Anti-angiogenic Factors sFlt-1 Research Articles

Alterations in fibrin formation and fibrinolysis in early onset-preeclampsia: Association with disease severity

Objective; Early-onset preeclampsia is a rare pregnancy-specific disorder associated with significantly increased maternal and fetal morbidity and mortality. Whilst it is known that even normotensive pregnancies are associated with changes in clot formation and dissolution, the nature of how these changes differ in those with early onset preeclampsia has not been well established.We sought to evaluate parameters of fibrin formation and fibrinolysis in individuals with early onset preeclampsia in comparison to both pregnant and non-pregnant controls. Furthermore, such parameters were correlated with markers of disease severity in this patient cohort, including the presence of multiorgan involvement, the rate of disease progression and the extent of the anti-angiogenic state in this condition. Study design; Patients with early onset preeclampsia (N = 20) and both pregnant (N = 16) and non -pregnant (N = 16) controls were recruited from the cohort at a large urban maternity hospital which saw over 15,000 deliveries during the study period. Platelet poor plasma was prepared from collected whole blood and analysed for parameters of fibrin formation and fibrinolysis (lagtime to and rate of fibrin formation; PAI-1; PAI-2; D-dimer; plasmin-antiplasmin; tPA) in addition to markers of angiogenesis (sFLT-1; Endoglin) using commercially available specific immunoassays. Results; The maximum rate of fibrin formation as well as PAI-1, PAI-2 and D-dimer levels were all significantly increased in those with early onset preeclampsia and pregnant controls when compared to non-pregnant controls without significant differences between the 2 former groups. Plasmin-antiplasmin levels were significantly reduced in a similar manner. tPA levels were significantly elevated in EOP compared to both pregnant and non-pregnant controls. EOP was associated with significantly increased anti-angiogenic factors (sFLT-1; Endoglin) when compared to both pregnant and non-pregnant controls. Conclusion; Markers of fibrin formation and fibrinolysis are significantly alerted in early onset preeclampsia; furthermore, certain markers correlate with disease severity in this patient cohort.

Placental hypoplasia and maternal organic vascular disorder in pregnant women with gestational hypertension and preeclampsia

Objective: This study aimed to investigate the etiology and pathology of preeclampsia (PE), a two-stage disorder involving uteroplacental dysfunction resulting from abnormal implantation and placentation, and gestational hypertension (GH), for which maternal organic vascular disorder is often an underlying factor.Methods: We assessed concentrations of oxygen free radicals (d-ROMs), maternal angiogenic factor (PlGF), and antiangiogenic factor (sFlt-1), placental hypoxic changes, oxidative DNA damage, and maternal organic vascular disorders in 23 women with PE (PE group), 13 with GH (GH group), and 16 with uncomplicated pregnancies (normal group). Intima-media thickness (IMT) of the carotid artery was assessed as a proxy for maternal organ vascular disorder. Immunohistochemical analysis was performed to measure the proportion of placental trophoblast cell nuclei staining positive for hypoxia-inducible factor-1α (HIF-1α), which reflects hypoxic changes, and 8-hydroxy-2′-deoxyguanosine (8-OHdG), which reflects oxidative DNA damage.Results: Maternal serum d-ROM concentrations were significantly increased in both GH and PE groups relative to the normal group. Maternal serum d-ROM concentrations were significantly increased in both GH and PE groups relative to the normal group. Maternal serum sFlt-1 concentrations, ratio of sFlt-1/PlGF, and proportions of HIF-1α-positive nuclei and 8-OHdG-positive nuclei were significantly higher in the PE group compared to GH and normal groups. IMT was significantly greater in GH and PE groups compared to the normal group, and was higher in the GH group compared to the PE group.Conclusions: Our findings suggest that placental hypoxic changes and oxidative DNA damage are severe in patients with PE and accompanied by an increase in antiangiogenic factors. Moreover, maternal organ vascular disorder was more severe in patients with GH compared to those with PE, as assessed by IMT.Key message: PE is a two-stage disorder that involves uteroplacental dysfunction, and organic vascular disorder underlies GH.

Maternal Flt-1 and endoglin expression by circulating monocyte subtype and polarization in preeclampsia and fetal growth restriction.

ObjectiveCirculating levels of the anti-angiogenic factors sFlt-1 and sEndoglin are elevated in preeclampsia (PE) and fetal growth restriction (FGR), mainly secreted from placental trophoblast. This study aims to identify the contributory role of monocyte Flt-1 and endoglin expression in PE and FGR.Study designA prospective cross-sectional study was conducted and patients recruited from four clinical groups including normal pregnancy, PE, FGR and PE + FGR. Peripheral blood samples and cord blood were collected from 54 pregnant women between 24–40 weeks of gestation. Monocyte subset distribution was assessed using CD14 and CD16 expression and the surface expression of Flt-1, endoglin, CD86 and CD163 assessed by flow cytometry. We compared these factors between (1) clinical groups. (2) monocyte subset (3) monocyte polarization and (4) gestational age.ResultsAcross all clinical groups, Flt-1 was mainly expressed by classical and intermediate monocytes, but no differences between clinical groups were observed. Surface expression of endoglin was higher on intermediate and non-classical monocytes and decreased in PE + FGR total monocytes. Flt-1 and endoglin expression correlated with increasing gestational age as well as higher CD86/CD163 ratio favouring M1 polarisation. The fetal monocyte endoglin expression was increased in FGR.ConclusionWe conclude that monocyte Flt-1 and endoglin expression increase with gestational age and with M1 polarization suggesting their upregulation with inflammatory changes in monocytes. Endoglin expression by M1 monocytes may play a part in increased cardiovascular risk associated with preeclampsia. Endoglin expression on fetal monocytes is increased in FGR as a likely response to placental injury.

Biology of preeclampsia: Combined actions of angiogenic factors, their receptors and placental proteins

Although massive efforts have been undertaken to elucidate the etiology of the pregnancy syndrome preeclampsia, its developmental origin remains a mystery. Most efforts of the last decade have focused on biomarkers to predict and/or diagnose preeclampsia, including the anti-angiogenic factor sFlt-1 (soluble fms-like tyrosin kinase-1), the angiogenic factor PGF (placental growth factor) and PP13 (placental protein 13). The origins of these marker proteins are still under debate, and so far their actions have only been describe separate from each other. This study will focus on the origins and actions of all three markers during pregnancy and outside pregnancy and will describe a scenario where all three markers act synergistically to rescue the mother from the deleterious effects of the debris that is released from the placenta during preeclampsia. This more holistic approach may open new avenues to think about maternal-fetal interactions and putative therapies.

Clinical Implications

Clinical Implications

169. Retinoic acid down regulates sFlt1 in decidual stromal cells independent of decidualization. Implications to preeclampsia

Introduction Retinoic acid (RA) plays a vital role during pre- and early pregnancy stages at the maternal-fetal interface (decidua). We recently showed that RA levels are reduced in the decidua of women with preeclampsia (PE). This finding, combined with the knowledge that PE decidual stromal cells (DSC) secrete elevated levels of the anti-angiogenic factor sFlt1 and that the promoter of this protein has a putative RA binding site, suggests the possibility that RA plays a direct role in regulating sFlt1 expression in DSC. This study was to determine the effects of RA on sFlt1 production in DSC. The relationship of these effects to DSC decidualization, known to be defective in PE, was also investigated. Methods Primary DSCs (n = 6 in each group, normotensive (NT) and PE) were treated with 0.1 μ M of RA, 1 μ M BMS493 (pan RA antagonist) or 0.5 mM cAMP (to induce decidualization) for 12 days. sFLT1 and PRL levels were determined by using both qPCR and ELISA. Group means were analyzed by Mann–Whitney U test. p ⩽ 0.05 is considered significant. Results RA treatment of NT-DSCs and PE-DSCs inhibited sFlt1 production to 44 ± 24% and 24 ± 13% of controls but did not affect DSC decidualization as quantified by prolactin (PRL) secretion. Independent of RA, cAMP-mediated decidualization reduced sFlt1 and increased PRL levels in NT-DSC but only moderately in PE-DSC. BMS493 blocked the effects of RA and, by itself, induced upregulation of sFlt1. Studies using decidualization defective stromal cells confirmed that reduction of sFlt1 by RA is independent of decidualization. Conclusion RA downregulates sFlt1 production in DSCs independent of decidualization. PE-DSCs are significantly more responsive to sFlt1 inhibition by RA than are NT-DSC. Our finding of reduced RA levels in PE decidua, combined with studies by us and others that PE women show defective decidualization, may help explain elevated sFlt1 expression in maternal PE decidua.

296. Loss of Placental NR4A2 may contribute to the pathogeneisis of pre-eclampsia

Introduction Placental dysfunction is a key contributor in the development of pre-eclampsia. Recently, we identified NR4A2 mRNA to be altered in the maternal circulation in pregnancies complicated by severe, early onset fetal growth restriction and pre-eclampsia (PE). Objective The current study aimed to determine whether NR4A2 expression was altered in the human placenta with hypoxic insult and pre-eclampsia. Methods Isolated primary cytotrophoblasts (n = 5) and placental explant tissue (n = 4) from normal term placentas were cultured under normoxia (8% oxygen) and hypoxia (1% oxygen) for 24 h. Expression of NR4A2 was assessed by qPCR. Placental tissue was collected from severe early onset PE (n = 49) and gestation matched controls (n = 47) to test NR4A2 mRNA expression. NR4A2 protein was assessed in PE (n = 31) and preterm (n = 22) placenta by Western blotting. Primary cytotrophoblasts were transfected with NR4A2 siRNA (n = 3) to silence gene expression (under normoxia and hypoxia for 48 h). sFLT-1 secretion was assessed by ELISA and expression of the sFLT-1 variants (e15a and i13) and cell survival associated genes by qPCR. Normality (Gaussian distribution) was assessed in each case and appropriate post hoc analysis was performed. Results NR4A2 mRNA expression was significantly reduced with hypoxia in trophoblast, but not placental explants. Additionally, NR4A2 mRNA expression was significantly reduced in PE placenta. However, there was no difference in NR4A2 protein expression between PE and controls. Silencing NR4A2 significantly increased anti-angiogenic factor sFLT-1 secretion, but not sFLT-1 mRNA expression. Furthermore, loss of NR4A2 in the trophoblast reduced pro-survival IGF2 gene expression and increased NOX4 (oxidative stress marker) expression. Conclusion NR4A2 is reduced in the trophoblast with hypoxia and mRNA expression is low in the PE placenta. Signs of placental dysfunction were observed with the silencing of NR4A2 in the trophoblast and an increase in sFLT-1 secretion suggests potential roles for NR4A2 in the pathogenesis of PE.

113. sFlt-1 leads to severe changes in placental differentiation and maternal and fetal vessel formation in transgenic sFlt-1 preeclampsia/ IUGR mouse models

Introduction The anti-angiogenic factor sFlt-1 is the main candidate in the progression of preeclampsia, a disease which causes placental dysfunctions often leading to IUGR. Mostly is known about the impairment of the endothelial cell function by sFlt-1, but how sFlt-1 leads to IUGR and affects the placenta and fetus is currently unknown as well as therapeutic agents against such diseases are missing. Objectives Therefore we have established two transgenic, inducible sFlt-1 mouse models: (1) maternal ubiquitous (sFlt-1/rtTA mice) and (2) placental (sFlt-1/tTA/TpbpaCre mice) overexpression of human sFlt-1. We hypothesize that sFlt-1 is involved in the development of IUGR by influencing placental development and function and is therefore a potential candidate for intervention strategies. Material and Methods We examined the effects of sFlt-1 on placental morphology and function at 18.5 dpc with morphometric and immunohistochemical analyses and transcript expression of placental marker genes, nutrient transporters and proteomic analyses. Results Ubiquitous overexpression of sFlt-1 led to IUGR of the fetuses as shown by reduced fetal weights and signs of retardation. In addition, we observed a severely impaired placental phenotype shown by enlarged maternal blood sinusoids, a reduced number of fetal vessels and an inadequate placental differentiation of the labyrinth. Glucose, fatty acid and amino acid transport seem to be negatively affected. Preliminary results of the placental overexpression of sFlt-1 revealed while expressing lower levels of sFlt-1 also IUGR. Conclusion We assume that sFlt-1 has an inhibitory effect on placental differentiation, especially on fetal vessel development. A possible reactive response could be an increase in maternal blood flow promoting dilatation of the maternal sinusoids to fulfil the nutrient requirements of the fetus. This ultimately resulted in an uteroplacental insufficiency leading to IUGR. Thus, we speculate that the alterations triggered by increased anti-angiogenesis upon sFlt-1 strongly may affect fetal outcome and programming.

190. Combination sulfasalazine and metformin are additive in reducing placental secretion of antiangiogenic factor sFlt

Introduction Preeclampsia is associated with increased placental secretion of antiangiogenic factors sFlt-1 and sENG and a reduction in angiogenic proteins placental growth factor secretion (PlGF) and vascular endothelial growth factor (VEGF). We have identified two medications, safe in pregnancy, that mitigate key aspects of preeclampsia in vitro. We have demonstrated metformin and sulfasalazine independently reduce placental sFlt-1 secretion and vessel dysfunction. In this study we investigate the possibility of combining these medications to reduce the dose required to mitigate key aspects of preeclampsia; sFlt-1 and sENG secretion and PlGF and VEGF expression, using primary human tissues. Methods Metformin and sulfasalazine, at low doses individually and in combination, were administered to primary trophoblasts and placental explants and sFlt-1 secretion assessed. At the mRNA level there are two sFlt-1 splice variants; the primate and placental specific isoform sFlt-1 e15a and the predominately vascular isoform sFlt-1 i13. Both were measured following combination treatment. In addition the expression of angiogenic factors PlGF and VEGFα were assessed. Results Combining low-dose metformin and sulfasalazine additively decreased sFlt-1 secretion from primary trophoblast compared to either drug alone. Similarly, an additive decrease in sFlt-1 e15a and i13 was identified following combination treatment. In placental explants, combination treatment also additively decreased sFlt-1 secretion. Low dose sulfasalazine potently upregulated PlGF expression in primary trophoblasts and this was maintained with the addition of metformin. Metformin and sulfasalazine individually increased VEGFα, and this effect was additive when the two drugs were combined. Conclusion Combination metformin and sulfasalazine additively reduced sFlt-1 secretion and the expression of its splice variants, sFlt-1 e15a and i13. Furthermore, in combination metformin and sulfasalazine upregulate angiogenic factor VEGFα. Therefore we conclude that they might be promising combination therapeutics that could reduce the burden of this devastating disease.

82. Disulfiram inhibits placental sFLT-1 secretion

Introduction Preeclampsia is associated with elevated placental secretion of the anti-angiogenic factor sFLT-1. Surprisingly, little is understood about its regulation, identifying such mechanisms may uncover therapeutic leads. Objectives Our objective was to determine the effects of disulfiram, a 20S subunit proteasome inhibitor, on placental secretion of sFLT-1 and endothelial dysfunction. Methods Primary trophoblast were isolated from normotensive term placenta, treated with disulfiram and mRNA expression of sFLT-1 variants and protein secretion assessed. To assess the effects of disulfiram on the 20S subunit of the proteasome, primary trophoblast were treated with disulfiram in the presence of Suc-LLVY-AMC fluorogenic substrate. Next, we investigated the effect of disulfiram on endothelial dysfunction using isolated primary human umbilical vein endothelial cells (HUVECs) treated with 5% preeclamptic serum +/− disulfiram. The effect of disulfiram on markers of endothelial dysfunction were measured via qRT-PCR for vascular cell adhesion molecule-1 (VCAM-1) and adhesion of fluorescently labelled peripheral blood mononuclear cells (PBMCs) to HUVECs. Results Disulfiram significantly reduced mRNA expression of the sFLT-1 variants, sFLT-1-i13 and sFLT-e15a. This translated into a significant and dose dependent reduction in the protein secretion of sFLT-1. In primary trophoblast disulfiram did not inhibit the 20S subunit of the proteasome, evidenced by no change in cellular fluorescence following addition of Suc-LLVY-AMC. Treating primary HUVECs with serum from preeclamptic women induced endothelial dysfunction with significantly increased mRNA expression of VCAM-1 as well as the adhesion of PBMCs to HUVECs. Disulfiram reduced VCAM-1, with expression reduced below control levels, however, did not alter the adhesion of PBMCs to primary HUVECs. Conclusions We have identified disulfiram to quench placental sFLT-1 secretion via a mechanism independent of inhibiting the 20S subunit of the proteasome. Understanding of the mechanisms by which disulfiram inhibits sFLT-1 secretion may assist in identifying therapeutic targets. Interestingly, disulfiram also reduced markers of endothelial dysfunction.

Apium graveolens Prevents Intrauterine Growth Restriction via Suppression of Antiangiogenic Factor Production

Preeclampsia is the worldwide leading cause of fetomaternal morbidity and mortality which involves the placental dysfunction. A poor placentation and formed of non-dilated spiral artery caused utero-placental circulation insufficiency, resulted in inadequate supply of nutrients and oxygen to support normal aerobic growth of the fetus. Apium graveolens or celery has been widely known as antioxidant, antiinflammation and antihypertensive with flavonoid-apigenin as main active compound. Apigenin can inhibit TNF-α, HIF-1α and nitric oxide blocking as major pathophysiological pathway of preeclampsia. This study was aimed to find how the Apium graveolens can improve intrauterine growth and its correlation with suppression of anti-angiogenic factor sFlt-1 in anti-Qa2 preeclampsia animal model. Twenty female BALB/c Mus musculus were divided into 4 groups: control, anti-Qa2 and anti-Qa2 with 500 and 1000 mg/kgBW celery herbs extract. The fetal weights and lengths, placental weights and serum sFlt-1 levels were measured and analyzed with One Way ANOVA and further tested with Least Significance Difference in 95% confidence interval. The result showed a difference between control and treatments group (p≤0.05) with 1000 mg/kgBW significantly increase intrauterine growth and decrease sFlt-1, then there is a negative correlation between intrauterine weight and serum sFlt-1. This study suggests that celery herbs extract (CHE) has an apigenin-flavonoid compound which can prevent intrauterine growth restriction (IUGR) via suppression of antiangiogenic factor production in preeclampsia mice model.

Effect of sildenafil citrate on circulating levels of sFlt-1 in preeclampsia.

To examine the effect of sildenafil on level of antiangiogenic proteins of preeclampsia. Firstly to examine the effect of sildenafil on serum biomarkers in a patient with preterm preeclampsia. Secondly, to examine the effect of sildenafil on sFlt-1 and soluble endoglin secretion from primary trophoblasts and placental explants. The clinical team administered 50 mg tds sildenafil to a 26-year-old primigravid woman with severe preeclampsia at the threshold of viability (24 3/7 weeks gestation) and we collected bloods to examine the effect of slidenafil on antiangiogenic factors sFlt-1 and soluble endoglin (sENG), pro-angiogenic factor PlGF and vascular cell adhesion molecule 1 (VCAM-1) and endothelin-1 (ET1). We administered sildenafil to human primary trophoblasts and placental explants and explored its effect on sFlt-1 and sENG secretion. We examined serum anti-angiogenic factors sFlt-1 and sENG, pro-angiogenic factor PlGF, the potent vasoconstrictor ET1 and VCAM-1 by ELISA. We explored the effect of sildenafil on sFlt-1 secretion from primary trophoblasts and sFlt-1 and sENG secretion from placental explants. We found a reduction in serum sFlt-1, stabilisation in sENG and PlGF in a patient with peri-viable preterm preeclampsia administered oral sildenafil 50 mg three times daily (tds). Furthermore there was an initial stabilisation in serum VCAM-1 and a decline in ET1 with sildenafil administration. This was concordant with stabilisation of clinical and biochemical features of preeclampsia. Interestingly, treating placental cells and tissues in vitro with sildenafil did not appear to change sFlt-1 or sENG secretion. Sildenafil administration was associated with a reduction in serum sFlt-1 and sENG secretion and increase in PlGF secretion in a patient with preterm preeclampsia, potentially via increasing placental perfusion rather than acting directly on the placenta.

AT1-AA (Angiotensin II Type 1 Receptor Agonistic Autoantibody) Blockade Prevents Preeclamptic Symptoms in Placental Ischemic Rats.

Women with preeclampsia produce AT1-AA (agonistic autoantibodies to the angiotensin II type 1 receptor), which stimulate reactive oxygen species, inflammatory factors, and hypertensive mechanisms (ET [endothelin] and sFlt-1 [soluble fms-like tyrosine kinase-1]) in rodent models of preeclampsia. The placental ischemic reduced uterine perfusion pressure (RUPP) rat model of preeclampsia exhibits many of these features. In this study, we examined the maternal outcomes of AT1-AA inhibition ('n7AAc') in RUPP rats. Blood pressure was higher in RUPP rats versus normal pregnant (NP) rats (123±2 versus 99±2 mm Hg, P<0.05), which was reduced in RUPP+'n7AAc' (105±3 versus 123±2 mm Hg, P<0.05 versus RUPP). Uterine artery resistant index was increased in RUPP versus NP rats (0.71±0.02 versus 0.49±0.02, P<0.05) and normalized in RUPP+'n7AAc' rats (0.55±0.03). Antiangiogenic factor sFlt-1 was elevated in RUPP versus NP rats (176±37 versus 77±15 pg/mL, P<0.05) but normalized in RUPP+'n7AAc' (86±9, P=0.05 versus RUPP). Plasma nitrate and nitrite were decreased (14±1 versus 20±1 µMNO3, P<0.05) and isoprostanes were elevated (20 117±6304 versus 2809±1375 pg/mL, P<0.05) in RUPP versus NP rats; and normalized in RUPP+'n7AAc' rats; (18±2 µMNO3; 4311±1 pg/mL). PPET-1 (preproendothelin-1) expression increased 4-fold in RUPP versus NP rats which were prevented with 'n7AAc'. Importantly, placental cytolytic natural killer cells were elevated in RUPP versus NP rats (8±2% versus 2±2% gated, P<0.05), which was prevented in RUPP+'n7AAc' total (3±1% gated, P<0.05) In conclusion, AT1-AA inhibition prevents the rise in maternal blood pressure and several pathophysiological factors associated with preeclampsia in RUPP rats and could be a potential therapy for preeclampsia.

112: Neutralization of Fas Ligand reduces placental inflammation and endothelial damage in an animal model of HELLP syndrome

The Fas ligand (FasL) system has an impact on inflammation and hypertension during pregnancy and is dysregulated in women with severe preeclampsia and HELLP syndrome. We have recently reported that endothelin antagonism impacted the Fas/FasL system, therefore in the current study we tested the hypothesis that neutralization of FasL in an animal model of HELLP syndrome decreases inflammation, improves endothelial damage and in turn improves hypertension. Mini-osmotic pumps infusing anti-angiogenic factors sFlt-1 and sEng were placed into normal pregnant (NP) rats on gestational day (GD) 12 to induce HELLP syndrome (n=15). On GD13, 7 of these HELLP rats were infused with 500ng/kg of MFL4 via the jugular vein to inhibit FasL. Untreated NP rats (n=5) served as controls. On GD19 mean arterial pressure (MAP) was measured and all rats were euthanized. Maternal tissues were collected, placentas were cultured under normal culture conditions and media collected (conditioned media - CM) and placed over human umbilical vein endothelial cells (HUVECs) for 24hrs. Administration of FasL to HP rats significant decreased MAP (p=0.03) compared to untreated HP rats. However there was not a significant change in LDH (p=0.164), AST (p=0.52) or platelets (p=0.746) due to attenuation of FasL in HELLP rats compared to untreated HELLP rats. Circulating FasL was significantly increased in HELLP rats compared to NP rats (p=0.0006) but was attenuated with infusion of MFL4 (p=0.0005). Placental protein expression of TNF-alpha, measured via ELISA, was significantly reduced due to MFL4 infusion in HELLP rats (p=0.0009). Finally, HUVECs exposed to CM from HP+MFL4 rats secreted significantly less endothelin-1 compared to HUVECs exposed to CM from HP placentas (p=0.004) and CM from NP placentas (p=0.02). These data suggest that neutralization of FasL decreases MAP and improves placental inflammation and endothelial damage in an animal model of HELLP syndrome. However FasL neutralization does not improve the symptomology of HELLP syndrome, suggesting that FasL may only contribute to the inflammatory pathway.

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

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

Is there any relationship between adipocytokines and angiogenesis factors to address endothelial dysfunction and platelet aggregation in untreated patients with preeclampsia?

Preeclampsia is a multisystem disorder and its etiology remains still unclear. Recent hypotheses rely on imbalance between angiogenic and antiangiogenic factors and disruption of endothelial function of spiral arteries. In addition; increased VTE (venous thromboembolism) risk is still unclear in preeclampsia. Our aim was to investigate the relationship between endothelial dysfunction, adipocytokines, platelet function, and vasculogenesis in preeclampsia. Plasma angiogenic (PlGF, VEGF), antiangiogenic factors (sflt-1, endoglin) with adipocytokines (leptin, adiponectin, ghrelin), endothelial dysfunction markers (vWF, NO), and platelet function markers (ADP and collagen induced platelet aggregation, P-selectin) were examined in 30 early-onset, 22 late-onset preeclampsia, and 27 healthy pregnants. Enzyme-linked immunosorbent assay (ELISA) was used to determine the serum biomarker levels except NO. NO levels were determined using colorimetric method. Endoglin, leptin, and vWF levels were increased in preeclampsia (P<0.001), whereas PlGF, P-selectin (P<0.001), and col-induced platelet aggregation slope (P<0.05) were decreased in the same counterpart as compared to healthy pregnants. Endoglin also correlated with sflt-1 in preeclamptic patients. Increase in the levels of antiangiogenic factors and leptin herewith decline in the level of other angiogenic factor PlGF, did not affect nitric oxide and platelet aggregation markers significantly. Increased levels of vWF and endoglin might be result of endothelial dysfunction, so our findings suggest that an impaired angiogenesis may address endothelial dysfunction, but not platelet aggregation for preeclampsia.

Punica granatum fruit extract inhibits the production of pro-inflammatory cytokines and angiogenic factors of HUVEC cells induced by plasma from patients with pre-eclampsia

Summary This study aims to find out whether the ethanol extract of Punica granatum fruit can inhibit pro-inflammatory cytokines (TNF-α and IL-6) and anti-angiogenic factors (sFlt-1 and sEng) of endothelial cells given plasma stimulus of pre-eclamptic patients. When the cells reached confluence, cells would be grouped into five groups (five replicates per group), i.e. endothelial cells exposed to the plasma of normal pregnancy, endothelial cells exposed to the plasma of patients with severe pre-eclampsia, endothelial cells exposed to the plasma of patients of severe pre-eclampsia and given the extract of P. granatum in various doses (14 ppm; 28 ppm, and 56 ppm). Once induced, measurement of pro-inflammatory cytokine levels and anti-angiogenic factors would be carried out by using the technique of enzyme-linked immunosorbent assay (ELISA). Phytochemical analysis showed that the extract contains phenolic, flavonoid and tannin. Plasma from patients with pre-eclampsia increased the levels of TNF-α, IL-6, sFlt-1 and sEng significantly compared with normal pregnancy plasma exposure group. The increase in the levels of TNF-α, sFlt-1 and sEng was significantly inhibited by the administration of extract at a dose of 56 ppm (P

Resveratrol inhibits release of soluble fms-like tyrosine kinase (sFlt-1) and soluble endoglin and improves vascular dysfunction \u2013 implications as a preeclampsia treatment

Preeclampsia is a disease of pregnancy associated with placental oxidative stress, inflammation and elevated release of anti-angiogenic factors sFlt-1 and soluble endoglin. These placental factors cause generalized maternal endothelial dysfunction. There are no treatments to halt disease progression; delivery is the only cure. Resveratrol modulates pathways involved in inflammation and oxidative stress and may offer a potential therapeutic for preeclampsia. Resveratrol reduced sFlt-1, sFlt-1 e15a and soluble endoglin secretion from primary trophoblasts and HUVECs and reduced mRNA expression of pro-inflammatory molecules NFκB, IL-6 and IL-1β in trophoblasts. IL-6, IL-1β and TNFα secretion were also significantly reduced. In HUVECs, resveratrol significantly increased mRNA of anti-oxidant enzymes HO-1, NQO1, GCLC and TXN but did not significantly alter HO-1 protein expression, whilst reducing HO-1 protein in trophoblast. Endothelial dysfunction was induced in HUVECs using TNFα, increasing expression of cell adhesion molecule VCAM1 and adhesion of peripheral blood mononuclear cells, both of which were increased further by resveratrol. In contrast, resveratrol significantly reduced TNFα-induced Endothelin-1 (a vasoconstrictor) and significantly increased the phosphorylation of endothelial nitric oxide synthase (eNOS). In summary, resveratrol decreases secretion of anti-angiogenic factors however its effects on the endothelium are mixed. Overall, it may have potential as a treatment for preeclampsia.

Microvascular function in pre-eclampsia is influenced by insulin resistance and an imbalance of angiogenic mediators.

In preeclampsia, maternal microvascular function is disrupted and angiogenesis is dysfunctional. Insulin resistance that occurs in some pregnancies also pathologically affects microvascular function. We wished to examine the relationship of angiogenic mediators and insulin resistance on microvascular health in pregnancy. We performed a nested, case–control study of 16 women who developed preeclampsia with 17 normal pregnant controls. We hypothesized that the impaired microvascular blood flow in preeclamptic women associated with an increased ratio of the antiangiogenic factors; (s‐endoglin [sEng] and soluble fms‐like tyrosine kinase‐1 [sFlt‐1]) and proangiogenic molecule (placental growth factor [PlGF]) could be influenced by insulin resistance. Serum samples taken after 28 weeks of gestation were measured for the angiogenic factors, insulin, and glucose alongside the inflammatory marker; tumor necrosis factor‐α and endothelial activation, namely; soluble vascular cell adhesion molecule 1, intercellular adhesion molecule‐1, and e‐selectin. Maternal microvascular blood flow, measured by strain gauge plethysmography, correlated with ratios of pro‐ and antiangiogenic mediators independently of preeclampsia. Decreased microvascular function measured in preeclampsia strongly correlated with both the antiangiogenic factor (sFlt‐1 + sEng): PlGF ratio and high levels of insulin resistance, and combining insulin resistance with antiangiogenic factor ratios further strengthened this relationship. In pregnancy, microvascular blood flow is strongly associated with perturbations in pro‐ and antiangiogenic mediators. In preeclampsia, the relationship of maternal microvascular dysfunction with antiangiogenic mediators is strengthened when combined with insulin resistance.

Clinical Implications

HomeHypertensionVol. 69, No. 3Clinical Implications Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBClinical Implications Originally published1 Mar 2017https://doi.org/10.1161/HYPERTENSIONAHA.117.09012Hypertension. 2017;69:383IntroductionHypertension in Former Extremely Low Birth Weight Children (p 443)Low birth weight and prematurity are forerunners of hypertension in adulthood. Few studies reported on plasma renin activity in preterm or full-term born children. In prematurely born children with birth weight <1000 g (extremely low birth weight) and healthy controls, we tested the hypothesis whether renin might modulate the pathogenesis of hypertension associated with preterm birth. In cases compared with controls, systolic blood pressure was found to be 7.5 mm Hg higher, but plasma renin activity was found to be 0.54 ng/mL per hour lower. Plasma renin activity decreased with systolic blood pressure but was unrelated to sodium excretion. Extremely low birth weight predisposes young children to a low-renin volume-expanded sodium-sensitive type of hypertension. Extremely low birth weight children might therefore benefit from a life course reduction in salt intake and, if hypertensive, are probably more responsive to diuretics than to inhibitors of the renin–angiotensin system, the currently recommended first-line treatment in children with high blood pressure. Above all, our study highlights the importance of screening for hypertension in all prematurely born infants to avoid the complications associated with high blood pressure later in life. Finally, information on birth weight and on the perinatal history of patients might provide clinically relevant cues to physicians managing hypertension in adults.Download figureDownload PowerPointInterferon Regulatory Factor 3 Modulates Atherosclerosis (p 510)Download figureDownload PowerPointAtherosclerosis is a chronic inflammatory disease in which endothelial dysfunction plays an important role. A major feature of endothelial dysfunction is enhanced adhesion molecule expression, leading to adherence and transmigration of inflammatory cells, including monocytes and macrophages. In this study, we demonstrate that IRF3 (interferon regulatory factor 3) plays an important role in this inflammatory response. IRF3 was found to promote endothelial secretion of vascular cell adhesion molecule-1 and expression of intercellular adhesion molecule-1 by directly binding to its gene promoter. IRF3 was found to be upregulated in endothelial cells in both human and mouse atherosclerotic plaques. Ablation of IRF3 decreased vascular infiltration of macrophages and ameliorated the development of atherosclerosis in apolipoprotein E–deficient mice. IRF3 ablation also improved the stability of atherosclerotic plaques. Because plaque stability is more significant than plaque size in predicting acute clinical events, IRF3 represents a promising target for the treatment of atherogenesis and for prevention of its untoward clinical outcomes.Proton Pump Inhibitors to Treat Preeclampsia (p 457)Preeclampsia is a major complication of pregnancy, responsible for 70 000 maternal deaths annually and far greater fetal losses. A treatment that can quench the disease process could be a major advance, saving the lives of many mothers and babies. Sadly, no treatment exists, except delivery. The preeclamptic placenta releases elevated levels of the antiangiogenic factors sFlt-1 (soluble fms-like tyrosine kinase-1) and soluble endoglin. They disseminate throughout the maternal circulation and cause widespread endothelial dysfunction and hypertension, which then leads to injury to multiple maternal organs. In this issue of Hypertension, Onda et al report preclinical studies showing proton pump inhibitors may have diverse biological effects that make them an unlikely candidate treatment for preeclampsia. Using many functional assays performed on primary human tissues, they show that proton pump inhibitors decrease placental (Figure) and endothelial secretion of sFlt-1 and soluble endoglin, and they decrease endothelial dysfunction. Furthermore, they found that the proton pump inhibitor esomeprazole vasodilates blood vessels isolated from pregnant women and potently decreases blood pressure in a transgenic animal model of preeclampsia where sFlt-1 is overexpressed in the placenta. Proton pump inhibitors are widely prescribed to pregnant women to relieve gastric acid reflux. Large epidemiological studies suggest that proton pump inhibitors are safe to give to pregnant women. Therefore, the exciting clinical implication is that proton pump inhibitors may have the potential to treat or prevent preeclampsia. The team has translated these findings into a phase II placebo-controlled clinical trial to see whether esomeprazole can be used to treat preterm preeclampsia.Download figureDownload PowerPoint Previous Back to top Next FiguresReferencesRelatedDetails March 2017Vol 69, Issue 3 Advertisement Article InformationMetrics © 2017 American Heart Association, Inc.https://doi.org/10.1161/HYPERTENSIONAHA.117.09012 Originally publishedMarch 1, 2017 PDF download Advertisement