Guro M. Johnsen

Preeclampsia and uteroplacental acute atherosis: immune and inflammatory factors

Acute atherosis (Aa) affects uteroplacental spiral arteries in 20-40% of cases of preeclampsia. Its hallmark is lipid-filled, CD68-positive foam cells. It usually develops in the decidua (the pregnancy endometrium) at the distal ends of arteries that are often unremodelled in their proximal segments. Aa resembles the early stages of atherosclerosis, which becomes symptomatic in the middle-aged and elderly, in contrast to the young age of pregnant women with Aa. Although the mechanisms of Aa are largely unknown, they are likely to resemble those of early atherosclerosis, which is an inflammatory lesion of the arterial wall. However, Aa is likely to have added pregnancy-specific features. Because it also occurs in normotensive pregnancies, complicated by foetal growth restriction, diabetes mellitus or autoimmune disease or even without any complications, we suggest that Aa is the final manifestation of several inflammatory processes. We revisit an old proposition that immunological incompatibility between mother and foetus may sometimes induce Aa. We propose that excessive inflammatory activation, of other aetiologies, primarily in the decidua basalis, may explain the different ways in which Aa occurs. We speculate that the subset of women who develop these lesions may be at an increased risk of atherosclerotic arterial disease later in life. We hypothesise that use of anti-atherogenic statins during established preeclampsia may ameliorate Aa, improve uteroplacental perfusion and enhance pregnancy outcome.

Docosahexaenoic acid stimulates tube formation in first trimester trophoblast cells, HTR8/SVneo

Angiogenesis is a key factor in the placentation process and vascular remodeling that involves several growth factors such as vascular endothelial growth factor (VEGF) and angiopoietin-like protein 4 (ANGPTL4). PPARs are involved in the placentation process but not much information is available on whether their ligands such as fatty acids have any effects on these processes. We therefore investigated the effect of fatty acids (arachidonic acid, 20:4 n-6(ARA), eicosapentaenoic acid, 20:5 n-3(EPA), docosahexaenoic acid, 22:6 n-3 (DHA) and oleic acid, 18:1 n-9 (OA)) on tube formation (as a measure of angiogenesis) on matrigel in the first trimester trophoblast cells, HTR8/SVneo. In addition we also investigated the effects of fatty acids on expression of genes involved in angiogenesis (VEGF and ANGPTL4) and lipid metabolism in these cells. Gene expression was determined after incubating these cells with different fatty acids for 24 h using real-time qRT-PCR, whereas VEGF and ANGPTL4 proteins were measured by respective ELISA kits. Of all the fatty acids tested, DHA increased tube formation to the greatest extent. DHA-induced increase in tube length was 583%, 247% and 70% over control, OA and EPA, respectively (p < 0.05). In addition, DHA stimulated cell proliferation by 150% of these cells. Of all fatty acids investigated, only DHA stimulated VEGF mRNA expression and protein secretion compared with control. Unlike DHA, other fatty acids (OA, EPA, ARA) stimulated ANGPTL4 mRNA expression and protein secretion in these cells. An inhibitor of VEGF decreased DHA stimulated tube formation in these cells. Altogether these data indicate that DHA may potently influence the placentation process by stimulating tube formation and this effect may be mediated in part via VEGF in first trimester trophoblast cells.

Long-chain Polyunsaturated Fatty Acid Transport across Human Placental Choriocarcinoma (BeWo) Cells

Long-chain polyunsaturated fatty acids (LCPUFAs) such as docosahexaenoic acid (DHA) and arachidonic acid (AA) are essential for proper development of fetal brain and retina. These LCPUFAs are selectively enriched in the fetal circulation compared with the maternal circulation. In the current study we investigated the transfer of LCPUFAs and a non-essential fatty acid (oleic acid, OA) in a transwell monolayer system of placental choriocarcinoma (BeWo) cells. We show that incubation with OA results in increased triglyceride accumulation and lipid droplet formation compared with that of DHA. The relative amount of transfer of DHA across the cell monolayer was approximately 4-fold greater compared with that of OA when these fatty acids were added individually at 100 muM. This reflects the different fates of these two fatty acids in their metabolism and subsequent transport across the placental trophoblasts to the fetus. When using a mixture of fatty acids mimicking the composition of plasma non-esterified fatty acids during the last trimester of pregnancy, the transfer of OA and the LCPUFAs (DHA and AA) into the basolateral reservoir was not significantly different, whereas the transfer of palmitic acid (PA) was approximately 3.5-fold higher than OA transfer. However, since the concentration of OA compared to LCPUFAs was 10-fold higher in the donor chamber, the relative transport of the LCPUFAs was higher compared with that of OA. In addition, we show that inhibiting esterification of fatty acids into acyl-CoA can modulate, in part, the degree of transport through the cells. In conclusion, the transwell model system closely mimics the mechanisms of differential fatty acid transport as observed in vivo. LCPUFAs were transported through the cells more efficiently than shorter fatty acids such as OA.

Long-chain Polyunsaturated Fatty Acids Stimulate Cellular Fatty Acid Uptake in Human Placental Choriocarcinoma (BeWo) Cells

Supplementation of long-chain polyunsaturated fatty acids (LCPUFAs) is advocated during pregnancy in some countries although very little information is available on their effects on placental ability to take up these fatty acids for fetal supply to which the fetal growth and development are critically dependent. To identify the roles of LCPUFAs on placental fatty acid transport function, we examined the effects of LCPUFAs on the uptake of fatty acids and expression of fatty acid transport/metabolic genes using placental trophoblast cells (BeWo). Following 24 h incubation of these cells with 100 microM of LCPUFAs (arachidonic acid, 20:4n-6, eicosapentaenoic acid, 20:5n-3, or docosahexaenoic acid, 22:6n-3), the cellular uptake of [(14)C] fatty acids was increased by 20-50%, and accumulated fatty acids were preferentially incorporated into phospholipid fractions. Oleic acid (OA, 18:1n-9), on the other hand, could not stimulate fatty acid uptake. LCPUFAs and OA increased the gene expression of ADRP whilst decreased the expression of ACSL3, ACSL4, ACSL6, LPIN1, and FABP3 in these cells. However, LCPUFAs but not OA increased expression of ACSL1 and ACSL5. Since acyl-CoA synthetases are involved in cellular uptake of fatty acids via activation for their channelling to lipid metabolism and/or for storage, the increased expression of ACSL1 and ACLS5 by LCPUFAs may be responsible for the increased fatty acid uptake. These findings demonstrate that LCPUFA may function as an important regulator of general fatty acid uptake in trophoblast cells and may thus have impact on fetal growth and development.

Circulating and Placental Growth-Differentiation Factor 15 in Preeclampsia and in Pregnancy Complicated by Diabetes Mellitus

Growth-differentiation factor 15 (GDF-15), a stress-responsive transforming growth factor-&bgr;-related cytokine, is emerging as a new risk marker in patients with cardiovascular disease. We explored GDF-15 in preeclampsia and in diabetic pregnancies, because these conditions are associated with augmented risk for cardiovascular disease, both in mother and in offspring. Plasma from pregnant women (n=267; controls: n=59, preeclampsia: n=85, diabetes mellitus: n=112, and superimposed preeclampsia in diabetes mellitus: n=11), fetal plasma (n=72), and amniotic fluid (n=99) were analyzed by immunoassay for GDF-15. Placental GDF-15 mRNA and protein expression levels were analyzed by quantitative real-time PCR and immunoblots in 78 and 18 pregnancies, respectively. Conditioned media from preeclamptic (n=6) and control (n=6) villous placenta explants were analyzed by immunoassay for GDF-15. Median maternal GDF-15 concentration was elevated in those with diabetes mellitus, as compared with controls (91 549 versus 79 875 ng/L; P=0.02). Median GDF-15 concentration was higher in patients with preeclampsia than in controls in term maternal blood samples (127 061 versus 80 319 ng/L; P<0.001). In the fetal circulation and amniotic fluid, GDF-15 was elevated in preeclampsia and superimposed preeclampsia in diabetes mellitus, as compared with controls. GDF-15 placental mRNA expression was elevated in preeclampsia, as compared with controls (P=0.002). Placenta immunoblots confirmed a single GDF-15 protein band, and a time-dependent increase in GDF-15 protein was detected in the conditioned media. Our study is the first to show that GDF-15 is dysregulated, both in preeclampsia and in diabetic pregnancies. The mechanisms and diagnostic implications of these findings remain to be explored.

Placental miR-1301 is dysregulated in early-onset preeclampsia and inversely correlated with maternal circulating leptin.

INTRODUCTION miRNAs are small non-coding RNAs important for the regulation of mRNA in many organs including placenta. Adipokines and specifically leptin are known to be dysregulated in preeclampsia, but little is known regarding their regulation by miRNAs during pregnancy. METHODS We performed high-throughput sequencing of small RNAs in placenta from 72 well-defined patients: 23 early-onset preeclampsia (PE), 26 late-onset PE and 23 controls. The regulation of some miRNAs was confirmed on qRT-PCR. Maternal circulating levels and placental mRNA of leptin, resistin and adiponectin were measured using Bio-Plex and qRT-PCR. RESULTS We found that miR-1301, miR-223 and miR-224 expression was downregulated in early-onset PE, but not in late-onset PE, compared to controls. In silico analysis predicted the leptin gene (LEP) to be a target for all three miRNAs. Indeed, we found significant correlation between maternal circulating levels of leptin and placental LEP expression. In addition, we found a significant inverse correlation between maternal circulating leptin/placental LEP expression and placental miR-1301 expression levels. Interestingly, placental expression of miR-1301 was also correlated with newborn weight percentile and inversely correlated with both maternal systolic and diastolic blood pressure prior to delivery. DISCUSSION Our results confirm that placenta is a major site of LEP expression during pregnancy. It further suggests that miR-1301 could be involved in the regulation of leptin during pregnancy and may play a role in early-onset PE. CONCLUSIONS miR-1301 is dysregulated in early-onset preeclampsia and could possibly play a role in the regulation of leptin during pregnancy.

Expression of Liver X Receptors in Pregnancies Complicated by Preeclampsia

Preeclampsia is a pregnancy-specific disorder associated with hyperlipidemia. Liver X receptor (LXR) alpha and LXRbeta are key regulators of lipid homeostasis. In the current study, we investigated expression of LXRalpha, LXRbeta and their target genes in human term placenta, decidua and subcutaneous adipose tissue from pregnancies complicated by preeclampsia. Furthermore, we analyzed the protein levels of LXRalpha and LXRbeta in placenta. We also analyzed lipid concentrations in term placental tissue. Gene expression of LXRalpha, LXRbeta and fatty acid transporter CD36 was significantly decreased in placental tissues, while increased expression was observed for LXRalpha in adipose tissue, from pregnancies complicated by preeclampsia. The placental protein level of LXRbeta was reduced, and there was a positive correlation between placental LXRbeta mRNA expression and placental free fatty acids in preeclampsia. Our results suggest a possible role for LXRbeta as a transcriptional regulator in preeclampsia.

CD74-Downregulation of Placental Macrophage-Trophoblastic Interactions in Preeclampsia

RATIONALE We hypothesized that cluster of differentiation 74 (CD74) downregulation on placental macrophages, leading to altered macrophage-trophoblast interaction, is involved in preeclampsia. OBJECTIVE Preeclamptic pregnancies feature hypertension, proteinuria, and placental anomalies. Feto-placental macrophages regulate villous trophoblast differentiation during placental development. Disturbance of this well-balanced regulation can lead to pathological pregnancies. METHODS AND RESULTS We performed whole-genome expression analysis of placental tissue. CD74 was one of the most downregulated genes in placentas from preeclamptic women. By reverse transcriptase-polymerase chain reaction, we confirmed this finding in early-onset (<34 gestational week, n=26) and late-onset (≥34 gestational week, n=24) samples from preeclamptic women, compared with healthy pregnant controls (n=28). CD74 protein levels were analyzed by Western blot and flow cytometry. We identified placental macrophages to express CD74 by immunofluorescence, flow cytometry, and RT-PCR. CD74-positive macrophages were significantly reduced in preeclamptic placentas compared with controls. CD74-silenced macrophages showed that the adhesion molecules ALCAM, ICAM4, and Syndecan-2, as well as macrophage adhesion to trophoblasts were diminished. Naive and activated macrophages lacking CD74 showed a shift toward a proinflammatory signature with an increased secretion of tumor necrosis factor-α, chemokine (C-C motif) ligand 5, and monocyte chemotactic protein-1, when cocultured with trophoblasts compared with control macrophages. Trophoblasts stimulated by these factors express more CYP2J2, sFlt1, TNFα, and IL-8. CD74-knockout mice showed disturbed placental morphology, reduced junctional zone, smaller placentas, and impaired spiral artery remodeling with fetal growth restriction. CONCLUSIONS CD74 downregulation in placental macrophages is present in preeclampsia. CD74 downregulation leads to altered macrophage activation toward a proinflammatory signature and a disturbed crosstalk with trophoblasts.

Expression profiling of autophagy associated genes in placentas of preeclampsia

Autophagy, a mechanism of cell survival during times of stress, may be active in normal placental maintenance, cushioning the fetus from strain during fluctuations in nutrient availability. Moreover, in cases of placental insufficiency, often present in preeclampsia, autophagy may be defective. We used published microarray datasets to analyze differential expression of autophagy pathway genes. No statistically significant difference in autophagy associated gene expression was found in preeclamptic vs. normal placenta samples. Thus although preeclampsia displays many of the features suggestive of altered autophagy, impaired placental autophagy as a cause of preeclampsia is not supported by whole placental tissue differential expression profiling.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.