Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Gene expression in primary human macrophages and foam cells

ABSTRACT: The aim of the experiment was to determine the effects of 48 hours of treatment with oxidized low density lipoprotein (oxLDL) on gene expression in primary human monocyte-derived macrophages. 6 independent biological replicates were processed. From each replicate half of the cells were treated with oxLDL and the other half were treated with the control buffer. For foam cell formation (after 7 days) macrophages were treated with oxLDL (50mcg/ml, enodtoxin free, Copper oxidized). Control macrophages were treated with a matching buffer without oxLDL. Treatment duration - 48 hours.

ORGANISM(S): Homo sapiens

SUBMITTER: Christopher O'Callaghan

PROVIDER: E-GEOD-54666 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Lipid-induced epigenomic changes in human macrophages identify a coronary artery disease-associated variant that regulates PPAP2B Expression through Altered C/EBP-beta binding.

Reschen Michael E ME Gaulton Kyle J KJ Lin Da D Soilleux Elizabeth J EJ Morris Andrew J AJ Smyth Susan S SS O'Callaghan Christopher A CA

PLoS genetics 20150402 4

Genome-wide association studies (GWAS) have identified over 40 loci that affect risk of coronary artery disease (CAD) and the causal mechanisms at the majority of loci are unknown. Recent studies have suggested that many causal GWAS variants influence disease through altered transcriptional regulation in disease-relevant cell types. We explored changes in transcriptional regulation during a key pathophysiological event in CAD, the environmental lipid-induced transformation of macrophages to lipi ...[more]

PMID: 25835000

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Project description:Foam cell formation from monocyte-derived macrophages is a hallmark of atheroscle-rotic lesions. Aspects of this process can be recapitulated in vitro by exposing MCSF-induced or platelet factor4 (CXCL4)-induced macrophages to oxidized (ox) or minimally modified (mm) low density lipoprotein (LDL). We measured gene expression in periph-eral blood mononuclear cells (PBMCs), monocytes and macrophages treated with CXCL1 (GRO-Î±) or CCL2 (MCP-1) as well as foam cells induced by native LDL, mmLDL or oxLDL using 22 Affymetrix gene chips. Using an advanced Bayesian error-pooling approach and a heterogeneous error model (HEM) with a false discovery rate (FDR) <0.05, we found 5,303 of 22,215 probe sets to be significantly regulated in at least one of the conditions. Among a subset of 917 candidate genes that were preselected for their known biological functions in macrophage foamcell differentiation, we found that 290 genes met the above statistical criteria for significant differential expression patterns. While many expected genes were found to be upregulated by LDL and oxLDL, very few were induced by mmLDL. We also found induction of unexpected genes, most strikingly MHC-II and other dendritic cell markers such as CD11c. The gene expression patterns in response to oxLDL were similar in MCSF-induced and CXCL4-induced macrophages. Our findings suggest that LDL and oxLDL, but not mmLDL, induce a dendritic cell-like phenotype in macrophages, suggesting that these cells may be able to present antigens and support an immune response. Experiment Overall Design: Human blood was drawn from the antecubital veins of healthy blood donors and provided as buffy coats by the Virginia Blood Services (Richmond, VA). The mononuclear fractions were pooled from four unidentified donors to decrease individual variations in monocytes. Mixed peripheral blood mononuclear cells (PBMCs) were isolated by Histopaque 1.077 (Sigma Diagnostics, Inc., St. Louis, MO). Following centrifugation, the mononuclear layer was removed and washed with PBS containing 0.02% ethylenediaminetetraacetate (EDTA). The pellet was resuspended in 1X H-lyse Buffer (R&D Systems Inc., Minneapolis, MN), and washed with wash buffer. PBMCs contain mainly monocytes and lymphocytes as well as platelets that tend to be associated with blood monocytes. From these PBMCs, monocytes were isolated using a negative selection monocyte isolation kit and LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the isolated fraction was > 97% as estimated by flow cytometry using anti-CD14 (data not shown). Although these cells are often called âuntouchedâ monocytes and thought to show little activation, the gene chip analysis conducted on these cells shows massive changes in gene expression compared to PBMCs (see below). Experiment Overall Design: Monocytes were cultured in Macrophage Serum-Free Medium (MSFM, Invitro-gen, Carlsbad, CA) in the presence of 1% media supplement nutridoma-HU (Roche Molecular Biochemicals, Indianapolis, IN) and 100 nM M-CSF for 6 days, after which the cells showed the expected morphological signs of macrophage differentiation. These macrophages were incubated either with 100 nM CCL2 or CXCL1 for 5 hours. CXCL1 was selected because we have previously found that it is an important arrest chemokine for monocytes in vitro and in atherosclerotic arteries in vivo. CCL2 was chosen because mice lacking CCL2 or its receptor CCR2 are relatively resistant to atherosclerosis, suggesting a role in macrophage recruitment, differentiation and/or survival. Human monocyte-derived macrophages (MDM) were also incubated with native LDL, oxidized LDL (oxLDL) or minimally modified LDL (mmLDL) (each at a concentration of 100 ug/ml) for 2 days to induce foam cell formation. Foam cell formation was verified by oil red O staining (figure 1) and by determining their cholesterol and cholesterol ester content. OxLDL and mmLDL were prepared from the same native LDL for each experiment as described. Control experiments were conducted on macrophages cultured in M-CSF without LDL for an additional 2 days. Two separate sets of monocytes were incubated with CXCL4 (100 nM) for 6 days, another procedure known to induce macrophage differentiation (29), with and without oxLDL to induce foam cell formation. RNA was extracted from cells in all 11 conditions (table 1) and gene expression was measured in duplicates at the University of Virginia Gene Expression Core Facility using Affymetrix equipment.

Project description:OBJECTIVE: The liver X receptor M-NM-1 (LXRM-NM-1) 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 LXRM-NM-1 reporter-gene assays. STX4 displayed the unique property to activate LXRM-NM-1 effectively but not its subtype LXRM-NM-2. The potential of STX4 to increase transcriptional activity as an LXRM-NM-1 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 LXRM-NM-1-ligand to study transcriptional regulation of anti-atherogenic processes in cell or ex vivo models, and provides a promising lead structure for pharmaceutical development. 2 treatment groups (STX4 vs. DMSO in foam cells), 3 biological replicates per treatment (6 samples in total), each representing one well from cell culture plate 2 treatment groups (STX4 vs. DMSO in macrophages), 4 biological replicates per treatment (8 samples in total), each representing one well from cell culture plate 2 treatment groups (T0901317 vs. DMSO in foam cells), 3-4 biological replicates per treatment (7 samples in total), each representing one well from cell culture plate 2 treatment groups (T0901317 vs. DMSO in macrophages), 4 biological replicates per treatment (8 samples in total), each representing one well from cell culture plate

Dataset Information

Gene expression in primary human macrophages and foam cells

Publications

Lipid-induced epigenomic changes in human macrophages identify a coronary artery disease-associated variant that regulates PPAP2B Expression through Altered C/EBP-beta binding.

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