Decidua basalis and acute atherosis: Expression of atherosclerotic foam cell associated proteins.

INTRODUCTION: Uteroplacental acute atherosis is frequently observed in preeclampsia, and shares features with early atherosclerotic lesions, including artery wall foam cells. The lipid-associated proteins FABP4 (fatty acid binding protein 4), perilipin-2, and LOX-1 (lectin-like oxidized LDL-receptor 1) are involved in atherosclerotic foam cell formation. Increased levels of these proteins have been associated with preeclampsia systemically and in placental tissue. Their role in acute atherosis is yet unidentified. Our aim was to describe the presence of these proteins in acute atherosis, and compare our findings to what is known in early atherosclerotic lesions. METHODS: Serial sections of decidua basalis tissue from 12 normotensive (4 with acute atherosis) and 23 preeclamptic pregnancies (16 with acute atherosis) were stained with HE and immunostained for CK7, CD68, FABP4, perilipin-2, and LOX-1. Artery wall and perivascular protein expression was assessed in 190 spiral artery sections; 55 with acute atherosis. RESULTS: Acute atherosis foam cells were commonly positive for perilipin-2 (55%), less often for FABP4 (13%), and never for LOX-1. LOX-1 was frequently observed in intramural trophoblasts of normal spiral arteries. Perivascularly, LOX-1 positivity of decidual stromal cells surrounding arteries with acute atherosis was significantly increased as compared to arteries lacking acute atherosis (38% vs. 15%, p < 0.001). DISCUSSION: We found that perilipin-2 and FABP4 are expressed by acute atherosis foam cells, similar to atherosclerosis, supporting possible shared pathways for foam cell generation. Unlike atherosclerosis, LOX-1 is not present in acute atherosis, possibly explained by pregnancy-specific routes to decidua basalis foam cell generation.

Lymphocyte characterization of decidua basalis spiral arteries with acute atherosis in preeclamptic and normotensive pregnancies.

Uteroplacental acute atherosis (AA) is a common spiral arterial lesion in preeclampsia, characterized by intramural foam cells, fibrinoid necrosis, and a perivascular immune cell infiltrate. A clear definition of this infiltrate is lacking. Therefore, our aim was to characterize lymphocytes in pre-defined zones regarding spiral arteries with or without AA, from preeclamptic and normotensive pregnancies. Lymphocytes were characterized in decidua basalis samples (n = 91), previously evaluated for AA, around spiral arteries in three pre-defined zones; 1) intramural, 2) perivascular and 3) interstitial. Adjacent serial sections were immunostained to identify different T-cell populations (CD3+, CD8+, FOXP3+), and NK-cells (CD56+). CD3+CD8- T-cells were also identified. These were presumed to be largely CD4+ T-cells. AA was associated with significantly higher intramural CD3+ cell concentrations in Zone 1, in both normotensives and preeclamptics. In preeclamptics only, this difference extended into Zone 2. Similar results were observed for CD3+CD8- cells. AA was also associated with increased intramural CD8+ concentration; however, the number of cells was low. Regulatory T-cells (FOXP3+) were generally scarce or absent in all pre-defined zones. Although intramural NK-cells (CD56+) were scarce, the intramural concentration was significantly lower in spiral arteries with AA compared to without AA in preeclamptics. Our main finding was that CD3+CD8-FoxP3- T-cells were associated with AA. We therefore suggest that T-cells, of a non-regulatory CD4+ subtype, could be involved in the formation of spiral artery AA in the decidua basalis. Whether AA gives rise to, or is partly mediated by increased T-cell concentration around the lesions, remains to be determined.

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 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.

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.

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.

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.

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.