Inflammatory Response In Preeclampsia Research Articles

LPS-induced PTGS2 manipulates the inflammatory response through trophoblast invasion in preeclampsia via NF-κB pathway.

Preeclampsia (PE) is a serious obstetric complication, in which trophoblast cell invasion and migration contribute to placental inflammation. In line with the discovery that mRNA prostaglandin endoperoxide synthase 2 (PTGS2) participates in the inflammatory responses in various disorders, our study aims to explore the role of PTGS2 in trophoblast invasion and further in inflammatory response in PE, ultimately providing new therapeutic targets. Bioinformatics analysis was exploited to examine PTGS2 expression in GSE40182 and find inflammatory response-relevant genes in downstream targets of PTGS2. HTR-8/SVneo cells were treated with lipopolysaccharide (LPS) and transfected with short hairpin RNA against PTGS2 (shPTGS2). Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and immunofluorence assays were performed to quantify the expressions of PTGS2 and involved genes (matrix metallopeptidase 2 (MMP-2), tissue inhibitors of metalloproteinase-2 (TIMP-2), p65, p-p65, IκB-α, p-IκB-α, PTGIS, CAV1, AGTR1). The migration and invasion of trophoblasts were detected through wound healing and Transwell assays. We screened out PTGS2 from GSE40182 dataset. LPS promoted cell migration and invasion, the expressions of PTGS2 and MMP-2, and reduced the expression of TIMP-2, while PTGS2 knockdown reversed all above effects of LPS. Activation of nuclear factor kappa-B (NF-κB) pathway was reinforced by LPS which also upregulated CAV1 and AGTR1 levels, and downregulated PTGIS level. Also, the effects of LPS were offset by PTGS2 knockdown. Altogether, PTGS2 silencing reverses the promoting effect of LPS on trophoblast invasion and inflammation in PE, making a breakthrough in the research regarding molecular mechanism of PE.

Modulatory effect of two regimens of magnesium sulfate on the systemic inflammatory response in pregnant women with imminent eclampsia.

This study compared the modulatory effect of two intravenous magnesium sulfate (MgSO4) regimens on the systemic inflammatory response in pregnant women diagnosed with imminent eclampsia. In a single-blind cross-sectional study, 33 women were allocated according to the Zuspan (n=16) and Sibai (n=17) MgSO4 regimens, and treated for 24h. Blood samples were collected pre-administration of the loading dose, at 24h of the maintenance dose of MgSO4, and at 48h, when patients were without treatment. Plasma was used to determine interleukin (IL)-1 beta (IL-1β), IL-6, IL-10, tumor necrosis factor-alpha (TNF-α), heat shock protein (Hsp70), and heme oxygenase-1 (HO-1) by ELISA. The treatment with the Zuspan's regimen didn't change plasma concentrations of TNF-α, IL-10, and Hsp70 in the three-time points studied. However, it decreased IL-1β at 24h and 48h and IL-6 at 48h, and increased HO-1 concentration at 48h. On the other hand, compared to the pre-treatment period, Sibai's regimen induced a significant decrease in TNF-α, IL-1β, IL-6, and Hsp70, while increased HO-1 levels both at 24h and 48h and, IL-10 concentration at 48h. Sibai's regimen determined an early and efficient immunoregulatory effect on systemic inflammatory response in preeclampsia, suggesting that the maintenance dose of two grams of MgSO4 was better than one gram in the treatment of imminent eclampsia.

Gene Expression of Pregnancy Neutrophils Differs for Protease versus Lipopolysaccharide Stimulation.

Neutrophils, which extensively infiltrate maternal systemic blood vessels in preeclampsia, express protease-activated receptor 1 (PAR-1) but only during pregnancy. Neutrophils are generally considered to be non-specific in their response, but the pregnancy-specific expression of PAR-1 could result in a gene expression profile unique to pregnancy, which could help explain why the maternal inflammatory response in preeclampsia is systemic rather than localized. We sought to determine if gene expression of pregnancy neutrophils would differ if stimulated by a protease versus bacterial lipopolysaccharide (LPS). We isolated neutrophils from normal pregnant women at 30 weeks’ gestation and cultured them with elastase or LPS. We used elastase because it is a protease elevated in women with preeclampsia, and it activates pregnancy neutrophils via PAR-1. RNA was isolated from the neutrophils for sequencing of the transcriptomes. We discovered many differences in the gene expression profiles. For example, exposure to elastase resulted in three times more uniquely expressed genes than LPS, and the number of significantly differentially upregulated and downregulated genes was greater for elastase. Analysis of canonical pathways revealed similarities for innate immunity but also differences. LPS treatment enriched more pathways, but elastase activated more genes in each pathway. Elastase treatment enriched the MAPK signaling pathway, whereas LPS did not. This is significant because MAPK is a key mediator of transcriptional responses. These findings indicate that protease stimulation of pregnancy neutrophils results in a different profile than stimulation with LPS, which may help explain why the sterile inflammatory response of preeclampsia is systemic and unique to pregnancy.

Immunomodulatory effect of vitamin D on the STATs and transcription factors of CD4+ T cell subsets in pregnant women with preeclampsia

This study evaluated the in vitro modulatory effect of vitamin D (VD) on T cells, by determining the expression of STATs and the transcription factors of each CD4+ T cell subsets. Twenty women with preeclampsia (PE) and 20 normotensive pregnant women were studied. Peripheral blood mononuclear cells were cultured with or without VD to analyse the STATs and transcription factors by flow cytometry, and cytokines production by ELISA. The plasma levels of VD were lower in the PE group. Treatment of cells with VD decreased STAT1/STAT4/T-bet, STAT3/RORγt, and increased STAT6/GATA-3 and STAT5/FoxP3 in preeclamptic women. Treatment with VD also decreased the levels of inflammatory cytokines and increased IL-10 and TGF-β. This hormone exerts immunomodulatory effects on the STAT signalling pathway, shifting the inflammatory profiles, Th1/Th17 cells to Th2/Treg profiles, and it can be suggested as a promising strategy to regulate the systemic inflammatory response in PE.

MOLECULAR PATHWAYS THAT PLAY A ROLE IN THE PREECLAMPSIA PATHOPHYSIOLOGY

Preeclampsia (PE) is an obstetric disease seen in approximately 4-5% of pregnancies progressing with hypertension and urinary proteinuria. It may cause maternal and fetal complications. Despite numerous researches in the field of PE, the underlying pathogenesis remains unclear. However, with these related studies, it has been concluded that there are many molecular mechanisms that trigger PE. Based on these molecular mechanisms, PE can be examined in two stages. The first stage is placental ischemia caused by abnormal placentation. In the second stage, necrotic and apoptotic factors released from the ischemic placenta into the circulation cause systemic inflammation and endothelial dysfunction. One of these factors released from placental cells is the antiangiogenic factor. Also, there is an imbalance in the antioxidant and prooxidant mechanisms that are thought to be due to recurrent ischemia reperfusion injury in PE. The systemic inflammatory response in PE is associated with the immunological response resulting from the contact of the maternal immune cells with trophoblasts. The aim of this review is to present the current molecular mechanisms implicating the pathway leading to PE. The development of new insights into the pathogenesis of PE in conclusion of a better understanding of the relevant molecular mechanisms will guide further studies.

Up-regulation of microRNA-135 or silencing of PCSK6 attenuates inflammatory response in preeclampsia by restricting NLRP3 inflammasome

ObjectiveNumerous studies have confirmed the correlation of microRNAs (miRNAs) with human disease, yet few have explored the role of miR-135 in preeclampsia (PE). This study intends to discuss miR-135’s function in inflammatory response in PE by modulating proprotein convertase subtilisin/kexin-6 (PCSK6) and NLR pyrin domain containing 3 (NLRP3).MethodsThe venous blood and placental tissues were collected from PE pregnant women and 25 normal ones. The levels of miR-135, PCSK6 and NLRP3 in placenta tissues of patients were detected. Hypoxia/reoxygenation HTR-8/SVneo and HPT-8 models were established to mimic PE in vitro, and cell proliferation, colony formation, apoptosis rate, invasion, migration and inflammation were detected through gain-of and loss-of-function assays.ResultsMiR-135 was down-regulated, and PCSK6 and NLRP3 were up-regulated in PE patients. Up-regulating miR-135 or silencing PCSK6 strengthened colony formation ability, viability, invasion and migration ability, and weakened apoptosis and inflammation of H/R-treated HTR-8/SVneo and HPT-8 cells. Inhibition of NLRP3 negated the effects of silenced PCSK6 in H/R-treated HTR-8/SVneo and HPT-8 cells.ConclusionsAltogether, we demonstrate that up-regulated miR-135 or reduced PCSK6 attenuates inflammatory response in PE by restricting NLRP3 inflammasome, which provides novel therapy for PE treatment.

Vitamin D stimulates miR-26b-5p to inhibit placental COX-2 expression in preeclampsia

Vitamin D insufficiency or deficiency during pregnancy has been associated with an increased risk of preeclampsia. Increased placental cyclooxygenase-2 (COX-2) activity was proposed to contribute to the inflammatory response in preeclampsia. This study was to investigate if vitamin D can benefit preeclampsia by inhibiting placental COX-2 expression. Placenta tissues were obtained from 40 pregnant women (23 normotensive and 17 preeclampsia). miR-26b-5p expression was assessed by quantitative PCR. Vitamin D receptor (VDR) expression and COX-2 expression were determined by immunostaining and Western blot. HTR-8/SVneo trophoblastic cells were cultured in vitro to test anti-inflammatory effects of vitamin D in placental trophoblasts treated with oxidative stress inducer CoCl2. 1,25(OH)2D3 was used as bioactive vitamin D. Our results showed that reduced VDR and miR-26b-5p expression, but increased COX-2 expression, was observed in the placentas from women with preeclampsia compared to those from normotensive pregnant women. Transient overexpression of miR-26b-5p attenuated the upregulation of COX-2 expression and prostaglandin E2 (PGE2) production induced by CoCl2 in placental trophoblasts. 1,25(OH)2D3 treatment inhibited CoCl2-induced upregulation of COX-2 in placental trophoblasts. Moreover, miR-26b-5p expression were significantly upregulated in cells treated with 1,25(OH)2D3, but not in cells transfected with VDR siRNA. Conclusively, downregulation of VDR and miR-26b-5p expression was associated with upregulation of COX-2 expression in the placentas from women with preeclampsia. 1,25(OH)2D3 could promote miR-26b-5p expression which in turn inhibited COX-2 expression and PGE2 formation in placental trophoblasts. The finding of anti-inflammatory property by vitamin D through promotion of VDR/miR-26b-5p expression provides significant evidence that downregulation of vitamin D/VDR signaling could contribute to increased inflammatory response in preeclampsia.

Placental Expression and Relative Role of Anti-inflammatory Annexin A1 and Animal Lectin Galectin-3 in the Pathogenesis of Preeclampsia.

Preeclampsia (PE) remains the major cause for maternal and foetal mortality and morbidity all over the world. Preeclampsia is associated with maternal, placental aggravated inflammatory response and generalized endothelial damage. AnnexinA1 (AnxA1) is glucocorticoid regulated protein regulates a wide range of cellular and molecular steps of the inflammatory response and is implicated in resolution of inflammation. Galectin-3 (Gal-3), β-galcotoside-binding lectin participates in many functions, both intra- and extracellularly. Recently it has been shown that galectin-3 modulates the inflammation. Role of AnxA1 and Galectin-3 is poorly studied in context with human reproductive disease like Preeclampsia. Therefore, the present study examined the expression of AnxA1 and Gal-3 which are involved in modulation of inflammation and their association in the placental bed of pregnancy with and without PE. The study group consisted of placental bed biopsy tissues obtained from pregnancies with PE (n = 30) and without (n = 30) PE. The expression of AnxA1 and Gal-3 in the placental bed tissues was evaluated quantitatively using Immunohisto-chemistry (IHC), western blot and mRNA expression analysis by quantitative RT-PCR. Our IHC, western blot and RT PCR analyses showed the increase in the expression of AnxA1 and Gal-3 in PE group compared with the normotensive control group (P < 0.001). The increased expression of AnxA1 and Gal-3 in placental bed may be associated with a systemic inflammatory response in PE, suggesting role of AnxA1 and Gal-3 in PE pathogenesis.

Glycan expression in chorionic villi from histocultures of women with early-onset preeclampsia: Immunomodulatory effects on peripheral natural killer cells

New evidence suggests that glycan expression in placental cells of women with invasive disorders of pregnancy differs from that in normal pregnant women. Hypothesizing that modifications of glycan expression could account for the course of preeclampsia, we established placental villous histocultures and compared glycan expression in women with preeclampsia with that in normal pregnant women and also in syncytialized BeWo cells, and we tested the effect of glycan expression on the functional phenotypes of circulating natural killer (NK) cells. Histocultures of third-trimester placentae from women with preeclampsia and full-term placentae from healthy pregnant women and BeWo choriocarcinoma cells were assessed for the expression of terminal glycans by lectin-binding assays. Circulating NK cells from nonpregnant healthy donors were tested in vitro for their cytotoxic activity and intracellular cytokine content. Histocultures from women with preeclampsia expressed significantly more mannose than did those from healthy pregnant women. Both histocultures and BeWo cells expressed terminal fucose, mannose, sialic acid, and N -acetylgalactosamine, although mean fluorescence intensity (MFI) expression was lower in choriocarcinoma cells than in cells from histocultures. Cocultures of circulating NK cells with K562 target cells resulted in a dose-dependent cytotoxicity effect, but the use of BeWo cells as target reduced cytotoxic activity; this reduction was not affected by syncytialization. Histocultures of placental villous tissue of women with preeclampsia expressed high levels of terminal mannose. We proposethat placental glycans may modulate the functional activity of circulating NK cells in the context of systemic inflammatory response in preeclampsia.

Increased TLR4 pathway activation and cytokine imbalance led to lipopolysaccharide tolerance in monocytes from preeclamptic women.

Preeclampsia (PE) is a pregnancy syndrome characterized by a systemic inflammatory response, and endogenous activation of monocytes. This study aimed to determine whether the activation of monocytes from preeclamptic women might interfere with the response to lipopolysaccharide (LPS)-in vitro stimulation. Fifty-two preeclamptic women and 32 normotensive (NT) pregnant women were included. Monocytes from peripheral blood were cultured with or without LPS. TLR4 expression was analyzed by flow cytometry, NF-κB activity was determined in nuclear extracts and cytokines production was evaluated by ELISA. Endogenous TLR4 ligands such as Hyaluronan, HMGB1 and Hsp70 were determined in plasma. The endogenous TLR4 expression and activation of NF-κB were statistically higher in monocytes from women with PE compared to NT group. Early-onset PE showed higher TLR4 expression compared to late-onset PE. Plasma levels of Hyaluronan, HMGB1, and Hsp70, as well as endogenous production of inflammatory cytokines, were elevated whilst lower production of IL-10 was observed in the PE group. After culture with LPS, monocytes presented lower NF-κB activation, TNF-α and IL-12 production in PE groups than in the NT group. The study demonstrates endogenous activation of monocytes from preeclamptic women, accompanied by higher expression of TLR4, NF-κB activation and elevated production of pro-inflammatory cytokines. The higher plasma levels of the TLR4 ligands hyaluronan, HMGB1 and hsp70, as well as the high concentration of TNF-α endogenously produced by monocytes, could induce the LPS tolerance phenomenon in these cells. These results suggest that monocytes play an important role in the maternal excessive systemic inflammatory response in PE.

MiRNA-203a-3p inhibits inflammatory response in preeclampsia through regulating IL24.

This study aims to investigate the protective role of miRNA-203a-3p in preeclampsia (PE) patients via inhibiting the inflammatory key protein IL24. Serum samples of 36 PE pregnant women and 30 normal pregnant volunteers hospitalized between 2015 and 2019 were collected to extract placental mononuclear cells and exosomes. Relative levels of microRNA-203a-3p and IL24 were examined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). In addition, the interaction between microRNA-203a-3p and IL24 was analyzed through bioinformatics analysis and Luciferase reporting assay. Finally, the underlying molecular mechanisms were further explored via immunofluorescence and Western blotting. Compared with normal pregnant volunteers, microRNA-203a-3p expression in serum exosomes and placental mononuclear cells of PE patients were dramatically reduced, while IL24 was conversely up-regulated, indicating a negative correlation between microRNA-203a-3p and IL24 levels. In addition, IL24, which was down-regulated in mononuclear macrophages overexpressing microRNA-203a-3p, was indicated as a target of microRNA-203a-3p. At the same time, microRNA-203a-3p was able to suppress the proliferation capacity of LPS-stimulated mononuclear macrophages, and it exerted anti-inflammatory effects via down-regulating IL24 in THP-1 cells. MicroRNA-203a-3p plays an anti-inflammatory role in PE pregnant women by down-regulating IL24 level.

Targeting senescence improves angiogenic potential of adipose-derived mesenchymal stem cells in patients with preeclampsia

BackgroundPreeclampsia is a pregnancy-specific hypertensive disorder characterized by impaired angiogenesis. We postulate that senescence of mesenchymal stem cells (MSC), multipotent cells with pro-angiogenic activities, is one of the mechanisms by which systemic inflammation exerts inhibitory effects on angiogenesis in preeclampsia.MethodsMSC were isolated from abdominal fat tissue explants removed during medically indicated C-sections from women with preeclampsia (PE-MSC, n = 10) and those with normotensive pregnancies (NP-MSC, n = 12). Sections of the frozen subcutaneous adipose tissue were assessed for inflammation by staining for tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein (MCP)-1. Viability, proliferation, and migration were compared between PE-MSC vs. NP-MSC. Apoptosis and angiogenesis were assayed before and after treatment with a senolytic agent (1 μM dasatinib) using the IncuCyte S3 Live-Cell Analysis System. Similarly, staining for senescence-associated beta galactosidase (SABG) and qPCR for gene expression of senescence markers, p16 and p21, as well as senescence-associated secretory phenotype (SASP) components, IL-6, IL-8, MCP-1, and PAI-1, were studied before and after treatment with dasatinib and compared between PE and NP.ResultsAfter in vitro exposure to TNF-alpha, MSC demonstrated upregulation of SASP components, including interleukins-6 and -8 and MCP-1. Staining of the subcutaneous adipose tissue sections revealed a greater inflammatory response in preeclampsia, based on the higher levels of both TNF-alpha and MCP-1 compared to normotensive pregnancies (p < 0.001 and 0.024, respectively). MSC isolated from PE demonstrated a lower percentage of live MSC cells (p = 0.012), lower proliferation (p = 0.005), and higher migration (p = 0.023). At baseline, PE-MSC demonstrated a senescent phenotype, reflected by more abundant staining for SABG (p < 0.001), upregulation of senescence markers and SASP components, as well as lower angiogenic potential (p < 0.001), compared to NP-MSC. Treatment with dasatinib increased significantly the number of apoptotic PE-MSC compared to NP-MSC (0.011 vs. 0.093) and decreased the gene expression of p16 and six SASP components. The mechanistic link between senescence and impaired angiogenesis in PE was confirmed by improved angiogenic potential of PE-MSC (p < 0.001) after dasatinib treatment.ConclusionsOur data suggest that MSC senescence exerts inhibitory effects on angiogenesis in preeclampsia. Senolytic agents may offer the opportunity for mechanism-based therapies.

Downregulation of vitamin D receptor and miR-126-3p expression contributes to increased endothelial inflammatory response in preeclampsia.

To investigate whether downregulation of miR-126-3p and vitamin D receptor (VDR) expression contributes to increased endothelial inflammatory response in preeclampsia. Maternal vessel miR-126-3p expression was assessed by in situ hybridization. VDR expression and VCAM-1 expression were determined by immunostaining. Subcutaneous adipose tissue sections from normotensive and preeclamptic pregnant women were used. HUVECs from normotensive deliveries were used to test anti-inflammatory effects of vitamin D and miR-126-3p in endothelial cells (ECs) treated with TNFα in vitro. 1,25(OH)2 D3 was used as bioactive vitamin D. Transient overexpression of miR-126-3p in ECs was induced by transfection of pre-mir-126 precursor. Endothelial VCAM-1 and SOCS-3 expression or production was determined by Western blotting or by ELISA, respectively. Reduced VDR and miR-126-3p expression, but increased VCAM-1 expression, was observed in maternal vessel endothelium in tissue sections from women with preeclampsia compared to normotensive pregnant controls. Transient overexpression of miR-126-3p not only attenuated upregulation of VCAM-1 expression and production, but also preserved downregulation of SOCS-3 expression, induced by TNFα in ECs. VDR expression and miR-126-3p expression were significantly upregulated in cells treated with 1,25(OH)2 D3 , but not in cells transfected with VDR siRNA. Downregulation of VDR and miR-126-3p expression was associated with upregulation of VCAM-1 expression in systemic vessel endothelium in preeclampsia. The finding of increased anti-inflammatory property by 1,25(OH)2 D3 through promotion of VDR and miR-126-3p expression in ECs provide plausible evidence that vitamin D deficiency and downregulation of VDR expression could contribute to increased inflammatory phenotypic changes in maternal vasculature in preeclampsia.

MiRNA-548c-5p downregulates inflammatory response in preeclampsia via targeting PTPRO.

Preeclampsia is a serious complication of pregnancy and leads to maternal hypertension and proteinuria. It remains a major health problem for mothers and babies across the world due to high maternal and fetal morbidity and mortality. Accumulated data have implicated the critical role of microRNA in preeclampsia. However, to date, the role of miR-548c-5p in preeclampsia remains vaguely understood. In this study, we first elucidate the role of miR-548c-5p and its underlying molecular mechanism in preeclampsia. Compared with healthy controls, miR-548c-5p was obviously downregulated in serum exosomes and placental mononuclear cells in patients with preeclampsia. Nonetheless, PTPRO was significantly upregulated and negatively associated with miR-548c-5p in placental mononuclear cells in patients with preeclampsia. PTPRO was a target of miR-548c-5p. PTPRO was downregulated in the miR-548c-5p-overexpressed macrophages. In addition, miR-548c-5p could inhibit the proliferation and activation of LPS-stimulated macrophages, as evidenced by decreased levels of inflammatory cytokines (IL-12 and TNF-α) and less nuclear translocation of pNF-κB in pTHP1 cells. MiR-548c-5p acts as an anti-inflammatory factor in preeclampsia. The axis of miR-548c-5p/PTPRO/NF-κB may provide novel targets for the diagnosis and treatment of preeclampsia.

122. Silibinin regulates the NF-κB pathway and NLRP1/NLRP3 inflammasomes induced by monosodium urate in monocytes from preeclamptic women and THP-1 cells

Introduction Preeclampsia (PE) is characterized by a state of abnormal activation of the innate immune system. It’s associated with hyperuricemia and elevated serum levels of inflammatory cytokines. Uric acid crystals can activate inflammasome, a multiprotein structure important for processing and release of inflammatory cytokines. The imbalance between pro- and anti-inflammatory cytokines in PE seems to be dependent on the deficiency of regulatory factors capable of modulating inflammatory response, which could be alleviated by administration of substances with anti-inflammatory and modulatory properties such as silibinin (SB). Objective The study aimed to evaluate the modulatory effect of silibinin on NLRP1 and NLRP3 inflammasomes and on NF-κB pathway activation in monocytes from preeclamptic women stimulated with monosodium urate (MSU). Methods Twenty preeclamptic women, 20 normotensive pregnant women (NT) and THP-1 cells were cultured with or without SB and MSU. Gene expression of NLRP1, NLRP3, Caspase-1, TLR4, MyD88, NF-κB, IL-1β, IL-18, TNF-α and IL-10 was performed by qPCR. Inflammatory cytokines and p65NF-κB activity were evaluated by ELISA. Results Monocytes of preeclamptic women presented higher endogenous activation of the inflammatory genes, as well as p65NF-κB basal activity and the inflammatory cytokines production than NT group. MSU stimulation increased the expression of these parameters, whereas SB treatment reduced them. Additionally, THP-1 cells had a similar immunological response profile of monocytes from preeclamptic women when cultured with or without MSU and SB. Conclusion These results suggest the MSU participation in the systemic inflammatory response, characteristic of preeclampsia, and that silibinin treatment is capable of modulating the sterile inflammation established in monocytes of preeclamptic women, demonstrating that this flavonoid plays a relevant role in the regulation of the inflammatory response in preeclampsia. Financial support FAPESP 2015/26147-3, 2016/18155-9 and 2016/22854-0

Increased serum levels of high mobility group protein B1 and calprotectin in pre-eclampsia.

To determine whether women with pre-eclampsia have serum levels of biomarkers indicative of an elevated systemic inflammatory response. The present cross-sectional study was conducted among pregnant women either with pre-eclampsia or without pre-eclampsia who were recruited at a single Chinese hospital between August 1, 2016, and April 30, 2017. Eligible women had no history of acute or chronic inflammation. Serum concentrations of high mobility group protein B1 (HMG-1), calprotectin, and Toll-like receptor 4 (TLR4) were measured and compared. There were 55 patients included (25 with pre-eclampsia and 30 without). The mean serum concentration of calprotectin was 2656.76±1724.56μg/L in the pre-eclampsia group versus 1877.33±905.69μg/L in the control group (P=0.036). Among patients with pre-eclampsia, elevated calprotectin levels were positively associated with the duration of hypertension in pregnancy (P=0.031) and were negatively associated with pregnancy duration at delivery (P=0.035). The mean serum concentration of HMG-1 was 72.48±27.57μg/L in the pre-eclampsia group versus 57.57±20.07μg/L in the control group (P=0.017). The mean serum concentration of TLR4 was 22.83±8.46μg/L in the pre-eclampsia group versus 18.83±6.79μg/L in the control group (P=0.057). Elevated levels of HMG-1 and calprotectin could reflect an excessive systemic inflammatory response in pre-eclampsia.

Estimation of asymmetric dimethylarginine (ADMA), placental growth factor (PLGF) and pentraxin 3 (PTX 3) in women with preeclampsia.

To evaluate the sequential trend of asymmetric dimethylarginine (ADMA), placental growth factor (PLGF) and pentraxin 3 (PTX 3) in pregnancies developing preeclampsia (PE) as compared to healthy pregnancy (HP) and to estimate their predictive value for development of PE later in pregnancy. Nested case control design, sampling was done in 13 women with PE and 21 age matched healthy pregnant women at 11-13 weeks, 20-22 weeks and 30-32 weeks of gestation. PLGF, ADMA, and PTX 3 were estimated temporally. Serum ADMA and PTX 3 levels were higher in PE than HP even at 11-13 weeks and remained elevated throughout the gestation. PTX 3 concentration increased in both the groups with advancing gestation however significant rise was observed only in HP group. PLGF levels also increased with advancing gestation in HP group while in PE, there was a rise till 20-22 weeks of gestation followed by fall at 30-32 weeks. PLGF levels were lower in PE at 30-32 weeks than healthy pregnancy. Area under curve (AUC) for ADMA and PTX 3 were: at 11-13 weeks; 95.95% and 83.33% and 20-22 weeks; 89.88% and 90.06% respectively. At 30-32 weeks, PLGF and ADMA demonstrated an AUC of 86.51% and 86.51% respectively. Abnormally elevated ADMA and PTX 3 levels precede the manifestation of PE and suggest endothelial dysfunction with exaggerated inflammatory response in PE. Both ADMA and PTX 3 can be used to segregate high risk women for development of PE than others in early pregnancy.

Cardiac myeloperoxidase activity is elevated in hypertensive pregnant rats.

Myeloperoxidase (MPO) is released from activated neutrophils. The inflammation in preeclampsia was found to be associated with endothelial dysfunction. We hypothesized that cardiac and circulating MPO levels are elevated in hypertensive pregnancy. Systolic and diastolic blood pressure and heart rate were measured on pregnancy days 14, 16, 18 and 20 in normal pregnant and hypertensive pregnant rats. Left and right ventricle weights, the number of viable fetuses, litter size, fetal and placenta weights were recorded on gestational day 21. Circulating and cardiac MPO activities, soluble fms-like tyrosine kinase-1 (sFlt-1) and vascular endothelial growth factor (VEGF) and nitric oxide (NO) were detected. The results showed increases in cardiac (left, but not right ventricle) and circulating MPO activities, and concomitantly lower number of viable fetuses, litter size, and fetal and placenta weights, and decreases in NO in hypertensive pregnant rats. Also, the increases in circulating sFlt-1 and VEGF were found in hypertensive pregnant group. In conclusion, maternal and fetal detrimental changes along with increases in circulating sFlt-1 and VEGF in hypertensive pregnancy may be associated with increases in cardiac and circulating MPO activities, confirming the causative role of inflammatory response in preeclampsia.

Up-regulation of miR-203 expression induces endothelial inflammatory response: Potential role in preeclampsia.

To determine whether miR-203 mediates endothelial inflammatory response in preeclampsia. Maternal vessel miR-203 expression was assessed by in situ hybridization. Suppressor of cytokine signaling-3 (SOCS-3) and ICAM expression was determined by immunostaining. Subcutaneous fat tissue sections from normal and preeclamptic pregnant women were used. miR-203-induced inflammatory response was evaluated by the measurements of IL-6, IL-8, ICAM, and VCAM expression and production and neutrophil adhesion in the endothelial cells (EC) transfected with miR-203 precursor, pre-miR-203. SOCS3 expression was also determined. Up-regulation of miR-203 and ICAM expression and down-regulation of SOCS-3 expression were demonstrated in maternal vessel endothelium in preeclampsia. Overexpression of miR-203 resulted in down-regulation of SOCS-3 expression and increases in the production of IL-6, IL-8, ICAM, and VCAM and neutrophil adhesion in ECs. As miR-203 is an inflammatory microRNA, increased miR-203 production/expression in ECs could diminish an anti-inflammatory activity and increase the endothelial inflammatory response in preeclampsia.

37 1,25(OH)2D3 suppresses oxidative stress – Induced microparticle release by placental trophoblasts

Introduction Emerging evidence showed that microparticles (MPs) derived from placental trophoblasts (TC) contribute to maternal vascular dysfunction and promote endothelial inflammatory response in preeclampsia (PE). Vitamin D insufficiency has been linked to placental dysfunction in pregnancy disorders such as PE and IUGR. Objective To determine: (1) if TC from PE placentas release more MPs than TC from normal placentas; (2) if vitamin D could suppress oxidative stress-induced MP release by placental TC; and (3) potential mechanism of vitamin D protective effects on placental TCs. Methods TC were isolated from normal and PE placentas by trypsin digestion and purified with Percoll gradient centrifugation. Freshly isolated TC (5×06cells/well) were seeded into 6 well plates and cultured with DMEM containing 5% FBS and antibiotics for 72h. For the oxidative stress experiment, cobalt chloride (CoCl 2 , a hypoxic mimicking agent) was used to induce TC oxidative stress and 1,25(OH)2D3 was used as bioactive vitamin D. MPs were isolated from culture supernatants by a two-step centrifugation procedure (420g for 20min and then 20,000g for 60min) and quantified by annexin V positive staining measured by flow cytometry with TruCount beads to determine absolute counts of MP for each sample. Data are expressed as annexin V positive MP/μg cell protein/well. Total cellular protein and MP protein were also collected and protein expression for caveolin-1, eNOS, pro-caspase-3, cleaved caspase-3, and ROCK1 were determined by Western blot. Data were analyzed by paired or unpaired t-test or ANOVA with Newman-Keuls test as a post hoc test. A p level Results TC isolated from PE placentas ( n =6) released significantly more MP than cells isolated from normal placentas ( n =6), 295.20±47.67 vs. 117.20±18.02/μg protein/well, p 2 compared to control TC, p 2 was significantly reduced in TC treated with 1,25(OH)2D3+CoCl 2 , p 2 . This phenomenon could be reversed when cells were treated with 1,25(OH)2D3. Moreover, reduced pro-caspase-3 and ROCK1 expression in TC induced by CoCl 2 could also be attenuated in cells treated with1,25(OH)2D3. Conclusions MP release was significantly increased in TC from PE placenta. 1,25(OH)2D3 suppresses oxidative stress-induced MP release by placental TC. Caveolin-1 is an essential structural component of caveolae and eNOS is associated with plasma membrane caveolae. Therefore, reduced caveolin-1 and increased eNOS expression together with reduced MP release in TC treated with 1,25(OH)2D3 demonstrate that vitamin D could protect placental TC from oxidative stress-induced injury during pregnancy. Since caspase-3 cleavage and ROCK1 activation are key steps of MP formation and release, suppression of caspase-3 cleavage and ROCK1 activation by vitamin D could be a plausible mechanism of vitamin D inhibition of increased MP release induced by oxidative stress.