increases in vasoconstrictors such as endothelin-1 and thromboxane A2. Enhanced mechanisms of vascular smooth muscle contraction, such as intracellular Ca2+ , protein kinase C, and Rho-kinase cause further increases in vasoconstriction. Changes in matrix metalloproteinases and extracellular matrix cause inadequate vascular remodeling and increased arterial stiffening, leading to further increases in vascular resistance and hypertension. Therapeutic options are currently limited, but understanding the molecular determinants of microvascular dysfunction could help in the design of new approaches for the prediction and management of preeclampsia.

Brain angiogenic gene expression in fetuses with congenital heart disease.

To assess potential differences in the expression of antiangiogenic and angiogenic factors and of genes associated with chronic hypoxia in cerebral tissue of euploid fetuses with congenital heart disease (CHD) vs those without. Cerebral tissue was obtained from 15 fetuses with CHD and 12 control fetuses that had undergone termination of pregnancy. Expression profiles of the antiangiogenic factor soluble fms-like tyrosine kinase-1 (sFlt-1), the angiogenic vascular endothelial growth factor-A (VEGF-A) and placental growth factor (PlGF), and of genes associated with chronic hypoxia were determined by real-time polymerase chain reaction in tissue from the frontal cortex and the basal ganglia of the fetuses. Expression of sFlt-1 was 48% higher in the frontal cortex (P= 0.0431) and 72% higher in the basal ganglia (P= 0.0369) of CHD fetuses compared with controls. The expression of VEGF-A was 60% higher (P= 0.0432) and that of hypoxia-inducible factor 2-alpha was 98% higher (P= 0.0456) in the basal ganglia of CHD fetuses compared with controls. No significant differences were observed between the two groups in the expression of PlGF and hypoxia-inducible factor 1-alpha. An overall dysregulation of angiogenesis with a net balance towards an antiangiogenic environment was observed in the cerebral tissue of fetuses with CHD, suggesting that these fetuses may have an intrinsic angiogenic impairment that could contribute to impaired brain perfusion and abnormal neurological development later in life. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

PKC Modulates sFlt1 Production by Human Placental Trophoblasts in Response to Oxidized LDL

Preeclampsia (PE) is a leading cause of maternal and perinatal mortality and mortality, and its prevalence is 4 fold higher in women with diabetes than those without: the underlying mechanism is unclear. The anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt1) plays a key role in PE pathogenesis. Evidence suggests that PKC activation, which is associated with the development of diabetic vascular complications, may mediate the enhanced sFlt1 release in non-trophoblast cells. We aimed to determine the role of PKC in the regulation of sFlt1 expression in placental trophoblasts, and whether diabetic conditions upregulate sFlt1 expression via the PKC pathway. Human trophoblasts HTR8/SVneo were treated in three separate experiments with PKC activator phorbol-12-myristate-13-acetate (PMA), or diabetes stimuli ’heavily oxidized, glycated’ low-density lipoproteins (HOG-LDL) vs. native LDL ± high glucose (30 mM), over 24h, with or without PKC inhibitor GF109203X (GX). Both sFlt1 mRNA expression (RT-PCR) and protein release (ELISA) were measured. Compared to the control, PMA (5 nM) increased sFlt1 mRNA expression (2.5 fold, p&amp;lt;0.001) and protein release (383.8 ± 27.4 vs. 118.3 ± 21.3 pg/ml, p&amp;lt;0.001) in trophoblasts; these effects were entirely abrogated by the pre-treatment with 5 uM GX for mRNA expression (by 54%, p&amp;lt;0.001) and protein release (115.5 ± 20.9 pg/ml, p&amp;lt;0.001) vs. PMA-treated cells. Similarly, HOG-LDL (50 µg/ml) increased sFlt1 mRNA expression (3.7 fold, p&amp;lt;0.001) and protein release (126.4 ± 16.3 vs. 55.1 ± 2.4 pg/ml, p&amp;lt;0.001), and the both effects were partially abolished by GX (by 33-37%, p&amp;lt;0.05). High glucose did not significantly affect sFlt1 expression, or alter the effect of HOG-LDL. The data suggest that modified lipoproteins may promote PE development in women with diabetes, at least in part via PKC-mediated upregulation and release of sFlt1. These findings provide new insights into disease mechanisms, and may lead to new targets for prevention and treatment of PE. Disclosure R.P. Chow: None. J. Zhao: None. J. Yu: None.

Melatonin enhances antioxidant molecules in the placenta, reduces secretion of soluble fms-like tyrosine kinase 1 (sFLT) from primary trophoblast but does not rescue endothelial dysfunction: An evaluation of its potential to treat preeclampsia.

Preeclampsia is one of the most serious complications of pregnancy. Currently there are no medical treatments. Given placental oxidative stress may be an early trigger in the pathogenesis of preeclampsia, therapies that enhance antioxidant pathways have been proposed as treatments. Melatonin is a direct free-radical scavenger and indirect antioxidant. We performed in vitro assays to assess whether melatonin 1) enhances the antioxidant response element genes (heme-oxygenase 1, (HO-1), glutamate-cysteine ligase (GCLC), NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), thioredoxin (TXN)) or 2) alters secretion of the anti-angiogenic factors soluble fms-like tyrosine kinase-1 (sFLT) or soluble endoglin (sENG) from human primary trophoblasts, placental explants and human umbilical vein endothelial cells (HUVECs) and 3) can rescue TNF-α induced endothelial dysfunction. In primary trophoblast melatonin treatment increased expression of the antioxidant enzyme TXN. Expression of TXN, GCLC and NQO1 was upregulated in placental tissue with melatonin treatment. HUVECs treated with melatonin showed an increase in both TXN and GCLC. Melatonin did not increase HO-1 expression in any of the tissues examined. Melatonin reduced sFLT secretion from primary trophoblasts, but had no effect on sFLT or sENG secretion from placental explants or HUVECs. Melatonin did not rescue TNF-α induced VCAM-1 and ET-1 expression in endothelial cells. Our findings suggest that melatonin induces antioxidant pathways in placenta and endothelial cells. Furthermore, it may have effects in reducing sFLT secretion from trophoblast, but does not reduce endothelial dysfunction. Given it is likely to be safe in pregnancy, it may have potential as a therapeutic agent to treat or prevent preeclampsia.

Combining metformin and esomeprazole is additive in reducing sFlt-1 secretion and decreasing endothelial dysfunction - implications for treating preeclampsia.

IntroductionThe discovery of new treatments that prevent or treat preeclampsia would be a major advance. Antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG) are secreted in excess from the placenta, causing hypertension, endothelial dysfunction, and multiorgan injury. We recently identified metformin and esomeprazole as potential treatments for preeclampsia. Both reduce placental and endothelial secretion of sFlt-1 and soluble endoglin, and reduce endothelial dysfunction.ObjectivesWe set out to assess whether combining metformin and esomeprazole would additively reduce sFlt-1 and soluble endoglin secretion and reduce endothelial dysfunction (verses drug alone). Metformin and esomeprazole were added to primary placental cells and tissues, and endothelial cells and their effects on sFlt-1 and soluble endoglin secretion were assessed in vitro. Tumor necrosis factor-α (TNF-α) was added to endothelial cells to induce dysfunction in vitro. We examined the ability of metformin + esomeprazole to rescue TNF-α induced vascular cell adhesion molecule-1 (VCAM-1) and Endothelin-1 (ET-1) expression, leukocyte adhesion (markers of endothelial dysfunction).ResultsCombining metformin and esomeprazole was additive at reducing sFlt-1 secretion and expression of sFlt-1 e15a mRNA isoform in primary cytotrophoblast, placental explants and endothelial cells. In contrast, no additive reduction in sENG was observed with combined metformin and esomeprazole. The low-dose combination of metformin + esomeprazole additively reduced TNF-α-induced VCAM-1 mRNA, but not VCAM-1 protein expression. There was no additive reduction when combining metformin and esomeprazole on TNF-α induced PBMC adhesion to endothelial cells. However, combining metformin and esomeprazole additively reduced ET-1 mRNA expression.ConclusionsIn conclusion combining metformin and esomeprazole additively reduced secretion of sFlt-1, and markers of endothelial dysfunction. The combination of metformin and esomeprazole may provide a more effective treatment or prevention for preeclampsia compared to either as single agents.

Analytical validation of soluble fms-like tyrosine and placental growth factor assays on B·R·A·H·M·S KRYPTOR Compact Plus automated immunoassay platform.

Preeclampsia is one of the leading hypertensive disorders of pregnancy. Angiogenic biomarkers such as anti-angiogenic factor soluble fms-like tyrosine kinase 1 (sFlt1) and pro-angiogenic factor placental growth factor (PlGF) are involved in the pathophysiology of preeclampsia. The aim of this study is to validate the analytical performance of sFlt1 and PlGF on the B·R·A·H·M·S KRYPTOR Compact Plus (ThermoFisher Scientific). We examined K2-EDTA plasma samples from 50 patients on B·R·A·H·M·S KRYPTOR Compact Plus, an automated immunoassay platform. QC materials were used to assess intra- and inter-precision of the assay. Lower limit of quantitation and interference studies were determined using pooled patient plasma. The sFlt1 and PlGF assays demonstrated an analytical measuring range of 90-69,000 pg/mL and 11-7000 pg/mL, respectively (r2 > 0.99). Lower limit of quantitation (20% CV) was interpolated to be 35 pg/mL for sFlt1 and 10 pg/mL for PlGF. Total precision for both assay displayed CVs of <10%. Interference studies showed that both assays were not significantly affected by hemolysis up to an H-index of 1100 for sFlt1 and 300 for PlGF; L- and I-index of 800 and 80 respectively for both assays. The Passing-Bablok regression analysis for sFlt1/PlGF yielded an equation of y = 1.05x + 0.02, and the Bland Altman analysis showed an average bias of 0.84. Plasma levels of sFlt1 and PlGF measured on the B·R·A·H·M·S KRYPTOR Compact Plus platform demonstrate excellent analytical performance and are acceptable as clinical grade assays.

Proton Pump Inhibitors Quench the Pathophysiological Characteristics of Preeclampsia in Both Human and Mouse Models and Represent an Exciting Novel Candidate Therapeutic

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.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).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.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.

P 22 Analytical evaluation of soluble fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF) on Brahms Kryptor automated immunoassay for the diagnosis of preeclampsia

Background Preeclampsia is one of the leading hypertensive disorders in pregnant women worldwide and is characterized by maternal vascular dysfunction. Current diagnosis of preeclampsia in the U.S. is based on non-specific tests such as new onset of hypertension and proteinuria after 20 weeks of gestation. More recently, two emerging biomarkers anti-angiogenic factor soluble fms-like tyrosine kinase 1 (sFlt-1) and pro-angiogenic factor placental growth factor (PlGF), have been demonstrated to be involved in the pathophysiology of preeclampsia. Studies have shown that the usefulness of sFlt-1/PlGF ratio in confirming or ruling out suspected preeclampsia cases. Thus, wider availability of the automated measurement of sFlt-1 and PlGF has the potential of improving the clinical management of preeclampsia. Methods This study examined K2-EDTA plasma samples from 50 patients on Brahms Kryptor, an automated immunoassay platform. QC materials were used to access intra- and inter-precision of the assay. Lower limit of quantitation and interference studies were determined using pooled patient plasma. Results The sFlt-1 and PlGF assays demonstrated an analytical measuring range of 90–69,000 pg/mL and 11–7000 pg/mL, respectively (r2 > 0.99). Lower limit of quantitation (20% CV) was interpolated to be 35 pg/mL for sFlt-1 and 10 pg/mL for PlGF. Total precision for both assay displayed CVs of y = 0.9939x-37.15 with an overall bias of 90.35 for sFlt-1; and y = 0.8936x + 2.562, with an overall bias of −39.644 for PlGF. The larger differences in overall bias based on individual tests however was greatly improved when a ratio of sFlt-1/PlGF is taken into account for method comparison, yielding an equation of y = 1.05x + 0.02, with an overall bias of 0.84. Conclusion The pre-eclamptic biomarkers, sFlt-1 and PlGF assayed on the Brahms Kryptor platform, demonstrate good analytical performance and are acceptable for clinical studies defining the role of these biomarkers for preeclampsia.

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.

Matrix Metalloproteinases in Normal Pregnancy and Preeclampsia.

Normal pregnancy is associated with marked hemodynamic and uterine changes that allow adequate uteroplacental blood flow and uterine expansion for the growing fetus. These pregnancy-associated changes involve significant uteroplacental and vascular remodeling. Matrix metalloproteinases (MMPs) are important regulators of vascular and uterine remodeling. Increases in MMP-2 and MMP-9 have been implicated in vasodilation, placentation, and uterine expansion during normal pregnancy. The increases in MMPs could be induced by the increased production of estrogen and progesterone during pregnancy. MMP expression/activity may be altered during complications of pregnancy. Decreased vascular MMP-2 and MMP-9 may lead to decreased vasodilation, increased vasoconstriction, hypertensive pregnancy, and preeclampsia. Abnormal expression of uteroplacental integrins, cytokines, and MMPs may lead to decreased maternal tolerance, apoptosis of invasive trophoblast cells, inadequate remodeling of spiral arteries, and reduced uterine perfusion pressure (RUPP). RUPP may cause imbalance between the antiangiogenic factors soluble fms-like tyrosine kinase-1 and soluble endoglin and the proangiogenic vascular endothelial growth factor and placental growth factor, or stimulate the release of inflammatory cytokines, hypoxia-inducible factor, reactive oxygen species, and angiotensin AT1 receptor agonistic autoantibodies. These circulating factors could target MMPs in the extracellular matrix as well as endothelial and vascular smooth muscle cells, causing generalized vascular dysfunction, increased vasoconstriction and hypertension in pregnancy. MMP activity can also be altered by endogenous tissue inhibitors of metalloproteinases (TIMPs) and changes in the MMP/TIMP ratio. In addition to their vascular effects, decreases in expression/activity of MMP-2 and MMP-9 in the uterus could impede uterine growth and expansion and lead to premature labor. Understanding the role of MMPs in uteroplacental and vascular remodeling and function could help design new approaches for prediction and management of preeclampsia and premature labor.

Imbalanced Angiogenesis in Peripartum Cardiomyopathy - Diagnostic Value of Placenta Growth Factor.

Concentrations of the anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt-1) are altered in peripartum cardiomyopathy (PPCM). In this study we investigated changes in the angiogenesis balance in PPCM.Methods and Results:Plasma concentrations of sFlt-1 and the pro-angiogenic placenta growth factor (PlGF) were determined in patients with PPCM during the post-partum phase (n=83), in healthy women at delivery (n=30), and in patients with acute heart failure (AHF; n=65). Women with cardiac failure prepartum or associated with any form of hypertension, including pre-eclampsia, were excluded. Compared with non-pregnant women, in women with AHF and PPCM, median PlGF concentrations were greater (19 [IQR 16-22] and 98 [IQR 78-126] ng/mL, respectively; P<0.001) and the sFlt-1/PlGF ratio was lower (9.8 [6.6-11.3] and 1.2 [0.9-2.8], respectively; P<0.001). The sFlt-1/PlGF ratio was lower in PPCM than in normal deliveries (1.2 [0.9-2.8] vs. 94.8 [68.8-194.1], respectively; P<0.0001). The area under the curve for PlGF (cut-off value: 50ng/mL) and/or the sFlt-1/PlGF ratio (cut-off value: 4) to distinguish PPCM from either normal delivery or AHF was >0.94. Median plasma concentrations of the anti-angiogenic factor relaxin-2 were lower in PPCM and AHF (0.3 [IQR 0.3-1.7] and 0.3 [IQR 0.3-1] ng/mL, respectively) compared with normal deliveries (1,807 [IQR 1,101-4,050] ng/mL; P<0.001). Plasma of PPCM patients shows imbalanced angiogenesis. High PlGF and/or low sFlt-1/PlGF may be used to diagnose PPCM.