Foam Cell Specific LXRα Ligand

Objective The liver X receptor α (LXRα) is a ligand-dependent nuclear receptor and the major regulator of reverse cholesterol transport in macrophages. This makes it an interesting target for mechanistic study and treatment of atherosclerosis. Methods and Results We optimized a promising stilbenoid structure (STX4) in order to reach nanomolar effective concentrations in LXRα reporter-gene assays. STX4 displayed the unique property to activate LXRα effectively but not its subtype LXRβ. The potential of STX4 to increase transcriptional activity as an LXRα ligand was tested with gene expression analyses in THP1-derived human macrophages and oxLDL-loaded human foam cells. Only in foam cells but not in macrophage cells STX4 treatment showed athero- protective effects with similar potency as the synthetic LXR ligand T0901317 (T09). Surprisingly, combinatorial treatment with STX4 and T09 resulted in an additive effect on reporter-gene activation and target gene expression. In physiological tests the cellular content of total and esterified cholesterol was significantly reduced by STX4 without the undesirable increase in triglyceride levels as observed for T09. Conclusions STX4 is a new LXRα-ligand to study transcriptional regulation of anti-atherogenic processes in cell or ex vivo models, and provides a promising lead structure for pharmaceutical development

Genom-weite Analyse von LXRα regulierten transkriptionalen Netzwerken in der Entwicklung humaner atherosklerotischer Schaumzellen

1\. Introduction 9 1.1 Cardiovascular disease - major cause of death 9 1.2 Major risk factors for cardiovascular disease 9 1.2.1 Aging 10 1.2.2 Blood lipids 10 1.2.2.1 Apolipoproteins 11 1.2.2.2 Lipid metabolism 11 1.2.3 Overweight and obesity 13 1.2.4 Genetics 14 1.3 Pathology of heart attacks and strokes -Atherosclerosis 14 1.3.1 Cause of atherosclerosis 16 1.3.1.1 Endothelial injury hypothesis 16 1.3.1.2 Lipid hypothesis 17 1.3.2 Role of macrophages 19 1.3.2.1 Inflammatory response 19 1.3.2.2 Foam cell formation 20 1.3.2.3 Apoptosis and necrosis of macrophages 22 1.4 Ligand dependent nuclear receptor 23 1.4.1 Nuclear receptor structure 24 1.4.2 Interaction of response element and DNA binding domain 25 1.4.3 Gene regulation mechanisms 25 1.4.4 Liver X receptor (LXR) 27 1.4.4.1 LXR physiology 28 1.4.4.2 LXR dependent reverse cholesterol transport (RCT) 30 1.4.4.3 LXR ligands 32 1.4.4.4 LXR drugs 33 1.5 Genome-wide transcription factor binding studies 33 1.5.1 Chromatin immunoprecipitation (ChIP) 34 1.5.2 ChIP-sequencing 35 1.5.2.1 Limitations of ChIP-sequencing 36 1.5.2.2 Data analyses 36 1.6 Gene regulatory networks 36 1.7 Aims of the thesis 37 2\. Materials and Methods 39 2.1 LXRα ligands 39 2.2 Cell culture experiments 39 2.2.1 THP1 cells 39 2.2.2 Primary human macrophages 39 2.2.3 HEK cells 40 2.2.4 Foam cell formation 40 2.2.5 Cholesterol and triglyceride analyses 41 2.2.6 LXR knockdown macrophages 41 2.3 Immunoblotting 41 2.4 Chromatin immunoprecipitation (ChIP) 42 2.4.1 ChIP- sequencing 45 2.4.2 Peak calling and filtering 46 2.4.3 Comparative ChIP- sequencing analysis 47 2.5 Formaldehyde assisted isolation of regulatory elements (FAIRE) 47 2.5.1 FAIRE-sequencing 48 2.6 Transcription factor binding site enrichment 48 2.7 LXRα motif analyses 49 2.8 Reporter-gene assays 49 2.9 Functional annotation of LXRα binding sites 51 2.9.1 Genomic distribution 51 2.9.2 Annotation of genes controlled by nearby peaks 51 2.10 Gene expression study 52 2.10.1 RNA purification, cDNA synthesis and qPCR 52 2.10.2 Genome- wide gene expression analysis 54 2.11 Correlation of LXRα binding and gene expression 55 2.12 Functional description and network analyses 55 2.12.1 Gene ontology analysis 56 2.12.2 Pathway analysis 56 2.12.3 Association of LXRα binding sites with GWAS 56 2.12.4 Functional network analysis 56 2.12.5 Database search for known LXRα target genes 57 2.13 Figures, equipment and reagents 57 2.13.1 Figures 57 2.13.2 Reagents 57 2.13.3 Cells and media 61 2.13.4 Equipments and consumables 61 2.13.5 Software/Internet tools 63 3\. Results 65 3.1 Atherosclerosis model and ligand treatment 65 3.1.1 Foam cells accumulate lipids 65 3.1.2 Ligand-dependent autoregulatory upregulation of LXRα 66 3.2 Genome-wide binding study 67 3.2.1 LXRα ChIP-seq 67 3.2.2 LXRα binding is ligand dependent 68 3.2.3 Shared and differential binding of LXRα to genomic loci in macrophages and foam cells 71 3.2.4 T0901317 sharpens LXRα peak enrichment at promoter sites 74 3.3 Functional characterization of LXRα binding 77 3.3.1 Gene expression profiles are mostly similar among cell models 77 3.3.2 High correlation of gene expression and LXRα binding 81 3.3.3 Main functions of LXRα in cholesterol metabolism and interaction with PPARα signaling pathway 83 3.3.4 LXRα binding sites are linked with disease associated SNPs 85 3.4 Network analyses 87 3.4.1 LXRα controls a network of responses to activating ligands 87 3.4.2 LXRα modulation in foam cells activates carbohydrate metabolism, molecular transport and lipid metabolism 88 3.4.3 Novel LXRα target genes with atheroprotective potential in LXR/RXR activation pathway 91 3.5 STLX4 - A new LXRα ligand 93 3.5.1 Chemically optimized stilbenoid activates LXRα 93 3.5.2 STLX4 targets specifically foam cells 94 3.5.3 STLX4 decreases cholesterol without undesired triglyceride increase 95 4\. Discussion 97 4.1 Significance of data 97 4.2 LXRα cistrome 98 4.2.1 Ligand requirement for LXRα binding 98 4.2.2 Different peak patterns T0901317 and between foam cell-specific loci 100 4.2.3 Motif requirement and positioning of LXRα 101 4.3 LXRα transcriptome 102 4.3.1 Cell type dominates gene expression 102 4.3.2 Tight interaction of LXRα and PPARα regulated pathways 103 4.4 Data integration 103 4.4.1 Disease-associated genetic variation data 104 4.5 Network analyses 104 4.5.1 Gene networks in foam cells vs. T0901317 treated foam cells 105 4.6 STLX4 - A novel disease specific LXRα ligand 107 4.6.1 STLX4 mode of action 107 4.6.2 Physiological effects of STLX4 108 4.7 Conclusions and future perspectives 109 5\. Summary 110 6\. Zusammenfassung 111 7\. References 112 8\. Abbreviations 138 9\. Publications 143 10\. Supplementary 144 10.1 Supplementary figures 144 10.2 Supplementary tables 152Atherosclerosis is the leading cause of cardiovascular diseases (CVD) and an enormous health burden. The ligand dependent nuclear receptor liver X receptor alpha (LXRα) is an important target for anti-atherosclerotic compounds due to its essential role in the adaptation of macrophages to lipid overload. Unfortunately, treatment with currently investigated LXRα modulating ligands is associated with deleterious side effects such as hypertriglyceridemia. Thus, it is of fundamental interest to investigate LXRα contribution to gene regulatory networks in macrophages, atherosclerotic foam cells and changes upon modulation with known anti-atherosclerotic LXRα ligands as well as the discovery of novel, selective LXRα modulators. Applying a highly integrative approach, global LXRα cistrome, epigenome and transcriptome data in macrophages, foam cells and with LXRα ligand T0901317 treated cell models were generated. Subsequent network analyses revealed that LXRα regulates broad networks via interaction with other factors, including PPARα and NFƙB. The diseased foam cell network could be successfully modulated by pharmacological intervention with T0901317 resulting in the discovery of 32 novel LXRα target genes with potential athero-beneficial effects, which in part explained the implications of disease-associated genetic variation data. The identified networks provide a powerful platform to investigate the complex biological foam cell system and will in future help to find new avenues for treating systematically atherosclerosis and related diseases. Additionally, the presented work debuts a novel LXRα ligand, STLX4. The optimized stilbenoid- based molecule binds preferably the LXRα-subtype and selectively induces its anti-atherogenic potential in diseased foam cells but not in macrophages. STLX4 has similar potency to reduce excess cholesterol in foam cells as T0901317 without the adverse increase in triglycerides. Thus, STLX4 is a novel LXRα ligand with outstanding potential for future pharmaceutical development.Atherosklerose ist die Hauptursache für die Entstehung von kardiovaskulären Erkrankungen und stellt ein großes Gesundheitsproblem unserer Gesellschaft dar. Der ligandenabhängige nukleare Rezeptor Leber X Rezeptor alpha (LXRα) ist ein wichtiger Zielrezeptor für anti-atherosklerotische Substanzen, da er von zentraler Bedeutung für die Bewältigung der Lipidüberladung von Makrophagen ist. Leider weisen aktuell erforschte LXRα-modulierende Substanzen Nebenwirkungen, wie zum Beispiel Hypertriglyzeridämie, auf. Daher ist es von besonderem Interesse, den Beitrag von LXRα zu genregulatorischen Netzwerken, sowie dessen anti-atherosklerotische Modulation in Makrophagen und Schaumzellen zu untersuchen. Darüber hinaus steht auch die Entwicklung neuer und selektiver LXRα-Modulatoren im Vordergrund. Durch die Anwendung eines überaus integrativen Ansatzes wurden genomweite Cistrom-, Epigenom- und Transkriptomdaten für LXRα in Makrophagen, Schaumzellen und mit dem LXRα liganden T0901317 behandelten Zellmodellen generiert. Die anschließende Netzwerkanalyse zeigte, dass LXRα weitreichende Netzwerke durch Interaktion mit anderen Faktoren, wie PPARα und NFƙB, reguliert. Das pathologische Schaumzellnetzwerk konnte durch die pharmakologische Intervention mit T0901317 moduliert werden und ermöglichte die Identifikation von 32 neuen LXRα- Zielgenen, die ein hohes athero-protektives Potential bergen und zum Teil die Auswirkungen von krankheitsassoziierten genetischen Variationsdaten erklären können. Die identifizierten Netzwerke sind eine hervorragende Basis für die Analyse der komplexen Biologie des Schaumzellsystems und werden in Zukunft dazu beitragen, neue Wege für die systematische Behandlung von Atherosklerose und ähnlichen Erkrankungen zu finden. Im Rahmen der vorliegenden Arbeit gelang es zudem, mit STLX4 einen neuen LXRα Liganden zu identifizieren. Das optimierte, auf einem Stilbenoid basierende Molekül, bindet bevorzugt den LXRα-Subtyp und induziert sein anti-atherogenes Potential nur in Schaumzellen und nicht in Makrophagen. Das Potential von STLX4, überschüssiges Cholesterin zu reduzieren, ist vergleichbar mit dem von T0901317, allerdings ruft STLX4 keinen unerwünschten Anstieg an Triglyzeriden hervor. STLX4 ist ein neuer LXRα-Ligand mit einem herausragenden Potential für weitere zukünftige pharmakologische Entwicklungen

STX4 specifically regulates gene expression in foam cells.

<p><b>A</b>, Genome-wide gene expression and subsequent gene set enrichment analysis (GSEA) of macrophages and foam cells after treatment with T09 (10 µM) or STX4 (10 µM). Regulation of lipid-derived Reactome and KEGG pathways is shown. <b>B</b>, Validation of gene expression microarray analysis by quantitative PCR. For all tested samples, array observations significantly correlated with qPCR results. <b>C</b>, Gene distance matrix of genome-wide gene expression analysis of macrophages and foam cells after treatment with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). Pairwise distances were calculated for comparison of two treatments and cell types. Colored squares show the distance in Euclidean space, ranging from exactly the same profile (black) to completely different (red). In macrophages STX4 expression is very different to that of T09 (P<0.0001), whereas in foam cells STX4-induced gene expression is similar to that of T09 (P<0.0001).</p

Physiological analyses of STX4 treatment confirm unique STX4 properties in foam cells.

<p><b>A</b>, Cholesterol content in foam cells after treatment for 48 h with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). Data are expressed as mean±SEM (n = 5–6). <b>B</b>, Cholesterol content in macrophages after treatment for 48 h with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). Data are expressed as mean±SEM (n = 5–6). <b>C</b>, Triglyceride content in foam cells after treatment for 48 h with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). <b>D</b>, Triglyceride content in macrophages after treatment for 48 h with DMSO (0.1%), T09 (10 µM) or STX4 (10 µM). Data are expressed as mean±SEM (n = 5–6). ***P<0.001 vs. DMSO; n.s., not significant.</p

STX4 is a novel selective LXRα agonist.

<p><b>A</b>, Chemical structure of STX4. <b>B</b>, Transcriptional activation of LXRα by T0901317 (T09), 22-R-hydroxycholesterol or STX4 in a reporter gene assay (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057311#pone-0057311-t001" target="_blank">Table 1</a>). Data are expressed as mean±SD (n = 3). <b>C</b>, Validation of LXRα specificity. Transcriptional activation of LXRβ by T09 or STX4 in a reporter gene assay. STX4 does not activate the LXRβ subtype (mean±SD, n = 3). <b>D</b>, Cytotoxicity of STX4 in macrophages (mean±SD, n = 3). STX4 does not reduce cellular viability up to 25 µM.</p

Results from competitive reporter-gene assays.

<p>Results from competitive reporter-gene assays.</p

STX4 specifically targets diseased, oxysterol-loaden foam cells.

<p><b>A</b>, Gene expression in foam cells after siRNA-mediated, single and combined LXRα/β knockdown (mean±SEM, n = 4, fold-change vs. DMSO, logarithmised). Left, efficiency of LXRα, LXRβ and LXRα/β knockdown (kd). Middle, LXRα, LXRβ and LXRα/β knockdown (kd). Middle, single and combined knockdown influence on gene expression of LXRα, LXRβ and ABCA1 upon T09 treatment. Right, single and combined knockdown influence on gene expression of LXRα, LXRβ and ABCA1 upon STX4 treatment. *P<0.05, **P<0.01, ***P<0.001 vs. negative siRNA. <b>B</b>, Gene expression in THP-1 macrophages and foam cells after treatment with T09 (10 µM) or STX4 (10 µM). Data are expressed as mean±SEM (n = 4). <b>C</b>, Western blot analysis of LXRα and APOE content in foam cells and STX4 treated foam cells. Bar plot displays the results of densitometry analysis (mean±SEM, n = 3). *P<0.05, **P<0.01 vs. foam cell.<b>D</b>, Transcriptional activation of LXRα by STX4 in the presence or absence of 200 nM T09 (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057311#pone-0057311-t001" target="_blank">table <b>1</b></a>). Data are expressed as mean±SD (n = 3). <b>E</b>, Gene expression in THP-1 macrophages after treatment with different concentrations of T09 or STX4 in the presence or absence of 1 µM T09. Data are expressed as mean±SEM (n = 2–3). *P<0.05, **P<0.01, ***P<0.001 vs. DMSO; n.s., not significant.</p