Endothelial Dysfunction In Preeclampsia Research Articles

Abstract 68: Soluble FMS-Like Tyrosine Kinase 1 induces Vascular Endothelial Dysfunction in Pregnant Female Mice

Preeclampsia (PE) is a hypertensive disorder of pregnancy clinically characterized by hypertension and increased risk of fetal growth restriction. Elevated levels of anti-angiogenic soluble fms-like tyrosine kinase 1 (sFlt-1) is a well-known hallmark of PE. In addition, emerging clinical evidence indicates that vascular endothelial dysfunction is a mediator of PE. Although these two facets of PE are associated, whether sFlt-1 is a crucial mediator in endothelial dysfunction in PE is unknown. We previously showed that the placenta-derived hormone leptin, whose levels characteristically increase in PE patients, induces the features of PE in pregnant mice, notably endothelial dysfunction. Novel preliminary data also shows that sFlt-1 induces an increase in placental leptin production, however, what remains to be explored is whether this pathway mediates endothelial dysfunction in PE pregnant mice. Therefore, we hypothesize that in pregnant mice, sFlt-1 induces vascular endothelial dysfunction via increasing leptin production. We bred BALB/c female mice for timed pregnancy (12-14 wk old). On gestational day (GD)11, pregnant females were implanted with subcutaneous (sc.) minipumps containing either sFlt-1 (1.2 µg/day, n=5) or saline (n=6). Uterine artery resistance index (UARI) was measured at GD17 via Doppler ultrasound. At GD18, vascular reactivity was measured in 2nd-order mesenteric arteries by wire myography. Our data showed that sFlt-1 increased UARI compared to sham mice ([PSV-EDV]/PSV; Mean±SEM;0.61±0.01 vs. 0.45±0.01, t test, p=0.0007). In addition, sFlt-1 reduced endothelial-dependent relaxation to acetylcholine (concentration-response, 1nM-10µM, 2-way ANOVA w/RM, p<0.0001), indicating endothelial dysfunction. sFlt-1 also reduced smooth muscle relaxation responses to sodium nitroprusside (p<0.05). To test whether sFlt-1 induces leptin production, we bred BALB/c mice as described above. At GD12, mice were treated with either sFlt-1 (sc.10 µg) or saline (n=6/group). After 24 h, mice were euthanized, and plasma and subcutaneous adipose tissue were collected. Our data showed an increase in plasma leptin in sFlt-1 group (ng/mL;5.0 ±0.2 vs. 2.9±0.1, t test, p<0.0001) and an upregulation in placental leptin gene expression (normalized to 18S; fold change to control; 7.8±1.8 vs. 1.1±0.3, t test, p<0.05). In conclusion, our data indicates that sFlt-1 induces vascular endothelial dysfunction and increases leptin plasma levels and gene expression in preeclampsia.

A Novel Stem Cell Model to Study Preeclampsia Endothelial Dysfunction

Preeclampsia is a common pregnancy complication affecting 5% to 7% of all pregnancies worldwide annually. While the pathogenesis is not fully understood, maternal endothelium dysfunction is thought to be a central component to preeclampsia development. Studies to dissect maternal endothelial dysfunction, particularly on a patient-specific basis, are hampered by limited access to systemic primary endothelial cells (ECs). The objective of this study was to establish a replenishable, patient-specific in vitro EC model to allow robust mechanistic studies to dissect endothelial dysfunction in preeclampsia. Induced pluripotent stem cells (iPSCs) from three women with a history of normotensive pregnancies were differentiated into ECs. The established ECs were exposed to pooled sera from normotensive pregnancies, preeclamptic pregnancies, normotensive postpartum for non-pregnant comparison and controls. Endothelial functions including nitric oxide (NO) release, cell migration, tube formation and viability were evaluated. Levels of NO release were significantly lower after incubation with preeclamptic sera compared to the fetal bovine serum (FBS) control, and normotensive and non-pregnant (postpartum) sera treatments were also lower than FBS but higher than preeclamptic sera treatments. Tube formation and cell migration were also impaired with preeclamptic sera compared to FBS controls. Cell viabilities remained unaffected by any sera treatment. Consistent outcomes were obtained across all three patient-specific lines treated with the same pooled sera. Establishment of patient-derived iPSC-ECs treated with pregnancy sera serves as a novel model to explore the interplay between individual maternal endothelial health and circulating factors that lead to endothelial dysfunction in preeclampsia.

The Correlation Between Neutrophil Elastase and Neutrophil-Lymphocyte Ratio in Endothelial Dysfunction of Preeclampsia

The Correlation Between Neutrophil Elastase and Neutrophil-Lymphocyte Ratio in Endothelial Dysfunction of Preeclampsia

Placental Protein 13 and Syncytiotrophoblast Basement Membrane Ultrastructures in Preeclampsia.

Background and Objectives: Preeclampsia has been linked to an inflammatory response that may be brought on by endothelial cell dysfunction. This paper investigates the pathomechanism of syncytiotrophoblast basement membrane (STBM) damage and Placental Protein 13 (PP13) release, which may have a role in systemic endothelial dysfunction in preeclampsia. Materials and Methods: This comparative cross-sectional study involves 54 preeclampsia patients (27 early-onset preeclampsia and 27 late-onset preeclampsia) and 27 pregnant women with normal blood pressure. An enzyme-linked immunosorbent assay was performed to evaluate maternal blood levels of PP13. Following birth, a portion of the placenta was collected for transmission electron microscope (TEM) and immunohistochemical (IHC) analysis. The data were analyzed using STATA version 15. Results: PP13 expression in the placental syncytiotrophoblast was significantly lower in the early-onset preeclampsia, compared to late-onset preeclampsia and normotensive pregnancy, group (p < 0.001). In contrast, serum PP13 levels were found to be the highest in the early-onset preeclampsia group, although no significant difference were found in mean maternal serum levels of PP13 between the three groups. The decreased PP13 expression in placental syncytiotrophoblast can be attributed to the greater extent of damage in the STBM in early-onset preeclampsia that leads to the release of a larger amount of PP13 into maternal circulation. The hypothesis aligns with the TEM analysis results. Preeclamptic pregnancies showed placental syncytiotrophoblast aponeurosis, whereas normotensive pregnancies did not. Placental lesions and STBM shedding were found to be more pronounced in early-onset preeclampsia compared to late-onset preeclampsia. Conclusions: PP13 and STBM damage may play a role in systemic endothelial dysfunction in preeclampsia.

Placental IL-1β inhibits PKM2 and leads to endothelial barrier injury in preeclampsia

Preeclampsia, a life-threatening hypertensive disorder of pregnancy, continues to be a major global health concern. Maternal endothelial dysfunction induced by components from the impaired placenta is a key hallmark of preeclampsia (PE). Increased levels of IL-1β have been observed in preeclamptic placentas, but its association with endothelial dysfunction has remained uncertain. Our hypothesis was that IL-1β released from the placenta links the placental abnormalities with maternal endothelial dysfunction in PE. Our investigation revealed elevated levels of IL-1β in preeclampsia placentas, which were negatively correlated with reduced VE-Cadherin expression in endothelial cells, indicating consistent endothelial dysfunction. Moreover, we identified the glycolytic enzyme pyruvate kinase 2 (PKM2) as a key regulator of glycolysis involved in the regulation of junctional VE-Cadherin in endothelial cells. Rescue assays further demonstrated that the overexpression of PKM2 was necessary to ameliorate endothelial barrier injury induced by IL-1β. Our study provides novel insights into the molecular mechanisms driving preeclampsia, highlighting the regulatory roles of IL-1β, PKM2, and VE-Cadherin in endothelial cells. These identified interactions may play a crucial role in the pathogenesis of preeclampsia, offering potential targets for therapeutic interventions to preserve endothelial integrity and alleviate the condition. This project was funded by Natural Science Foundation of Shandong (ZR2022MH195 and ZR2023MH211), and the Graduate Student Research Grant from Weifang Medical University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 12569: The Roles of microRNA in Preeclampsia Induced Fetal Sex-Specific Endothelial Dysfunction

Introduction: Preeclampsia (PE) is a hypertensive disorder that complicates 5-10% of human pregnancies and leads to high fetal morbidity and mortality. Maternal obesity increases the risks of PE by 3-fold in association with elevated circulating pro-inflammatory cytokines which are related to endothelial dysfunction observed in PE. MiR146a-5p is a cardiovascular disease-associated miRNA that regulates endothelial cell responses. We have previously reported fetal sex-specific fetal endothelial dysfunction in PE. Although miR146a-5p has been reported to be dysregulated in PE HUVECs, the role of miR146a-5p in PE-dysregulated fetal sex-specific endothelial dysfunction is unclear. Hypothesis: MiR146a-5p differently mediate PE-induced sex-specific fetal endothelial dysfunction in lean and obese pregnancies. Methods: Human umbilical vein endothelial cells (unpassaged, P0-HUVECs; 5-7 days of culture, P1-HUVECs) were isolated immediately after delivery from normotensive (NT; 39±0.5 wks) and PE (38±0.5 wks) pregnancies (lean and OB) with female (F) and male (M) fetuses. Expression of miR146a-5p was examined in P0-HUVECs using RT-qPCR (n=8-10/group). Bioinformatics analysis was performed with our existing RNAseq data to identify PE-dysregulated miR146a-5p target genes in HUVECs. Endothelial monolayer integrity (MI) responses to cytokines were examined in P1 HUVECs with and without miR146a-5p overexpression and knockdown (n=5-7/group/assay). Results: PE upregulated miR146a-5p in lean F-HUVECs, while downregulated miR146a-5p in obese M-HUVECs. PE differentially dysregulated vascular/endothelial function-associated miR146a-5p target genes (e.g., TNFα-/TGFβ-signaling pathways) in F&amp;M HUVECs. PE further reduced the TNFα-weakened MI in F and M HUVECs in both lean and OB pregnancies. TGFβ1 strengthened MI only in F-HUVECs, which was abolished in lean PE-F-HUVECs and was further decreased in OB PE-F-HUVEC. Knockdown of miR146a-5p rescues the PE-impaired MI responses to TNF-α and TGF-β1 in lean F-HUVECs but only rescues PE-impaired MI responses to TGF-β1 in OB F-HUVECs. Conclusions: MiR146a-5p plays important roles in mediating PE-induced fetal sex-specific fetal endothelial dysfunction in lean and obese pregnancies.

Increased 8-OHdG in umbilical cord blood represents the risk of preeclampsia with Intra-Uterine Growth Restriction (IUGR)

Link of Video Abstract: https://youtu.be/VsAy9X8XIAk Background: Preeclampsia is a pregnancy-related disease involving multi-organs, characterized by increased blood pressure in the second trimester of pregnancy. Preeclampsia can have short-term and long-term consequences for the mother and the fetus, including intra-uterine growth restriction (IUGR). One of the markers of DNA damage due to oxidative stress due to endothelial dysfunction in preeclampsia is the increase in 8-OHdG levels in cord blood. This study aims to see an increase in 8-OHdG levels in preeclampsia. Methods: This study was a cross-sectional study with a total of 41 patients with preeclampsia and 41 patients with healthy pregnant women at Wahidin Sudirohusodo Hospital and its network. Samples were taken using the purposive sampling method according to the inclusion criteria. This 8-OHdG level was examined using the ELISA kit at the HUMRC laboratory of Hasanuddin University Hospital. The data is then processed statistically using SPSS. Results: This study found 6 patients with PE + IUGR, 31 with PE, and 41 without PE. The examination results of 8-OHdG levels found significant differences between the 3 groups PE + IUGR, PE, and normal pregnancies, which showed a significant relationship between elevated 8-OHdG levels and the incidence of PE accompanied by IUGR (p = 0.006) Conclusion: Elevated 8-OHdG levels are found in preeclampsia, especially in preeclampsia accompanied by IUGR.

The Evaluation of Adropin and Autotaxin as Potential Markers of Endothelial Dysfunction in Preeclampsia.

Endothelial dysfunction (ED) plays a prominent role in the pathogenesis of preeclampsia (PE). There is a need for non-invasive methods to assess endothelial function in preeclamptic patients. In the present study, adropin, autotaxin (ATX), and lysophosphatidic acid (LPA) were evaluated as indicators of ED. Patients diagnosed with PE and healthy pregnant women (n = 42 for each group) were compared. After measuring flow-mediated dilation (FMD), the participants were stratified as ED (+) or ED (-) based on a cut-off value of 6.5%. The PE patients were divided as early/late onset PE and severe/mild PE. Adropin, ATX, and LPA levels were measured, and their relevance to ED was evaluated. Student t, Mann-Whitney U, or ANOVA tests were used for statistics, as appropriate. Adropin levels were diminished in the ED (+) group, whereas ATX and LPA levels were increased. The decrease in adropin levels was more pronounced in severe PE, showing a positive correlation with the FMD. In the logistic regression model, adropin was the only parameter that was an independent variable for the FMD test (P < .001). Adropin measurements in serum may be of value for disease follow-up in patients with PE.

Human umbilical cord mesenchymal stem cell derived exosomes (HUCMSC-exos) recovery soluble fms-like tyrosine kinase-1 (sFlt-1)-induced endothelial dysfunction in preeclampsia

BackgroundPreeclampsia is a unique multisystem disorder that affects 5–8% of pregnancies. A high level of soluble fms-like tyrosine kinase-1 (sFlt-1) is a hallmark of preeclampsia that causes endothelial dysfunction. Exosomes derived from mesenchymal stem cells (MSCs) have been indicated to improve endothelial performances by transporting signals to target cells. We hypothesized that exosomes derived from MSCs have potential effects against preeclampsia.MethodsWe collected human umbilical cord MSC-derived exosomes (HUCMSC-exos) by ultracentrifugation. The size and morphology of the exosomes were examined using a transmission electron microscope and nanoparticle tracking analysis. Pregnant mice were injected with murine sFlt-1 adenovirus to build the preeclampsia-like mouse model and then treated with HUCMSC-exos. Human umbilical vein endothelial cells (HUVECs) were infected with lentiviruses expressing tet-on-sFlt-1 to obtain cells overexpressing sFlt-1. Cell proliferation and migration assays were used to measure the endothelial functions. The exosomes enriched proteins underlying mechanisms were explored by proteomic analysis.ResultsIn the current study, we successfully collected the cup-shaped HUCMSC-exos with diameters of 30–150 nm. In the sFlt-1-induced preeclampsia mouse model, HUCMSC-exos exhibited beneficial effects on adverse birth events by decreasing blood pressure and improving fetal birth weight. In addition, preeclamptic dams that were injected with HUCMSC-exos had rebuilt dense placental vascular networks. Furthermore, we observed that HUCMSC-exos partially rescued sFlt-1-induced HUVECs dysfunction in vitro. Proteomics analysis of HUCMSC-exos displayed functional enrichment in biological processes related to vesicle-mediated transport, cell communication, cell migration, and angiogenesis.ConclusionWe propose that exosomes derived from HUCMSCs contain abundant Versican and play beneficial roles in the birth outcomes of sFlt-1-induced preeclamptic mice by promoting angiogenesis.Graphical

Placental expression of striatin & endothelial nitric oxide synthase in women with & without pre-eclampsia.

Striatin is a multi-domain scaffolding protein essential for activating endothelial nitric oxide synthase (eNOS). However, its role in pre-eclampsia remains use explored. Hence, this study aimed to investigate the association between striatin and eNOS in regulating nitric oxide (NO) production in the placenta of women with and without pre-eclampsia. Forty pregnant women each without (controls) and with pre-eclampsia (cases) were enrolled in the study. Blood striatin and NO concentrations were detected by the ELISA. Protein expression of striatin, phosphorylated eNOS (peNOS), inducible NOS (iNOS) and phosphorylated NF-κB were measured in the placental tissues by Western blotting. Twenty-four-hour urinary protein and serum urea, uric acid and creatinine were analyzed as an autoanalyzer. Placental histology was analyzed by haematoxylin and eosin staining. Compared to normotensive pregnant women, the levels of serum NO and striatin were decreased in pre-eclamptic women. The protein expression of striatin and peNOS was significantly reduced (P<0.05) while p65NF-κB and iNOS were upregulated considerably (P<0.05) in the placenta of cases compared to controls. Our results show for the first time that decreased striatin expression was associated with decreased peNOS protein expression in the placental tissue of pre-eclamptic women. Interestingly, no significant difference was found in blood striatin or NO levels between controls and cases. Thus, therapies that improve placental striatin expression are attractive possibilities, both for prevention as well as treatment of endothelial dysfunction in pre-eclampsia.

Potential role of uric acid to activate NLRP3 inflammasome triggering endothelial dysfunction in preeclampsia

Preeclampsia (PE) is a pregnancy-related disorder associated with increasing maternal death rates and affecting 2–8% of pregnant women worldwide. Arterial hypertension and proteinuria, followed by other maternal dysfunctions are classic clinical parameters to identify this disease. Furthermore, hyperuricemia is strictly related to PE, and high plasmatic levels of uric acid have been associated with disease severity. The inflammation in the endothelium caused by danger signals, such as uric acid crystals, is persistent and stimulates the release of inflammatory cytokines, and activates the NLRP3 inflammasome. The mechanisms underlying endothelial dysfunction induced by the metabolism of uric acid, and consequently increased levels of inflammation and oxidative stress in women with PE could be ameliorated with interventions related to inhibition of the NLRP3 activation MSU-mediated and may improve the prognostics of this disease.

Abstract 11411: Anti-MiR-510-3p Alleviates Endothelial Dysfunction in Preeclampsia Induced Rat Model B\by Regulating PTEN and Its Signalling Pathways

Introduction: Preeclampsia (PE) is a pregnancy complication associated with multi-organ endothelial dysfunction. The hypoxia resulting from vascular remodelling lowers blood flow in placenta leading to high mortality and morbidity in PE. Studies on molecular and signalling pathways involved in PE could pave way for early diagnosis and treatment. miRNAs are crucial regulators of gene expression in various diseases including PE. Our previous studies reported high expression of miR-510 in PE patients compared to normal. Hypothesis: We hypothesize that miR-510-3p targets PTEN in regulation of PI3K/AKT/eNOS/mTOR axis in PE and miR-510-3p could be a potential biomarker and therapeutic target. We elucidate the therapeutic role of antimiR-510-3p in PE and its interaction with miR-510-3p and associated signalling pathways in PE induced rat model. Methodology: The proliferation, migration, invasion and apoptosis of HTR/SVNeo cells were analyzed for miR-510-3p, PTEN and antimiR-510-3p. Luciferase assay confirmed PTEN as target for miR-510-3p. The expression of miR-510-3p and PTEN/PI3K/AKT/eNOS/mTOR axis was analysed using qPCR and western blot. Deoxycorticosterone (DOCA) (12.5 mg/kg before mating and 6.5 mg/kg after mating twice a week) and 0.9% salt ad libitum were used to develop a PE induced rat model (SD rats). By quantifying miR-510-3p levels in PE group, the doses of antimiR-510-3p for the dose-dependent study were determined. The final effective dose was fixed from the dose-response curve. To examine the hypoxia and hypertrophy, histological analysis was performed in paraformaldehyde fixed tissues. Results: Significant changes were observed in the BP, proteinuria and other biochemical parameters between PE and control rats. Our results suggest that miR-510-3p targets PTEN leading to endothelial dysfunction, hypoxia and hypertrophy in PE, while treatment with antimiR-510-3p exhibits a significant reduction in PE in PE induced rat model. The gene and protein expression analysis revealed a significant correlation between miR-510-3p, PTEN and antimiR-510-3p in PE. Conclusion: Thus our findings from in vitro and in vivo suggest miR-510-3p as a potential biomarker and therapeutic target and antimiR-510-3p as a novel therapeutic molecule for PE.

Abstract 050: Endothelial Cell Mineralocorticoid Receptor Deletion Improves Endothelial Function, Blood Pressure And Fetal Growth In The RUPP Mouse Model Of Preeclampsia

Preeclampsia (PE) is a hypertensive disorder of pregnancy that causes adverse pregnancy outcomes. Progesterone in pregnancy increases endothelial mineralocorticoid receptor (ECMR) expression, which we showed mediates endothelial function in hypertensive female mice, however, whether ablating endothelial dysfunction in PE improves pregnancy outcomes is unknown. We hypothesized that improving endothelial function via ECMR deletion increases fetal growth and ablates hypertension in the reduced uterine perfusion pressure (RUPP) PE mouse. Pregnant ECMR-intact (WT) and -deficient (KO) mice underwent RUPP or sham surgery on GD13 and sacrificed on GD18 (WT Sham=9, WT RUPP=5, KO Sham=6, KO RUPP=6). Vascular function was measured via wire myography on 2 nd order mesenteric arteries and thoracic aorta. Systolic blood pressure (BP) was measured by radiotelemetry in a subset of mice. RUPP did not decrease maternal weights in either WT or KO mice (P&gt;0.05) but decreased pup weight (726±20 WT+Sham vs 660±11mg WT+RUPP, *P&lt;0.05) and fetal demise (16±4 vs 28±4% resoprtions/litter, *P&lt;0.05) in WT pregnant mice, however, not in KO mice (710±15 KO+Sham vs 693±19 mg KO+RUPP, P&gt;0.05, and 13±5 vs 28±12% resoprtions/litter, P&gt;0.05), indicating KO protected fetal growth. RUPP reduced endothelial-dependent, acetylcholine-mediated vascular relaxation (10 -9 -3x10 -5 M) in WT, but not KO pregnant mice (*P&lt;0.05, 2-way ANOVA w/ RM), but not aorta ACh-mediated relaxation (P&gt;0.05). Nitric oxide synthase (NOS) inhibitor LNAME ablated differences in ACh-mediated relaxation between WT Sham and WT RUPP (P&gt;0.05), with no differences observed in KO Sham or KO RUPP, in both aorta and mesenteric arteries, indicating RUPP impaired endothelial function via decreasing NOS activity. Preliminary data demonstrates RUPP increased BP on GD15-17 in WT (127±21 mmHg, N=3), however, not in KO pregnant mice (111±4, N=2). RUPP also ablated circadian rhythm in WT, but not KO, pregnant mice from GD15-18. Collectively, these data indicate that ECMR activation induces reduced pup weight, endothelial dysfunction and hypertension in the RUPP model which may lead to clinical studies for consideration of specific MR antagonists as therapeutic options for endothelial dysfunction in PE pregnancies.

Pentoxifylline as a therapeutic option for pre-eclampsia: a study on its placental effects.

Recently pentoxifylline, a non-selective phosphodiesterase inhibitor and adenosine receptor antagonist, has attracted much interest for the treatment of the increased vascular resistance and endothelial dysfunction in pre-eclampsia. We therefore investigated the placental transfer, vascular effects and anti-inflammatory actions of pentoxifylline in healthy and pre-eclamptic human placentas. The placental transfer and metabolism of pentoxifylline were studied using ex vivo placenta perfusion experiments. In wire myography experiments with chorionic plate arteries, pentoxifyllines vasodilator properties were investigated, focusing on the cGMP and cAMP pathways and adenosine receptors. Its effects on inflammatory factors were also studied in placental explants. Pentoxifylline transferred from the maternal to foetal circulation, reaching identical concentrations. The placenta metabolized pentoxifylline into its active metabolite lisofylline (M1), which was released into both circulations. In healthy placentas, pentoxifylline potentiated cAMP- and cGMP-induced vasodilation, as well as causing vasodilation by adenosine A1 antagonism and via NO synthase and PKG. Pentoxifylline also reduced inflammatory factors secretion. In pre-eclamptic placentas, we observed that its vasodilator capacity was preserved, however not via NO-PKG but likely through adenosine signalling. Pentoxifylline neither potentiated vasodilation through cAMP and cGMP, nor suppressed the release of inflammatory factors from these placentas. Pentoxifylline is transferred across and metabolized by the placenta. Its beneficial effects on the NO pathway and inflammation are not retained in pre-eclampsia, limiting its application in this disease, although it could be useful for other placenta-related disorders. Future studies might focus on selective A1 receptor antagonists as a new treatment for pre-eclampsia.

Endothelial Cell Mineralocorticoid Receptor (ECMR) deletion improves fetal growth and vascular function in the RUPP mouse model of preeclampsia

Preeclampsia (PE), a hypertensive disorder of pregnancy, induces adverse pregnancy outcomes including fetal growth restriction (FGR) and afflicts ~5‐10% of pregnancies. Compelling clinical data indicate that PE risk and severity in pregnant women is strongly associated with endothelial dysfunction. Previous work by our group shows that deletion of endothelial cell mineralocorticoid receptors (ECMR) improves endothelial function in premenopausal hypertensive female mice, however whether ECMR promotes endothelial dysfunction in PE is unknown. We tested the hypothesis that ECMR deletion improves FGR and endothelial function in a mouse model of PE, the reduced uterine perfusion pressure (RUPP) mouse. Pregnant ECMR‐intact (WT) and ECMR‐deficient (KO) mice were randomized to RUPP or sham surgery on GD13 and sacrificed on GD18 (WT Sham=6, WT RUPP=5, KO Sham=7, KO RUPP=6). Vascular function was measured via wire myography on 2nd order mesenteric arteries. Conscious blood pressure was measured by radiotelemetry throughout pregnancy in a subset of mice. RUPP surgery did not decrease maternal weights in either WT or KO mice (P&gt;0.05) at GD18. RUPP surgery significantly decreased GD18 pup weight, a measure of FGR, (726±20mg WT+Sham vs 660±11mg WT+RUPP, *P&lt;0.05) and trended to decrease placental efficiency (pup/placenta weight) (8.23±0.23 WT+Sham vs 7.45±0.22 WT+RUPP P=0.13) in WT pregnant mice. ECMR deletion protected pregnant mice both from RUPP‐induced reductions in pup weight (710±20mg KO+Sham vs 690±20mg KO+RUPP) and decreases in placental efficiency (7.63±0.19 KO+Sham vs 7.76±0.31 KO+RUPP). RUPP reduced acetylcholine‐mediated relaxation (10‐9‐3x10‐5 M) in mesenteric arteries of WT pregnant mice but not KO pregnant mice (*P&lt;0.05, 2‐way ANOVA, repeated measures), indicating RUPP induced endothelial dysfunction in WT mice only. Preincubation with nitric oxide (NO) synthase inhibitor LNAME ablated differences in Ach‐mediated relaxation between WT+sham and WT+RUPP mice (P&gt;0.05), indicating that reductions in endothelial function in WT+RUPP mice were mediated by reduced NO. However, ECMR deletion as a variable increased Ach‐mediated relaxation in the presence of LNAME in both Sham and RUPP mice (*P&lt;0.05). Therefore, ECMR deletion may have protected pregnant mice from endothelial dysfunction via a non‐NO mechanism. RUPP did not increase a‐receptor agonist phenylephrine (Phe)‐induced constriction in WT mice, however, ECMR deletion as a variable decreased Phe‐mediated constriction independent of RUPP surgery indicating that ECMR deletion reduces smooth muscle‐mediated vasoconstriction in pregnant sham and RUPP mice. These data indicate that specific deletion of ECMR improves vascular function to increase vasodilation and prevent constriction in PE pregnancy. Preliminary data further indicates that systolic blood pressure increased from GD8‐18 (pre‐post RUPP) in WT+RUPP but decreased in KO+RUPP pregnant mice (2.7 vs ‐8.6 DmmHg, respectively, N=1). Lastly RUPP surgery trended to increase plasma levels of the ECMR agonist corticosterone in WT pregnant mice (2.03±0.4pg/ml WT+Sham vs 3.33±0.95pg/ml WT+RUPP). Collectively, these data indicate that ECMR activation promotes FGR, vascular dysfunction and hypertension in the RUPP model of PE which may lead to clinical studies for the consideration of specific MR antagonists as therapeutic options for PE pregnancies.

Long noncoding TUG1 promotes angiogenesis of HUVECs in PE via regulating the miR-29a-3p/VEGFA and Ang2/Tie2 pathways.

Preeclampsia (PE) is a pregnancy-specific disease that is associated with oxidative stress-induced endothelial dysfunction. Long noncoding RNAs (lncRNAs) are related to PE progression. The purpose is to study whether lncRNA taurine-upregulated gene 1 (TUG1) takes part in endothelial dysfunction in PE. The placenta tissues were collected from PE patients and normal subjects. Human umbilical vein endothelial cells (HUVECs) were suffered from hypoxia-reoxygenation (H/R). TUG1, miR-29a-3p and vascular endothelial growth factor A (VEGFA) were detected via qRT-PCR. soluble fms-related tyrosine kinase-1 (sFLT1) and soluble endoglin (sENG) levels were detected by ELISA. Cell proliferation, migration, invasion and angiogenesis were examined via MTT, wound healing analysis, transwell and tube formation analysis. The proteins in VEGFA and angiopoietin 2 (Ang2)/tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2) signaling were measured by western blot. The binding relationship was analyzed via Starbase, Jefferson and dual-luciferase reporter analysis. TUG1 and VEGFA levels were downregulated, and levels of miR-29a-3p, sFLT1 and sENG were increased in PE patients. TUG1 abundance was reduced in H/R-stimulated HUVECs, and TUG1 overexpression increased proliferation, migration, invasion and angiogenesis, and activated the VEGFA and Ang2/Tie2 signaling in H/R-stimulated HUVECs. TUG1 sponged miR-29a-3p, and miR-29a-3p overexpression reversed the function of TUG1 on H/R-induced HUVECs dysfunction. MiR-29a-3p knockdown attenuated H/R-induced inhibition of proliferation, migration, invasion, angiogenesis and activation of the VEGFA and Ang2/Tie2 signaling in HUVECs. VEGFA and Ang2 were targeted by miR-29a-3p, and VEGFA or Ang2 silence weakened the role of miR-29a-3p knockdown in H/R-caused HUVECs dysfunction. TUG1 facilitates proliferation, migration, invasion and angiogenesis in H/R-stimulated HUVECs via activating the VEGFA and Ang2/Tie2 signaling by regulating miR-29a-3p.

P-045. VEGFR/SFlt1 is associated to disruption of oxidative stress leading to endothelial dysfunction in preeclampsia

The deformation properties of rocks ultimately become anisotropic depending on the orientations of mineral particles and sedimentary structures. Characterization of deformation anisotropy is important for accurate understanding of the deformation characteristics of structures constructed inside rocks (e.g., rock foundation and tunnel). The simplest anisotropic elastic model is transversely isotropic elasticity, and in recent years, many methods for rationally specifying this elastic constant have been proposed.Although the most general linear elastic model for describing deformation anisotropy is orthotropic elasticity, no efficient method currently exists for determining the orthotropic elastic constants because the number of elastic constants required for the model description is extremely large. A rational determination method for orthotropic elasticity is necessary when considering application at construction sites and coupling with other phenomena such as differences in water content. In this study, a new method was developed for obtaining all 12 constants of orthotropic elasticity by three uniaxial compression tests on specimens sampled in three directions orthogonal to each other. For each specimen, the strain tensor was measured, and the strains in the elastic region were added and analyzed to identify the principal orientation of orthotropic elasticity. Thereafter, nine elastic constants are specified using the stress–strain relationship of each specimen. The methodology is described herein. The validity of the proposed method was evaluated on the basis of results from verification experiments performed with Neogene soft sedimentary rocks.

In silico study of gene-gene interaction of PIK3R1and PIGF deleterious SNPs in correlation to the preeclampsia risk and its haplotype effect on antihypertensive treatment

Introduction: The PIK3R1 (phosphoinositide-3-kinase regulatory subunit 1) and PlGF (Placenta growth factor)genes share some common pathways with eNOS,( Endothelial nitric oxide synthase) that plays a vital role in angiogenesis of blood vessels and regulating endothelial function. The PIK3R1 gene encodes the receptor for the p85α regulatory subunit of the phosphoinositide-3-kinase (PI3K), that involved in endothelial cell migration. The PlGF gene encodes for the placenta growth factor, a homolog of vascular endothelial growth factor (VEGFA) that is involved in angiogenesis. PIK3R1 and/or PlGF mutations may cause dysregulation of eNOS contributing to endothelial dysfunction in preeclampsia. This study aims to analyze the effect of mutation of the PIK3R1 and PlGF genes on the structure and function of PIK3R1 and PGF protein that may have an important role in pathogeneses of preeclampsia. Methodology: The data on human PIK3R1and PGF genes were retrieved from dbSNP/NCBI. Ten prediction algorithms; SIFT, PROVEAN, Polyphen, SNAP2, SNPs&GO, PANTHER PhD-SNP, I-Mutant, Mutpred, and Hope were used to analyzing the effect of nsSNPs on functions and structure of the PGF and PIK3R1 protein. STRING and KEGG databases were used for PGF and PIK3R1 protein-protein interaction. Results and Discussion: As per the dbSNP database, the humanPIK3R1gene contained 365 missense mutations. A total 3nsSNPs (T239M, S229W, E47K) and 2 nsSNPs (H125Y, V59G) were predicted to have the most damaging effects on the structure and function of PIK3R1 and PGF respectively. STRING and KEGG revealed that PIK3R1and PGF had strong interactions with proteins involved in the VEGF signaling pathway and PI3K-Akt signaling pathway. PIK3R1 is confirmed to be linked to important diseases like preeclampsia and can affect its treatment response. Conclusion: Gene-gene interaction is an important factor in preeclampsia treatment, effect of mutation of the PIK3R1 and PlGF genes on the structure and function of PIK3R1 and PGF protein may have an important role in pathogeneses of preeclampsia. Also, these are linked to its treatment effect. This document gives formatting instructions for authors preparing papers for publication in the journal. Authors are encouraged to prepare manuscripts directly using this template. This template demonstrates the format requirements for the Journal.

Second and third trimester serum levels of HtrA1 in pregnancies affected by pre-eclampsia

IntroductionAltered placental expression of high temperature requirement factor A1 (HtrA1) is implicated in abnormal trophoblastic invasion and endothelial dysfunction in pre-eclampsia (PE). Serum levels of HtrA1 have been proposed as a novel biomarker to improve the prediction of PE. This study assesses serum HtrA1 levels in prospectively collected samples of women who developed PE compared to normotensive pregnancies. MethodsThis was a case-control study of serum HtrA1 levels in second and third trimester samples in women who later developed preterm or term PE compared to controls. Overall, 300 serum samples were drawn from a prospective observational study of adverse pregnancy outcomes in three different gestational age windows (19–24, 30-34 and 35–37 weeks) at the Fetal Medicine Research Institute, King's College Hospital, London. Serum HtrA1 levels were determined by enzyme-linked immunosorbent assay (ELISA) by a blinded laboratory professional. Median HtrA1 MoM values, adjusted for gestational age and maternal characteristics, were compared between cases and controls at each gestational age group. ResultsWomen who later developed PE, compared to controls, had significantly higher maternal weight and more frequently had chronic hypertension or a history of PE in a previous pregnancy. In normotensive pregnancies, serum HtrA1 increased with increasing gestational age, whereas, in PE pregnancies HtrA1 levels remained stable, but were not significantly different from control pregnancies at any gestational age. DiscussionSerum HtrA1 levels are not significantly different in women who develop PE compared to controls.

INVESTIGATION OF SFLT-1 AND VEGF EXPRESSION IN NORMOTENSIVE AND PREECLAMPTIC PLACENTA. AN IMMUNOHISTOCHEMICAL STUDY

Objectives: The pathogenesis of preeclampsia is still not clear, but endothelial dysfunction believed to be one of the most encountered problems during placenta development in preeclamptic patients. Both vascular endothelial growth factor (VEGF) and its antagonist, soluble Fms-Like tyrosine kinase-1 (sFlt-1), have roles in vascular function. In this study, we have investigated immunohistochemical expression of VEGF and sFlt-1 in term placenta of normotensive and preeclampsia patients. Methods: Totally twenty term placentas were obtained from pregnant women of which 10 preeclampsia patient and 10 normotensive. Placentas were dissected and tissue samples were subjected to routine tissue processing protocol, then embedded in paraffin blocks. Serial sections were obtained from paraffin blocks and stained with H&amp;amp;E and PAS for routine histopathology. VEGF and sFlt-1 immunohistochemistry was performed to the sections. Results: When compared to control group, severe pathological changes were observed in preeclamptic placentas. Increase in number of syncytial knots and intervillous bridges, hemorrhage in interstitium, dilatation and congestion in villous capillaries, increase in fibrin accumulation in villus stroma, and increase in thickening of basement membrane were very clear. VEGF expression was significantly higher in normotensive placentas compared to preeclamptic. On the other hand, sFlt-1 expression was significantly increased in preeclamptic placentas. Conclusions: When the VEGF and sFlt-1 expression was considered, higher expression of sFlt-1 at preeclampsia, but decrease in VEGF expression, might be related to endothelial dysfunction in preeclampsia. Overall, this study demonstrates, imbalance between VEGF and sFlt-1 is one of the major reason of endothelial dysfunction in preeclamptic placenta.