Preeclamptic Serum Research Articles

Exploring the Therapeutic Potential of C-Type Natriuretic Peptide for Preeclampsia.

Preeclampsia is a serious condition of pregnancy, complicated by aberrant maternal vascular dysfunction. CNP (C-type natriuretic peptide) contributes to vascular homeostasis, acting through NPR-B (natriuretic peptide receptor-B) and NPR-C (natriuretic peptide receptor-C). CNP mitigates vascular dysfunction of arteries in nonpregnant cohorts; this study investigates whether CNP can dilate maternal arteries in ex vivo preeclampsia models. Human omental arteries were dissected from fat biopsies collected during cesarean section. CNP, NPR-B, and NPR-C mRNA expression was assessed in arteries collected from pregnancies complicated by preeclampsia (n=6) and normotensive controls (n=11). Using wire myography, we investigated the effects of CNP on dilation of arteries from normotensive pregnancies. Arteries were preconstricted with either serum from patients with preeclampsia (n=6) or recombinant ET-1 (endothelin-1; vasoconstrictor elevated in preeclampsia; n=6) to model vasoconstriction associated with preeclampsia. Preconstricted arteries were treated with recombinant CNP (0.001-100 µmol/L) or vehicle and vascular relaxation assessed. In further studies, arteries were preincubated with NPR-B (5 µmol/L) and NPR-C (10 µmol/L) antagonists before serum-induced constriction (n=4-5) to explore mechanistic signaling. CNP, NPR-B, and NPR-C mRNAs were not differentially expressed in omental arteries from preeclamptic pregnancies. CNP potently stimulated maternal artery vasorelaxation in our model of preeclampsia (using preeclamptic serum). Its vasodilatory actions were driven through the activation of NPR-B predominantly; antagonism of this receptor alone dampened CNP vasorelaxation. Interestingly, CNP did not reduce ET-1-driven omental artery constriction. Collectively, these data suggest that enhancing CNP signaling through NPR-B offers a potential therapeutic strategy to reduce systemic vascular constriction in preeclampsia.

Serum Collected from Preeclamptic Pregnancies Drives Vasoconstriction of Human Omental Arteries-A Novel Ex Vivo Model of Preeclampsia for Therapeutic Development.

New-onset maternal hypertension is a hallmark of preeclampsia, driven by widespread endothelial dysfunction and systemic vasoconstriction. Here, we set out to create a new ex vivo model using preeclamptic serum to cause injury to the endothelium, mimicking vascular dysfunction in preeclampsia and offering the potential to evaluate candidate therapeutic interventions. Human omental arteries were collected at caesarean section from normotensive pregnant patients at term (n = 9). Serum was collected from pregnancies complicated by preterm preeclampsia (birth < 34 weeks’ gestation, n = 16), term preeclampsia (birth > 37 weeks’ gestation, n = 5), and healthy gestation-matched controls (preterm n = 16, term n = 12). Using wire myography, we performed ex vivo whole vessel assessment where human omental arteries were treated with increasing doses of each serum treatment (2–20%) and vasoreactivity was assessed. All pregnant serum treatments successfully drove vasoconstriction; no significant difference was observed in the degree of vasoconstriction when exposed to preeclamptic or control serum. We further demonstrated the ability of esomeprazole (a candidate therapeutic for preeclampsia; 0.1–100 µM) to drive vasorelaxation of pre-constricted vessels (only with serum from preeclamptic patients). In summary, we describe a novel human physiological model of preeclamptic vascular constriction. We demonstrate its exciting potential to screen drugs for their therapeutic potential as treatment for vasoconstriction induced by preeclampsia.

Quantitative proteomic analysis of serum-purified exosomes identifies putative pre-eclampsia-associated biomarkers

BackgroundThe high incidence of pre-eclampsia, which affects 2–7% of all pregnancies, remains a major health concern. Detection of pre-eclampsia before the appearance of clinical symptoms is essential to allow early intervention, and would benefit from identification of plasma/serum biomarkers to help guide diagnosis and treatment. Liquid biopsy has emerged as a promising source of protein biomarkers that circumvents some of the inherent challenges of proteome-wide analysis of plasma/serum. In this respect, purified exosomes have the added benefit of being carriers of intercellular communication both in physiological and pathological conditions.MethodsWe compared the protein complement of purified exosomes from three different collections of control and pre-eclamptic serum samples, obtained at the end of the second trimester of pregnancy and at delivery. We employed shotgun label-free proteomics to investigate differential protein expression, which was then validated by targeted proteomics.ResultsWe developed a purification method that yielded highly enriched exosome preparations. The presence of specific pregnancy protein markers suggested that a significant proportion of purified exosomes derived from tissues related to pregnancy. Quantitative proteomic analyses allowed us to identify 10, 114 and 98 differentially-regulated proteins in the three sample collections, with a high degree of concordance. Functional analysis suggested that these proteins participate in biological processes related to pre-eclampsia, including angiogenesis, inflammation and cell migration. The differential abundance of 66 proteins was validated by targeted proteomics. Finally, we studied the impact of the pre-eclampsia-associated exosomes in the proteome using an in vitro cellular model.ConclusionsWe have identified and validated differential exosomal proteins in liquid biopsy of pregnant women that open new possibilities for early detection of pre-eclampsia. Additionally, the functional impact of the proteome composition of purified pre-eclamptic exosomes in target cells provides new information to better understand changes in embryo-maternal interactions and, consequently, the pathogenesis of this disease.

The Preeclamptic Environment Promotes the Activation of Transcription Factor Kappa B by P53/RSK1 Complex in a HTR8/SVneo Trophoblastic Cell Line.

Preeclampsia is a pregnancy disorder associated with shallow placentation, forcing placental cells to live in hypoxic conditions. This activates the transcription factor kappa B (NFκB) in maternal and placental cells. Although the role of NFκB in preeclampsia is well documented, its mechanism of activation in trophoblastic cells has been never studied. This study investigates the mechanism of NFκB activation in a first trimester trophoblastic cell line (HTR8/SVneo) stimulated by a medium containing serum from preeclamptic (PE) or normotensive (C) women in hypoxic (2% O2) or normoxic (8% O2) conditions. The results indicate that in HTR8/SVneo cells, the most widely studied NFκB pathways, i.e., canonical, non-canonical and atypical, are downregulated in environment PE 2% O2 in comparison to C 8% O2. Therefore, other pathways may be responsible for NFκB activation. One such pathway depends on the activation of NFκB by the p53/RSK1 complex through its phosphorylation at Serine 536 (pNFκB Ser536). The data generated by our study show that inhibition of the p53/RSK1 pathway by p53-targeted siRNA results in a depletion of pNFκB Ser536 in the nucleus, but only in cells incubated with PE serum at 2% O2. Thus, the p53/RSK1 complex might play a critical role in the activation of NFκB in trophoblastic cells and preeclamptic placentas.

LOX-1 expression is reduced in placenta from pregnancies complicated by preeclampsia and in hypoxic cytotrophoblast.

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is upregulated in the maternal vasculature in preeclampsia, and contributes to oxidative stress and endothelial dysfunction. However, its function in the placenta is unclear. This paper investigated LOX-1 expression in models of placental dysfunction and preeclampsia, and whether candidate therapeutics for preeclampsia could alter its expression. Placentas were collected from preterm pregnancies and cases of preterm preeclampsia and fetal growth restriction. Blood was collected from participants whose pregnancies were complicated by preterm fetal growth restriction and/or preeclampsia. Primary cytotrophoblast and placental explant tissue were cultured under hypoxic (1% O2) or normoxic (8% O2) conditions. Cytotrophoblast were exposed to 10% preeclamptic or control serum. Cytotrophoblast and preeclamptic explant tissue were treated with 100µM esomeprazole, lansoprazole or rabeprazole. LOX-1 expression was assessed in all samples via qPCR. LOX-1 expression was reduced in placentas from cases of preterm preeclampsia, but not fetal growth restriction, compared to controls. LOX-1 expression was reduced in cytotrophoblast under hypoxia, but not in explant tissue. Treatment with preeclamptic serum in vitro did not alter cytotrophoblast LOX-1 expression. Circulating LOX-1 mRNA was unaltered in patients with fetal growth restriction, preeclampsia, and fetal hypoxia, compared to controls. Treatment with esomeprazole or lansoprazole in vitro increased placental LOX-1 expression. LOX-1 expression is reduced in preeclamptic placentas and hypoxic cytotrophoblast. Esomeprazole and lansoprazole increase placental LOX-1 expression. These findings demonstrate a role for LOX-1 in the placenta, and improve our understanding of maternal adaptations in pregnancy complications.

Reduction of serum-induced endothelial STAT3(Y705) activation is associated with preeclampsia.

Preeclampsia is associated with maternal morbidity and mortality during pregnancy, and also an increased cardiovascular disease (CVD) risk later in life. During preeclampsia, alterations in secreted placental factors leading to systemic maternal endothelial dysfunction are evident. However, little is known about the associated endothelial intracellular signaling. STAT3 is a latent cytoplasmic transcription factor involved in endothelial cell differentiation, survival, and angiogenesis. We aimed to test if preeclampsia and preeclampsia-related placental factors could alter serum-induced STAT3(Y705) activation in endothelial cells. Furthermore, if altered serum-induced endothelial STAT3 (Y705) activation is related to post-preeclamptic CVD risk. HUVECs were used as a model of maternal endothelium. Experiments entailed addition of 20% human pregnancy serum as well as addition of recombinant PlGF, sFLT1 and VEGF-A165a to the cells. Levels of pSTAT3(Y705) related to STAT3 levels were evaluated by immunoblotting analysis. Our results show that preeclamptic serum induces significantly lower STAT3(Y705) phosphorylation compared with uncomplicated pregnancy serum (P=0.0089) in endothelial cells. Furthermore, STAT3(Y705) phosphorylation was not changed upon addition of PlGF, sFLT1, or VEGF-A165a together with pregnancy sera compared with sera alone. Finally, sera from women with previous preeclampsia and current hypertension and carotid atherosclerotic plaques show significantly lower STAT3(Y705) phosphorylation capabilities compared with healthy women with previous uncomplicated pregnancies 8-18years after deliveries (P=0.029). Reduction in serum-induced endothelial STAT3(Y705) activation may play an important role in the preeclampsia-associated endothelial dysfunction. Additionally, reduced endothelial STAT3(Y705) phosphorylation may contribute to increased post-preeclamptic CVD risk 8-18years after delivery.

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.

82. Disulfiram inhibits placental sFLT-1 secretion

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

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.

Angiotensin II Type I Receptor Agonistic Autoantibody Induces Podocyte Injury via Activation of the TRPC6- Calcium/Calcineurin Pathway in Pre-Eclampsia

Background/Aims: Angiotensin II type I receptor agonistic autoantibody (AT1-AA) is closely related to pre-eclampsia, which is characterized by proteinuria and hypertension. AT1-AA has been shown to enhance the effect of AngII in pre-eclampsia, such as production of endothelin-1, activation of ROS, and vasoconstriction, which are considered to be associated with hypertension; however, whether or not AT1-AA participates in podocyte damage leading to the generation of proteinuria has not been reported. In this study we investigated the role of pre-eclamptic serum AT1-AA on podocytes and the mechanism underlying the generation of proteinuria. Methods: The levels of AT1-AA isolated from pre-eclamptic sera were determined by an enzyme-linked immunosorbent assay. Human podocytes were cultured in vitro and treated with various concentrations of AT1-AA. Whether or not an ERK1/2 inhibitor and TRPC6 siRNA inhibit the effect of AT1-AA on podocytes was determined. Western blot was used to detect the expression of podocyte-specific proteins (nephrin, synaptopodin, and podocin) and the phosphorylation of ERK1/2 and TRPC6. The arrangement of F-actin was observed by immunofluorescence. A Calcineurin Cellular Activity Assay Kit was used to detect calcineurin activity. Changes in the intracellular Ca²⁺ concentration was determined by confocal laser. Results: AT1-AA induced a decrease in podocyte-specific protein expression and calcineurin activity and increased expression of p-ERK1/2 and TRPC6 protein and the intracellular Ca²⁺ concentration. Immunofluorescence revealed rearrangement of F-actin. PD98059, an inhibitor of ERK1/2, and TRPC6 siRNA attenuated the decreased expression of podocyte-specific proteins and decreased intracellular Ca²⁺ concentration. The expression of TRPC6 was reduced following the addition of ERK1/2 inhibitor. Conclusion: AT1-AA induced podocyte damage in a dose-dependent manner. The underlying mechanism might involve activation of the TRPC6 –calcium/calcineurin pathway. This study provides new details regarding podocyte injury and the mechanism underlying the generation of proteinuria in pre-eclampsia.

Multiple soluble TGF-β receptors in addition to soluble endoglin are elevated in preeclamptic serum and they synergistically inhibit TGF-β signalling

Multiple soluble TGF-β receptors in addition to soluble endoglin are elevated in preeclamptic serum and they synergistically inhibit TGF-β signalling

Effect of the microenvironment on the placental behaviour: Response of the chorionic villi to normal and preeclamptic pregnant serum

Effect of the microenvironment on the placental behaviour: Response of the chorionic villi to normal and preeclamptic pregnant serum

High levels of HtrA4 observed in preeclamptic serum induce endothelial cell cycle arrest and senescence and inhibit endothelial progenitor cell differentiation for repair

High levels of HtrA4 observed in preeclamptic serum induce endothelial cell cycle arrest and senescence and inhibit endothelial progenitor cell differentiation for repair

Aggregated transthyretin is specifically packaged into placental nano-vesicles in preeclampsia

In preeclampsia, the serum levels of transthyretin, a carrier protein for thyroxine, are elevated. Transthyretin isolated from preeclamptic serum is also aggregated and can induce preeclampsia-like symptoms in pregnant IL10−/− mice. Using western blotting, immunofluorescence, ELISA and qRT-PCR, we investigated the production of transthyretin by preeclamptic placentae and whether transthyretin is carried into the maternal circulation via placental extracellular vesicles. Both total and aggregated transthyretin were present in higher levels in preeclamptic placentae compared to normotensive placentae (p < 0.05, n = 7), however the levels of transythretin mRNA were not significantly different (n = 8). Preeclamptic placentae secreted similar levels of total transthyretin compared to normotensive placentae (2352 ± 2949 ng/mL vs. 3250 ± 1864 ng/mL, mean ± SD, p > 0.05, n = 8), however in preeclampsia, a significant proportion is vesicle-associated (~48% vs 0%). Increased levels of aggregated transthyretin were specifically associated to preeclamptic nano-vesicles (p < 0.02, n = 8). This study showed that the placenta actively produces transthyretin and in preeclampsia, a significant amount is extruded into the maternal circulation via placental exracellular vesicles. The increased aggregation of transthyretin in preeclampsia occurs at the post-transcriptional level and while preeclamptic nano-vesicles may be removing a toxic aggregated protein from the placenta, they may also be delivering aggregated transthyretin to specific maternal organs, contributing to the pathogenesis of preeclampsia.

Multiple Soluble TGF-β Receptors in Addition to Soluble Endoglin Are Elevated in Preeclamptic Serum and They Synergistically Inhibit TGF-β Signaling.

Preeclampsia (PE) can be classified into early-onset (<34 weeks of gestation) and late-onset (>34 weeks of gestation) subtypes. Soluble endoglin, an auxiliary receptor for transforming growth factor (TGF)-β ligands, is increased in PE circulation and believed to inhibit TGF-β action by sequestering the ligands. However, soluble endoglin, with a low affinity to TGF-β ligands, has been demonstrated to have little effect by itself on TGF-β action. We examined whether multiple soluble TGF-β receptors are elevated in PE circulation and whether they synergistically block TGF-β signaling. TGF-β receptors were measured using enzyme-linked immunosorbent assay in sera collected from preeclamptic pregnancies and gestation-age-matched controls. TGF-β signaling was assessed using an in vitro bioassay and a tube formation assay. TGF-β type I, II, and III receptors were all identified in pregnant serum; all were substantially elevated in early-onset but not late-onset PE. Endoglin was increased in both subtypes. At the greatest concentrations detected in PE, none of these soluble TGF-β receptors alone, including endoglin, inhibited TGF-β signaling. However, when all four soluble receptors were present, signaling of both TGF-β1 and TGF-β2 was substantially reduced. Removal of any one of these soluble receptors alleviated TGF-β1 inhibition; however, removal of soluble TGFβRIII was necessary to relieve TGF-β2 inhibition. Multiple soluble TGF-β receptors are present in pregnant circulation and elevated in early-onset PE; they synergistically inhibit TGF-β signaling, which might be more likely to occur in early-onset than late-onset PE. Reducing soluble TGFβRIII, rather than endoglin, would be more effective in alleviating the inhibition of both TGF-β1 and TGF-β2 signaling in PE.

The effects of hydroxychloroquine on endothelial dysfunction.

Hydroxychloroquine is an anti-malarial drug which, due to its anti-inflammatory and immunomodulatory effects, is widely used for the treatment of autoimmune diseases. In a model of systemic lupus erythematosus hydroxychloroquine has been shown to exert protective endothelial effects. In this study, we aimed to investigate whether hydroxychloroquine was endothelial protective in an in vitro model of TNF-α and preeclamptic serum induced dysfunction. We showed that hydroxychloroquine significantly reduced the production of TNF-α and preeclamptic serum induced endothelin-1 (ET-1). Hydroxychloroquine also significantly mitigated TNF-α induced impairment of angiogenesis. These findings support the further assessment of hydroxychloroquine as an adjuvant therapy in preeclampsia.

96 Inflammatory stress and deposition markers in the serum and on adipose tissue in normal and hypertensive pregnancy

Introduction Not only in hypertensive pregnancy but also in normal course, homeostatic inflammation on adipocyte and adipose tissue has been confirmed by basic pilot study. We previously showed inflammatory and anti-inflammatory genetic action of the adipose tissue under the new culture method in preeclamptic serum compared with normal pregnancy (Naruse K, et al. Mediators Inflamm, 2015). Objectives In this study, we measured Interleukins, proteases and cell death markers on the inflammatory pathway, in the culture supernatants of visceral adipose tissue with normal and preeclamptic serum. Additionally, other markers for lipid oxidative stress and adipose storage were measured in the serum from normal pregnancy and preeclampsia. Methods All samples were taken under informed consent of the patients. Subcutaneous fat tissue from repeated cesarean section and omentum (visceral fat) from probe laparotomy for ovarian cancer were dissected, vessels were removed and captured in Peptide Hydrogel (BD Biosciences, Inc.). After 24 h starvation, tissues were cultured additional 24 h with medium containing 10% of maternal serum from severe preeclampsia (n = 5) and gestational age-matched healthy pregnant maternal serum (n = 5). Ratio of M30 (Apoptosis marker)/M65 (all cell death marker including necrosis) protein were measured with ELISA after correction with original serum concentrations of the molecules. Concentration of LDH (cytotoxicity marker) was measured with specific assay. Interleukin (IL)-6 and Matrix Metalloproteinase (MMP)-2 were measured with SearchLightR Multiplex Immunoassay. On the serum samples taken from 1st, 2nd and 3rd trimester of normal pregnancy and at onset of severe preeclampsia (n = 10 each), Malondialdehyde (MDA: a lipid marker for oxidative stress) were measured with Thiobarbituric Acid Reactive Substances (TBARS) Assay and cell death-inducing DFFA-like effector c (CIDEC/FSP27: lipid storage and adipocyte apoptosis marker) were measured with ELISA. Results In adipose tissue culture, M30/M65 ratio showed trend to increase in preeclampsia than normal pregnancy (p = 0.053). However, LDH was significantly increased in normal pregnancy than preeclampsia (p Conclusions In the adipose tissue, increase of apoptosis was shown after addition of preeclamptic serum. On the other hand, total tissue injury seemed to be stronger in normal pregnancy than preeclampsia, suggesting that adipose tissue act as not only an inflammatory focus but as a systemic buffer for acute inflammation in severe preeclampsia. Inflammatory stress on adipose tissue in normal pregnant subjects and preeclampsia were shown firstly by MDA concentrations. Taken together, adipose tissue is proposed as a key organ of variated pathophysiology of preeclampsia.

Role of activin A in the pathogenesis of endothelial cell dysfunction in preeclampsia.

Circulating markers for endothelial activation such as endothelin-1 (ET-1), ICAM-1 and VCAM-1 are elevated in women with preeclampsia. Using human umbilical vein endothelial cells (HUVECs) as an in vitro model of the maternal vasculature, we show that activin A and preeclamptic serum upregulate ET-1, ICAM-1, and VCAM-1 in HUVECs. Further, we show that follistatin, a specific binding protein for activin, mitigates the upregulation of ET-1, ICAM-1 and VCAM-1 in HUVECs exposed to either activin A or preeclamptic serum. These data are consistent with activin A contributing to the pathophysiology of preeclampsia and suggest that therapies targeting activin signalling are worth exploring.

260-POS

Objectives To know the difference of eNOS expression in the exposure of normal serum with preeclamptic serum and Syzygium cumini extract. Methods This study used HUVEC cultures that were already exposed to preeclamptic serum as HUVEC PE model that has dysfunction. Endothelial cells were taken from umbilical cord of female newborns and culture made until a confluent culture was formed. HUVEC cultures were divided into six groups: (1) HUVECs+ Normal Serum (Control), (2) HUVECs+ Preeclamptic serum (HUVECs PE model), (3) HUVECs PE Model+ S.cumini extract 50 ppm, (4) HUVECs PE Model+ S. cumini extract 100 ppm, (5) HUVECs PE Model+ S. cumini extract 200 ppm, (6) HUVECs PE Model+ S. cumini extract 400 ppm. The cultures were incubated for 24 h; immunocytochemistry-painted using primary antibody was used. Endothelial NOS expression was observed using microscopy. Results When eNOS expression in group 1 was compared to group 2 and 5, there were significant differences; on the other hand there were no significant differences between group 1 and group 3, 4, and 6. Group 2 was significantly different from group 5 and 6, not significantly different from group 3 and 4. Group 3 was significantly different from group 5 and 6 but not significantly different from group 4. In group 4, 5 and 6 there were significant differences among each other. In the observation of HUVEC culture that was being exposed by S. cumini it was found that higher S. cumini dosage is given, the higher eNOS expression. Conclusions There were significant differences of eNOS expression between HUVECs preeclamptic model exposed to S. cumini. In HUVECs preeclamptic model exposed by S. cumini, the higher S. cumini dosage the higher eNOS expression. Disclosures K. Keman: None. S. Candra WB: None. T. Olfah: None. T. Olfah: None.

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Objectives Increased insulin resistance in preeclampsia has been regarded as a key mechanism for the establishment of the disease. Additionally, inflammatory action including adipocytokine production and homeostatic inflammation is shown as a critical pathway of pathophysiology of preeclampsia. To investigate the inflammatory pathway within tissue, we established new culture system of adipose tissue on the bottom of the plate (”gel-bottom capture”) with preeclampsia serum, and quantified the expressions of inflammation-related genes using paneled-array method. Methods Visceral fat (omentum) was obtained from the patients at reproductive age that had omentectomy with gynecological reason, which confirmed no metastasis of cancer. Tissue was captured on the bottom of culture plate using Peptide Hydrogel (BD Biosciences, Inc.) and incubated at 37 °C for 24 h with 10% serum from five severe preeclampsia or five gestational age-matched normotensive women. After extractions of total mRNA, 370 genes set related to inflammation were analyzed with inflammation-targeted PCR array (RT 2 Profiler™ PCR Array, Qiagen Inc.). Results The viability of the tissue was not altered after 24 h culture. 30 genes related inflammation in the adipose tissue showed statistically significant increase or decrease in preeclamptic culture compared to normal pregnancy. Altered genes were related to chemokine, inflammatory action including toll-like receptor and ligands, insulin resistance and adipogenesis, which plays a central role in systemic inflammation of preeclampsia. However, beneficial genes in regulation of Th2 predominance, anti-oxidative stress and insulin sensitivity were also defined. Conclusions Under our newly innovated culture method, it was firstly revealed that adipose tissue effects on both systemic inflammation and protection against inflammation or oxidative stress under preeclamptic serum. It suggested that the adipose tissue acts as a key organ of the pathogenesis of preeclampsia. Disclosures J. Akasaka: None. K. Naruse: None. N. Koike: None. A. Shigemitsu: None. K. Iwai: None. H. Kobayashi: None.