Preeclampsia Serum Research Articles

Altered serum soluble furin and prorenin receptor levels in pregnancies with pre-eclampsia and fetal growth restriction

ObjectiveThe proprotein convertase furin is known to be involved in the processing of pro-B-type natriuretic peptide (proBNP) and prorenin receptor (PRR), suggesting that it has a potential function in blood pressure regulation. We investigated the role of furin in the etiology of pre-eclampsia and its related disorder, unexplained fetal growth restriction (FGR) without hypertension. MethodsWe evaluated serum and placental furin levels in pre-eclampsia, FGR and uncomplicated pregnancy. Additionally, we investigated the correlation between the serum furin levels and products of furin enzymatic activity or clinical parameters. ResultsWe demonstrated that the maternal circulation in cases of pre-eclampsia and FGR had lower levels of soluble furin than uncomplicated pregnancies. Both NT-proBNP and soluble PRR were elevated in pre-eclampsia, whereas only soluble PRR was at higher levels in unexplained FGR. Linear regression analysis revealed a negative correlation between the serum furin level and that of NT-proBNP or soluble PRR. While we observed that the serum furin or soluble PRR level correlated with blood pressure, a stronger correlation was observed with birth and placental weights. Further to this, the FURIN mRNA levels were significantly reduced in placental pre-eclamptic placentas as well as in FGR cases. ConclusionThese data suggest the possibility that reduced levels of furin may be the result of a negative feedback from the activation of the renin-angiotensin pathway that leads to feto-placental dysfunction with or without maternal hypertension. This may represent an etiologic pathway of pre-eclampsia and unexplained FGR.

Model for Early Prediction of Preeclampsia: A Nested Case Controlled Study in Indian Women.

Preeclampsia (PE) affects 5-7% of the pregnancies worldwide, and is one of the most dreaded disorders of pregnancy contributing to maternal and neonatal mortality. PE is mostly presented in the third trimester of pregnancy. Here, we used serum placental growth factor (PIGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) to develop a model for predicting PE in Indian women in early second trimester. In this case-control study, a total 1452 healthy pregnant women were recruited. Blood samples were collected at the following gestational weeks (GWs), 12-20 (GW1), 21-28 (GW2) and 29-term (GW3), and post-delivery. Body mass index (BMI) was calculated by anthropometric measurements. Serum sFlt-1, PIGF and VEGF were analyzed by ELISA. A predictive model for PE was developed using multivariable logistic regression analysis. In PE cases, serum PlGF and VEGF levels were significantly lower at each GW, while serum sFlt-1 was lower only at GW1, relative to age-matched controls, (n = 132/group). Age-matched comparison between PE cases and controls indicated that sFlt-1 was associated with decreased PE outcome (Odds ratio. OR = 0.988, CI = 0.982-0.993), whereas sFlt-1/PlGF ratio (OR = 1.577, CI = 1.344-1.920) and BMI (OR = 1.334, CI = 1.187-1.520) were associated with increased PE outcome. Logistic regression was used to develop a predictive model for PE at GW1. Using testing dataset, model was externally validated which resulted in 88% accuracy in predicting PE cases at 0.5 probability cutoff. Prediction model using sFlt-1, sFlt-1/PlGF ratio and BMI may be useful to predict PE as early as 12-20weeks in women with optimal sensitivity and specificity.

Tissue Transglutaminase Impairs HTR-8/SVneo Trophoblast Cell Invasion via the PI3K/AKT Signaling Pathway

Objectives: The pathogenesis of preeclampsia (PE) is associated with impaired trophoblast invasion, which results in placental insufficiency. Our earlier studies demonstrated that tissue transglutaminase (tTG) is highly expressed in human PE serum. However, whether tTG participates in trophoblast invasion remains unclear. The aim of the present study was to determine the role and mechanism of tTG in regulating matrix metalloproteinase (MMP)-2/MMP-9 expression to reduce trophoblast invasiveness in PE. Methods: HTR-8/SVneo cells were transfected with a lentivirus vector and small interfering RNA targeting tTG. The protein level was detected by Western blotting. Cell proliferation and apoptosis were assessed by MTS and flow cytometry assays, respectively. Cell invasion was investigated by Transwell assay. In addition, the influence of tTG on PI3K and AKT mRNA levels in HTR-8/SVneo cells was evaluated using reverse transcription-quantitative PCR. Results: tTG-overexpression inhibited HTR-8/SVneo cell proliferation and invasion and promoted apoptosis. In addition, upregulation of tTG induced an increase of PI3K and phosphorylated AKT and a decrease of MMP-2 and MMP-9 expression. tTG-knockdown significantly promoted the proliferation and invasion of HTR-8/SVneo cells and inhibited the apoptosis. Furthermore, the PI3K expression level was reduced, and the MMP-2/MMP-9 protein levels were increased. Conclusion: Taken together, the present study demonstrated that tTG-overexpression inhibited HTR-8/SVneo cell invasion via reducing the expression of MMP-2 and MMP-9 by activating PI3K/AKT signaling pathway, which may lead to the occurrence or development of PE. The present data provide new insights into modulation of tTG expression as a potential therapeutic target for PE.

Association between Serum C-reactive Protein and Pre-eclampsia

Among the common disorders of pregnancy, Pre-eclampsia is important one which causes significant maternal and perinatal morbidity and mortality. Its incidence is still high in the developing countries. The triad of high blood pressure, edema and albuminuria is neither specific nor sensitive enough; therefore, a reliable biochemical marker is needed to solve the problem. C-reactive protein(CRP), a marker of tissue damage and inflammation, is elevated in serum in overt preeclampsia. The present study is aimed to explore the association of high maternal serum C-reactive protein (CRP) level with preeclampsia and correlation with the severity of pre-eclamptic process. A total of 60 pregnant women constituting 30 pre-eclamptic (case) and 30 normal (control) pregnant women in the third trimester were enrolled in this study. Both the groups were matched for their age, parity and other baseline characteristics. More than three quarters (76.70%) of the case group exhibited raised serum CRP, which was 20% in control group (p=0.001). CRP was elevated about 13 fold higher than that in the normal pregnant women. The mean systolic and diastolic blood pressure were significantly higher in case group (154±12 mm of Hg) vs (107±7 mm of Hg) in control group (p<_0.001) and serum level of CRP bears linear relationship with both systolic and diastolic blood pressures. Preeclamptic women with higher serum CRP level were at a significantly (p<0.001) lower gestational age than control. Twenty two (73.30%) cases had gestational age <37 weeks (p=0.302) and 66.70% control group had gestational age > 37 weeks. The hypothesis of the study was supported by the study findings that maternal CRP concentration was higher in women with preeclampsia and was correlated with disease progression as evidenced by the investigative analysis. Faridpur Med. Coll. J. Jan 2020;15(2): 58-61

AT1‐AAs cause Vascular Endothelial Mitochondrial Oxidative Stress associated with Preeclampsia

Preeclampsia (PE) is characterized by new onset hypertension during pregnancy and is associated with autoantibodies to the angiotensin II type I receptor (AT1-AA) and oxidative stress. There is growing evidence for mitochondrial (mt) dysfunction in PE, however the causes for mt dysfunction are still being identified. Although we have demonstrated a role for AT1-AA in renal and placental mt dysfunction in pregnant rats, the role of AT1-AA in mt dysfunction in PE patients is unknown. We hypothesized that PE placentas exhibit impaired mt function and that circulating factors, such as the AT1-AA, cause endothelial mt dysfunction. To test our hypothesis, placentas and sera were collected from PE patients and normal pregnant (NP) controls immediately after delivery. Mt were isolated using differential centrifugation and respiration and ROS were measured using an Oxygraph-2K and flow cytometry. AT1-AA inhibitor peptide (n’7AAc’) was used to examine a role for the autoantibody to cause endothelial mt dysfunction in response to PE sera. A student's t-test was used for statistical analysis. Mean arterial pressure in PE patients was higher in both PE<34 weeks (110±6.04 mmHg, n=6, p<0.05) and PE>34 weeks (100.2±4.52 mmHg, n=4) compared to NP controls (94.5±2.67 mmHg, n=8). Placental mt maximal respiration was reduced in both PE<34 (333.5±89.8 pmol/sec/mg, n=4) and PE>34 (168.2±51.32 pmol/sec/mg, n=5) compared to NP (457.1 ±131.4 pmol/sec/mg, n=7). MtROS in PE placentas was reduced in both PE<34 (26.18±3.1 % gated, n=4, p<0.05) and PE>34 (71.19±2.99 % gated, p<0.05, n=4) compared to NP (100±6.05 % gated, n=4). HUVECs incubated with 10% sera from PE patients exhibited reduced maximal respiration in both PE<34 weeks (20.40±3.83 pmol O2/million cells, n=6, p<0.05) and PE >34 (46.5±17.13, pmol O2/million cells, n=4)] compared to HUVECs treated with NP sera (49.11±8.61, pmol O2/million cells, n=8) and increased mt ROS in both PE<34 weeks (93.25±1.54 % gated, n=6, p<0.05) and PE>34 weeks (88.25±1.71 % gated, n=4) compared to NP controls (83.13±3.38 % gated, n=8). Inhibition of AT1-AA activity via the inhibitor peptide, ‘n7AAc’, reduced mtROS with PE sera in both PE<34 weeks to 82.7±0.17, % gated, n=3, (p<0.05) and PE >34 to 77.4±1.21, % gated, n=3, (p<0.05) compared to HUVECS without ’n7AAc’. Collectively, PE mt dysfunction occurs in the placenta and in response to circulating factors which can be normalized by blockade of circulating AT1-AA, indicating a potential new therapeutic target that can be utilized to better treat PE.

Relation of Serum and Salivary Total Sialic Acid and Total Sialic Acid/Total Protein Ratio with Clinical Parameters of Disease Progression in Preeclampsia.

Sialic acid is a terminal component of carbohydrate chains of glycoproteins and glycolipids. The present study estimated total sialic acid (TSA) and its ratio with total proteins (TP), in serum and saliva of preeclampsia. The study further investigated the association of these parameters with clinical variables of disease progression. 50 preeclampsia patients (32 mild preclampsia and 18 severe preeclampsia cases) and 50 pregnant controls were included in the study. Serum and salivary free sialic acid, protein bound sialic acid and TP were measured spectrophotometrically. Serum and salivary TSA and its ratio with TP were calculated. There was a significant increase in serum TSA and its ratio with TP in preeclampsia compared to the controls. The increase reflected with the severity of the disease. Serum TSA and TSA/TP showed a significant positive correlation with blood pressure, proteinuria and a significant negative correlation with infant birth weight. In saliva, there was no statistical difference between TSA in preeclampsia and controls. Salivary TSA/TP increased significantly in preeclampsia. However the increase was not in accordance to the disease severity. Salivary TSA and TSA/TP were not significantly associated with any of the clinical parameters of disease progression. Significant increase in seum TSA reflects the disturbance in sialyation of serum proteins in preeclampsia, that could not be depicted in the saliva of these patients. Disturbance in serum protein sialyation is further exaggerated with the severity of the disease. Serum TSA and TSA/TP and not the respective salivary parameters, could serve as useful indicators in assessment of clinical progression of the disease.

Expression and significance of Parkinson disease protein 7 in placental, serum and umbilical cord blood in preeclampsia.

To study the role of changes in the expression of human Parkinson's disease protein 7 (PARK7/DJ-1) in preeclampsia. We selected 120 gravidas, including 60 cases of severe preeclampsia group and control group, and divided into early onset preeclampsia group (< 34 weeks), late onset preeclampsia group (≥ 34 weeks) and control group according to the onset of pregnancy. The expression level of DJ-1 was detected by ELISA. The expression level of DJ-1 in placenta tissue of gravidas was detected by Western-blot and RT-PCR. The level of DJ-1 in serum and cord blood of preeclampsia group was higher than that of control group. The relative level of DJ-1 protein and DJ-1 mRNA in placenta tissue of preeclampsia group was higher than that of control group. The expression level of DJ-1 in serum, umbilical cord blood and placenta tissue increased in preeclampsia patients, suggesting that DJ-1 may take part in the pathophysiology process of preeclampsia.

Preeclampsia serum increases CAV1 expression and cell permeability of human renal glomerular endothelial cells via down-regulating miR-199a-5p, miR-199b-5p, miR-204

IntroductionTo gain insight into mechanisms of preeclampsia (PE)-dependent proteinuria, this study focused on whether preeclampsia serum (PES) could induce hyperpermeability in human renal glomerular endothelial cells (HRGECs) via the miRNAs-Caveolin-1 (CAV-1)-dependent pathway. MethodsBioinformatics approach was used to identify miRNAs targeting CAV1. Normal pregnancy serum (NPS) and severe PES were used to treat HRGECs monolayer to demonstrate if PES could induce the expression of identified miRNAs. A luciferase reporter assay was used to determine whether CAV1 was a direct target of miR-199a-5p, miR-199b-5p, and miR-204. The relationship between the expression of miR-199a-5p, miR-199b-5p, miR-204, and CAV1 in HRGECs was determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. The gain-of-function and loss‐of‐function experiments were performed on HRGECs to investigate the effects of miR-199a-5p, miR-199b-5p, miR-204 on HRGECs permeability. ResultsWe identified that CAV1 3′UTR has putative binding sites for miR-199a-5p, miR-199b-5p, and miR-204, whereas miR-199a-5p does not appear to be a direct regulator of CAV1. We detected that PE serum downregulated the expression of miR-199a-5p, miR-199b-5p and miR-204, increased expression of CAV1 and increased cell monolayer permeability in HRGECs. The level of CAV1 and permeability decreased when miR-199b-5p or miR-204, but not miR-199a-5p, were overexpressed. DiscussionmiR-199b-5p and miR-204 may play a role in PES-induced increasing permeability of HRGECs by regulating CAV1 expression.

MicroRNA-27a inhibits trophoblast cell migration and invasion by targeting SMAD2: Potential role in preeclampsia.

Preeclampsia (PE) is a severe idiopathic obstetric complication that occurs worldwide. Insufficient trophoblast invasion is a characteristic of the pathogenesis of PE. MicroRNA-27a (miR-27a) has been reported to be highly expressed in PE placentas. The aim of the present study was to investigate the role and underlying mechanisms of miR-27a in the pathogenesis of PE. The expression level of miR-27a was evaluated in the placenta and serum from patients with PE and healthy pregnant women. Cell Counting Kit-8 and flow cytometry assays were performed to detect human HTR-8/SVneo trophoblast proliferation and apoptosis after miR-27a overexpression or inhibition. In addition, Transwell assays were used to measure cell migration and invasion. A luciferase reporter assay was performed to determine the interaction between miR-27a and SMAD2. The present results suggested that miR-27a expression level was significantly increased in PE placentas and serum. In addition, miR-27a overexpression suppressed cell migratory and invasive abilities, impaired proliferation and promoted apoptosis in human trophoblasts. It was demonstrated that miR-27a may target SMAD and contribute to trophoblast invasion. Collectively, the results of the present study suggested that miR-27a inhibited trophoblast cell migration and invasion by targeting SMAD2, thus presenting a promising therapeutic target for PE.

Expression imbalance of IL-17/IL-35 in peripheral blood and placental tissue of pregnant women in preeclampsia

ObjectivesThe possible mechanism of preeclampsia is investigated in this study to facilitate the exploration of the future remediation of this disease by analysing the changes of IL-17 and IL-35 in peripheral blood and placental tissue of pregnant women with preeclampsia (PE). Materials and methodsThe study was conducted using 45 healthy pregnant women as the control group and 90 pregnant women in the preeclampsia group, including 45 cases with severe preeclampsia and 45 cases with mild preeclampsia. All of 135 pregnant women underwent caesarean delivery. IL-17 and IL-35 concentrations in the serum were measured by ELISA, and IL-17 and IL-35 expression in placental specimens was detected by immunohistochemistry. ResultsThere were no statistically significant differences in age among the three study groups. Serum IL-17 levels were significantly higher in PE patients than in healthy pregnant women (P < 0.01). The ratio of positive staining for IL-17 was markedly higher in mild PE tissues (84.44%; 38/45) and severe PE tissues (86.67%; 39/45) than in healthy pregnant tissues (35.56%; 16/45) (P < 0.01). The strong positive rates for IL-17 were markedly higher in mild PE tissues (48.89%; 22/45) and severe PE tissues (68.89%; 31/45) than in healthy pregnant tissues (13.33%; 6/45) (P < 0.01). No differences between mild PE tissues and severe PE tissues were noted in both positive case rates and strong positive rates. Consistent with this finding, the ratio of strong positive staining for IL-35 was higher in healthy pregnant tissues (66.67%; 30/45) than in mild PE tissues (33.11%; 14/45) and severe PE tissues (26.67%; 12/45) (P < 0.01). ConclusionsThe abnormal increase in serum and placental of IL-17 has an association with the formation and development of PE. IL-35 expression is significantly lower in severe PE placenta tissue and serum compared with normal pregnant women. These results suggested that IL-17/IL-35 imbalance may play a role in the pathophysiology of PE.

Evidence for lysosomal biogenesis proteome defect and impaired autophagy in preeclampsia

ABSTRACT The etiology of preeclampsia (PE), a serious pregnancy complication, remains an enigma. We have demonstrated that proteinopathy, a pathologic feature of neurodegenerative diseases, is a key observation in the placenta and serum from PE patients. We hypothesize that the macroautophagy/autophagy machinery that mediates degradation of aggregated proteins and damaged organelles is impaired in PE. Here, we show that TFEB (transcription factor EB), a master transcriptional regulator of lysosomal biogenesis, and its regulated proteins, LAMP1, LAMP2, and CTSD (cathepsin D), were dysregulated in the placenta from early and late onset PE deliveries. Primary human trophoblasts and immortalized extravillous trophoblasts (EVTs) showed reduced TFEB expression and nuclear translocation as well as lysosomal protein content in response to hypoxia. Hypoxia-exposed trophoblasts also showed decreased PPP3/calcineurin phosphatase activity and increased XPO1/CRM1 (exportin 1), events that inhibit TFEB nuclear translocation. These proteins were also dysregulated in the PE placenta. These results are supported by observed lysosomal ultrastructural defects with decreased number of autolysosomes in hypoxia-treated primary human trophoblasts. Autophagy-deficient human EVTs exhibited poor TFEB nuclear translocation, reduced lysosomal protein expression and function, and increased MTORC1 activity. Sera from PE patients induced these features and protein aggregation in EVTs. Importantly, trophoblast-specific conditional atg7 knockout mice exhibited reduced TFEB expression with increased deposition of protein aggregates in the placenta. These results provide compelling evidence for a regulatory link between accumulation of protein aggregates and TFEB-mediated impaired lysosomal biogenesis and autophagy in the placenta of PE patients. Abbreviation: atg7: autophagy related 7; CTSD: cathepsin D; ER: endoplasmic reticulum; EVTs: extravillous trophoblasts; KRT7: keratin 7; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; mSt: mStrawberry; MTORC1: mechanistic target of rapamycin complex 1; NP: normal pregnancy; NPS: normal pregnancy serum; PE: preeclampsia; PES: preeclampsia serum; p-RPS6KB: phosphorylated ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; XPO1/CRM1: exportin 1

Preeclamptic patient-derived circulating cell-free DNA activates the production of inflammatory cytokines via toll-like receptor 9 signalling in the human placenta.

Preeclampsia, a pregnancy-specific syndrome, is associated with maternal systemic and placental inflammatory responses. Cell-free DNA (cfDNA) and cf-foetal DNA (cffDNA) in the blood are elevated in patients with preeclampsia and act as danger signals. Placenta-derived foetal DNA induces inflammatory responses and pregnancy complications in mice. However, whether extracellular DNA from the placenta really causes inflammatory responses remains unclear. Therefore, we investigated the effect of serum cfDNA and placental cffDNA on inflammatory responses using normal pregnant women and preeclampsia patients. Sera were taken from normal pregnant women and preeclampsia patients, and human trophoblast cell line Sw.71 cells were treated with serum with or without toll-like receptor 9 (TLR9; a sensor of exogenous DNA) inhibitor and genome elimination reagent. For cffDNA collection, placental tissue from the participants was cultured, and the released cffDNA was administrated to Sw.71 cells. The amount of serum cfDNA was higher in preeclampsia patients than in normal pregnant women. Treatment of preeclampsia serum stimulated inflammatory cytokine secretion, which was inhibited by a genome elimination reagent. Expression levels of TLR9 and amount of cffDNA from the placenta were higher in preeclampsia patients than of normal pregnant women. Preeclampsia-derived cffDNA increased inflammatory cytokine levels compared with normal pregnant derived cffDNA. In human trophoblast cells, preeclampsia patient-derived cfDNA increased inflammatory cytokine levels via TLR9. Preeclampsia placenta released more cffDNA, which stimulated inflammatory cytokine. We suggest that elevated circulating cfDNA and cffDNA induces placental inflammatory responses, resulting in accelerated pathological features of preeclampsia.

PERBEDAAN RERATA RASIO KALSIUM MAGNESIUM DAN RERATA RASIO NATRIUM KALIUM SERUM

The high incidence of preeclampsia and eclampsia causes the importance of early detection especially eclampsia which is the main cause of maternal morbidity and mortality and bad perinatal outcome. The etiology was unknown, but is related to changes in electrolyte status. Electrolytes such as calcium (Ca2+), Magnesium (Mg2+), sodium (Na+) and potassium (K+) play an important role in pre-eclampsia and eclampsia because they contribute significantly in vascular smooth muscle function. This study was done to analyze the differences in mean levels of calcium magnesium ratio and sodium potassium ratio of maternal serum in severe preeclampsia and eclampsia. We performed an observasional comparative with cross sectional study on 16 women with severe preeclampsia and 16 women with eclampsia who met the inclusion criteria and there were no exclusion criteria. The samples were recruited in Dr. M Djamil general hospital Padang, Solok District Hospital, and Pariaman District Hospital from May 2015 to January 2016. The levels of calcium serum were examined by atomic absorption spectrophotometry (AAS), magnesium levels were examined by enzymatic metode, sodium and potassium levels were examined by ion selection electrode (ISE). The differences in mean levels of calcium magnesium ratio and sodium potassium ratio between the two groups was analyzed by using independent t test. The mean levels of calcium magnesium ratio in severe preeclampsia was significantly higher than eclampsia. The mean levels of sodium potassium ratio in severe preeclampsia was significantly lower than eclampsia.Keywords: Calcium magnesium ratio, sodium potassium ratio, severe preeclampsia, eclampsia

Internalization and Transportation of Endothelial Cell Surface KCa2.3 and KCa3.1 in Normal Pregnancy and Preeclampsia.

Altered redox state modulates the expression levels of endothelial KCa2.3 and KCa3.1 (KCas) in normal pregnancy (NP) and preeclampsia (PE), thereby regulating vascular contractility. The mechanisms underlying KCas endocytosis and transportation remain unknown. We investigated the regulation of KCas expression in plasma membrane (PM) during NP and PE. Cultured human uterine artery endothelial cells were incubated in serum from normal nonpregnant women and women with NP or PE, or in oxidized LDL-, or lysophosphatidylcholine- (LPC-) containing a medium for 24 hours. NP serum elevated PM levels of KCas and reduced caveolin-1 and clathrin levels. PE serum, oxidized LDL, or LPC reduced PM levels of KCas and elevated caveolin-1, clathrin, Rab5c, and early endosome antigen-1 (EEA1) levels. Reduced KCas levels by PE serum or LPC were reversed by inhibition of caveolin-1, clathrin, or EEA1. Catalase and glutathione peroxidase 1 (GPX1) knockdown elevated PM-localized KCas levels and reduced caveolin-1 and clathrin levels. Elevated KCa2.3 levels upon catalase and GPX1 knockdown were reversed by PEG-catalase treatment. An H2O2 donor reduced clathrin and Rab5c. In contrast, elevated clathrin, caveolin-1, or colocalization of caveolin-1 with KCa3.1 by PE serum or LPC was reversed by NADPH oxidase inhibitors or antioxidants. A superoxide donor xanthine+xanthine oxidase elevated caveolin-1 or Rab5c levels. We concluded that KCas are endocytosed in a caveola- or a clathrin-dependent manner and transported in a Rab5c- and EEA1-dependent manner during pregnancy. The endocytosis and transportation processes may slow down via H2O2-mediated pathways in NP and may be accelerated via superoxide-mediated pathways in PE.

Decreased Ang-(1–7) and Downregulated Intrarenal RAS May Contribute to the Direct Podocyte Injury With Proteinuria in Preeclampsia

The mechanisms of proteinuria development in preeclampsia (PE) are still enigmatic. Renin-angiotensin system (RAS) components may play a role. Maternal serum and urinary concentrations of angiotensin-(1-7) [Ang-(1-7)], angiotensin II (Ang II), and angiotensinogen in women with PE (n - 14), gestational hypertension (n - 14), and normal pregnancy were quantified. The alteration in these concentrations was used to evaluate their relationships with podocyturia and proteinuria in PE. In addition, the podocytes cultured in vitro were interfered in serum of preeclamptic and normotensive pregnant women, with or without Ang- (1-7). The morphologic change in podocyte was observed using a microscope. The changes in podocyte-specific proteins (nephrin, CD2-associated protein [CD2AP]), the cytoskeletal protein F-actin, the tight junction protein (ZO-1), and Mas receptor (MasR) were examined by immunofluorescence. Western blot was used to examine the expression and variation of MasR. We found that the concentrations of RAS components were associated with prepartal urinary podocyte number, random urine albumin/creatinine ratio, blood pressure, and renal function. The expression of nephrin, F-actin, ZO-1, and MasR on podocytes interfered in serum of PE was significantly decreased compared to normal control and normal pregnant serum group in vitro, yet their expression was significantly increased after coculture by 10-6 mol/L Ang-(1-7) and the preeclamptic serum. The expression of CD2AP had no significant difference. We concluded that decreased Ang-(1-7) and downregulated intrarenal RAS contributed to the direct podocyte injury with proteinuria in PE.

Status of sFlt‐1 in Pregnant Women with Preeclampsia and its Impact on GRP78 in Trophoblast Cells

IntroductionsFlt‐1 is secreted in large amounts by placental trophoblasts, released into maternal circulation and reduces bioavailability of angiogenic molecule, VEGF. Plenty of evidence suggest that production of excess sFlt‐1 is a consequence of placental hypoxia leading to stressed placenta noted in preeclampsia (PE). Placental stress has been categorized into oxidative, immunologic and endoplasmic reticulum (ER) stress. Placental ER stress arises due to failure of unfolded protein response (UPR) affects its central regulator, GRP78, which dissociates from transmembrane sensors, PERK like ER kinase, IRE1 (Inositol‐Requiring Enzyme 1) and ATF6 (Activated Transcription Factor 6). The present study was aimed to unravel whether sFlt‐1 had any correlation with GRP78. Also, in vitro experiments were performed to explore effect of sFlt‐1 on GRP78.MethodsSera from 60 recruited women [(30 PE patients and 30 Normotensive, Non‐proteinuric (NT, NP; controls)] were used to estimate levels of sFlt‐1 and GRP78 proteins (ELISA; R and D system). Trophoblast cells (BeWo cells; human choriocarcinoma cell line) were subjected to various treatments with varying sFlt‐1 concentrations and expression of GRP78 was analysed using immunofluorescence (IF), western blot and qRT‐PCR at 8, 14 and 24 hours. The BeWo cells of group 1 and group 2 were exposed to sera from normotensive pregnant women (low sFlt‐1), and preeclamptic pregnant women (high sFlt‐1). The BeWo cells of group 3 were exposed to normotensive sera with recombinant sFlt‐1 (NT+re‐sFlt‐1). Statistical tests used were Paired t, Pearson correlation coefficient, One‐way ANOVA, Kruskal‐Wallis, Wilcoxon signed‐rank.ResultsSerum sFlt‐1 levels were found to be significantly elevated in women with preeclampsia [11295.25 (2936.2–37818) pg/ml] than in controls [2936.2 (1180.43–6706.6) pg/ml] (p=0.0001). GRP78 levels were also raised significantly in PE patients (1103.26 ± 104.27) ng/ml as compared to controls (1018.61 ± 125.51) ng/ml (p=0.023). Patients with increased sFlt‐1 in their sera had higher GRP78 levels suggesting a positive correlation. Significant expression of GRP78 was observed at 8 h when BeWo cells were treated with PE sera and NT sera with recombinant sFlt‐1 by IF. GRP78 expression was observed as early as 8 h, reached peak at 14 h, started diminishing after 14 h and was significantly upregulated when BeWo cells were treated with PE sera as compared to NT sera at both protein (p&lt;0.0001) and mRNA (p&lt;0.0001) levels. Expression of GRP78 was significantly raised at 8 and 14 h when BeWo cells were treated with NT+re‐sFlt‐1 as compared to NT sera at both protein (p&lt;0.0001) and mRNA (p&lt;0.0001) levels.ConclusionPositive correlation was observed between increased maternal serum levels of sFlt‐1 and GRP78 in preeclamptic patients. Upregulation of GRP78 in trophoblast cells exposed to group 2 and 3 indicate towards an important role which may be played by sFlt‐1 in inducing ER stress in Preeclampsia.Ethical clearance IESC/T‐323Support or Funding InformationA‐159 AIIMS, New DelhiThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vascular endothelial growth factor alleviates endoplasmic reticulum stress via PERK, IRE1, and ATF6 pathways in trophoblast cells

Introduction: In developing countries, preeclampsia is still a leading cause of maternal mortality and morbidity, affecting nearly 8%–10% of pregnancies and overall death. Apart from various stresses that placenta undergoes while pregnancy, endoplasmic reticulum (ER) stress has been the center of attraction for several researchers all across the globe. Imbalance in circulating pro- and anti-angiogenic agents in maternal serum has also been linked with the upregulation of stress at subcellular level. The present study is an attempt to demonstrate the role of pro-angiogenic factor in mitigating ER stress in trophoblast cells. Material and Methods: Evaluation of expression of ER stress markers (eIF2α, X-box binding protein-1, and ATF6) at various time points was done after exposure of varying concentration (s) of pro-angiogenic factor (from preeclamptic mothers) to trophoblast cells (BeWo cells). Expression was also analyzed when BeWo cells were exposed to recombinant vascular endothelial growth factor (VEGF) along with serum from preeclamptic mothers. Molecular techniques used were immunofluorescence staining and Western blot analysis. Results: Immunofluorescence staining and Western blot analysis demonstrated upregulated expression of studied ER stress markers in BeWo cells when they were exposed to Preeclampsia (PE) sera. Exogenous addition of recombinant VEGF along with preeclamptic sera significantly reduced the expression of ER stress markers. Discussion and Conclusion: In the present study, significantly reduced expression of ER stress markers in BeWo cells indicates an interrelationship of angiogenic factor and molecular transmembrane sensors. Further experimentations thus may provide a strong base for the modulation of ER stress sensors, which could be effective in minimizing ER stress in preeclamptic pregnancies and thus would bring a new hope to numerous women worldwide.

Association between polymorphism in Cyclophilin A gene and its serum and placental expression in Han Chinese women with severe preeclampsia.

Cyclophilin A (CypA) plays important roles in inflammation and oxidative stress and is significantly increased in serum of preeclampsia (PE) patients. We aimed to investigate CypA genetic polymorphism and its serum and placenta expressions in severe PE of Han Chinese women. A case-control study of 82 severe PE patients and 179 healthy pregnancies was conducted. Single-nucleotide polymorphism (SNP) sites of rs3735481, rs9638978 and rs11984372 were analyzed by TaqMan assay. CypA serum levels were determined by enzyme-linked immunosorbent assay (ELISA). CypA mRNA levels and protein expressions in placentas were assessed by quantitative real-time polymerase chain reaction (PCR), western blot, and immunofluorescence assay, respectively. There were significantly lower frequency of rs3735481 allele C (odds ratio (OR): 0.60, 95% confidence interval (CI): 0.36-0.98; p = .04), and significantly higher frequency of rs9638978 allele A in severe PE especially in early onset PE patients (OR: 2.23, 95% CI: 1.35-3.71, p = .002). Frequency of rs9638978 AA genotype was significantly higher in early onset PE (p < .001). CypA serum levels were significantly higher in severe PE especially in early onset PE (p < .001). Meanwhile, CypA serum levels were significantly lower in carriers with the AC genotype of rs3735481 (p = .019) and significantly higher in carriers with the AA genotype of rs9638978 (p = .017). CypA mRNA levels and protein expressions were found to be significantly increased in PE placentas (both p < .01). The CypA genetic polymorphisms of rs3735481 and rs9638978 may be associated with severe PE, and rs9638978 AA genotype may be associated with an increasing risk of early onset severe PE in Han Chinese women. High CypA levels in serum and placenta may contribute to the pathogenesis of severe PE. Our results may provide a new clue for the etiology of severe PE.

Preeclampsia serum induces human glomerular vascular endothelial cell hyperpermeability via the HMGB1-Caveolin-1 pathway

To explore new ideas about the pathogeny of preeclampsia (PE) proteinuria, this study focused on whether severe PE serum (PES) could induce high-molecular-weight protein (HMWP) hyperpermeability in glomerular endothelial cells (GEC) via the HMGB1-Caveolin-1 (CAV-1) pathway. Normal pregnancy serum (NPS) and severe PES were used to treat primary human GEC monolayer for 24 h. The CAV-1 inhibitor methyl-beta-cyclodextrin (MBCD), the HMGB1 inhibitor glycyrrhizicacid (GA), recombinant HMGB1 (rHMGB1) were also used to treat GEC monolayer that were stimulated by NPS or severe PES. The dynamic permeability of GEC to HMWP was detected by Evans blue-labeled BSA and CAV-1 expression in GEC was analyzed by immunofluorescence staining and Western blotting. We detected HMGB1 expression in placenta and serum in normal pregnancy and severe PE. The results showed that severe PES significantly promoted GEC hyperpermeability and CAV-1 expression. By inhibiting CAV-1 expression, MBCD reversed severe PES-induced GEC monolayer permeability. HMGB1 expression in PE placenta and serum was significantly increased. Compared with that in normal placenta, HMGB1expression was increased in the cytoplasm of syncytiotrophoblast cells in PE placenta. GA decreased the severe PES-induced hyperpermeability and CAV-1 expression in GEC. rHMGB1 induced high expression levels of CAV-1 and HMWP hyperpermeability in GEC. In conclusion, HMGB1 is increased in severe PE patients and induces the expression of CAV-1 in GEC. High expression of CAV-1 in GEC can promote HMWP hyperpermeability, which may contribute to the development of PE proteinuria.

Altered Redox State Modulates Endothelial KCa2.3 and KCa3.1 Levels in Normal Pregnancy and Preeclampsia.

Altered redox state has been related to the development of normal pregnancy (NP) and preeclampsia (PE). Endothelial KCa2.3 and KCa3.1 (KCas) play an important role in vasodilation, and KCas levels are affected by oxidative stress. We investigated the mechanisms of oxidative stress-mediated KCas expression modulation during NP and PE. Human uterine microvascular endothelial cells were incubated in serum from normal nonpregnant women (n = 13) and women with NP (n = 24) or PE (n = 15), or in vascular endothelial growth factor (VEGF), oxidized low-density lipoprotein (ox-LDL), progesterone, or estradiol-17β (E2)-containing medium for 24 h. NP serum elevated H2O2 levels via reducing catalase and glutathione peroxidase 1 levels, thereby enhancing KCas levels via a H2O2/fyn/extracellular signal-regulated kinase (ERK)-mediated pathway. VEGF enhanced H2O2 and KCas levels and KCa3.1 currents. KCas were upregulated and KCas activation-induced endothelium-dependent relaxation (EDR) was augmented in vessels from pregnant mice and rats. Whereas PE serum, ox-LDL, progesterone, or soluble fms-like tyrosine kinase 1 (sFlt-1) elevated superoxide levels via elevating NADPH oxidase 2 (NOX2) and NOX4 levels and reducing superoxide dismutase (SOD) 1 levels, thereby downregulating KCas. sFlt-1 inhibited EDR. PE serum- or progesterone-induced alterations in levels of KCas were reversed by polyethylene glycol-SOD, NOX inhibition, or E2. Innovation and Conclusions: This is the first study of how endothelial KCas levels are modulated during NP and PE. KCas were upregulated by soluble serum factors such as VEGF via H2O2 generation in NP, and were downregulated by serum factors such as progesterone and ox-LDL via superoxide generation in PE, which may contribute to hemodynamic adaptations in NP or to the development of PE.