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European Lipoprotein Club: Report of the 31st ELC Annual Conference, Tutzing, 8–11 September 2008
Atherosclerosis 205 (2009) 41–47
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Meeting report
European Lipoprotein Club: Report of the 31st ELC Annual Conference, Tutzing, 8–11 September 2008 1. Introduction The 31st meeting of the European Lipoprotein Club was held from 8 to 11 September at the Evangelische Akademie in Tutzing Germany and attended by some 120 participants from all over Europe and the United States. It was the first meeting under the chairmanship of Arnold von Eckardstein (Zurich, Switzerland) who followed Marten Hofker (Groningen, The Netherlands) and it was the last co-organized by Ko Willem van Dijk (Leiden, The Netherlands), who in the past was responsible for the compilation of the annual report, and Gertrud Schuster (Davis, USA) who will be followed in 2009 by Monique Mulder (Rotterdam, The Netherlands) and Rachel Fisher (Stockholm, Sweden). Also under the new chairmanship the structure of the meeting was kept as in the past: The program was composed of six scientific sessions each of which was introduced by the lecture of an invited speaker. All other oral presentations as well as the poster presentations were selected by rating of abstracts through the ELC committee members. As in the past, two types of awards were presented to two young scientists. The young investigator award was presented to Christina Christofferson (Copenhagen, Denmark) for her presentation entitled “The signal peptide anchors apolipoprotein M in plasma lipoproteins and prevents rapid clearance of apolipoprotein M from plasma”. The poster award was presented to Philip Gordts (Leuven, Belgium) for his poster entitled: “Inactivation of the NPxYxxL Motif of LRP1 Reduces Lipoprotein Clearance and Affects Adipogenesis.” The winner of the poster prize was also given the opportunity to orally present his poster in the “Varia” session. 2. Key note lecture After the welcome by Arnold von Eckardstein the scientific part of this year’s meeting was opened by a key note lecture from Michael R. Hayden (University of British Columbia, Vancouver, Canada) on “The Ins and Outs of Cholesterol in Cells: The Pivotal Role of ABCA1”. He reported results of several collaborative studies from his laboratory on the role of the ATP binding cassette transporter A1 (ABCA1) for the regulation of cholesterol homeostasis and function of various organs. The now nearly 10 years old discovery of ABCA1 as the defective gene in patients with Tangier disease by Michael Hayden and other researchers has been a breakthrough for lipoprotein and lipid research and added an important piece of a puzzle to our understanding of cellular cholesterol homeostasis and atherosclerosis. In his lecture Michael Hayden focused on published and unpublished data that have been obtained by careful analysis of conditional knock-out mouse models in which ABCA1 was tissue-specifically 0021-9150/$ – see front matter doi:10.1016/j.atherosclerosis.2008.12.003
deleted in either liver, intestine, pancreatic  cells, or the brain. The knock-out of ABCA1 in the liver and intestine reduced HDLcholesterol levels by 80 and 30%, respectively. The crossed animals with a combined knock-out of hepatic and intestinal ABCA1 had 90% lower HDL-cholesterol levels. By contrast to previous observations that hepatic overexpression of ABCA1 increases atherosclerosis, the conditional knock-out of ABCA1 in the liver was found to aggravate atherosclerosis in apoE deficient mice. Mice with specific inactivation of ABCA1 in  cells showed severe impairment of glucose tolerance and defective insulin secretion. Analysis of the islet cholesterol levels revealed cholesterol accumulation in the islets. Interestingly, systemic knock-out of ABCA1 resulted in a much milder glucose intolerance and normal islet cholesterol levels, probably because these animals have much lower plasma cholesterol levels than the -cell specific knock-out. Michael Hayden therefore concluded that the susceptibility to cholesterol accumulation in  cells and hence impaired insulin secretion is influenced by ABCA1 function and plasma cholesterol levels. The importance of ABCA1 for regular -cell function is also indicated by the suggestion of increased risk for diabetes in humans with mutations or polymorphisms that interfere with ABCA1 function. Interestingly also the LDL receptor was found to contribute to -cell failure. It is the main entrance route for cholesterol into  cells. Feeding with a fat-rich Western diet-induced glucose tolerance in wild type and apoE deficient mice but not in LDL receptor deficient mice. In agreement with a permissive role of the LDL receptor in -cell failure are also genetic data on reduced prevalence of diabetes in heterozygous familial hypercholesterolemia. Finally Michael Hayden presented data on the effects of brain-specific ABCA1 deletion. These mice showed reduced cholesterol levels in the cortex and hippocampus as well as in plasma (−30% compared to wild type mice). Michael Hayden and coworkers found enhanced selective uptake of cholesterol from HDL through the blood brain barrier by SR-BI. The lack of ABCA1 in the brain was also associated with disturbed synapse formation and dysfunction in the sensori- and locomotoric systems. Altogether the data presented by Michael Hayden show the importance of ABCA1 for normal organ function beyond the liver and macrophages. 3. Session I Lipid and Lipoprotein Metabolism: chaired by Athina Kalopissis (Paris, France) and Jörg Heeren (Hamburg, Germany). As the invited speaker, Stephen G. Young (Los Angeles) presented the discovery of a new player in the lipolytic processing of chylomicrons, GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1). GPIHBP1 is an endothelial cell glycoprotein of the lymphocyte antigen 6 (Ly6) family, expressed on the luminal surface of capillaries in heart, adipose tissue, and skeletal muscle. These same tissues express high
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levels of LPL (lipoprotein lipase), the enzyme that is responsible for the hydrolysis of the triglycerides in chylomicrons and VLDL. LPL is synthesized by the parenchymal cells of adipose tissue and striated muscle and is then translocated onto the luminal surface of capillaries, where it has been assumed to bind to negatively charged heparan sulfate proteoglycans (HSPGs) through electrostatic interactions with the positively charged heparin-binding domains of LPL. It had been widely assumed that LPL, anchored to HSPGs, hydrolyzes chylomicron triglycerides and, within minutes, transforms chylomicrons into remnants, which are then taken up by tissues, largely via the LDL receptor. Now, it appears that GPIHBP1 might play an important role in binding LPL and chylomicrons on the surface of capillaries. GPIHBP1 was originally identified by Ioka and coworkers by expression cloning as a protein that promotes the binding of HDL to cultured cells. It therefore came as a surprise that Gpihbp1−/− mice, characterized by the laboratory of S. Young, display milky plasma and extremely high plasma triglyceride levels, even on a low-fat chow diet. This observation alone suggested that GPIHBP1 is important for the lipolytic processing of triglyceriderich lipoproteins by LPL. Subsequent studies with transfected CHO cells revealed that GPIHBP1 avidly binds LPL and chylomicrons, suggesting a new model in which GPIHBP1 might serve as a platform for the interaction of LPL and chylomicrons on the surface of endothelial cells. Clearly, efficient lipolytic processing of chylomicrons requires GPIHBP1. However, the new model for lipolysis does not necessarily exclude a role for HSPGs in binding LPL. The authors showed that GPIHBP1-bound LPL can be released by heparin. When Gpihbp1+/+ or Gpihbp1+/− mice are injected with heparin, LPL appears in the plasma very quickly, with LPL levels peaking in 1 min. In Gpihbp1−/− mice, heparin releases LPL into the plasma, but the release is slower, peaking in 15 min. This observation led to the hypothesis that there may be more than one pool of heparinreleasable LPL. Perhaps there is an endovascular GPIHBP1-bound pool of LPL that is highly relevant to lipolysis and a second pool, located in subendothelial spaces and bound to HSPGs, that is not relevant to lipolysis. The latter pool, which is present in Gpihbp1−/− mice, appears to be released into plasma more slowly after heparin. Interestingly, the LPL that is released by heparin in Gpihbp1−/− mice is enzymatically active and quickly lowers the markedly elevated plasma triglyceride levels in these mice. GPIHBP1 contains a prominent acidic domain, rich in aspartate and glutamate residues, near its amino terminus. Anne Beigneux, Stephen Young, and coworkers showed that the acidic domain plays an important role in the binding of LPL and chylomicrons. A mutant GPIHBP1 in which the aspartates and glutamates between residues 38 and 48 are replaced with alanine cannot bind LPL or chylomicrons, suggesting that electrostatic interactions are important for binding of ligands. These studies have identified a new player in lipolysis, GPIHBP1. Understanding the function of this new molecule will undoubtedly add to our understanding of plasma triglyceride metabolism and the pathogenesis of hypertriglyceridemias. To seek evidence that GPIHBP1 plays an essential role in triglyceride (TG) metabolism in humans as it is in mice and might be responsible for severe chylomicronemia, Geesje Dallinga-Thie (Amsterdam, The Netherlands) sequenced the human GPIHBP1 gene in 60 patients with plasma TG levels above the 95th percentile. One patient was identified with a homozygous missense mutation, resulting in a glutamine to a proline substitution at position 115 in the ly6 domain, which is essential for LPL binding. This mutation was not found in 100 controls. An intravenous injection of heparin failed to release LPL from the endothelial cell surface, suggesting that GPIHBP1-Q115P is not able to bind LPL. Furthermore, experiments using CHO cells transfected with normal GPIHBP1 or with GPIHBP1-Q115P in combination with a human LPL expression vector revealed further evidence that the Q115P mutant could not bind LPL or chylomicrons. Thus, a homozygous Q115P mutation in
GPIHBP1 was identified and leads to a severe hypertriglyceridemic phenotype. Leanne Hodson (Oxford, United Kingdom) studied in eight healthy men the contribution of chylomicron-triglyceride (CMTG), VLDL-TG and plasma non-esterified fatty acids (NEFA) to TG stored in adipose tissue. Three nutritionally similar meals were given at 5 h intervals. The efficiency of adipose tissue FA uptake increased with each meal, CM-TG was the main FA source and the fractional extraction increased from 21% after the 1st meal to 35% after the 2nd meal to 47% after the 3rd meal. Thus, the spillover of CM-TG decreased after each meal, mainly through increased FA re-esterification. Uptake of VLDL-derived FA was more important after the last meal, with spillover seen only in the fasted state. The decrease in spillover fraction of CM-derived FA with sequential meals over 24 h suggests induction of fat storage by meal repetition. HDL and their main protein, apolipoprotein (apo) AI exert antiatherogenic effects within the arterial wall. Lucia Rohrer (Zurich, Switzerland) studied transport of 125 I-HDL by endothelial cells cultured on inserts. The cells bound, internalised and translocated HDL from the apical to the basolateral compartment in a specific manner without degrading the protein moiety of the transported particles. Using RNA interference, reduced 125 I-HDL transport was obtained after silencing SR-BI and ABCG1, respectively but not all after knockdown of ABCA1. In addition the role of endothelial lipase (EL) in HDL transport through endothelial cells was shown by: (i) decreased binding and cell association of HDL after heparin and heparinase treatment, which releases EL from membranes; (ii) inhibition of EL activity reduced HDL cell association; (iii) EL knockdown by siRNA diminished HDL transport. In the second part of this session Ernst Steyrer (Graz, Austria) described the role of Phosphatidylethanolamine–Methyltransferase (PEMT) for lipid droplet biogenesis. The core of lipid droplets (LD) is covered by a phospholipid monolayer. Since the phosphatidylcholine:phosphatidylethanolamine ratio changes during adipocyte differentiation, the aim of this study was to investigate a potential role of PEMT for lipid droplet biogenesis. In 3T3-L1 cells, adipocyte differentiation was associated with an increase in the expression and activity of PEMT. Fluorescence microscopy revealed that a PEMT-EGFP fusion protein localized to LD. As demonstrated by fat cell morphometry, mice deficient in PEMT are resistant to high fat diet-induced hypertrophy of fat cells suggesting that phospholipid remodeling is crucial for lipid droplet biogenesis in adipocytes. Furthermore, PEMT deficiency resulted in lipid accumulation within enterocytes (chylomicron retention) under high fat conditions indicating that PEMT is also involved in chylomicron synthesis. Thomas Grewal (Sydney, Australia) gave new insights into the molecular mechanisms regulating intracellular transport of caveolin. It has recently been shown that caveolin requires cholesterol to exit the Trans Golgi Network (TGN) in order for it to be transferred to the plasma membrane. In elegant cell culture studies he could show that cholesterol from late endosomal compartments is the driving force and delivered to the TGN to promote caveolin export from the TGN to the cell surface. The cholesterol-dependent export of caveolin requires the activity of cytoplasmic phospholipase A2 (cPLA2), which is targeted to the TGN to promote Golgi vesiculation in a cholesterol-dependent manner. The phospholipid-binding protein Annexin A6 inhibits cholesterol export from late endosomes, thereby reducing the cholesterol-dependent association of cPLA2 with the TGN. This correlates with the reduced release of arachidonic acid in Annexin A6 overexpressing cells, and it was concluded that high expression levels of Annexin A6 downregulate the activity of cholesterol-dependent cPLA2 to block caveolin transport to the cell surface. Herbert Stangl (Vienna, Austria) investigated the role of HDL uptake and subsequent re-secretion in cholesterol efflux. Earlier
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studies in macrophages identified apoAI in structures that appeared to be coated pits or coated vesicles. In these studies inhibition of receptor-mediated endocytosis interfered with cholesterol efflux to apoAI, suggesting that efflux involves the internalisation of apoAI via clathrin-coated pits. As demonstrated by living cell microscopy, electron microscopy and efflux experiments using radiolabeled HDL Dr. Stangl showed that (1) HDL holo-particle uptake is mediated by scavenger receptor class B1 (SR-BI) and (2) HDL retroendocytosis can mediate cholesterol efflux. Carlos Vrins (Amsterdam, Netherlands) investigated the role of HDL in trans-intestinal cholesterol efflux (TICE). This pathway has been recognized as a major route of sterol output and describes the direct transport from blood via enterocytes to feces. With the prominent role of HDL in the hepatobilary route of reverse cholesterol transport, the aim of this study was to determine whether this lipoprotein also is involved as a cholesterol donor for TICE. Intestine perfusions were first performed in SR-BI deficient mice, which have elevated serum HDL-cholesterol levels by ligating the bile ducts to prevent the entry of bile into the intestine and subsequently perfusing the intestine with model bile to quantify TICE. The rate of TICE in these mice is 2-fold higher than that found in wildtype (WT) animals. Next, TICE was analysed in mice lacking both ABCA1 and SR-BI. These double knock-out mice (DKO) mice have no circulating HDL cholesterol, but, interestingly, their rate of TICE is similar to WT. When HDL with radiolabeled cholesterol was injected into the DKO, no radioactivity could be found in the intestinal lumen during intestine perfusions. However, a similar result was obtained in WT. These data indicate that HDL is not directly involved in TICE. Egon Demetz (Innsbruck, Austria) analysed the effect of hepatic SR-BI knockdown mediated by RNA interference (RNAi) on the development of atherosclerosis in NZW rabbits. The work was presented by the co-author of this study, Ivan Tancevski. Compared to mice, rabbits express cholesteryl ester transfer protein (CETP), display a more human-like lipoprotein profile and are more prone to develop atherosclerosis. To downregulate SR-BI specifically in the liver, a plasmid coding for small hairpin RNA against rabbit SRBI was associated with a poly-lysine-based shuttle vector enriched with galactose. Specific downregulation was demonstrated by realtime PCR. To study the development of atherosclerosis, NZW rabbits were fed a Western type diet and treated either with a control vector or a vector mediating the downregulation of hepatic SR-BI. By analyzing atherosclerotic lesion area it was shown that downregulation of hepatic SR-BI resulted in the reduction of heavy lesions in the aorta of rabbits, suggesting that hepatic SR-BI expression is associated with the progression of atherosclerosis in this CETP-expressing species. 4. Session II Gene Regulation: chaired by Paolo Parini (Stockholm, Sweden) and Gertrud Schuster (Davis, USA). Beatrice Desvergne (Lausanne, Switzerland) gave an overview of their recent findings on the “PPAR-mediated activities of phthalates and metabolic disorders”. PPARs are activated by a wide variety of compounds, whereby possible functions of some endogeous ligands as endocrine disruptors are still not clarified. Most recently mono-ethylhexyl-phthalate (MEHP) was identified as endocrine disrupting chemical. Phthalates are widely used in the plastic industry as PVC softener and are found in daily products such as food packaging or cosmetics. MEHP is the active metabolite of di-2ethylhexyl phthalate (DEHP), the most abundant phthalate in the environment. MEHP and DEHP induce peroxisome proliferative products. MEHP and Rosiglitazone bind in a very similar fashion to the pocket of PPAR-␥’s ligand binding domain; whereby MEHP induces adipogenesis in vitro to a lower extent than Rosiglitazone. It appears that MEHP and Rosiglitazone induces the formation of
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different PPAR-␥ associated transcription complexes by recruiting distinct co-factors. Whereas Med-1 can be recruited by both, Src-1 and p300 are recruited only by Rosiglitazone, and PGC1a more efficiently by MEHP. This results in the regulation of a different set of target genes. Because MEHP can also activate PPAR␣ and PPAR, in vivo studies were required to evaluate its metabolic impact. First observations show that DEHP protects from diet-induced obesity in wild type mice, in a mechanism involving PPAR␣ in the liver. The different behavior of rodent and human PPAR␣ then raises the question of species-specific (mouse versus human) activity of DEHP, further explored by the group of B. Desvergne. These results leave us with the question of whether there is a potential role of phthalates in human obesity. The study by Jesper Schou (Copenhagen, Denmark) evaluated genetic alterations in the ABCG1 promoter and the risk of ischemic heart disease (IHD). In studies of ABCG1−/− versus ABCG1+/+ mice, total body expression of ABCG1 protected against atherosclerosis, without affecting HDL cholesterol or other lipid or lipoprotein levels. They found that individuals with the ABCG1-376C>T variant, located in a putative SP-1 binding site, had an increased risk of developing IHD, independent of HDL cholesterol and other lipid and lipoprotein levels. Tomas Jakobsson (Stockholm, Sweden) discussed his findings that the co-repressor complex subunit GPS2 is selectively required for the LXR-induced transcription of ABCG1, and revealed fundamental differences between ABCG1 and ABCA1 genes regarding LXR coregulator requirement. Also, LXR-induced cholesterol efflux in THP-1 cells is eliminated by depletion of GSP2. Sophie Lestavel, from Lille, France, presented a study in which PPARalpha has been identified as an intestinal specific target gene of Liver X Receptors. LXR ligands were administrated to wild type and LXRalpha/beta double knock-out mice, both on a C57Bl6/J background, and gene expression analysis was used to identify intestine-specific LXR target genes. Chronic administration of LXR synthetic agonists led to a significant increase of PPARalpha mRNA levels in the small intestine but not in the liver. This was paralleled by up-regulation of PPARalpha protein. Administration of a synthetic LXR agonist to wild type mice resulted in the induction of several PPARalpha target genes and this effect was completely blunted in PPARalpha-deficient mice, demonstrating the biological relevance of this LXR-PPARalpha transcriptional cascade. The session was concluded by a presentation of Arie Van Tol, Rotterdan, The Netherlands, entitled Acute Elevation of Plasma phosholipid transfer protein (PLTP) activity Strongly Increases Pre-existing Atherosclerosis. A transgenic mouse model in which the conditional expression of human PLTP by the tetracyclineresponsive gene system was developed in order to study the effects of an acute increase in plasma PLTP activity on the metabolism of apolipoprotein B-containing lipoproteins and on diet-induced pre-existing atherosclerosis. Induced expression of PLTP strongly increased plasma VLDL triglycerides in LDL receptor knock-out mice, while VLDL secretion was not affected. The elevation was secondary to PLTP-dependent inhibition of VLDL catabolism, which is caused, at least partly, by a decreased lipoprotein lipase activity. Acute overexpression of PLTP also reduced HDL and resulted in a more atherogenic lipoprotein profile leading to an increase in size of pre-existing atherosclerotic lesions. In addition, the lesions contain more macrophages and less collagen relative to controls, suggesting a less stable lesion phenotype. 5. Session III Functional Genomics and Genetics: chaired by Anne Tybjaerg Hansen (Copenhagen, Denmark) and Florian Kronenberg (Innsbruck, Austria).
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A. Jake Lusis (Los Angeles, USA) provided an overview of his perspective concerning the metabolic syndrome and how epidemiology and systems biology can interact to unravel the pathogenesis of this epidemic disease. Although the single components of the metabolic syndrome are subject to high heritability, this high heritability is not due to a few genes with large effects. Besides environmental influences and sex differences it is rather due to combinations of many different genes, each of which contributes a tiny fraction of risk. Genome-wide association studies during recent months have without doubt contributed some novel genes to metabolic syndrome traits (e.g. MC4R, FTO, MLXIPL, and TCF7L2), but these common polymorphisms explain only a small fraction of 5–10% of the genetic effect size. He showed examples that classical inbred and recombinant inbred populations of mice can be used for high resolution mapping including systems biology approaches by collecting phenotypic, transcriptomic, proteomic and metabolomic data at once and by applying whole genome association approaches and mathematical modeling of biological networks. This can dramatically increase the mapping resolution compared to classical linkage analysis. Instead of using a genome-centric view of complex diseases by modeling genetic variation with clinical endpoints, an integrative approach models the influence of genetic variability on gene expression and clinical endpoints and thereby allows a network analysis. The hierarchical clustering of differentially expressed and coexpressed genes allows the identification of highly connected elements of hub genes with major contributions to the pathogenesis of the metabolic syndrome. Jake Lusis’ lab has tested some of the predicted genes using a series of transgenic experiments. The results have been highly encouraging, as a high fraction of the genes predicted to be involved in these traits have been confirmed. In these studies, gene expression serves as a kind of intermediary phenotype between genotype and the complex phenotypes. This global analysis of networks is a challenge, which can hopefully be better handled by a systems biology approach. Christiane Lundegaard (Copenhagen, Denmark) and coworkers investigated the ability of mutations in APOA1 to predict risk of ischemic heart disease (IHD) and death in the general population. Among three nonsynonymous mutations identified by resequencing APOA1 in 200 individuals, a common mutation (1 in 500 in the general population), A164S, predicted a 3-fold increase in risk of IHD, and a reduction in mean survival time of almost 10 years in the Copenhagen City Heart Study (n = 10,500, 31 years follow-up). This is the first report of a common mutation in APOA1, which predicts an increased risk of IHD and MI, and decreased longevity in the general population independent of lipid and lipoprotein levels. Stefano Romeo (Dallas, TX, USA) and coworkers sequenced the ANGPTL3, 5, 6 genes in participants in the Dallas Heart Study (DHS) (n = 3351), and identified several nonsynonymous sequence variants in ANGPTL3 and ANGPTL5 which associated with low plasma levels of triglycerides. When expressed in HEK-293 cells the majority of these mutations were associated with a failure of the proteins to be secreted from cells. Those who where secreted failed to inhibit lipoprotein lipase in vitro. These data suggest a non-redundant role of Angiopoietin-like proteins in triglycerides metabolism. Katharina Leidl (Regensburg, Germany) and coworkers compared lipid species in monocytes, lymphocytes, granulocytes, platelets and red blood cells of healthy volunteers using electrospray ionization tandem mass spectrometry. In an extensive study, the authors clearly showed that the lipid pattern of the different circulating blood cells matched with various aspects of cell specific functions related to shape, size and cellular life span in the vascular system.
6. Session IV Lipoprotein Function: chaired by Franco Bernini (Parma, Italy) and Dilys Freeman (Glasgow, UK). This session was opened with an invited lecture given by Tomas Vaisar (Seattle, US). Epidemiological and clinical studies demonstrated that low levels of high-density lipoprotein (HDL) cholesterol are an independent risk factor for premature cardiovascular disease (CAD). A primary mechanism by which HDL protects against atherosclerosis is by promoting reverse cholesterol transport (RCT). However HDL has other biological activities that contribute to its antiatherogenic properties, including antioxidant and antiinflammatory effects. Dr. Vaisar underlined that the proteome component of HDL plays critical roles in mediating these biological activities suggesting that HDL-cholesterol levels may not be the only determinant of the cardioprotective role of HDL. Using mass spectrometric analysis he showed that HDL3 of subjects with CAD is enriched in several proteins, including apolipoproteins E (apoE), complement C3 and apoC-IV. Based on this data he suggested that altered protein composition might affect the antiatherogenic and anti-inflammatory properties of HDL. They also investigated whether combination therapy with statin and niacin, which raises HDL levels, might affect these changes observed in HDL3 of subjects with CAD. They found that combination therapy significantly decreased the amount of apoE in HDL3 while the abundance of apoF, apoJ and PLTP levels increased. No significant changes were found in apoAI levels of HDL after niacin/statin treatment. Taken together these results demonstrated that statin/niacin combination therapy remodels the HDL3 proteome, resulting in a protein composition that resembles that of healthy subjects. Dr. Vaisar concluded that combined statin/niacin therapy may partially reverse the changes in the protein composition seen in HDL3 in CAD subjects. His results suggested the possibility that quantifying the HDL proteome by MS could provide insights into the therapeutic efficacy of antiatherosclerotic interventions. Moreover he provided evidence that HDL protein composition differences result in a specific “fingerprint” in MALDI-TOF mass spectrum of HDL, which is reproducible and sensitive to proteome changes. Using this approach they demonstrated that HDL2 from subjects with CAD is enriched in Lp(a) and apoC-III while level of apoC-I decrease. Collectively Dr. Vaisar’s data suggest that the proteomic fingerprint of HDL could potentially be used for diagnostic purposes. Johannes Levels (Amsterdam, Netherlands) presented a low dose challenge of endotoxin (1 ng/kg body weight) to healthy men (n = 20) in order to investigate changes in the array of proteins carried by high-density lipoprotein particles over 24 h. The technique of SELDI-TOF mass spectrometry carried out on HDL, captured using antibodies to apolipoprotein AI, was shown to be a feasible high throughput method to analyse a time series of change in HDL protein profile. The endotoxin challenge induced profound changes in HDL composition. Hierarchical clustering analysis of protein changes indicated that individuals fell in to one of three distinct clusters that were independent of their plasma HDL-cholesterol concentration. Martin Merkel (Hamburg, Germany) presented data on apoA5 metabolism in mouse models comprising apoA5 transgenic (apoA5tr) mice bred on an apoE deficient (E0) or wild type (WT) background. The presence of the apoA5 transgene reduced plasma VLDL triglyceride levels more on the E0 than on a WT background. Turnover studies showed an apoA5 mediated faster lipolysis of VLDL TG from 14 C-TG-VLDL, whereas the plasma removal of 125 I-TC protein labelled VLDL was not affected. In the aortic root, apoA5 expression reduced atherosclerotic lesion size by more than 50% in both genders. It was concluded, that apoA5 may be atheroprotective by accelerating plasma TG hydrolysis.
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Andrea Dichlberger (Vienna, Austria) identified and studied apoAV in the chicken, an excellent model for lipid transport due to the high level of lipid transport to the yolk. ApoAV is found in the circulation of laying hens in both lipid-associated and lipidfree forms. They observed that human apoAV interacted with the lipoprotein receptor LR8 and the human LDLR, and that structural mutations in apoAV can influence receptor binding. Chicken apoAV is taken up into the chicken oocyte and in LR8-293 cells chicken apoAV is endocytosed by LR8. It was proposed that reduced receptor-mediated clearance of apoAV-containing particles may lead to elevated plasma triglyceride levels. 7. Session V Inflammation and Vascular Biology: chaired by Ko Willems van Dijk (Leiden, The Netherlands) and Ken Lindstedt (Helsinki, Finland). The session on “Inflammation and Vascular Biology” started with a talk by the invited speaker, Ziad Mallat (Paris, France), on “Regulatory T-cell immunity in the control of Atherosclerosis”. Prof. Mallat introduced evidence that Th1-driven responses are detrimental to the atherosclerotic process, whereas the role of the Th2 pathway in the development of atherosclerosis remains controversial. In contrast, natural Treg cells (nTregs), characterized by the expression of CD4 and CD25, develop in the thymus and home in peripheral tissues to maintain self-tolerance. nTregs prevent autoimmunity by inhibiting pathogenic T cells. Furthermore, a subset of induced Tregs (iTregs) is also generated in the periphery during an active immune response where they maintain self-tolerance by secreting IL-10. Tregs suppress atherosclerosis by cell contact-dependent suppression, by consumption and limitation of growth factors (IL-2), and by producing inhibitory cytokines (IL-10 and TGF-). Secondly, Prof. Mallat stated that reduced phagocytic removal of apoptotic vascular SMCs and macrophages may play a major role in the perpetuation of the inflammatory response in atherosclerosis. The mechanisms involve a reduction in the immuno-suppressive response that follows the ingestion of apoptotic bodies, i.e. increased expression of IL-10 and TGF, or a promotion of inflammatory responses attributable to the high concentration of proinflammatory oxidized phospholipids on apoptotic cell membranes. Milk fat globule EGF-like Factor 8 (Mfge8), a glycoprotein involved in phagocytic clearance of apoptotic cells, is required for efficient removal of apoptotic cells during both early and advanced atherosclerosis and promotes an atheroprotective regulatory T-cell immune response. Moreover, deficiency in Mer receptor tyrosine kinase (mertk), a receptor expressed in bone marrow-derived macrophages and involved in apoptotic cell clearance, leads to an increased accumulation of apoptotic cells, promotes a proinflammatory immune response, and accelerates lesion development. Finally, Prof. Mallat concluded that the critical role of naturally occurring Tregs in atherosclerosis has initiated studies aiming at the promotion of Treg response in vivo. Thus, supplementation with Tregs might lead to the induction of immune suppression and a reduction in pathogenic Tcell-mediated responses, ultimately altering plaque development and/or composition. Jeppe Zacho (Copenhagen, Denmark) showed that CRP levels >3 mg/l versus <1 mg/l were associated with a 1.6-fold and 1.3fold increased risk of ischemic heart disease (IHD) and ischemic cerebrovascular disease (ICVD). However, although genotype combinations of 4 different CRP polymorphisms were associated with up to a 64% increase in CRP levels, they were not associated with increased risk of IHD or ICVD. Dr. Zacho concluded that it would be likely that a common process, like inflammation, causes both IHD, ICVD, and CRP elevations. Florence Bietrix (Amsterdam, Netherlands) showed that inhibition of glycosphingolipid (GSL) synthesis with AMP-DNM (25 mg/kg
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daily) in apoE3 Leiden mice on a high cholesterol diet for 18 weeks induced a 2-fold increase in their atherosclerotic lesions. However, no effect was seen on cholesterolemia/triglyceridemia. By increasing the AMP-DNM concentration to 50 and 100 mg/kg daily, however, Bietrix et al observed a dose dependent decrease in plasma cholesterol and trigycerides already after 6 weeks of treatment including a strong reduction in the atherosclerotic lesion size. In addition, the percentage of plasma HDL was increased with a decrease of the LDL/VLDL fraction. Triglyceride and cholesterol levels in the liver were reduced and the bile secretion was increased. The authors concluded that GSL synthesis inhibition have unexpected effects on lipoprotein metabolism in ApoE3 Leiden mice. Rachel Fisher (Stockholm, Sweden) showed that obese women with high liver fat (LFAT) content had more inflammation and increased levels of ceramides in their subcutaneous adipose tissue compared to equally obese women with normal LFAT. Gene expression analysis indicated that the increase in ceramide was independent of de novo ceramide synthesis. However, the authors found that the high LFAT subjects had higher gene expression levels of sphingomyelinases in their adipose tissue and that mRNA levels of one particular sphingomyelinase, SMPD3, correlated with concentrations of sphingomyelins and ceramides in adipose tissue. Dr. Fisher concluded that increased ceramide levels in inflamed adipose tissue may depend on increased SMPD3 activity. Nikolaos Kadoglou (Athens, Greece) presented the effects of exercise training on atherogenesis in apoE-deficient mice. Mice were put on a high-fat diet for 16 weeks and subsequently performed 1 h of treadmill running for 1 h per day, 5 days a week for 6 weeks. As compared to controls, the exercising mice did not differ in body weight or plasma lipid levels. However, plaque area was decreased significantly, and the plaques presented a more “stable” phenotype. These data support a role for exercise in the management of atherosclerosis burden. The paper by Patrick Rensen (Leiden, Netherlands) investigated the effects of the CETP-inhibitor torcetrapib in APOE*3-Leiden.CETP transgenic mice. In this mouse model, torcetrapib decreased plasma total cholesterol levels and increased HDL-cholesterol levels. Torcetrapib alone decreased atherosclerotic lesion size but did not add to the antiatherogenic effect of atorvastatin. Moreover, torcetrapib induced a more “unstable” plaque phenotype, characterized by more macrophages and less collagen, which may explain some of the negative effects seen in the human torcetrapib trial. Janna van Diepen (Leiden, Netherlands) presented her studies on the association between inflammation and TG metabolism. Mice expressing a constitutive active isoform of IKK-beta in hepatocytes only (LIKK) were cross bred with APOE*3-Leiden mice and VLDL metabolism was analysed. The LIKK mice exhibit a constitutively activated NF-kB response, which on the APOE*3-Leiden background, resulted in increased cholesterol and TG levels. After careful in vivo analyses, the increased plasma TG could be linked to an increased VLDL-TG production rate with no difference in clearance, indicating that hepatic inflammation may play a causal role in dyslipidemia. Pirkka-Pekka Laurila (Helsinki, Finland) presented a description of the phenotype of upstream stimulatory factor 1-deficient (Usf1−/−) mice. Usf1 is a ubiquitous transcription factor that has been linked to familial combined hyperlipidemia in human genetic association studies. On a Western type diet, Usf1−/− mice displayed a decreased plasma TG, which could be explained by up-regulated transcription of LPL and apoA5. On a chow diet, plasma cholesterol was increased, simultaneously with an increase in plasma apoA1 levels. This effect of Usf1-deficiency on ApoA1 transcription was replicated in human cells treated with Usf1 siRNA constructs. Genome-wide transcription analysis of Usf1−/− adipose tissue revealed several clues for a potential antiatherogenic effect of USF1 downregulation.
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Lucia Fuentes (Lille, France) provided evidence for a role of the cell cycle inhibitor protein p16-ink4a in determining macrophage phenotype. Recently, the locus encoding p16-ink4a has been genetically linked to CHD and type 2 diabetes. It was first demonstrated that p16-ink4a was expressed exclusively in classical macrophages and not in so-called IL-4 activated M2 macrophages. Next, adenovirus mediated overexpression of p16 resulted in an M1 phenotype, whereas siRNA mediated downregulation of p16 resulted in an M2 phenotype. Moreover, bone marrow-derived P16−/− macrophages presented a functional M2 phenotype. These data clearly implicate p16 in the determination of macrophage phenotype. Variation in p16 could thus affect the balance between proinflammatory (M1) and anti-inflammatory (M2) macrophages. 8. Session topic VI Varia: chaired by Albert Groen (Amsterdam, The Netherlands) and Arnold von Eckardstein (Zurich, Switzerland). Claudia Coomans reported on the stimulatory effect of central insulin on fat storage in white adipose tissue. It was shown that this effect of central insulin was dependent on the activation of KATP -channels in the brain. By blocking the activity of these KATP channels in the brain selectively, they were able to show that the stimulatory effect of peripheral insulin on fat storage in adipose tissue is completely abolished when insulin signalling in the brain is blocked. Feeding mice a high fat diet induces both peripheral and central insulin insensitivity. Indeed feeding mice a high fat diet completely abrogated the effect of insulin on FA uptake by WAT underlining the surprising importance of the brain in peripheral effects of insulin. Maria Pia Adorni and co workers investigated the mechanism by which probucol affects xanthoma regression. Probucol is an inhibitor of ABCA1 and this leads to a decrease in the cholesterol content of plasma membranes. The level of cholesterol in the plasma membrane of macrophages induces membrane ruffling and migration capacity of the cells. Adorni et al demonstrated that probucol treatment of acetyl-LDL loaded macrophages restores migration of the cells and reduces membrane ruffling suggesting a possible role of ABCA1 in promoting cholesterol membrane ruffling and in inhibiting migration in the absence of an extracellular acceptor. Knocking-down expression of ABCA1 in J774 macrophages had similar effects. They conclude that the effects of probucol on ABCA1 function may underlie its efficacy in reducing cutaneous and tendinous xanthoma in patients with different forms of hypercholesterolemia. Tommaso Fasano reported on the role of PCSK9 as a modulator of LDL-receptor function and on its relationship with LDL-R internalisation. Gain of function mutant D374Y-PCSK9 was incubated with fibroblasts or lymphocytes derived from ARH patients. Lymphocytes from these patients are not able to internalise the LDLreceptor. Despite the inability of these cells to internalise PCSK9, the amount of LDL-R on the surface decreased, suggesting that PCSK9 can be internalised via an alternative route and may affect LDL-R processing in the cells. Apolipoprotein M is an interesting novel apolipoprotein which seems to be involved in formation of pre--HDL. The protein has a molecular weight of around ∼25 kD. In plasma, apoM is mainly associated with HDL and to a lesser degree with LDL, VLDL and chylomicrons. Interestingly, plasma apoM has retained its N-terminal signal peptide. Christina Christoffersen and coworkers investigated whether the N-terminal signal peptide of apoM is involved in its association with lipoproteins. This indeed turned out to be the case. Transgenic mice producing mutant human apoM without signal peptide showed no detectable apoM levels in plasma. The protein was rapidly cleared by the kidneys
and indeed, ligation of kidney arteries induced a strong accumulation of the mutant protein in plasma. It is concluded that the hydrophobic signal peptide anchors the protein in lipoproteins. The prize for the best poster was given to Philip Gordts from Leuven (Belgium). He analysed lipoprotein metabolism, atherosclerosis and adipose tissue of LDL receptor knock-out mice (LDLR−/−) which in addition carry an inactivating mutation in the NPxYxxL motif of the LDL receptor related protein 1 (LRP1). Compared to pure LDLR−/− mice, mice with additional LRP1 inactivation showed more severe and mixed hyperlipidemia and more atherosclerosis in the aorta which is characterized by a higher SMC content and MMP2 activity. In addition the LRP1 knock-in mice showed a decreased expression of adipocyte markers such as GLUT4, LPL and FABP4. In conclusion LRP1 appears to modulate lipoprotein clearance, atherosclerosis and adipogenesis. Paolo Parini and his colleagues from Stockholm (Sweden) analysed the metabolic impact of gene regulation of ACAT2 by the two nuclear hormone receptors HNF1␣ and HNF4␣ which are mutated in patients with Maturity Onset Diabetes of the Youth MODY3 and MODY1, respectively. While patients with MODY3 had normal lipoprotein composition, those with MODY1 had lower levels of VLDL and LDL esterified cholesterol. Mutagenesis revealed one important HNF4␣ binding site in the human ACAT2 promoter. ChIP assays and protein-protein interaction studies showed that HNF4␣ directly or indirectly (via HNF1␣) can bind to the ACAT2 promoter. The authors conclude that the lower levels of esterified cholesterol in VLDL and LDL particles in patients with MODY1 may reflect the lower ACAT2 activity in these patients. This may translate into lower risk of atherosclerosis. Ronit Shiri-Sverdlov (Maastricht, The Netherlands) investigated the influence of scavenger receptors CD36 and SR-A in diet-induced steatohepatitis. In earlier work she has shown that the cholesterol content of the diet plays an important role in the induction of inflammation in the fatty-liver. Formation of foamy Kupffer cells seems to lie at heart of this finding. By knocking out expression of the scavenger receptors CD36 and SR-A via bone marrow transplantation procedures, Shiri-Sverdlov and coworkers provide additional evidence for this hypothesis. Mice lacking CD36 and SR-A in the haematopoetic compartment showed significantly reduced hepatic inflammation when fed a high cholesterol/high fat diet. These findings were confirmed by measuring levels of proinflammatory cytokines in plasma of these mice. The authors conclude that uptake of modified lipoproteins plays an important role in diet-induced hepatosteatosis. In cooperation with Oliver Weingärtner and cardiologists from the University Hospital of Saarland (Homburg, Germany) Dieter Lütjohann (Bonn, Germany) investigated the effects of a diet rich in plant sterol esters (PSE) on endothelial function, cerebral lesion size, and atherosclerosis in mice as well as associations of plant sterol plasma concentrations with aortic stenosis in humans. PSE supplementation impaired endothelium-dependent vasorelaxation and increased cerebral lesion size after middle cerebral artery occlusion. After randomization of apoE−/− mice to Western-type diet with the addition of PSE or ezetimibe, both animal groups showed equal plasma cholesterol levels but the PSE group showed less reduction in atherosclerosis plaque size than animals randomized to ezetimibe. Plant sterol plasma concentration strongly correlated with increased atherosclerotic lesion formation (r = 0.50; P < 0.01). Furthermore, patients eating PSE-supplemented margarine (n = 10) were found to have increased plasma concentrations and 5-fold higher sterol concentrations in aortic valve tissue. The authors conclude that food supplementation with PSE impairs endothelial function, aggravates ischemic brain injury, effects atherogenesis in mice, and leads to increased tissue sterol concentrations in humans.
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Acknowledgements The 30th ELC meeting was generously supported by its longstanding main sponsor Roche (Switzerland), and additional sponsorship by Deutsche Forschungsgemeinschaft (Germany), Sanofi Aventis (France). Merck, Sharpe and Dohme (USA), AstraZeneca (Sweden), Glaxo-SmithKline (UK), and Boehringer Ingelheim (Germany). We also gratefully acknowledge Dr. Joachim Ziegenhorn for the local organization. The 32nd ELC meeting is scheduled 7–10 September 2009 in Tutzing, Germany. For information about the preliminary program and abstract forms, please contact Dr. Arnold von Eckardstein, chairman of the ELC ([email protected]). Updates and forms will be published on the website of the ELC: http://www.elctutzing.org. Franco Bernini a Dilys Freeman b Albert Groen c Jörg Heeren d Athina Kalopissis e Florian Kronenberg f Ken Lindstedt g Paolo Parini h
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Gertrud Schuster i Anne Tybjaerg Hansen j Ko Willems van Dij k Arnold von Eckardstein l,∗ a Parma, Italy b Glasgow, United Kingdom c Amsterdam, The Netherlands d Hamburg, Germany e Paris, France f Innsbruck, Austria g Helsinki, Finland h Stockholm, Sweden i Davis, USA j Copenhagen, Denmark k Leiden, The Netherlands l University Hospital Zurich, Institute of Clinical Chemistry, Raemistrasse 100, CH 8091 Zurich, Switzerland ∗ Corresponding
author. Tel.: +41 44 255 2260; fax: +41 44 255 4590. E-mail address: [email protected] (A. von Eckardstein) Available online 7 December 2008
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Meeting report
European Lipoprotein Club: Report of the 31st ELC Annual Conference, Tutzing, 8–11 September 2008 1. Introduction The 31st meeting of the European Lipoprotein Club was held from 8 to 11 September at the Evangelische Akademie in Tutzing Germany and attended by some 120 participants from all over Europe and the United States. It was the first meeting under the chairmanship of Arnold von Eckardstein (Zurich, Switzerland) who followed Marten Hofker (Groningen, The Netherlands) and it was the last co-organized by Ko Willem van Dijk (Leiden, The Netherlands), who in the past was responsible for the compilation of the annual report, and Gertrud Schuster (Davis, USA) who will be followed in 2009 by Monique Mulder (Rotterdam, The Netherlands) and Rachel Fisher (Stockholm, Sweden). Also under the new chairmanship the structure of the meeting was kept as in the past: The program was composed of six scientific sessions each of which was introduced by the lecture of an invited speaker. All other oral presentations as well as the poster presentations were selected by rating of abstracts through the ELC committee members. As in the past, two types of awards were presented to two young scientists. The young investigator award was presented to Christina Christofferson (Copenhagen, Denmark) for her presentation entitled “The signal peptide anchors apolipoprotein M in plasma lipoproteins and prevents rapid clearance of apolipoprotein M from plasma”. The poster award was presented to Philip Gordts (Leuven, Belgium) for his poster entitled: “Inactivation of the NPxYxxL Motif of LRP1 Reduces Lipoprotein Clearance and Affects Adipogenesis.” The winner of the poster prize was also given the opportunity to orally present his poster in the “Varia” session. 2. Key note lecture After the welcome by Arnold von Eckardstein the scientific part of this year’s meeting was opened by a key note lecture from Michael R. Hayden (University of British Columbia, Vancouver, Canada) on “The Ins and Outs of Cholesterol in Cells: The Pivotal Role of ABCA1”. He reported results of several collaborative studies from his laboratory on the role of the ATP binding cassette transporter A1 (ABCA1) for the regulation of cholesterol homeostasis and function of various organs. The now nearly 10 years old discovery of ABCA1 as the defective gene in patients with Tangier disease by Michael Hayden and other researchers has been a breakthrough for lipoprotein and lipid research and added an important piece of a puzzle to our understanding of cellular cholesterol homeostasis and atherosclerosis. In his lecture Michael Hayden focused on published and unpublished data that have been obtained by careful analysis of conditional knock-out mouse models in which ABCA1 was tissue-specifically 0021-9150/$ – see front matter doi:10.1016/j.atherosclerosis.2008.12.003
deleted in either liver, intestine, pancreatic  cells, or the brain. The knock-out of ABCA1 in the liver and intestine reduced HDLcholesterol levels by 80 and 30%, respectively. The crossed animals with a combined knock-out of hepatic and intestinal ABCA1 had 90% lower HDL-cholesterol levels. By contrast to previous observations that hepatic overexpression of ABCA1 increases atherosclerosis, the conditional knock-out of ABCA1 in the liver was found to aggravate atherosclerosis in apoE deficient mice. Mice with specific inactivation of ABCA1 in  cells showed severe impairment of glucose tolerance and defective insulin secretion. Analysis of the islet cholesterol levels revealed cholesterol accumulation in the islets. Interestingly, systemic knock-out of ABCA1 resulted in a much milder glucose intolerance and normal islet cholesterol levels, probably because these animals have much lower plasma cholesterol levels than the -cell specific knock-out. Michael Hayden therefore concluded that the susceptibility to cholesterol accumulation in  cells and hence impaired insulin secretion is influenced by ABCA1 function and plasma cholesterol levels. The importance of ABCA1 for regular -cell function is also indicated by the suggestion of increased risk for diabetes in humans with mutations or polymorphisms that interfere with ABCA1 function. Interestingly also the LDL receptor was found to contribute to -cell failure. It is the main entrance route for cholesterol into  cells. Feeding with a fat-rich Western diet-induced glucose tolerance in wild type and apoE deficient mice but not in LDL receptor deficient mice. In agreement with a permissive role of the LDL receptor in -cell failure are also genetic data on reduced prevalence of diabetes in heterozygous familial hypercholesterolemia. Finally Michael Hayden presented data on the effects of brain-specific ABCA1 deletion. These mice showed reduced cholesterol levels in the cortex and hippocampus as well as in plasma (−30% compared to wild type mice). Michael Hayden and coworkers found enhanced selective uptake of cholesterol from HDL through the blood brain barrier by SR-BI. The lack of ABCA1 in the brain was also associated with disturbed synapse formation and dysfunction in the sensori- and locomotoric systems. Altogether the data presented by Michael Hayden show the importance of ABCA1 for normal organ function beyond the liver and macrophages. 3. Session I Lipid and Lipoprotein Metabolism: chaired by Athina Kalopissis (Paris, France) and Jörg Heeren (Hamburg, Germany). As the invited speaker, Stephen G. Young (Los Angeles) presented the discovery of a new player in the lipolytic processing of chylomicrons, GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1). GPIHBP1 is an endothelial cell glycoprotein of the lymphocyte antigen 6 (Ly6) family, expressed on the luminal surface of capillaries in heart, adipose tissue, and skeletal muscle. These same tissues express high
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levels of LPL (lipoprotein lipase), the enzyme that is responsible for the hydrolysis of the triglycerides in chylomicrons and VLDL. LPL is synthesized by the parenchymal cells of adipose tissue and striated muscle and is then translocated onto the luminal surface of capillaries, where it has been assumed to bind to negatively charged heparan sulfate proteoglycans (HSPGs) through electrostatic interactions with the positively charged heparin-binding domains of LPL. It had been widely assumed that LPL, anchored to HSPGs, hydrolyzes chylomicron triglycerides and, within minutes, transforms chylomicrons into remnants, which are then taken up by tissues, largely via the LDL receptor. Now, it appears that GPIHBP1 might play an important role in binding LPL and chylomicrons on the surface of capillaries. GPIHBP1 was originally identified by Ioka and coworkers by expression cloning as a protein that promotes the binding of HDL to cultured cells. It therefore came as a surprise that Gpihbp1−/− mice, characterized by the laboratory of S. Young, display milky plasma and extremely high plasma triglyceride levels, even on a low-fat chow diet. This observation alone suggested that GPIHBP1 is important for the lipolytic processing of triglyceriderich lipoproteins by LPL. Subsequent studies with transfected CHO cells revealed that GPIHBP1 avidly binds LPL and chylomicrons, suggesting a new model in which GPIHBP1 might serve as a platform for the interaction of LPL and chylomicrons on the surface of endothelial cells. Clearly, efficient lipolytic processing of chylomicrons requires GPIHBP1. However, the new model for lipolysis does not necessarily exclude a role for HSPGs in binding LPL. The authors showed that GPIHBP1-bound LPL can be released by heparin. When Gpihbp1+/+ or Gpihbp1+/− mice are injected with heparin, LPL appears in the plasma very quickly, with LPL levels peaking in 1 min. In Gpihbp1−/− mice, heparin releases LPL into the plasma, but the release is slower, peaking in 15 min. This observation led to the hypothesis that there may be more than one pool of heparinreleasable LPL. Perhaps there is an endovascular GPIHBP1-bound pool of LPL that is highly relevant to lipolysis and a second pool, located in subendothelial spaces and bound to HSPGs, that is not relevant to lipolysis. The latter pool, which is present in Gpihbp1−/− mice, appears to be released into plasma more slowly after heparin. Interestingly, the LPL that is released by heparin in Gpihbp1−/− mice is enzymatically active and quickly lowers the markedly elevated plasma triglyceride levels in these mice. GPIHBP1 contains a prominent acidic domain, rich in aspartate and glutamate residues, near its amino terminus. Anne Beigneux, Stephen Young, and coworkers showed that the acidic domain plays an important role in the binding of LPL and chylomicrons. A mutant GPIHBP1 in which the aspartates and glutamates between residues 38 and 48 are replaced with alanine cannot bind LPL or chylomicrons, suggesting that electrostatic interactions are important for binding of ligands. These studies have identified a new player in lipolysis, GPIHBP1. Understanding the function of this new molecule will undoubtedly add to our understanding of plasma triglyceride metabolism and the pathogenesis of hypertriglyceridemias. To seek evidence that GPIHBP1 plays an essential role in triglyceride (TG) metabolism in humans as it is in mice and might be responsible for severe chylomicronemia, Geesje Dallinga-Thie (Amsterdam, The Netherlands) sequenced the human GPIHBP1 gene in 60 patients with plasma TG levels above the 95th percentile. One patient was identified with a homozygous missense mutation, resulting in a glutamine to a proline substitution at position 115 in the ly6 domain, which is essential for LPL binding. This mutation was not found in 100 controls. An intravenous injection of heparin failed to release LPL from the endothelial cell surface, suggesting that GPIHBP1-Q115P is not able to bind LPL. Furthermore, experiments using CHO cells transfected with normal GPIHBP1 or with GPIHBP1-Q115P in combination with a human LPL expression vector revealed further evidence that the Q115P mutant could not bind LPL or chylomicrons. Thus, a homozygous Q115P mutation in
GPIHBP1 was identified and leads to a severe hypertriglyceridemic phenotype. Leanne Hodson (Oxford, United Kingdom) studied in eight healthy men the contribution of chylomicron-triglyceride (CMTG), VLDL-TG and plasma non-esterified fatty acids (NEFA) to TG stored in adipose tissue. Three nutritionally similar meals were given at 5 h intervals. The efficiency of adipose tissue FA uptake increased with each meal, CM-TG was the main FA source and the fractional extraction increased from 21% after the 1st meal to 35% after the 2nd meal to 47% after the 3rd meal. Thus, the spillover of CM-TG decreased after each meal, mainly through increased FA re-esterification. Uptake of VLDL-derived FA was more important after the last meal, with spillover seen only in the fasted state. The decrease in spillover fraction of CM-derived FA with sequential meals over 24 h suggests induction of fat storage by meal repetition. HDL and their main protein, apolipoprotein (apo) AI exert antiatherogenic effects within the arterial wall. Lucia Rohrer (Zurich, Switzerland) studied transport of 125 I-HDL by endothelial cells cultured on inserts. The cells bound, internalised and translocated HDL from the apical to the basolateral compartment in a specific manner without degrading the protein moiety of the transported particles. Using RNA interference, reduced 125 I-HDL transport was obtained after silencing SR-BI and ABCG1, respectively but not all after knockdown of ABCA1. In addition the role of endothelial lipase (EL) in HDL transport through endothelial cells was shown by: (i) decreased binding and cell association of HDL after heparin and heparinase treatment, which releases EL from membranes; (ii) inhibition of EL activity reduced HDL cell association; (iii) EL knockdown by siRNA diminished HDL transport. In the second part of this session Ernst Steyrer (Graz, Austria) described the role of Phosphatidylethanolamine–Methyltransferase (PEMT) for lipid droplet biogenesis. The core of lipid droplets (LD) is covered by a phospholipid monolayer. Since the phosphatidylcholine:phosphatidylethanolamine ratio changes during adipocyte differentiation, the aim of this study was to investigate a potential role of PEMT for lipid droplet biogenesis. In 3T3-L1 cells, adipocyte differentiation was associated with an increase in the expression and activity of PEMT. Fluorescence microscopy revealed that a PEMT-EGFP fusion protein localized to LD. As demonstrated by fat cell morphometry, mice deficient in PEMT are resistant to high fat diet-induced hypertrophy of fat cells suggesting that phospholipid remodeling is crucial for lipid droplet biogenesis in adipocytes. Furthermore, PEMT deficiency resulted in lipid accumulation within enterocytes (chylomicron retention) under high fat conditions indicating that PEMT is also involved in chylomicron synthesis. Thomas Grewal (Sydney, Australia) gave new insights into the molecular mechanisms regulating intracellular transport of caveolin. It has recently been shown that caveolin requires cholesterol to exit the Trans Golgi Network (TGN) in order for it to be transferred to the plasma membrane. In elegant cell culture studies he could show that cholesterol from late endosomal compartments is the driving force and delivered to the TGN to promote caveolin export from the TGN to the cell surface. The cholesterol-dependent export of caveolin requires the activity of cytoplasmic phospholipase A2 (cPLA2), which is targeted to the TGN to promote Golgi vesiculation in a cholesterol-dependent manner. The phospholipid-binding protein Annexin A6 inhibits cholesterol export from late endosomes, thereby reducing the cholesterol-dependent association of cPLA2 with the TGN. This correlates with the reduced release of arachidonic acid in Annexin A6 overexpressing cells, and it was concluded that high expression levels of Annexin A6 downregulate the activity of cholesterol-dependent cPLA2 to block caveolin transport to the cell surface. Herbert Stangl (Vienna, Austria) investigated the role of HDL uptake and subsequent re-secretion in cholesterol efflux. Earlier
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studies in macrophages identified apoAI in structures that appeared to be coated pits or coated vesicles. In these studies inhibition of receptor-mediated endocytosis interfered with cholesterol efflux to apoAI, suggesting that efflux involves the internalisation of apoAI via clathrin-coated pits. As demonstrated by living cell microscopy, electron microscopy and efflux experiments using radiolabeled HDL Dr. Stangl showed that (1) HDL holo-particle uptake is mediated by scavenger receptor class B1 (SR-BI) and (2) HDL retroendocytosis can mediate cholesterol efflux. Carlos Vrins (Amsterdam, Netherlands) investigated the role of HDL in trans-intestinal cholesterol efflux (TICE). This pathway has been recognized as a major route of sterol output and describes the direct transport from blood via enterocytes to feces. With the prominent role of HDL in the hepatobilary route of reverse cholesterol transport, the aim of this study was to determine whether this lipoprotein also is involved as a cholesterol donor for TICE. Intestine perfusions were first performed in SR-BI deficient mice, which have elevated serum HDL-cholesterol levels by ligating the bile ducts to prevent the entry of bile into the intestine and subsequently perfusing the intestine with model bile to quantify TICE. The rate of TICE in these mice is 2-fold higher than that found in wildtype (WT) animals. Next, TICE was analysed in mice lacking both ABCA1 and SR-BI. These double knock-out mice (DKO) mice have no circulating HDL cholesterol, but, interestingly, their rate of TICE is similar to WT. When HDL with radiolabeled cholesterol was injected into the DKO, no radioactivity could be found in the intestinal lumen during intestine perfusions. However, a similar result was obtained in WT. These data indicate that HDL is not directly involved in TICE. Egon Demetz (Innsbruck, Austria) analysed the effect of hepatic SR-BI knockdown mediated by RNA interference (RNAi) on the development of atherosclerosis in NZW rabbits. The work was presented by the co-author of this study, Ivan Tancevski. Compared to mice, rabbits express cholesteryl ester transfer protein (CETP), display a more human-like lipoprotein profile and are more prone to develop atherosclerosis. To downregulate SR-BI specifically in the liver, a plasmid coding for small hairpin RNA against rabbit SRBI was associated with a poly-lysine-based shuttle vector enriched with galactose. Specific downregulation was demonstrated by realtime PCR. To study the development of atherosclerosis, NZW rabbits were fed a Western type diet and treated either with a control vector or a vector mediating the downregulation of hepatic SR-BI. By analyzing atherosclerotic lesion area it was shown that downregulation of hepatic SR-BI resulted in the reduction of heavy lesions in the aorta of rabbits, suggesting that hepatic SR-BI expression is associated with the progression of atherosclerosis in this CETP-expressing species. 4. Session II Gene Regulation: chaired by Paolo Parini (Stockholm, Sweden) and Gertrud Schuster (Davis, USA). Beatrice Desvergne (Lausanne, Switzerland) gave an overview of their recent findings on the “PPAR-mediated activities of phthalates and metabolic disorders”. PPARs are activated by a wide variety of compounds, whereby possible functions of some endogeous ligands as endocrine disruptors are still not clarified. Most recently mono-ethylhexyl-phthalate (MEHP) was identified as endocrine disrupting chemical. Phthalates are widely used in the plastic industry as PVC softener and are found in daily products such as food packaging or cosmetics. MEHP is the active metabolite of di-2ethylhexyl phthalate (DEHP), the most abundant phthalate in the environment. MEHP and DEHP induce peroxisome proliferative products. MEHP and Rosiglitazone bind in a very similar fashion to the pocket of PPAR-␥’s ligand binding domain; whereby MEHP induces adipogenesis in vitro to a lower extent than Rosiglitazone. It appears that MEHP and Rosiglitazone induces the formation of
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different PPAR-␥ associated transcription complexes by recruiting distinct co-factors. Whereas Med-1 can be recruited by both, Src-1 and p300 are recruited only by Rosiglitazone, and PGC1a more efficiently by MEHP. This results in the regulation of a different set of target genes. Because MEHP can also activate PPAR␣ and PPAR, in vivo studies were required to evaluate its metabolic impact. First observations show that DEHP protects from diet-induced obesity in wild type mice, in a mechanism involving PPAR␣ in the liver. The different behavior of rodent and human PPAR␣ then raises the question of species-specific (mouse versus human) activity of DEHP, further explored by the group of B. Desvergne. These results leave us with the question of whether there is a potential role of phthalates in human obesity. The study by Jesper Schou (Copenhagen, Denmark) evaluated genetic alterations in the ABCG1 promoter and the risk of ischemic heart disease (IHD). In studies of ABCG1−/− versus ABCG1+/+ mice, total body expression of ABCG1 protected against atherosclerosis, without affecting HDL cholesterol or other lipid or lipoprotein levels. They found that individuals with the ABCG1-376C>T variant, located in a putative SP-1 binding site, had an increased risk of developing IHD, independent of HDL cholesterol and other lipid and lipoprotein levels. Tomas Jakobsson (Stockholm, Sweden) discussed his findings that the co-repressor complex subunit GPS2 is selectively required for the LXR-induced transcription of ABCG1, and revealed fundamental differences between ABCG1 and ABCA1 genes regarding LXR coregulator requirement. Also, LXR-induced cholesterol efflux in THP-1 cells is eliminated by depletion of GSP2. Sophie Lestavel, from Lille, France, presented a study in which PPARalpha has been identified as an intestinal specific target gene of Liver X Receptors. LXR ligands were administrated to wild type and LXRalpha/beta double knock-out mice, both on a C57Bl6/J background, and gene expression analysis was used to identify intestine-specific LXR target genes. Chronic administration of LXR synthetic agonists led to a significant increase of PPARalpha mRNA levels in the small intestine but not in the liver. This was paralleled by up-regulation of PPARalpha protein. Administration of a synthetic LXR agonist to wild type mice resulted in the induction of several PPARalpha target genes and this effect was completely blunted in PPARalpha-deficient mice, demonstrating the biological relevance of this LXR-PPARalpha transcriptional cascade. The session was concluded by a presentation of Arie Van Tol, Rotterdan, The Netherlands, entitled Acute Elevation of Plasma phosholipid transfer protein (PLTP) activity Strongly Increases Pre-existing Atherosclerosis. A transgenic mouse model in which the conditional expression of human PLTP by the tetracyclineresponsive gene system was developed in order to study the effects of an acute increase in plasma PLTP activity on the metabolism of apolipoprotein B-containing lipoproteins and on diet-induced pre-existing atherosclerosis. Induced expression of PLTP strongly increased plasma VLDL triglycerides in LDL receptor knock-out mice, while VLDL secretion was not affected. The elevation was secondary to PLTP-dependent inhibition of VLDL catabolism, which is caused, at least partly, by a decreased lipoprotein lipase activity. Acute overexpression of PLTP also reduced HDL and resulted in a more atherogenic lipoprotein profile leading to an increase in size of pre-existing atherosclerotic lesions. In addition, the lesions contain more macrophages and less collagen relative to controls, suggesting a less stable lesion phenotype. 5. Session III Functional Genomics and Genetics: chaired by Anne Tybjaerg Hansen (Copenhagen, Denmark) and Florian Kronenberg (Innsbruck, Austria).
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A. Jake Lusis (Los Angeles, USA) provided an overview of his perspective concerning the metabolic syndrome and how epidemiology and systems biology can interact to unravel the pathogenesis of this epidemic disease. Although the single components of the metabolic syndrome are subject to high heritability, this high heritability is not due to a few genes with large effects. Besides environmental influences and sex differences it is rather due to combinations of many different genes, each of which contributes a tiny fraction of risk. Genome-wide association studies during recent months have without doubt contributed some novel genes to metabolic syndrome traits (e.g. MC4R, FTO, MLXIPL, and TCF7L2), but these common polymorphisms explain only a small fraction of 5–10% of the genetic effect size. He showed examples that classical inbred and recombinant inbred populations of mice can be used for high resolution mapping including systems biology approaches by collecting phenotypic, transcriptomic, proteomic and metabolomic data at once and by applying whole genome association approaches and mathematical modeling of biological networks. This can dramatically increase the mapping resolution compared to classical linkage analysis. Instead of using a genome-centric view of complex diseases by modeling genetic variation with clinical endpoints, an integrative approach models the influence of genetic variability on gene expression and clinical endpoints and thereby allows a network analysis. The hierarchical clustering of differentially expressed and coexpressed genes allows the identification of highly connected elements of hub genes with major contributions to the pathogenesis of the metabolic syndrome. Jake Lusis’ lab has tested some of the predicted genes using a series of transgenic experiments. The results have been highly encouraging, as a high fraction of the genes predicted to be involved in these traits have been confirmed. In these studies, gene expression serves as a kind of intermediary phenotype between genotype and the complex phenotypes. This global analysis of networks is a challenge, which can hopefully be better handled by a systems biology approach. Christiane Lundegaard (Copenhagen, Denmark) and coworkers investigated the ability of mutations in APOA1 to predict risk of ischemic heart disease (IHD) and death in the general population. Among three nonsynonymous mutations identified by resequencing APOA1 in 200 individuals, a common mutation (1 in 500 in the general population), A164S, predicted a 3-fold increase in risk of IHD, and a reduction in mean survival time of almost 10 years in the Copenhagen City Heart Study (n = 10,500, 31 years follow-up). This is the first report of a common mutation in APOA1, which predicts an increased risk of IHD and MI, and decreased longevity in the general population independent of lipid and lipoprotein levels. Stefano Romeo (Dallas, TX, USA) and coworkers sequenced the ANGPTL3, 5, 6 genes in participants in the Dallas Heart Study (DHS) (n = 3351), and identified several nonsynonymous sequence variants in ANGPTL3 and ANGPTL5 which associated with low plasma levels of triglycerides. When expressed in HEK-293 cells the majority of these mutations were associated with a failure of the proteins to be secreted from cells. Those who where secreted failed to inhibit lipoprotein lipase in vitro. These data suggest a non-redundant role of Angiopoietin-like proteins in triglycerides metabolism. Katharina Leidl (Regensburg, Germany) and coworkers compared lipid species in monocytes, lymphocytes, granulocytes, platelets and red blood cells of healthy volunteers using electrospray ionization tandem mass spectrometry. In an extensive study, the authors clearly showed that the lipid pattern of the different circulating blood cells matched with various aspects of cell specific functions related to shape, size and cellular life span in the vascular system.
6. Session IV Lipoprotein Function: chaired by Franco Bernini (Parma, Italy) and Dilys Freeman (Glasgow, UK). This session was opened with an invited lecture given by Tomas Vaisar (Seattle, US). Epidemiological and clinical studies demonstrated that low levels of high-density lipoprotein (HDL) cholesterol are an independent risk factor for premature cardiovascular disease (CAD). A primary mechanism by which HDL protects against atherosclerosis is by promoting reverse cholesterol transport (RCT). However HDL has other biological activities that contribute to its antiatherogenic properties, including antioxidant and antiinflammatory effects. Dr. Vaisar underlined that the proteome component of HDL plays critical roles in mediating these biological activities suggesting that HDL-cholesterol levels may not be the only determinant of the cardioprotective role of HDL. Using mass spectrometric analysis he showed that HDL3 of subjects with CAD is enriched in several proteins, including apolipoproteins E (apoE), complement C3 and apoC-IV. Based on this data he suggested that altered protein composition might affect the antiatherogenic and anti-inflammatory properties of HDL. They also investigated whether combination therapy with statin and niacin, which raises HDL levels, might affect these changes observed in HDL3 of subjects with CAD. They found that combination therapy significantly decreased the amount of apoE in HDL3 while the abundance of apoF, apoJ and PLTP levels increased. No significant changes were found in apoAI levels of HDL after niacin/statin treatment. Taken together these results demonstrated that statin/niacin combination therapy remodels the HDL3 proteome, resulting in a protein composition that resembles that of healthy subjects. Dr. Vaisar concluded that combined statin/niacin therapy may partially reverse the changes in the protein composition seen in HDL3 in CAD subjects. His results suggested the possibility that quantifying the HDL proteome by MS could provide insights into the therapeutic efficacy of antiatherosclerotic interventions. Moreover he provided evidence that HDL protein composition differences result in a specific “fingerprint” in MALDI-TOF mass spectrum of HDL, which is reproducible and sensitive to proteome changes. Using this approach they demonstrated that HDL2 from subjects with CAD is enriched in Lp(a) and apoC-III while level of apoC-I decrease. Collectively Dr. Vaisar’s data suggest that the proteomic fingerprint of HDL could potentially be used for diagnostic purposes. Johannes Levels (Amsterdam, Netherlands) presented a low dose challenge of endotoxin (1 ng/kg body weight) to healthy men (n = 20) in order to investigate changes in the array of proteins carried by high-density lipoprotein particles over 24 h. The technique of SELDI-TOF mass spectrometry carried out on HDL, captured using antibodies to apolipoprotein AI, was shown to be a feasible high throughput method to analyse a time series of change in HDL protein profile. The endotoxin challenge induced profound changes in HDL composition. Hierarchical clustering analysis of protein changes indicated that individuals fell in to one of three distinct clusters that were independent of their plasma HDL-cholesterol concentration. Martin Merkel (Hamburg, Germany) presented data on apoA5 metabolism in mouse models comprising apoA5 transgenic (apoA5tr) mice bred on an apoE deficient (E0) or wild type (WT) background. The presence of the apoA5 transgene reduced plasma VLDL triglyceride levels more on the E0 than on a WT background. Turnover studies showed an apoA5 mediated faster lipolysis of VLDL TG from 14 C-TG-VLDL, whereas the plasma removal of 125 I-TC protein labelled VLDL was not affected. In the aortic root, apoA5 expression reduced atherosclerotic lesion size by more than 50% in both genders. It was concluded, that apoA5 may be atheroprotective by accelerating plasma TG hydrolysis.
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Andrea Dichlberger (Vienna, Austria) identified and studied apoAV in the chicken, an excellent model for lipid transport due to the high level of lipid transport to the yolk. ApoAV is found in the circulation of laying hens in both lipid-associated and lipidfree forms. They observed that human apoAV interacted with the lipoprotein receptor LR8 and the human LDLR, and that structural mutations in apoAV can influence receptor binding. Chicken apoAV is taken up into the chicken oocyte and in LR8-293 cells chicken apoAV is endocytosed by LR8. It was proposed that reduced receptor-mediated clearance of apoAV-containing particles may lead to elevated plasma triglyceride levels. 7. Session V Inflammation and Vascular Biology: chaired by Ko Willems van Dijk (Leiden, The Netherlands) and Ken Lindstedt (Helsinki, Finland). The session on “Inflammation and Vascular Biology” started with a talk by the invited speaker, Ziad Mallat (Paris, France), on “Regulatory T-cell immunity in the control of Atherosclerosis”. Prof. Mallat introduced evidence that Th1-driven responses are detrimental to the atherosclerotic process, whereas the role of the Th2 pathway in the development of atherosclerosis remains controversial. In contrast, natural Treg cells (nTregs), characterized by the expression of CD4 and CD25, develop in the thymus and home in peripheral tissues to maintain self-tolerance. nTregs prevent autoimmunity by inhibiting pathogenic T cells. Furthermore, a subset of induced Tregs (iTregs) is also generated in the periphery during an active immune response where they maintain self-tolerance by secreting IL-10. Tregs suppress atherosclerosis by cell contact-dependent suppression, by consumption and limitation of growth factors (IL-2), and by producing inhibitory cytokines (IL-10 and TGF-). Secondly, Prof. Mallat stated that reduced phagocytic removal of apoptotic vascular SMCs and macrophages may play a major role in the perpetuation of the inflammatory response in atherosclerosis. The mechanisms involve a reduction in the immuno-suppressive response that follows the ingestion of apoptotic bodies, i.e. increased expression of IL-10 and TGF, or a promotion of inflammatory responses attributable to the high concentration of proinflammatory oxidized phospholipids on apoptotic cell membranes. Milk fat globule EGF-like Factor 8 (Mfge8), a glycoprotein involved in phagocytic clearance of apoptotic cells, is required for efficient removal of apoptotic cells during both early and advanced atherosclerosis and promotes an atheroprotective regulatory T-cell immune response. Moreover, deficiency in Mer receptor tyrosine kinase (mertk), a receptor expressed in bone marrow-derived macrophages and involved in apoptotic cell clearance, leads to an increased accumulation of apoptotic cells, promotes a proinflammatory immune response, and accelerates lesion development. Finally, Prof. Mallat concluded that the critical role of naturally occurring Tregs in atherosclerosis has initiated studies aiming at the promotion of Treg response in vivo. Thus, supplementation with Tregs might lead to the induction of immune suppression and a reduction in pathogenic Tcell-mediated responses, ultimately altering plaque development and/or composition. Jeppe Zacho (Copenhagen, Denmark) showed that CRP levels >3 mg/l versus <1 mg/l were associated with a 1.6-fold and 1.3fold increased risk of ischemic heart disease (IHD) and ischemic cerebrovascular disease (ICVD). However, although genotype combinations of 4 different CRP polymorphisms were associated with up to a 64% increase in CRP levels, they were not associated with increased risk of IHD or ICVD. Dr. Zacho concluded that it would be likely that a common process, like inflammation, causes both IHD, ICVD, and CRP elevations. Florence Bietrix (Amsterdam, Netherlands) showed that inhibition of glycosphingolipid (GSL) synthesis with AMP-DNM (25 mg/kg
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daily) in apoE3 Leiden mice on a high cholesterol diet for 18 weeks induced a 2-fold increase in their atherosclerotic lesions. However, no effect was seen on cholesterolemia/triglyceridemia. By increasing the AMP-DNM concentration to 50 and 100 mg/kg daily, however, Bietrix et al observed a dose dependent decrease in plasma cholesterol and trigycerides already after 6 weeks of treatment including a strong reduction in the atherosclerotic lesion size. In addition, the percentage of plasma HDL was increased with a decrease of the LDL/VLDL fraction. Triglyceride and cholesterol levels in the liver were reduced and the bile secretion was increased. The authors concluded that GSL synthesis inhibition have unexpected effects on lipoprotein metabolism in ApoE3 Leiden mice. Rachel Fisher (Stockholm, Sweden) showed that obese women with high liver fat (LFAT) content had more inflammation and increased levels of ceramides in their subcutaneous adipose tissue compared to equally obese women with normal LFAT. Gene expression analysis indicated that the increase in ceramide was independent of de novo ceramide synthesis. However, the authors found that the high LFAT subjects had higher gene expression levels of sphingomyelinases in their adipose tissue and that mRNA levels of one particular sphingomyelinase, SMPD3, correlated with concentrations of sphingomyelins and ceramides in adipose tissue. Dr. Fisher concluded that increased ceramide levels in inflamed adipose tissue may depend on increased SMPD3 activity. Nikolaos Kadoglou (Athens, Greece) presented the effects of exercise training on atherogenesis in apoE-deficient mice. Mice were put on a high-fat diet for 16 weeks and subsequently performed 1 h of treadmill running for 1 h per day, 5 days a week for 6 weeks. As compared to controls, the exercising mice did not differ in body weight or plasma lipid levels. However, plaque area was decreased significantly, and the plaques presented a more “stable” phenotype. These data support a role for exercise in the management of atherosclerosis burden. The paper by Patrick Rensen (Leiden, Netherlands) investigated the effects of the CETP-inhibitor torcetrapib in APOE*3-Leiden.CETP transgenic mice. In this mouse model, torcetrapib decreased plasma total cholesterol levels and increased HDL-cholesterol levels. Torcetrapib alone decreased atherosclerotic lesion size but did not add to the antiatherogenic effect of atorvastatin. Moreover, torcetrapib induced a more “unstable” plaque phenotype, characterized by more macrophages and less collagen, which may explain some of the negative effects seen in the human torcetrapib trial. Janna van Diepen (Leiden, Netherlands) presented her studies on the association between inflammation and TG metabolism. Mice expressing a constitutive active isoform of IKK-beta in hepatocytes only (LIKK) were cross bred with APOE*3-Leiden mice and VLDL metabolism was analysed. The LIKK mice exhibit a constitutively activated NF-kB response, which on the APOE*3-Leiden background, resulted in increased cholesterol and TG levels. After careful in vivo analyses, the increased plasma TG could be linked to an increased VLDL-TG production rate with no difference in clearance, indicating that hepatic inflammation may play a causal role in dyslipidemia. Pirkka-Pekka Laurila (Helsinki, Finland) presented a description of the phenotype of upstream stimulatory factor 1-deficient (Usf1−/−) mice. Usf1 is a ubiquitous transcription factor that has been linked to familial combined hyperlipidemia in human genetic association studies. On a Western type diet, Usf1−/− mice displayed a decreased plasma TG, which could be explained by up-regulated transcription of LPL and apoA5. On a chow diet, plasma cholesterol was increased, simultaneously with an increase in plasma apoA1 levels. This effect of Usf1-deficiency on ApoA1 transcription was replicated in human cells treated with Usf1 siRNA constructs. Genome-wide transcription analysis of Usf1−/− adipose tissue revealed several clues for a potential antiatherogenic effect of USF1 downregulation.
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Lucia Fuentes (Lille, France) provided evidence for a role of the cell cycle inhibitor protein p16-ink4a in determining macrophage phenotype. Recently, the locus encoding p16-ink4a has been genetically linked to CHD and type 2 diabetes. It was first demonstrated that p16-ink4a was expressed exclusively in classical macrophages and not in so-called IL-4 activated M2 macrophages. Next, adenovirus mediated overexpression of p16 resulted in an M1 phenotype, whereas siRNA mediated downregulation of p16 resulted in an M2 phenotype. Moreover, bone marrow-derived P16−/− macrophages presented a functional M2 phenotype. These data clearly implicate p16 in the determination of macrophage phenotype. Variation in p16 could thus affect the balance between proinflammatory (M1) and anti-inflammatory (M2) macrophages. 8. Session topic VI Varia: chaired by Albert Groen (Amsterdam, The Netherlands) and Arnold von Eckardstein (Zurich, Switzerland). Claudia Coomans reported on the stimulatory effect of central insulin on fat storage in white adipose tissue. It was shown that this effect of central insulin was dependent on the activation of KATP -channels in the brain. By blocking the activity of these KATP channels in the brain selectively, they were able to show that the stimulatory effect of peripheral insulin on fat storage in adipose tissue is completely abolished when insulin signalling in the brain is blocked. Feeding mice a high fat diet induces both peripheral and central insulin insensitivity. Indeed feeding mice a high fat diet completely abrogated the effect of insulin on FA uptake by WAT underlining the surprising importance of the brain in peripheral effects of insulin. Maria Pia Adorni and co workers investigated the mechanism by which probucol affects xanthoma regression. Probucol is an inhibitor of ABCA1 and this leads to a decrease in the cholesterol content of plasma membranes. The level of cholesterol in the plasma membrane of macrophages induces membrane ruffling and migration capacity of the cells. Adorni et al demonstrated that probucol treatment of acetyl-LDL loaded macrophages restores migration of the cells and reduces membrane ruffling suggesting a possible role of ABCA1 in promoting cholesterol membrane ruffling and in inhibiting migration in the absence of an extracellular acceptor. Knocking-down expression of ABCA1 in J774 macrophages had similar effects. They conclude that the effects of probucol on ABCA1 function may underlie its efficacy in reducing cutaneous and tendinous xanthoma in patients with different forms of hypercholesterolemia. Tommaso Fasano reported on the role of PCSK9 as a modulator of LDL-receptor function and on its relationship with LDL-R internalisation. Gain of function mutant D374Y-PCSK9 was incubated with fibroblasts or lymphocytes derived from ARH patients. Lymphocytes from these patients are not able to internalise the LDLreceptor. Despite the inability of these cells to internalise PCSK9, the amount of LDL-R on the surface decreased, suggesting that PCSK9 can be internalised via an alternative route and may affect LDL-R processing in the cells. Apolipoprotein M is an interesting novel apolipoprotein which seems to be involved in formation of pre--HDL. The protein has a molecular weight of around ∼25 kD. In plasma, apoM is mainly associated with HDL and to a lesser degree with LDL, VLDL and chylomicrons. Interestingly, plasma apoM has retained its N-terminal signal peptide. Christina Christoffersen and coworkers investigated whether the N-terminal signal peptide of apoM is involved in its association with lipoproteins. This indeed turned out to be the case. Transgenic mice producing mutant human apoM without signal peptide showed no detectable apoM levels in plasma. The protein was rapidly cleared by the kidneys
and indeed, ligation of kidney arteries induced a strong accumulation of the mutant protein in plasma. It is concluded that the hydrophobic signal peptide anchors the protein in lipoproteins. The prize for the best poster was given to Philip Gordts from Leuven (Belgium). He analysed lipoprotein metabolism, atherosclerosis and adipose tissue of LDL receptor knock-out mice (LDLR−/−) which in addition carry an inactivating mutation in the NPxYxxL motif of the LDL receptor related protein 1 (LRP1). Compared to pure LDLR−/− mice, mice with additional LRP1 inactivation showed more severe and mixed hyperlipidemia and more atherosclerosis in the aorta which is characterized by a higher SMC content and MMP2 activity. In addition the LRP1 knock-in mice showed a decreased expression of adipocyte markers such as GLUT4, LPL and FABP4. In conclusion LRP1 appears to modulate lipoprotein clearance, atherosclerosis and adipogenesis. Paolo Parini and his colleagues from Stockholm (Sweden) analysed the metabolic impact of gene regulation of ACAT2 by the two nuclear hormone receptors HNF1␣ and HNF4␣ which are mutated in patients with Maturity Onset Diabetes of the Youth MODY3 and MODY1, respectively. While patients with MODY3 had normal lipoprotein composition, those with MODY1 had lower levels of VLDL and LDL esterified cholesterol. Mutagenesis revealed one important HNF4␣ binding site in the human ACAT2 promoter. ChIP assays and protein-protein interaction studies showed that HNF4␣ directly or indirectly (via HNF1␣) can bind to the ACAT2 promoter. The authors conclude that the lower levels of esterified cholesterol in VLDL and LDL particles in patients with MODY1 may reflect the lower ACAT2 activity in these patients. This may translate into lower risk of atherosclerosis. Ronit Shiri-Sverdlov (Maastricht, The Netherlands) investigated the influence of scavenger receptors CD36 and SR-A in diet-induced steatohepatitis. In earlier work she has shown that the cholesterol content of the diet plays an important role in the induction of inflammation in the fatty-liver. Formation of foamy Kupffer cells seems to lie at heart of this finding. By knocking out expression of the scavenger receptors CD36 and SR-A via bone marrow transplantation procedures, Shiri-Sverdlov and coworkers provide additional evidence for this hypothesis. Mice lacking CD36 and SR-A in the haematopoetic compartment showed significantly reduced hepatic inflammation when fed a high cholesterol/high fat diet. These findings were confirmed by measuring levels of proinflammatory cytokines in plasma of these mice. The authors conclude that uptake of modified lipoproteins plays an important role in diet-induced hepatosteatosis. In cooperation with Oliver Weingärtner and cardiologists from the University Hospital of Saarland (Homburg, Germany) Dieter Lütjohann (Bonn, Germany) investigated the effects of a diet rich in plant sterol esters (PSE) on endothelial function, cerebral lesion size, and atherosclerosis in mice as well as associations of plant sterol plasma concentrations with aortic stenosis in humans. PSE supplementation impaired endothelium-dependent vasorelaxation and increased cerebral lesion size after middle cerebral artery occlusion. After randomization of apoE−/− mice to Western-type diet with the addition of PSE or ezetimibe, both animal groups showed equal plasma cholesterol levels but the PSE group showed less reduction in atherosclerosis plaque size than animals randomized to ezetimibe. Plant sterol plasma concentration strongly correlated with increased atherosclerotic lesion formation (r = 0.50; P < 0.01). Furthermore, patients eating PSE-supplemented margarine (n = 10) were found to have increased plasma concentrations and 5-fold higher sterol concentrations in aortic valve tissue. The authors conclude that food supplementation with PSE impairs endothelial function, aggravates ischemic brain injury, effects atherogenesis in mice, and leads to increased tissue sterol concentrations in humans.
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Acknowledgements The 30th ELC meeting was generously supported by its longstanding main sponsor Roche (Switzerland), and additional sponsorship by Deutsche Forschungsgemeinschaft (Germany), Sanofi Aventis (France). Merck, Sharpe and Dohme (USA), AstraZeneca (Sweden), Glaxo-SmithKline (UK), and Boehringer Ingelheim (Germany). We also gratefully acknowledge Dr. Joachim Ziegenhorn for the local organization. The 32nd ELC meeting is scheduled 7–10 September 2009 in Tutzing, Germany. For information about the preliminary program and abstract forms, please contact Dr. Arnold von Eckardstein, chairman of the ELC ([email protected]). Updates and forms will be published on the website of the ELC: http://www.elctutzing.org. Franco Bernini a Dilys Freeman b Albert Groen c Jörg Heeren d Athina Kalopissis e Florian Kronenberg f Ken Lindstedt g Paolo Parini h
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Gertrud Schuster i Anne Tybjaerg Hansen j Ko Willems van Dij k Arnold von Eckardstein l,∗ a Parma, Italy b Glasgow, United Kingdom c Amsterdam, The Netherlands d Hamburg, Germany e Paris, France f Innsbruck, Austria g Helsinki, Finland h Stockholm, Sweden i Davis, USA j Copenhagen, Denmark k Leiden, The Netherlands l University Hospital Zurich, Institute of Clinical Chemistry, Raemistrasse 100, CH 8091 Zurich, Switzerland ∗ Corresponding
author. Tel.: +41 44 255 2260; fax: +41 44 255 4590. E-mail address: [email protected] (A. von Eckardstein) Available online 7 December 2008