The influence of maternal obesity on macrophage subsets in the human decidua.

Obesity is seen as a low grade inflammatory state, and is associated with adverse pregnancy outcomes. Disturbed macrophage characteristics might be essential in obesity associated pregnancy pathology via effects on the regulation of angiogenesis and placental development. This study aims to address the effects of maternal obesity on macrophage subsets in the decidua of women with term uncomplicated pregnancies. Macrophages were isolated from the decidua basalis and decidua parietalis of women with pre-gravid BMI < 25 (control) and BMI > 30 (obese). Macrophages were characterized and quantified using multi-color flow cytometry. Placentas of 10 obese and 10 control women after an uncomplicated term pregnancy were included. The decidua parietalis, but not decidua basalis, showed significantly lower levels of M1-type (HLA-DR+, CD163-) macrophages (p < 0.05) in obese women (4,3% of total macrophages) compared to control women (5,3% of total macrophages). The lower levels of M1 macrophages, considered to be pro-inflammatory, might indicate a mechanism to compensate for the pro-inflammatory environment in obese women to ensure healthy pregnancy outcomes.

Prenatal exposure to tobacco smoke sex dependently influences methylation and mRNA levels of the Igf axis in lungs of mouse offspring.

Prenatal smoke exposure is a risk factor for abnormal lung development and increased sex-dependent susceptibility for asthma and chronic obstructive pulmonary disease (COPD). Birth cohort studies show genome-wide DNA methylation changes in children from smoking mothers, but evidence for sex-dependent smoke-induced effects is limited. The insulin-like growth factor (IGF) system plays an important role in lung development. We hypothesized that prenatal exposure to smoke induces lasting changes in promoter methylation patterns of Igf1 and Igf1r, thus influencing transcriptional activity and contributing to abnormal lung development. We measured and compared mRNA levels along with promoter methylation of Igf1 and Igf1r and their protein concentrations in lung tissue of 30-day-old mice that had been prenatally exposed to cigarette smoke (PSE) or filtered air (control). Body weight at 30 days after birth was measured as global indicator of normal development. Female PSE mice showed lower mRNA levels of Igf1 and its receptor (Igf1: P = 0.05; Igf1r: P = 0.03). Furthermore, CpG-site-specific methylation changes were detected in Igf1r in a sex-dependent manner and the body weight of female offspring was reduced after prenatal exposure to smoke, while protein concentrations were unaffected. Prenatal exposure to smoke induces a CpG-site-specific loss of Igf1r promoter methylation, which can be associated with body weight. These findings highlight the sex-dependent and potentially detrimental effects of in utero smoke exposure on DNA methylation and Igf1 and Igf1r mRNA levels. The observations support a role for Igf1 and Igf1r in abnormal development.

In utero sFlt-1 exposure differentially affects gene expression patterns in fetal liver.

The soluble fms-like tyrosine kinase factor 1 (sFlt-1) is a major contributor to antiangiogenesis during preeclampsia. However, little is known about the effects of sFlt-1 on fetal health. In this study we aim to evaluate the effects of the sFlt-1 concentration during pregnancy on fetal liver physiology. We used adenoviral gene delivery in Sprague-Dawley dams (seven females, 10 weeks old) during mid-gestation (gestational day 8) with adenovirus overexpressing sFlt-1, and age-matched controls (six females, 10 weeks old) with empty adenoviral virus in order to quantify the sFlt-1 concentrations in pregnant dams. Dams exposed to adenoviral sFlt-1 delivery were subdivided into a low (n=4) and high sFlt-1 (n=3) group based on host response to the virus. One-way analysis of variance showed that fetuses (five per dam) exposed to high sFlt-1 concentrations in utero show fetal growth restriction (1.84±0.043 g high sFlt-1 v. 2.32±0.036 g control; mean (M)±s.e.m.; P<0.001), without hypertension or proteinuria in the dams. In continuation, the microarray analysis of the fetal liver of the high sFlt-1 group showed significant enrichment of key genes for fatty acid metabolism and Ppara targets. In addition, using pyrosequencing, we found that the Ppara enrichment in the high sFlt-1 group is accompanied by decreased methylation of its promoter (1.89±0.097 mean % methylation in high sFlt-1 v. 2.26±0.095 mean % methylation in control, M±s.e.m., P<0.02). Our data show that high sFlt-1 concentrations during pregnancy have detrimental effects on the fatty acid metabolism genes and the Ppara targets in the fetal liver.

Cholesterol transport by the placenta: placental liver X receptor activity as a modulator of fetal cholesterol metabolism?

Cholesterol is an important sterol in mammals. Defects in cholesterol synthesis or intracellular routing have devastating consequences already in utero: the Smith-Lemli-Opitz syndrome, desmosterolosis and Niemann-Pick C1 disease provide examples of severe human inherited diseases caused by mutations in cholesterol metabolism genes. On the other hand, elevated plasma cholesterol concentrations are associated with the development of atherosclerosis which represents a major health risk in Western societies. Moreover, several studies indicate that development of atherosclerosis may already start during fetal life. Hence, a carefully balanced regulation of cholesterol metabolism appears of critical importance for both the development of the fetus and health of the adult. In the adult, the liver X receptor is a key regulator of cholesterol metabolism. Its target genes regulate cellular cholesterol efflux and thereby modulate whole-body cholesterol fluxes. LXR and several of its target genes have recently been demonstrated to be expressed in the placenta, which would provide a means to control delivery of maternal cholesterol to the fetus. Here we discuss the potential role of the placenta in the regulation of fetal cholesterol homeostasis and strategies to influence maternal-fetal cholesterol transfer.

Maternal-fetal cholesterol transport in the second half of mouse pregnancy does not involve LDL receptor-related protein 2.

AIM: LDL receptor-related protein type 2 (LRP2) is highly expressed on both yolk sac and placenta. Mutations in the corresponding gene are associated with severe birth defects in humans, known as Donnai-Barrow syndrome. We here characterized the contribution of LRP2 and maternal plasma cholesterol availability to maternal-fetal cholesterol transport and fetal cholesterol levels in utero in mice. METHODS: Lrp2+/- mice were mated heterozygously to yield fetuses of all three genotypes. Half of the dams received a 0.5% probucol-enriched diet during gestation to decrease maternal HDL cholesterol. At E13.5, the dams received an injection of D7-labelled cholesterol and were provided with 1-13 C acetate-supplemented drinking water. At E16.5, fetal tissues were collected and maternal cholesterol transport and fetal synthesis quantified by isotope enrichments in fetal tissues by GC-MS. RESULTS: The Lrp2 genotype did not influence maternal-fetal cholesterol transport and fetal cholesterol. However, lowering of maternal plasma cholesterol levels by probucol significantly reduced maternal-fetal cholesterol transport. In the fetal liver, this was associated with increased cholesterol synthesis rates. No indications were found for an interaction between the Lrp2 genotype and maternal probucol treatment. CONCLUSION: Maternal-fetal cholesterol transport and endogenous fetal cholesterol synthesis depend on maternal cholesterol concentrations but do not involve LRP2 in the second half of murine pregnancy. Our results suggest that the mouse fetus can compensate for decreased maternal cholesterol levels. It remains a relevant question how the delicate system of cholesterol transport and synthesis is regulated in the human fetus and placenta.

The ABC of hepatic and intestinal cholesterol transport.

The liver and (small) intestine are key organs in maintenance of cholesterol homeostasis: both organs show active de novo cholesterogenesis and are able to transport impressive amounts of newly synthesized and diet-derived cholesterol via a number of distinct pathways. Cholesterol trafficking involves the concerted action of a number of transporter proteins, some of which have been identified only recently. In particular, several ATP-binding cassette (ABC) transporters fulfil critical roles. For instance, the ABCG5/ABCG8 couple is crucial for hepatobiliary and intestinal cholesterol excretion, while ABCA1 is essential for high-density lipoprotein formation and, hence, for inter-organ trafficking of the highly water-insoluble cholesterol molecules. Very recently, the Niemann-Pick C1-like 1 protein has been identified as a key player in cholesterol absorption by the small intestine and may represent a target of the cholesterol absorption inhibitor ezetimibe. Alterations in hepatic and intestinal cholesterol transport affect circulating levels of atherogenic lipoproteins and thus the risk for cardiovascular disease. This review specifically deals with the processes of hepatobiliary cholesterol excretion and intestinal cholesterol absorption as well as the interactions between these important transport routes. During the last few years, insight into the mechanisms of hepatic and intestinal cholesterol transport has greatly increased not in the least by the identification of involved transporter proteins and the (partial) elucidation of their mode of action. In addition, information has become available on (transcription) factors regulating expression of the encoding genes. This knowledge is of great importance for the development of a tailored design of novel plasma cholesterol-lowering strategies.

Lipotoxicity and the role of maternal nutrition.

Intrauterine malnutrition predisposes the offspring towards the development of type 2 diabetes and cardiovascular disease. To explain this association, the Developmental Origins of Health and Disease hypothesis was introduced, meaning that subtle environmental changes during embryonic and foetal development can influence post-natal physiological functions. Different mechanisms, including epigenetics, are thought to be involved in this foetal programming, but the link between epigenetics and disease is missing. There is increasing evidence that ectopic lipid accumulation and/or lipotoxicity is induced by foetal programming. The aim of this review is to provide insights into the mechanisms underlying lipotoxicity through programming, which contributes to the increase in hepatic and cardiac metabolic risk.

Maternal western diet primes non-alcoholic fatty liver disease in adult mouse offspring.

AIM: Metabolic programming via components of the maternal diet during gestation may play a role in the development of different aspects of the metabolic syndrome. Using a mouse model, we aimed to characterize the role of maternal western-type diet in the development of non-alcoholic fatty liver disease (NAFLD) in the offspring. METHODS: Female mice were fed either a western (W) or low-fat control (L) semisynthetic diet before and during gestation and lactation. At weaning, male offspring were assigned either the W or the L diet, generating four experimental groups: WW, WL, LW and LL offspring. Biochemical, histological and epigenetic indicators were investigated at 29 weeks of age. RESULTS: Male offspring exposed to prenatal and post-weaning western-style diet (WW) showed hepatomegaly combined with accumulation of hepatic cholesterol and triglycerides. This accumulation was associated with up-regulation of de novo lipid synthesis, inflammation and dysregulation of lipid storage. Elevated hepatic transaminases and increased expression of Tnfa, Cd11, Mcp1 and Tgfb underpin the severity of liver injury. Histopathological analysis revealed the presence of advanced steatohepatitis in WW offspring. In addition, alterations in DNA methylation in key metabolic genes (Ppara, Insig, and Fasn) were detected. CONCLUSION: Maternal dietary fat intake during early development programmes susceptibility to liver disease in male offspring, mediated by disturbances in lipid metabolism and inflammatory response. Long-lasting epigenetic changes may underlie this dysregulation.

Influence of relative NK-DC abundance on placentation and its relation to epigenetic programming in the offspring.

Normal placentation relies on an efficient maternal adaptation to pregnancy. Within the decidua, natural killer (NK) cells and dendritic cells (DC) have a critical role in modulating angiogenesis and decidualization associated with pregnancy. However, the contribution of these immune cells to the placentation process and subsequently fetal development remains largely elusive. Using two different mouse models, we here show that optimal placentation and fetal development is sensitive to disturbances in NK cell relative abundance at the fetal-maternal interface. Depletion of NK cells during early gestation compromises the placentation process by causing alteration in placental function and structure. Embryos derived from NK-depleted dams suffer from intrauterine growth restriction (IUGR), a phenomenon that continued to be evident in the offspring on post-natal day 4. Further, we demonstrate that IUGR was accompanied by an overall reduction of global DNA methylation levels and epigenetic changes in the methylation of specific hepatic gene promoters. Thus, temporary changes within the NK cell pool during early gestation influence placental development and function, subsequently affecting hepatic gene methylation and fetal metabolism.

The liver X receptor (LXR) and its target gene ABCA1 are regulated upon low oxygen in human trophoblast cells: a reason for alterations in preeclampsia?

OBJECTIVES: The Liver X receptors (LXR) alpha and beta and their target genes such as the ATP-binding cassette (ABC) transporters have been shown to be crucially involved in the regulation of cellular cholesterol homeostasis. The aim of this study was to characterize the role of LXR alpha/beta in the human placenta under normal physiological circumstances and in preeclampsia. STUDY DESIGN: We investigated the expression pattern of the LXRs and their target genes in the human placenta during normal pregnancy and in preeclampsia. Placental explants and cell lines were studied under different oxygen levels and pharmacological LXR agonists. MAIN OUTCOME MEASURES: Gene expressions (Taqman PCR) and protein levels (Western Blot) were combined with immunohistochemistry to analyze the expression of LXR and its target genes. RESULTS: In the human placenta, LXRA and LXRB expression increased during normal pregnancy. This was paralleled by the expression of their prototypical target genes, e.g., the cholesterol transporter ABCA1. Interestingly, early-onset preeclamptic placentae revealed a significant upregulation of ABCA1. Culture of JAr trophoblast cells and human first trimester placental explants under low oxygen lead to increased expression of LXRA and ABCA1 which was further enhanced by the LXR agonist T0901317. CONCLUSIONS: LXRA together with ABCA1 are specifically expressed in the human placenta and can be regulated by hypoxia. Deregulation of this system in early preeclampsia might be the result of placental hypoxia and hence might have consequences for maternal-fetal cholesterol transport.

Pharmacological activation of LXR in utero directly influences ABC transporter expression and function in mice but does not affect adult cholesterol metabolism.

Cholesterol is critical for several cellular functions and essential for normal fetal development. Therefore, its metabolism is tightly controlled during all life stages. The liver X receptors-alpha (LXRalpha; NR1H3) and -beta (LXRbeta; NR1H2) are nuclear receptors that are of key relevance in coordinating cholesterol and fatty acid metabolism. The aim of this study was to elucidate whether fetal cholesterol metabolism can be influenced in utero via pharmacological activation of LXR and whether this would have long-term effects on cholesterol homeostasis. Administration of the LXR agonist T0901317 to pregnant mice via their diet (0.015% wt/wt) led to induced fetal hepatic expression levels of the cholesterol transporter genes Abcg5/g8 and Abca1, higher plasma cholesterol levels, and lower hepatic cholesterol levels compared with controls. These profound changes during fetal development did not affect cholesterol metabolism in adulthood nor did they influence coping with a high-fat/high-cholesterol diet. This study shows that the LXR system is functional in fetal mice and susceptible to pharmacological activation. Despite massive changes in fetal cholesterol metabolism, regulatory mechanisms involved in cholesterol metabolism return to a "normal" state in offspring and allow coping with a high-fat/high-cholesterol diet.

Hyperlipidemia and atherosclerosis associated with liver disease in ferrochelatase-deficient mice.

Erythropoietic protoporphyria (EPP) is an inherited disorder of heme synthesis caused by deficiency of the mitochondrial enzyme ferrochelatase. EPP in humans is associated with liver disease, hypertriglyceridemia, and a low level of high density lipoprotein (HDL) cholesterol. To explore consequences of ferrochelatase deficiency in lipid metabolism, we have analyzed hepatic lipid content and plasma lipoprotein levels in chow-fed BALB/c mice homozygous ( fch/fch) or heterozygous ( fch/1) for a point mutation in the ferrochelatase gene and in wild-type controls (1/1). Livers of fch/fch mice show bile duct proliferation and biliary fibrosis, but bile formation is not impaired. The free cholesterol content of fch/fch livers is significantly increased when compared with fch/1 and 1/1 livers. Plasma cholesterol in fch/fch mice (9.9 +/- 6.4 mM) is elevated when compared with fch/1 and 1/1 mice (2.9 +/- 0.2 and 2.5 +/- 0.3 mM, respectively), because of an increased cholesterol content in the very low density lipoprotein-sized fractions, whereas HDL cholesterol is reduced. The ratio of cholesteryl ester to free cholesterol is 4.3 +/- 0.6, 3.3 +/- 0.3, and 0.3 +/- 0.1 in the plasma of 1/1, fch/1, and fch/fch mice, respectively. The latter is not due to reduced lecithin:cholesterol acyltransferase activity in plasma of fch/fch mice but to the presence of lipoprotein-X (Lp-X), a particle composed of bile-type lipids usually seen only in cholestatic conditions. Expression of mdr2, essential for biliary phospholipid/cholesterol secretion, is increased in fch/fch livers. In spite of this, biliary phospholipid/cholesterol secretion is reduced relative to that of bile salts. It is postulated that an inability of bile salts to stimulate lipid secretion adequately leads to formation of Lp-X in this noncholestatic condition. Distinct atherosclerotic lesions were found in aged fch/fch mice.Thus, ferrochelatase deficiency in mice leads to liver disease associated with altered hepatic lipid metabolism, a characteristic hyperlipidemia, and development of atherosclerosis.-Bloks, V. W., T. Plösch, H. van Goor, H. Roelofsen, J. Baller, R. Havinga, H. J. Verkade, A. van Tol, P. L. M. Jansen, and F. Kuipers. Hyperlipidemia and atherosclerosis associated with liver disease in ferrochelatase-deficient mice. J. Lipid Res. 2001. 42: 41;-50.

Crustacean hyperglycaemic hormone (CHH)-like peptides and CHH-precursor-related peptides from pericardial organ neurosecretory cells in the shore crab, Carcinus maenas, are putatively spliced and modified products of multiple genes.

About 24 intrinsic neurosecretory neurons within the pericardial organs (POs) of the crab Carcinus maenas produce a novel crustacean hyperglycaemic hormone (CHH)-like peptide (PO-CHH) and two CHH-precursor-related peptides (PO-CPRP I and II) as identified immunochemically and by peptide chemistry. Edman sequencing and MS revealed PO-CHH as a 73 amino acid peptide (8630 Da) with a free C-terminus. PO-CHH and sinus gland CHH (SG-CHH) share an identical N-terminal sequence, positions 1-40, but the remaining sequence, positions 41-73 or 41-72, differs considerably. PO-CHH may have different precursors, as cDNA cloning of PO-derived mRNAs has revealed several similar forms, one exactly encoding the peptide. All PO-CHH cDNAs contain a nucleotide stretch coding for the SG-CHH(41-76) sequence in the 3'-untranslated region (UTR). Cloning of crab testis genomic DNA revealed at least four CHH genes, the structure of which suggest that PO-CHH and SG-CHH arise by alternative splicing of precursors and possibly post-transcriptional modification of PO-CHH. The genes encode four exons, separated by three variable introns, encoding part of a signal peptide (exon I), the remaining signal peptide residues, a CPRP, the PO-CHH(1-40)/SG-CHH(1-40) sequences (exon II), the remaining PO-CHH residues (exon III) and the remaining SG-CHH residues and a 3'-UTR (exon IV). Precursor and gene structures are more closely related to those encoding related insect ion-transport peptides than to penaeid shrimp CHH genes. PO-CHH neither exhibits hyperglycaemic activity in vivo, nor does it inhibit Y-organ ecdysteroid synthesis in vitro. From the morphology of the neurons it seems likely that novel functions remain to be discovered.