Plasma Cross-Gestational Sphingolipidomic Analyses Reveal Potential First Trimester Biomarkers of Preeclampsia

Introduction Preeclampsia (PE) is a gestational disorder, manifested in the second half of pregnancy by maternal hypertension, proteinuria and generalized edema. PE is a major cause of maternal and fetal morbidity and mortality, accounting for nearly 40% of all premature births worldwide. Bioactive sphingolipids are emerging as key molecules involved in etiopathogenesis of PE, characterized by maternal angiogenic imbalance and symptoms of metabolic syndrome. The aim of this study was to compare the cross-gestational profile of circulating bioactive sphingolipids in maternal plasma from preeclamptic (PE) versus normotensive control (CTL) subjects with the goal of identifying sphingolipids as candidate first trimester biomarkers of PE for early prediction of the disease. Methods A prospective cohort of patients was sampled at the first, second and third trimester of pregnancy for each patient (11–14, 22–24, and 32–36 weeks´ gestation). A retrospective stratified study design was used to quantify different classes of sphingolipids in maternal plasma. We used a reverse-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS/MS) approach for determining different sphingolipid molecular species (sphingosine-1-phosphate (S1P), dihydro-sphingosine-1-phosphate (DH-S1P), sphingomyelins (SM) and ceramides (Cer)) in cross-gestational samples of human plasma from PE (n = 7, 21 plasma samples across pregnancy) and CTL (n = 7, 21 plasma samples across pregnancy) patients. Results Plasma levels of angiogenic S1P did not change significantly in control and in preeclamptic patients´ group across gestation. DH-S1P was significantly decreased in second trimester plasma of PE patients in comparison to their first trimester, which could contribute to reduced endothelial barrier observed in PE. The major ceramide species (Cer 16:0 and Cer 24:0) tended to be up-regulated in plasma of control and PE subjects across gestation. The levels of a less abundant plasma ceramide species (Cer 14:0) were significantly lower in first trimester plasma of PE patients when compared with their gestational-matched control samples (p = 0.009). Major plasma sphingomyelin species (SM 16:0, SM 18:1 and SM 24:0) tended to be higher in control pregnancies across gestation. However, in PE patients, SM 16:0, SM 18:0 and SM 18:1 showed significant up-regulation across gestation, pointing to atherogenic properties of the sphingomyelins and particularly the potential contribution of SM 18:0 to the disease development. In addition, two major sphingomyelins, SM 16:0 and SM 18:0, were significantly lower in first trimester plasma of PE patients versus first trimester samples of respective controls (p = 0.007 and p = 0.002, respectively). Conclusions Cross-gestational analysis of maternal plasma of preeclamptic and normotensive women identifies differences in the biochemical profile of major sphingolipids (DH-S1P, sphingomyelins and ceramides) between these two groups. In addition, first trimester maternal plasma sphingolipids (Cer 14:0, SM 16:0 and SM 18:0) may serve in the future as early biomarkers of PE occurrence and development

Impairment of Angiogenic Sphingosine Kinase-1/Sphingosine-1-Phosphate Receptors Pathway in Preeclampsia.

Preeclampsia (PE), is a serious pregnancy disorder characterized in the early gestation by shallow trophoblast invasion, impaired placental neo-angiogenesis, placental hypoxia and ischemia, which leads to maternal and fetal morbidity and mortality. Here we hypothesized that angiogenic sphingosine kinase-1 (SPHK1)/sphingosine-1-phosphate (S1P) receptors pathway is impaired in PE. We found that SPHK1 mRNA and protein expression are down-regulated in term placentae and term chorionic villous explants from patients with PE or severe PE (PES), compared with controls. Moreover, mRNA expression of angiogenic S1PR1 and S1PR3 receptors were decreased in placental samples of PE and PES patients, whereas anti-angiogenic S1PR2 was up-regulated in chorionic villous tissue of PES subjects, pointing to its potential atherogenic and inflammatory properties. Furthermore, in in vitro (JAR cells) and ex vivo (chorionic villous explants) models of placental hypoxia, SPHK1 mRNA and protein were strongly up-regulated under low oxygen tension (1% 02). In contrast, there was no change in SPHK1 expression under the conditions of placental physiological hypoxia (8% 02). In both models, nuclear protein levels of HIF1A were increased at 1% 02 during the time course, but there was no up-regulation at 8% 02, suggesting that SPHK1 and HIF1A might be the part of the same canonical pathway during hypoxia and that both contribute to placental neovascularization during early gestation. Taken together, this study suggest the SPHK1 pathway may play a role in the human early placentation process and may be involved in the pathogenesis of PE

Levels of Key Enzymes of Methionine-Homocysteine Metabolism in Preeclampsia

Objective. To evaluate the role of key enzymes in the methionine-homocysteine metabolism (MHM) in the physiopathology of preeclampsia (PE). Methods. Plasma and placenta from pregnant women (32 controls and 16 PE patients) were analyzed after informed consent. Protein was quantified by western blot. RNA was obtained with RNA purification kit and was quantified by reverse transcritase followed by real-time PCR (RT-qPCR). Identification of the C677T and A1298C methylenetetrahydrofolate reductase (MTHFR) single-nucleotide polymorphisms (SNPs) and A2756G methionine synthase (MTR) SNP was performed using PCR followed by a high-resolution melting (HRM) analysis. S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH) were measured in plasma using high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). The SNP association analysis was carried out using Fisher’s exact test. Statistical analysis was performed using a Mann-Whitney test. Results. RNA expression of MTHFR and MTR was significantly higher in patients with PE as compared with controls. Protein, SAM, and SAH levels showed no significant difference between preeclamptic patients and controls. No statistical differences between controls and PE patients were observed with the different SNPs studied. Conclusion. The RNA expression of MTHFR and MTR is elevated in placentas of PE patients, highlighting a potential compensation mechanism of the methionine-homocysteine metabolism in the physiopathology of this disease

<i>SPHK1</i> mRNA and protein levels are down-regulated in term human placentae of preeclamptic patients.

<p><b>(A)</b> qRT-PCR: bar graph represents relative <i>SPHK1</i> gene expression normalized to 18S in CTL (n = 16) and PE (n = 17) subjects <b>(B)</b> SPHK1 protein expression from control (CTL) and preeclamptic (PE) placentae was determined by Western blot. Ponceau staining was used as a loading control <b>(C)</b> Bar graph represents relative SPHK1 protein abundance normalized to Ponceau stain. Mean ±S.E.M, *p ≤ 0.05, Control (CTL): n = 16, Preeclamptic (PE): n = 17.</p

NFAT5 is up-regulated by hypoxia: possible implications in preeclampsia and intrauterine growth restriction

During gestation, low oxygen environment is a major determinant of early placentation process, while persistent placental hypoxia leads to pregnancy-related complications such as preeclampsia (PE) and intrauterine growth restriction (IUGR). PE affects 5%-8% of all pregnancies worldwide and is a cause of maternal and fetal morbidity and mortality. During placental development, persistent hypoxia due to poor trophoblast invasion and reduced uteroplacental perfusion leads to maternal endothelial dysfunction and clinical manifestation of PE. Here we hypothesized that nuclear factor of activated T cells-5 (NFAT5), a well-known osmosensitive renal factor and recently characterized hypoxia-inducible protein, is also activated in vivo in placentas of PE and IUGR complications as well as in the in vitro model of trophoblast hypoxia. In JAR cells, low oxygen tension (1% O2) induced NFAT5 mRNA and increased its nuclear abundance, peaking at 16 h. This increase did not occur in parallel with the earlier HIF1A induction. Real-time PCR and Western blot analysis confirmed up-regulation of NFAT5 mRNA and NFAT5 nuclear content in human preeclamptic placentas and in rabbit placentas of an experimentally induced IUGR model, as compared with the control groups. In vitro lambda protein phosphatase (lambda PPase) treatment revealed that increased abundance of NFAT5 protein in nuclei of either JAR cells (16 h of hypoxia) or PE and IUGR placentas is at least partially due to NFAT5 phosphorylation. NFAT5 downstream targets aldose reductase (AR) and sodium-myo-inositol cotransporter (SMIT; official symbol SLC5A3) were not significantly upregulated either in JAR cells exposed to hypoxia or in placentas of PE- and IUGR-complicated pregnancies, suggesting that hypoxia-dependent activation of NFAT5 serves as a separate function to its tonicity-dependent stimulation. In conclusion, we propose that NFAT5 may serve as a novel marker of placental hypoxia and ischemia independently of HIF1A

Impairment of Angiogenic Sphingosine Kinase-1/Sphingosine-1-Phosphate Receptors Pathway in Preeclampsia - Fig 1

<p><b>SPHK1 is up-regulated at low oxygen tension (1% PO</b>_<b>2</b><b>) in human choriocarcinoma JAR cells (A)</b> Bar diagram shows the relative mRNA expression of <i>SPHK1</i> transcripts in JAR cells during time course (3–16h) under 21% O₂ and 1% O₂ exposure. Data was normalized against 18S gene. Bar graph represents mean ±S.E.M., * indicates p ≤ 0.05, n = 3. <b>(B, C)</b> Representative Western blots of cytosolic SPHK1 and nuclear HIF1A in JAR cells under control (21% O₂) and hypoxic conditions (1% O₂ and 8% O₂)- time course. β-Actin blots are shown as loading controls, n = 4 independent experiments.</p