Placental Soluble Fms-like Tyrosine Kinase-1 Research Articles

Regulation of uterus and placenta remodeling under high estradiol levels in gestational diabetes mellitus models†.

The present study aimed to investigate the regulation of placentas and uterus remodeling and involvement of E2 in gestational diabetes mellitus (GDM). To achieve this, we established in vitro and in vivo models for GDM placentas by culturing BeWo cells under hyperglycemic concentration, and treating pregnant rats with streptozotocin (STZ). We evaluated the expression of angiogenesis-related proteins. The expression of the anti-angiogenic factor, excess placental soluble fms-like tyrosine kinase 1 (sFLT1) was increased in our in vitro GDM model compared to the control. Moreover, the expressions of placental sFLT1 and the von willebrand factor (VWF) were also significantly elevated in the placenta of STZ-treated rats. These data indicate the disruption of angiogenesis in the GDM placentas. The expression levels of connexin 43 (Cx43), a component of the gap junction and collagen type I alpha 2 chain (Col1a2), a component of the extracellular matrix, were decreased in the GDM uterus. These results suggest that uterus decidualization and placental angiogenesis are inhibited in GDM rats. Our results also showed upregulation of the expression of genes regulating E2 synthesis as well as estrogen receptors (ER) in vivo models. Accordingly, the concentration of E2 measured in the culture medium under hyperglycemic conditions, as well as in the serum and placenta of the STZ-treated rats, was significantly elevated compared to the control groups. These results suggest that the dysregulated remodeling of the placenta and uterus may result in the elevation of E2 and its signaling pathway in the GDM animal model to maintain pregnancy.

INDICATORS OF ANGIOGENESIS IN PREGNANT WOMEN WITH ARTERIAL HYPERTENSION

The role of the factors of angiogenesis and hormones of pregnancy in pregnant women with hypertension has been studied inadequately and their interaction in such patients is not fully elucidated at present time. Aim. To detect peculiarities of angiogenesis factors and placental hormones in pregnant women with arterial hypertension I and II grades in comparison with results of healthy pregnant women. Materials and methods. Conducting a prospective study of 88 pregnant women, which included: analysis of clinical and anamnestic characteristics and study of the peculiarities of pregnancy in women with chronic hypertension in the first trimester of pregnancy (11-12 weeks). The open prospective controlled study involved 61 pregnant women with chronic arterial hypertension (CAH) 1-2 degree, who were included in the first - the main group. The second control group included 27 healthy pregnant women with physiological pregnancies. Among the angiogenesis indices, the placental growth factor (PlGF), as a pro-angiogenic factor and placental soluble fms-like tyrosine kinase (sFlt-1) was determined as an anti-angiogenic factor, sFlt-1/PlGF ratio was also estimated. Statistical analysis was done by using «STATISTICA® for Windows 6.0» (Stat Soft Inc., № AXXR712D833214FAN5). Results. In the first trimester of pregnancy (11-12 weeks of gestation) in women with CAH is determined by a shift in the balance between pro- and antiangiogenic factors, manifested by statistically significant (p˂0.05) increase in sFlt-1 levels (1700, 9 pg / ml) and a decrease in PlGF levels in3.7 times (9.1 pg / ml) and, accordingly, an increase in the levels of the coefficient K in 5.3 times (sFlt-1 / PlGF) (184.5). The mean values of estradiol, progesterone and chorionic gonadotropin levels in pregnant women with CAH did not differ statistically significantly from those of the control group (p˃0.05). These changes indicate the presence of angiogenesis disorders in pregnant women with chronic hypertension, starting from the first trimester of gestation. Conclusions. Pregnant women with CAH in І trimester have disturbances in balance between pro-angiogenic and anti-angiogenic factors with prevalence of sFlt-1 and reducing of PlGF in serum plasma. Due to such disbalance in pregnant women with CAH of 1 and 2 grade changed cooperation between angiogenesis factors and hormones from direct to inverse. It may lead to development of placental insufficiency in future. That’s why treatment/prophylaxis of it should be administered to pregnant women with CAH in 1 trimester.

Chemical optimization of siRNA for safe and efficient silencing of placental sFLT1

Preeclampsia (PE) is a rising, potentially lethal complication of pregnancy. PE is driven primarily by the overexpression of placental soluble fms-like tyrosine kinase 1 (sFLT1), a validated diagnostic and prognostic marker of the disease when normalized to placental growth factor (PlGF) levels. Injecting cholesterol-conjugated, fully modified, small interfering RNAs (siRNAs) targeting sFLT1 mRNA into pregnant mice or baboons reduces placental sFLT1 and ameliorates clinical signs of PE, providing a strong foundation for the development of a PE therapeutic. siRNA delivery, potency, and safety are dictated by conjugate chemistry, siRNA duplex structure, and chemical modification pattern. Here, we systematically evaluate these parameters and demonstrate that increasing 2′-O-methyl modifications and 5′ chemical stabilization and using sequence-specific duplex asymmetry and a phosphocholine-docosanoic acid conjugate enhance placental accumulation, silencing efficiency and safety of sFLT1-targeting siRNAs. The optimization strategy here provides a framework for the chemical optimization of siRNAs for PE as well as other targets and clinical indications.

PREGNANCY OUTCOMES IN WOMEN WITH EXTREMELY HIGH SFLT-1/PIGF RATIO: CASE SERIES

Preeclampsia (PE) is a multisystem disorder, usually defined as the development of hypertension and proteinuria after 20 weeks of pregnancy. The sFlt-1/PlGF ratio has been widely studied as a diagnostic and prognostic marker of preeclampsia and other manifestations of placental dysfunction. A sFlt-1/PlGF ratio greater than 85 for early PE, <34 weeks of gestation suggests a high risk of PE requiring close clinical monitoring. Our main aim was to evaluate the maternal and perinatal outcomes of pregnancies with an extremely high sFlt-1/PlGF ratio. The analysis included data on placental growth factor and soluble fms-like tyrosine kinase serum levels, measured during 2017–2020 in 128 pregnant women. Here we present 8 cases of women with a numerical ratio greater than 850. In all 100% of cases, the signs of obstetric angiogenic catastrophe requiring imminent delivery developed soon. We observed a trend for worsening perinatal outcomes in women with an extremely high sFlt-1/PlGF of ≥850.

Peculiarities of blood flow in the uterine arteries, factors of angiogenesis, hormonal profile and their relationships in pregnant women with hypertension

The objective: to assess and establish the relationships between Doppler blood flow indicators in the uterine arteries, angiogenesis factors and hormones of the mother-placenta-fetus system in pregnant women with arterial hypertension I-II degree.Materials and methods. A prospective study of 88 pregnant women at 11–12 weeks of gestation was carried out: 61 pregnant women with chronic arterial hypertension (CH) of the I–II degree (main group) and 27 healthy pregnant women with physiological pregnancy (control group). All patients have been examined: blood flow Doppler in the left and right uterine arteries – pulsation index (PI), resistance index (IR) and systolic-diastolic ratio (SDR). Chorionic gonadotropin (HCG), progesterone (PG), estradiol (E) and angiogenesis indices (placental growth factor (PlGF) as a pro-angiogenic factor) and placental-soluble fms-like tyrosine kinase (sFlt-1) as an anti-angiogenic factor and the ratio of sFlt-1/PlGF) were determined in blood. Statistical analysis was performed using the program “STATISTICA 13”.Results. There was no statistically significant difference in SDR and IP indicators between the groups. PI values in the right uterine artery in pregnant women of the main group were significantly higher than the indicator of healthy pregnant women (1.73 (1.65; 1.8) units versus 1.33 (1.1; 1.49) units, respectively). The PI values in the left uterine artery in pregnant women with CAH also significantly differed from those in women with the physiological course of pregnancy (1.7 (1.66; 1.79) units and 1.35 (1.22; 1.51) units, respectively). A significant correlation between SDR indicators and markers of angiogenesis and hormonal profile in women of the main group has not been established. In pregnant women with CAH, an inverse relationship was observed between PI and PlGF (mean strength R=-0.34 for PI in the right uterine artery and R= -0.41 for PI in the left uterine artery), respectively, between PI and sFlt-1/PlGF – direct relationship (average strength R=+ 0.37 for PI in the right uterine artery and R=+0.43 for PI in the left uterine artery). In women with the physiological course of pregnancy and childbirth, the PI value in the right uterine artery correlated with the sFlt-1/PlGF ratio (direct relationship) and PlGF level (feedback). PI in the left uterine artery had a direct relationship with the value of the sFlt-1/PlGF ratio. In pregnant women with CAH, the PlGF level decreases, while the PI value, on the contrary, increases.Conclusions. The significant differences between the indicators of SDS and IR in pregnant women with CAH and pregnant women without CAH have not been established. In women with CAH, significantly higher PI values were found in the right and left uterine arteries compared to those in women without CAH. The course of pregnancy in women with CAH is accompanied by a shift in the balance between pro- and anti-angiogenic factors, as evidenced by a statistically significant increase in sFlt-1 level against the background of a decrease in PlGF level, as well as a corresponding increase in the sFlt-1/PlGF ratio as compared with pregnant women without CAH. There are no correlations between indicators of SDR and IR in the uterine arteries, markers of angiogenesis and hormonal profile in pregnant women with and without CAH. Nevertheless, PI values in the right and left uterine arteries correlate with the PlGF level (feedback) and the sFlt-1/PlGF ratio (direct relationship) in pregnant women with CAH. This indicates the presence of disorders in this contingent of women in the I trimester of pregnancy.

First-trimester placental soluble fms-like tyrosine kinase 1 (sFlt-1) for the prediction of preeclampsia

The placental soluble fms-like tyrosine kinase 1 (sFlt-1) is a maternal biomarker strongly associated with preeclampsia (PE) in the second- and third-trimester of pregnancy. However, a controversy exists regarding its role in the first-trimester: the largest studies have reported no association with PE, while a recent systematic review suggests that first-trimester sFlt-1 is decreased in women who develop early-onset PE. Our goal is to estimate the predictive value of first-trimester sFlt-1 for the prediction of PE.

Factors of angiogenesis and placental hormones in pregnant women with arterial hypertension

The changes of the factors of angiogenesis and hormones of pregnancy in pregnant women with hypertension of I and II stages has been studied inadequately and their interaction in such patients is not fully elucidated at present time. Aim. To detect peculiarities of angiogenesis factors and placental hormones in pregnant women with arterial hypertension I and II grades in comparison with results of healthy pregnant women. Materials and methods. A one-time study of pregnant women was performed in pregnancy of 12–13 weeks in all groups. 88 pregnant women were inspected. The 1st basic group consisted of 28 pregnant women with AH of I grade; the 2nd group consisted of 33 pregnant women with AH of II grade; the comparison group included 27 women without AH. The following hormones were determined: HCG, PG, E. Among the angiogenesis indices, the placental growth factor (PGF), as a pro-angiogenic factor and placental soluble fms-like tyrosine kinase (sFlt-1) was determined as an anti-angiogenic factor, sFlt-1/PGF ratio was also estimated. Statistical analysis was done by using “Statistica for Windows 13” (StatSoft Inc., № JPZ804I382130ARCN10-J). Results. Pregnant women in 1st group were the youngest: the average age was 26.4 ± 0.8 years versus 33.8 ± 0.6 years in 2nd group and 32.0 ± 1.0 years in control group, P ˂ 0.001. PGF levels were decreased in both 1st (17.5 ± 3.4 pg/ml) and 2nd groups (13.5 ± 2.5 pg/ml) in comparison with results in 3rd group (46.1 ± 7.0 pg/ml), P ˂ 0.001. We didn’t find statistical differences between results of women with AH of I and II degree, P ˃ 0.05. Contrary to previous results, sFlt-1 levels were increased in both 1st and 2nd groups, P ˂ 0.05: 1703.1 ± 99.2 pg/ml in 1st group and 1673.7 ± 93.5 pg/ml in 2nd group versus 1384.4 ± 77.1 pg/ml in control group, but they weren’t different between basic groups, P ˃ 0.05. As a result, sFlt-1/PGF ratio level was increased in both 1st (273.3 ± 54.6 units) and 2nd (415.8 ± 79.0 units) groups in comparison with control (46.6 ± 9.4 units), P ˂ 0.001. We didn’t set statistical differences in hormones levels among pregnant patients. PGF level was connected with E (r = -0.54, P ˂ 0.01) and HCG (r = -0.42, P ˂ 0.05) levels in pregnant women with AH of I degree. It was connected with HCG level in control group, but this correlation was contrary: r = +0.63, P ˂ 0.001. Conclusions. Pregnant women with AH in I trimester have disturbances in balance between pro-angiogenic and anti-angiogenic factors with prevalence of sFlt-1 and reducing of PGF in blood serum. Due to such disbalance in pregnant women with AH of I grade correlation between angiogenesis factors and hormones changed from direct to inverse.

Assessment of the SFlt-1 and sFlt-1/25(OH)D Ratio as a Diagnostic Tool in Gestational Hypertension (GH), Preeclampsia (PE), and Gestational Diabetes Mellitus (GDM)

Background Placental soluble fms-like tyrosine kinase-1 (sFlt-1), an antagonist of vascular endothelial growth factor, is considered an etiological factor of endothelial damage in pregnancy pathologies. An increase in the sFlt-1 level is associated with alterations of endothelial integrity. In contrast, vitamin D exerts a protective effect and low concentrations of 25(OH)D may have an adverse effect on common complications of pregnancy, such as gestational hypertension (GH), preeclampsia (PE), and gestational diabetes mellitus (GDM). The aim of this study was to analyze the levels of sFlt-1 in Polish women with physiological pregnancies and pregnancies complicated by GH, PE, and GDM. Moreover, we analyzed relationships between the maternal serum sFlt-1 level and the sFlt-1 to 25(OH)D ratio and the risk of GH and PE. Material and Methods The study included 171 women with complicated pregnancies; among them are 45 with GH, 23 with PE, and 103 with GDM. The control group was comprised of 36 women with physiological pregnancies. Concentrations of sFl-1 and 25(OH)D were measured before delivery, with commercially available immunoassays. Results Women with GH differed significantly from the controls in terms of their serum sFlt-1 levels (5797 pg/ml vs. 3531 pg/ml, p = 0.0014). Moreover, a significant difference in sFlt-1 concentrations was found between women with PE and those with physiological pregnancies (6074 pg/ml vs. 3531 pg/ml, p < 0.0001). GDM did not exert a statistically significant effect on serum sFlt-1 levels. Both logistic regression and ROC analysis demonstrated that elevated concentration of sFlt-1 was associated with greater risk of GH (AUC = 0.70, p = 0.0001) and PE (AUC = 0.82, p < 0.0001). Also, the sFlt-1 to 25(OH)D ratio, with the cutoff values of 652 (AUC = 0.74, p < 0.0001) and 653 (AUC = 0.88, p < 0.0001), respectively, was identified as a significant predictor of GH and PE. Conclusions Determination of the sFlt-1/25(OH)D ratio might provide additional important information and, thus, be helpful in the identification of patients with PE and GH, facilitating their qualification for intensive treatment and improving the neonatal outcomes.

A cross sectional study to assess the sFlt-1:PlGF ratio in pregnant women with and without preeclampsia

BackgroundPreeclampsia is a multisystem disorder characterized by vascular endothelial malfunction occurring after 20 weeks of gestation. Placental soluble fms-like tyrosine kinase-1 (sFlt-1) is an antiangiogenic factor and placental growth factor (PlGF) is a potent angiogenic factor. The imbalance between these factors during placenta and fetal development has been shown to play a role in endothelial damage in preeclampsia.Preeclampsia is the leading cause of maternal mortality in Nepal. This study was designed to compare the sFlt1:PLGF ratio in pregnant women with and without preeclampsia attending Tribhuvan University Teaching Hospital (TUTH).MethodAn observational cross-sectional study was performed in the Gynecology and Obstetrics Department of TUTH involving forty-four subjects with preeclampsia and forty-four age- and gestational-week-matched normal pregnant subjects as controls. Blood pressure, urinary protein levels, serum sFlt-1 levels, serum PlGF levels and the sFlt-1:PlGF ratio was compared in both the cases and control. The concentrations of sFlt-1 and PlGF were measured with commercially available ELISA kits. SPSS ver. 20.0 was used to analyze the data.ResultsThere was no significant difference in age or gestational age in either study group. The ratio of the sFlt-1 and PlGF concentrations was significantly higher in women with preeclampsia (31.6 ± 9.6) than in the controls (3.2 ± 1.3). Likewise, diastolic blood pressure was significantly associated (p-value 0.000), whereas the severity of proteinuria was not associated (p-value 0.773) with the sFlt-1:PlGF ratio in women with preeclampsia. The significantly higher ratio (35.51 ± 8.1 versus 25.4 ± 8.7) was found in women with preeclampsia who developed complications than the group of women with preeclampsia who did not develop complication.ConclusionThe sFlt-1:PlGF ratio is significantly higher in Nepalese women with preeclampsia than in normal controls and this finding can be applied for further planned clinical trials.

Angiogenic Dysregulation in Pregnancy-Related Hypertension-A Role for Metformin.

In the face of escalating maternal and fetal health threats, hypertensive pregnancy disorders (HPDs) is one of the leading cause of maternal and fetal morbidity and mortality. The range of HPDs include white-coat hypertension, chronic hypertension, gestational hypertension, mild-to-moderate and severe preeclampsia and eclampsia. Current evidence implicates an imbalance of circulating anti- and angiogenic factors in HPDs emanating from the placental vasculature, impacting on angiogenesis. Delivery of the fetus is thus far the only curative measure, albeit with increased risk. Resultant endothelial dysfunction caused by the excessive production of placental soluble fms-like tyrosine kinase-1 has been the basis of many studies to find a safer treatment strategy. Metformin, used historically in the treatment of diabetes mellitus has also found its therapeutic reach in many other disease states. These include, but are not limited to, improving blood flow in certain cancer types, treatment of polycystic ovarian disease, improving vasodilation, and reducing inflammation. Metformin is used to treat hyperglycemic endothelial dysfunction through the enhancement of the nitric oxide system, endothelin-derived hyperpolarizing factor and sirtuin 1. Similarly, endothelial dysfunction in preeclampsia and other HPDs leads to a hypoxic state and elevated blood pressures. Dubbed as the new "aspirin" of current times, the retardation of the antiangiogenic status by metformin provides an exciting and promising alternate strategy in treating these pregnancy disorders.

Disulfiram inhibits placental soluble FMS-like tyrosine kinase-1 and soluble endoglin secretion independent of the proteasome.

Preeclampsia is associated with intermittent placental hypoxia, inflammation and the release of antiangiogenic factors, namely sFLT-1 and sEng. These factors cause maternal endothelial dysfunction and the manifestation of clinical disease. Disulfiram is a dehydrogenase inhibitor used to treat alcoholism and has been suggested as a proteasome inhibitor. Inhibiting the proteasome has been previously shown to reduce FLT-1 gene expression. Thus, we aim to investigate whether disulfiram alters the secretion of sFLT-1 and sEng and reduces endothelial dysfunction. METHODS AND RESULTS: We assessed the effects of disulfiram on primary cytotrophoblast and human umbilical vein endothelial cells (HUVECs). Disulfiram significantly reduced mRNA expression of membrane bound FLT-1 and sFLT-1 variants in primary cytotrophoblasts, which translated into a significant reduction in the protein secretion of sFLT-1. Additionally, sFLT-1 was reduced in primary HUVECs treated with disulfiram, whilst sEng was only reduced in primary cytotrophoblasts. Next, we investigated the effect of disulfiram on endothelial dysfunction using primary HUVECs treated with 5% preeclamptic serum ± disulfiram. Serum from preeclamptic women induced endothelial dysfunction evidenced by increased mRNA expression of vascular cell adhesion molecule-1 (VCAM-1) and adhesion of peripheral blood mononuclear cells (PBMCs) to HUVECs. The addition of disulfiram reduced VCAM-1 mRNA expression, however did not affect the adhesion of PBMCs to endothelial cells. Lastly, we assessed the effects of disulfiram on the 20S subunit of the proteasome and found disulfiram did not inhibit this subunit in either primary cytotrophoblast or HUVECs. CONCLUSIONS: Disulfiram quenches sFLT-1 and sEng via mechanisms independent of the 20S subunit of the proteasome. Understanding of the mechanisms by which disulfiram inhibits antiangiogenic secretion may reveal insights into the pathogenesis and potential therapeutic targets for preeclampsia.

Vitamin D Supplementation Prevents Placental Ischemia Induced Endothelial Dysfunction by Downregulating Placental Soluble FMS-Like Tyrosine Kinase-1.

Maternal vitamin D deficiency in pregnancy has been associated with an increased risk of preeclampsia. Vascular endothelial dysfunction is a major phenotype of pregnancies with preeclampsia, contributing to increased maternal hypertension and proteinuria. We sought to determine whether vitamin D supplementation would alleviate preeclampsia associated endothelial dysfunction and explore the underlying mechanism using the reduced uterine perfusion pressure (RUPP) rat model. RUPP operated rats were supplemented with 1,25(OH)2D (RUPP+VD) on day 1, 7, and 14 of pregnancy by subcutaneous injection. On day 19 of pregnancy, after the measurement of blood pressure and urine collection, maternal blood serum and placenta samples were collected. 1,25(OH)2D treatment significantly improved endothelial dysfunction by reducing apoptosis and increasing nitric oxide (NO) production in blood vessels of RUPP operated rats compared to untreated RUPP rats. 1,25(OH)2D significantly down-regulated the expression of placental soluble FMS-like tyrosine kinase-1 (sFlt-1) in RUPP rats. Furthermore, the circulating sFlt-1 levels in maternal serum were positively correlated with the expression of placental sFlt-1 and were restored to a normal pregnant level by 1,25(OH)2D treatment in RUPP rats. Incubation of endothelial cell line with rat serum from RUPP+VD group significantly increased NO production and decreased caspase-3 activity compared with serum from untreated RUPP rats. Moreover, neutralization of sFlt-1 using the specific antibody mimicked the effect of 1,25(OH)2D, which abolished the deleterious effect of RUPP rat's serum on NO production and apoptosis. These results suggest that vitamin D supplementation is protective against RUPP induced endothelial dysfunction by downregulating placental sFlt-1, which can possibly alleviate preeclampsia associated symptoms.

Placental soluble fms-like tyrosine kinase expression in small for gestational age infants and risk for adverse outcomes

IntroductionSoluble fms-like tyrosine kinase 1 (sFLT-1) is an anti-angiogenic factor implicated in the pathogenesis of preterm preeclampsia. We evaluated sFLT-1 expression and placental pathology in pregnancies complicated by small for gestational age (SGA) infants (<10th percentile), without evidence of preeclampsia. MethodsClinical and histologic data were compared between groups with high or low sFLT-1 expression determined by immunohistochemistry on archived placentas. ResultsNineteen of 69 placentas showed high sFLT-1 expression. The high sFLT-1 group had higher predelivery median systolic blood pressure (BP); 140 (interquartile range (IQR) 133–152) vs. 126 (118–139) mm Hg (p = 0.003), and median diastolic BP; 87 (78–94) vs. 77.5 (71–86) mm Hg (p = 0.02). Abnormal umbilical Doppler abnormalities were more prevalent; 89.5% vs. 46% (p = 0.001). These pregnancies delivered earlier; 31.9 weeks (28.3–34.7 weeks) vs. 37.1 weeks (33.7–38.7 weeks) (p < 0.001), and infants had lower birthweight; 980 grams (520–1545 grams) vs. 2087.5 grams (1455–2340 grams) (p < 0.001). Placental-weight to fetal-weight ratios, a marker of vascular insufficiency, was increased in the high sFlt-1 group: 0.18 (0.14–0.28) vs 0.15 (0.13–0.18), p = 0.03. Placentas with high sFLT-1 showed more decidual vasculopathy; 42.1% vs. 10.0% (p = 0.005), infarction; 36.8% vs. 14.0% (p = 0.048), distal villous hypoplasia; 78.9% vs. 36.0% (p = 0.001), and fetal thrombotic vasculopathy; 47.4% vs. 16.0% (p = 0.01). DiscussionPlacental sFLT-1 expression is upregulated in approximately 28% of non-preeclamptic pregnancies complicated by SGA infants. These pregnancies showed increased placental vascular pathology, more umbilical Doppler abnormalities, and earlier delivery with lower birthweight. A subgroup of non-preeclamptic fetal growth restriction with upregulated sFlt-1 expression may share a common pathogenic pathway with preterm preeclampsia. This subgroup is worthy of additional study.

Trophoblast mitochondrial function is impaired in preeclampsia and correlates negatively with the expression of soluble fms-like tyrosine kinase 1.

Preeclampsia is a common medical complication of pregnancy that is associated with significant morbidity and mortality to the mother and the baby. Extensive human epidemiological and experimental studies suggest that excess placental soluble fms-like tyrosine kinase 1, a potent anti-angiogenic factor leads to the maternal hypertension, proteinuria and other systemic complications of preeclampsia. To evaluate the mechanisms of the aberrant placental sFlt1 expression, we studied the role of mitochondrial dysfunction as one possible etiological factor. Here, using human placental samples, we demonstrate that both the activity and expression of a mitochondrial electron transport chain enzyme cytochrome C oxidase (COX) is diminished in the syncytiotrophoblast layer of the chorionic villi from preeclamptic subjects. In addition, there was an inverse correlation between mitochondrial COX enzyme activity and placental sFlt1 expression. Functional in situ enzyme chemistry with electron microscopy also confirmed impaired mitochondrial function in preeclampsia. Ultrastructural and morphometric analysis of mitochondria using electron microscopy demonstrated, that mitochondria are smaller in both the syncytiotrophoblast and cytotrophoblast layers of preeclamptic tissue. The etiology of the mitochondrial dysfunction in preeclampsia as a cause or effect of the placental anti-angiogenic state remains to be elucidated in future studies.

Metformin as a prevention and treatment for preeclampsia: effects on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion and endothelial dysfunction

Preeclampsia is associated with placental ischemia/hypoxia and secretion of soluble fms-like tyrosine kinase 1 and soluble endoglin into the maternal circulation. This causes widespread endothelial dysfunction that manifests clinically as hypertension and multisystem organ injury. Recently, small molecule inhibitors of hypoxic inducible factor 1α have been found to reduce soluble fms-like tyrosine kinase 1 and soluble endoglin secretion. However, their safety profile in pregnancy is unknown. Metformin is safe in pregnancy and is also reported to inhibit hypoxic inducible factor 1α by reducing mitochondrial electron transport chain activity. The purposes of this study were to determine (1) the effects of metformin on placental soluble fms-like tyrosine kinase 1 and soluble endoglin secretion, (2) to investigate whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion are regulated through the mitochondrial electron transport chain, and (3) to examine its effects on endothelial dysfunction, maternal blood vessel vasodilation, and angiogenesis. We performed functional (in vitro and ex vivo) experiments using primary human tissues to examine the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from placenta, endothelial cells, and placental villous explants. We used succinate, mitochondrial complex II substrate, to examine whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion were mediated through the mitochondria. We also isolated mitochondria from preterm preeclamptic placentas and gestationally matched control subjects and measured mitochondrial electron transport chain activity using kinetic spectrophotometric assays. Endothelial cells or whole maternal vessels were incubated with metformin to determine whether it rescued endothelial dysfunction induced by either tumor necrosis factor-α (to endothelial cells) or placenta villous explant-conditioned media (to whole vessels). Finally, we examined the effects of metformin on angiogenesis on maternal omental vessel explants. Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary endothelial cells, villous cytotrophoblast cells, and preterm preeclamptic placental villous explants. The reduction in soluble fms-like tyrosine kinase 1 and soluble endoglin secretion was rescued by coadministration of succinate, which suggests that the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin were likely to be regulated at the level of the mitochondria. In addition, the mitochondrial electron transport chain inhibitors rotenone and antimycin reduced soluble fms-like tyrosine kinase 1 secretion, which further suggests that soluble fms-like tyrosine kinase 1 secretion is regulated through the mitochondria. Mitochondrial electron transport chain activity in preterm preeclamptic placentas was increased compared with gestation-matched control subjects. Metformin improved features of endothelial dysfunction relevant to preeclampsia. It reduced endothelial cell messenger RNA expression of vascular cell adhesion molecule 1 that was induced by tumor necrosis factor-α (vascular cell adhesion molecule 1 is an inflammatory adhesion molecule up-regulated with endothelial dysfunction and is increased in preeclampsia). Placental conditioned media impaired bradykinin-induced vasodilation; this effect was reversed by metformin. Metformin also improved whole blood vessel angiogenesis impaired by fms-like tyrosine kinase 1. Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary human tissues, possibly by inhibiting the mitochondrial electron transport chain. The activity of the mitochondrial electron transport chain was increased in preterm preeclamptic placenta. Metformin reduced endothelial dysfunction, enhanced vasodilation in omental arteries, and induced angiogenesis. Metformin has potential to prevent or treat preeclampsia.

Difference of concentration of placental soluble fms-like tyrosine kinase-1(sFlt-1), placental growth factor (PlGF), and sFlt-1/PlGF ratio in severe preeclampsia and normal pregnancy.

BackgroundPlacental soluble fms-like tyrosine kinase-1 (sFlt-1) which is an antagonist of vascular endothelial growth factor and placental growth factor (PIGF), is considered as one of etiology factors cause endothelial damage in preeclampsia due to increase of sFlt-1 level that change vascular endothelial integrity. This study aims to analyze the difference of sFlt-1 and PlGF concentration in severe preeclampsia and normal pregnancy, and the correlation between both in occurrence of severe preeclampsia.MethodThis is case control study involving 18 subjects with severe preeclampsia and 19 subjects with normal pregnancy as controls who met inclusion and exclusion criteria. Concentration of sFlt-1 and PlGF are measured with ELISA. Statistical analysis is performed with Chi square test, Fisher’s exact test, T test, Mann–Whitney test, and Spearman’s rank correlation test.ResultsThis study results in no significant difference in characteristics of gestational age, and parity in both study groups. Median concentration of sFlt-1 in severe preeclampsia is higher (20,524.75 pg/mL) compared with normal pregnancy (6820.4 pg/mL). Concentration of PlGF is lower in severe preeclampsia (47 pg/mL) compared with normal pregnancy (337 pg/mL). sFlt-1 concentration is higher in severe preeclampsia compared to normal pregnancy. PlGF concentration is lower in severe preeclampsia compared to normal pregnancy. Ratio of sFlt-1 and PlGF concentration is significantly correlated in both severe preeclampsia and normal pregnancy.ConclusionsThere is a significant negative correlation between the concentration of sFLt-1 and PlGF in normal pregnancy.

English

Objective: Preeclampsia is still a problem in Indonesia due to the high maternal and neonatal morbidity and mortality. Placental soluble fms-like tyrosine kinase-1 (sFlt-1), an antagonist of vascular endothelial growth factor (VEGF), and placental growth factor (PIGF), is considered as one of the etiology factors that cause endothelial damage in preeclampsia due to increased sFlt-1 level modulating vascular endothelial integrity. This study aims to analyze sFlt-1 and sFlt-1 mRNA levels in severe preeclampsia and normal pregnancy, and the correlation between both in occurrence of severe preeclampsia. Methods: This is a cross-sectional analytic observational study involving 18 subjects with severe preeclampsia and 19 subjects with normal pregnancy as controls who met inclusion and exclusion criteria. Levels of sFlt-1 and sFlt-1 mRNA were measured with ELISA and RT PCR. Statistical analysis was performed with Chi square test, Fisher’s exact test, T-test, Mann-Whitney test, and Spearman’s rank correlation test. Results: This study showed no significant difference (p&gt;0.05) in characteristics of maternal age, gestational age, and parity in both study groups. Mean level of sFlt-1 mRNA in severe preeclampsia was higher (6.3404 pg/mL) compared to its level in normal pregnancy (5.9701 pg/mL). There is a not significant (p&gt;0.05) positive correlation between sFlt-1 mRNA with sFlt-1 level in normal pregnancy, and a not significant (p&gt;0.05) negative correlation between both level in severe preeclampsia. Conclusions: sFlt-1 mRNA levels in severe preeclampsia are higher than its level in normal pregnancy. There is no correlation between sFlt-1 mRNA level and sFLt-1 protein level in severe preeclampsia. There is a not significant positive correlation between sFlt-1 mRNA with sFlt-1 level in normal pregnancy, and a not significant negative correlation between both in severe preeclampsia. Keywords: Normal pregnancy, severe preeclampsia, sFlt-1 mRNA DOI: 10.15850/ijihs.v3n2.586

Heme Oxygenase-1 Is Not Decreased in Preeclamptic Placenta and Does Not Negatively Regulate Placental Soluble fms-Like Tyrosine Kinase-1 or Soluble Endoglin Secretion.

Elevated placental release of the antiangiogenic factors, soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG), is central to the pathophysiology of preeclampsia. It is widely accepted that heme oxygenase-1 (HO-1) is decreased in preeclamptic placenta and negatively regulates sFlt-1 and sENG production. We set out to verify these contentions. There was no difference in HO-1 mRNA or protein levels in preterm preeclamptic placentas (n=17) compared with gestationally matched controls (n=27). In silico analysis of microarray studies did not identify decreased placental HO-1 expression in preeclamptic placenta. Silencing HO-1 in primary trophoblasts did not affect sFlt-1 protein secretion after 24 or 48 hours. Silencing nuclear factor (erythroid-derived 2)-like 2 (transcription factor that upregulates HO-1) in trophoblasts also did not affect sFlt-1 secretion. Administering tin protoporphyrin IX dichloride (HO-1 inhibitor) or cobalt protoporphyrin (HO-1 inducer) into placental explants did not affect sFlt-1 or sENG secretion. Silencing HO-1 in 2 types of primary endothelial cells (human umbilical vein endothelial and uterine microvascular endothelial cells) significantly increased sFlt-1 secretion but not sENG secretion. However, HO-1 silencing selectively increased mRNA expression of sFlt-1 i13 (generically expressed sFlt-1 variant) but not of sFlt-1 e15a (sFlt-1 variant mainly expressed in placenta). Furthermore, adding tin protoporphyrin IX dichloride decreased sFlt-1, whereas adding HO-1 inducers (cobalt protoporphyrin, dimethyl fumarate, and rosiglitazone) either had no effect or increased sFlt-1 or sENG secretion (these trends are opposite to what is expected). We conclude that HO-1 expression is not decreased in preeclamptic placenta and HO-1 does not negatively regulate placental sFlt-1 and sENG secretion in placental or endothelial cells.

Increased ADAM10 expression in preeclamptic placentas is associated with decreased expression of hydrogen sulfide production enzymes

Placental soluble fms-like tyrosine kinase 1 (sFlt1) release is increased in preeclampsia, which might be associated with increased shedding of Flt-1 through a disintegrin and metalloproteinase 10 (ADAM10). Here, we explored whether ADAM10 expression is regulated by hydrogen sulfide (H2S) in placenta. Pro- and active-ADAM10 expression were significantly increased in preeclamptic placentas and inversely correlated to H2S production enzymes cystathionine β-synthase and cystathionine γ-lyase expression. H2S significantly suppressed pro- and active-ADAM10 expression whilst inhibited sFlt-1 release. Knockdown of ADAM10 caused a decrease in sFlt-1 release. Our data suggest that dysregulation of H2S production may contribute to excessive Flt-1shedding in preeclampsia.

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Objectives Upregulation of placental soluble fms-like tyrosine kinase 1 (sFlt1) contributes to the pathogenesis of preeclampsia. Our aim was to evaluate novel upstream pathways that regulate placental sFlt1 production. Methods We screened a library of natural compounds ( N = 502) in primary cytotrophoblasts and placental villous cultures and characterized molecules that had biological activity in the nanomolar range. sFlt1 production was assayed by ELISA. Immunoblot analysis, Co-immunoprecipitation, RT-PCR were employed in Characterizing the sFlt1, HIF AND hsp27 proteins and RNA. Pregnancy-induced hypertension rats were used as animal models. Results Here we report three compounds in the cardiac glycoside family, ouabain, gitoxigenin and digitoxin that inhibited placental sFlt1 production at nanomolar concentrations in vitro. We further characterized ouabain and demonstrate that it inhibits sFlt1 mRNA and protein expression in human placental cytotrophoblasts and explant cultures in a dose and time dependent manner. Ouabain down-regulated sFlt1 production by inhibiting hypoxia inducible factor 1 (HIF-1 α ) protein expression in the placenta. Furthermore, we found that phosphorylation of heat shock protein 27 (HSP27) was necessary for ouabain to inhibit HIF-1 α translation. In a rat model of pregnancy-induced hypertension, ouabain reduced mean arterial pressure and enhanced placental HSP27 phosphorylation without any adverse effects on pups. Conclusions Collectively we show that ouabain inhibits placental sFlt1 production through a HIF-1 α / HSP27 dependent pathway. Further studies are needed to explore the usefulness of targeting HIF-1 α /HSP27 pathway in preeclampsia. Disclosures S. Rana: None. A. Rajakumar: None. C. Geahchan: None. S. Salahuddin: None. A. Cerdeira: None. S.D. Burke: None. E. George: None. J. Granger: None. S. Karumanchi: co-inventor on multiple patents for preeclampsia markers and reports service as a consultant to Siemens Diagnostics, and has financial interest in Aggamin LLC.