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[306-POS]
Abstracts / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 5 (2015) 53–156
Therefore, we tested the hypothesis that MR-proADM can serve as a biomarker for preeclampsia. Methods: We selected 30 women with preeclampsia and 30 normotensive controls from a prospective study that collected blood and tissue samples as patients presented to Duke University Hospital for delivery. Exclusion criteria were: mild preeclampsia (n = 5), postpartum plasma collection (n = 12), twin pregnancies (n = 3), and smokers (n = 7). Endoglin, placental growth factor (PlGF), and MR-proADM concentrations were quantified in plasma using a commercial ELISA kit or immunofluorescent assay. Endoglin, PlGF, and MR-proADM were then analyzed as biomarkers of severe preeclampsia by comparing the areas under the curve (AUC) of receiver operating characteristic (ROC) curves generated by logistic regression models of these analytes. Results: MR-proADM concentrations were blunted in plasma from women with severe preeclampsia (Matson et al., 2014). In our dataset, endoglin (AUC = 0.73) and MRproADM (AUC = 0.69) were similarly effective at classifying women with severe preeclampsia and controls, while PlGF was more informative (AUC = 0.85). Endoglin and MRproADM together (AUC = 0.80) discriminate patients from controls better than they do individually, as do PlGF and MR-proADM together (AUC = 0.87). Conclusions: MR-proADM can classify women with severe preeclampsia and controls and may provide a biomarker for severe preeclampsia. However, analysis of MR-proADM levels at earlier gestational ages is needed to determine whether MR-proADM can predict the development of preeclampsia later in pregnancy. Disclosures: B. Matson: Research Support Recipient: Ferring Innovation Fellowship. N. Karpinich: None. A. Murtha: None. W. Valdar: None. C.A. Grotegut: None. K.M. Caron: Research Support Recipient; Commercial Interest: Ferring Pharmaceuticals, Inc. doi:10.1016/j.preghy.2014.10.311
[306-POS] Pregnancy and magnesium Ragnar Rylander (BioFact Environmental Health Research Center, Lerum, Sweden) Objectives: To review the role of magnesium in pregnancy. Methods: Analysis of published reports with emphasis on recent work on cellular mechanisms and magnesium responsive genes (MgRGs), plus controlled trials of magnesium (Mg) supplementation to prevent gestational hypertension. Results: In a study on women with normal pregnancies, MgRGs were up regulated as compared to non-pregnant women, suggesting a higher Mg demand. They also had lower plasma levels of Mg and an increased urinary excretion of Mg. Systolic and diastolic blood pressure increases during pregnancy were related to the urinary excretion of Mg. Among women with PE, the expression of the MgRG SLC41A1 – a Na/Mg exchanger – was overexpressed in 54.2% of the placentas as compared to 9.5% in normal
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placentas. Brain and muscle levels of Mg were lower among women with PE. In three supplementation studies, Mg reduced the occurrence of gestational hypertension, particularly the diastolic blood pressure. Conclusions: Data from genetic, cellular, and clinical studies support a conclusion that Mg plays an important role in pregnancy and that Mg deficiency could be a major factor for the pathogenesis in complicated pregnancies. It is unlikely that Mg is the sole agent involved and other nutritional factors are probably also of importance. Further studies are needed to assess the relationship with Mg and formulate requirements for intervention programs. Disclosures: R. Rylander: None. doi:10.1016/j.preghy.2014.10.312
[307-POS] Cx43 phosphorylation and the functionality of Cx43 gap junctions are moderated by cyclic nucleotide activity in UAECs and HUVECs Bryan Ampey, Ian Bird, Ron Magness (University of Wisconsin, Madison, Madison, WI, USA) Objectives: Gap junctions (GJ) are intercellular channels that contribute to the robust vasodilation found in pregnancy in several vascular beds. GJs allow for the coordination of endothelial cell responses by directing cell-cell communication via facilitated diffusion of signaling molecules (e.g. cAMP, cGMP). The phosphorylation of GJ protein Cx43 at serine (Ser) residues regulates both open (Ser365) and closed gating (Ser368), thus allowing the passage of signaling molecules between cells. Although the phosphorylation events of cAMP are well studied, little is known about cGMPs role on gap junction intracellular communication (GJIC) and function. Hypothesis: The inhibition of cAMP and cGMP-mediated pathways will modulate the phosphorylation and expression of Cx43 at their respective phosphorylation sites and thus increase or reduce GJIC in pregnant ovine uterine artery endothelial cells (P-UAECs) and human umbilical vein endothelial cells (HUVECs). Methods: P-UAECs were pretreated with the respective cAMP and cGMP nucleotide cyclase inhibitors SQ22536 and ODQ (0.1–100 lM) followed by treatment with 100 lM of ATP or Forskolin, or 1 lM–1 mM 8-Bromo-cAMP or 8-Bromo-cGMP and analyzed using ELISA, western analysis, or scrape-loading/dye transfer technique. Results: ELISA revealed that the inhibitors SQ22536 and ODQ specifically and completely abrogated (P<0.01) cAMP and cGMP productions, respectively. Western analysis showed that SQ22536, decreased (P<0.05) the ATP, Forskolin, and cAMP-induced phosphorylation at Ser365 in PUAECs and HUVECs, while GJIC function was reduced. ODQ decreased (P<0.05) ATP and cGMP-induced elevation at Ser365 in P-UAECs and HUVECs. In contrast, ODQ alone had no effect on GJIC. Conclusions: cAMP via adenylyl clycase and cGMP via guanylyl cyclase differentially regulate Cx43 phosphorylation and expression, which is important in regulating GJIC.
Therefore, we tested the hypothesis that MR-proADM can serve as a biomarker for preeclampsia. Methods: We selected 30 women with preeclampsia and 30 normotensive controls from a prospective study that collected blood and tissue samples as patients presented to Duke University Hospital for delivery. Exclusion criteria were: mild preeclampsia (n = 5), postpartum plasma collection (n = 12), twin pregnancies (n = 3), and smokers (n = 7). Endoglin, placental growth factor (PlGF), and MR-proADM concentrations were quantified in plasma using a commercial ELISA kit or immunofluorescent assay. Endoglin, PlGF, and MR-proADM were then analyzed as biomarkers of severe preeclampsia by comparing the areas under the curve (AUC) of receiver operating characteristic (ROC) curves generated by logistic regression models of these analytes. Results: MR-proADM concentrations were blunted in plasma from women with severe preeclampsia (Matson et al., 2014). In our dataset, endoglin (AUC = 0.73) and MRproADM (AUC = 0.69) were similarly effective at classifying women with severe preeclampsia and controls, while PlGF was more informative (AUC = 0.85). Endoglin and MRproADM together (AUC = 0.80) discriminate patients from controls better than they do individually, as do PlGF and MR-proADM together (AUC = 0.87). Conclusions: MR-proADM can classify women with severe preeclampsia and controls and may provide a biomarker for severe preeclampsia. However, analysis of MR-proADM levels at earlier gestational ages is needed to determine whether MR-proADM can predict the development of preeclampsia later in pregnancy. Disclosures: B. Matson: Research Support Recipient: Ferring Innovation Fellowship. N. Karpinich: None. A. Murtha: None. W. Valdar: None. C.A. Grotegut: None. K.M. Caron: Research Support Recipient; Commercial Interest: Ferring Pharmaceuticals, Inc. doi:10.1016/j.preghy.2014.10.311
[306-POS] Pregnancy and magnesium Ragnar Rylander (BioFact Environmental Health Research Center, Lerum, Sweden) Objectives: To review the role of magnesium in pregnancy. Methods: Analysis of published reports with emphasis on recent work on cellular mechanisms and magnesium responsive genes (MgRGs), plus controlled trials of magnesium (Mg) supplementation to prevent gestational hypertension. Results: In a study on women with normal pregnancies, MgRGs were up regulated as compared to non-pregnant women, suggesting a higher Mg demand. They also had lower plasma levels of Mg and an increased urinary excretion of Mg. Systolic and diastolic blood pressure increases during pregnancy were related to the urinary excretion of Mg. Among women with PE, the expression of the MgRG SLC41A1 – a Na/Mg exchanger – was overexpressed in 54.2% of the placentas as compared to 9.5% in normal
151
placentas. Brain and muscle levels of Mg were lower among women with PE. In three supplementation studies, Mg reduced the occurrence of gestational hypertension, particularly the diastolic blood pressure. Conclusions: Data from genetic, cellular, and clinical studies support a conclusion that Mg plays an important role in pregnancy and that Mg deficiency could be a major factor for the pathogenesis in complicated pregnancies. It is unlikely that Mg is the sole agent involved and other nutritional factors are probably also of importance. Further studies are needed to assess the relationship with Mg and formulate requirements for intervention programs. Disclosures: R. Rylander: None. doi:10.1016/j.preghy.2014.10.312
[307-POS] Cx43 phosphorylation and the functionality of Cx43 gap junctions are moderated by cyclic nucleotide activity in UAECs and HUVECs Bryan Ampey, Ian Bird, Ron Magness (University of Wisconsin, Madison, Madison, WI, USA) Objectives: Gap junctions (GJ) are intercellular channels that contribute to the robust vasodilation found in pregnancy in several vascular beds. GJs allow for the coordination of endothelial cell responses by directing cell-cell communication via facilitated diffusion of signaling molecules (e.g. cAMP, cGMP). The phosphorylation of GJ protein Cx43 at serine (Ser) residues regulates both open (Ser365) and closed gating (Ser368), thus allowing the passage of signaling molecules between cells. Although the phosphorylation events of cAMP are well studied, little is known about cGMPs role on gap junction intracellular communication (GJIC) and function. Hypothesis: The inhibition of cAMP and cGMP-mediated pathways will modulate the phosphorylation and expression of Cx43 at their respective phosphorylation sites and thus increase or reduce GJIC in pregnant ovine uterine artery endothelial cells (P-UAECs) and human umbilical vein endothelial cells (HUVECs). Methods: P-UAECs were pretreated with the respective cAMP and cGMP nucleotide cyclase inhibitors SQ22536 and ODQ (0.1–100 lM) followed by treatment with 100 lM of ATP or Forskolin, or 1 lM–1 mM 8-Bromo-cAMP or 8-Bromo-cGMP and analyzed using ELISA, western analysis, or scrape-loading/dye transfer technique. Results: ELISA revealed that the inhibitors SQ22536 and ODQ specifically and completely abrogated (P<0.01) cAMP and cGMP productions, respectively. Western analysis showed that SQ22536, decreased (P<0.05) the ATP, Forskolin, and cAMP-induced phosphorylation at Ser365 in PUAECs and HUVECs, while GJIC function was reduced. ODQ decreased (P<0.05) ATP and cGMP-induced elevation at Ser365 in P-UAECs and HUVECs. In contrast, ODQ alone had no effect on GJIC. Conclusions: cAMP via adenylyl clycase and cGMP via guanylyl cyclase differentially regulate Cx43 phosphorylation and expression, which is important in regulating GJIC.