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Foam Cell Formation In Vivo Converts Macrophages to a Pro-Fibrotic Phenotype.

ABSTRACT: Formation of foam cell macrophages, which sequester extracellular modified lipids, is a key event in atherosclerosis. How lipid loading affects macrophage phenotype is controversial, with evidence suggesting either pro- or anti-inflammatory consequences. To investigate this further, we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in the subcutaneous granulomas of fed-fat ApoE null mice and normal chow fed wild-type mice in vivo. Consistent with previous studies, LXR/RXR pathway genes were significantly over-represented among the genes up-regulated in foam cell macrophages. Unexpectedly, the hepatic fibrosis pathway, associated with platelet derived growth factor and transforming growth factor-? action, was also over-represented. Several collagen polypeptides and proteoglycan core proteins as well as connective tissue growth factor and fibrosis-related FOS and JUN transcription factors were up-regulated in foam cell macrophages. Increased expression of several of these genes was confirmed at the protein level in foam cell macrophages from subcutaneous granulomas and in atherosclerotic plaques. Moreover, phosphorylation and nuclear translocation of SMAD2, which is downstream of several transforming growth factor-? family members, was also detected in foam cell macrophages. We conclude that foam cell formation in vivo leads to a pro-fibrotic macrophage phenotype, which could contribute to plaque stability, especially in early lesions that have few vascular smooth muscle cells.

SUBMITTER: Thomas AC

PROVIDER: S-EPMC4510387 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Foam Cell Formation In Vivo Converts Macrophages to a Pro-Fibrotic Phenotype.

Thomas Anita C AC Eijgelaar Wouter J WJ Daemen Mat J A P MJ Newby Andrew C AC

PloS one 20150721 7

Formation of foam cell macrophages, which sequester extracellular modified lipids, is a key event in atherosclerosis. How lipid loading affects macrophage phenotype is controversial, with evidence suggesting either pro- or anti-inflammatory consequences. To investigate this further, we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in the subcutaneous granulomas of fed-fat ApoE null mice and normal chow fed wild-type mice in vivo. Consistent with previous studies, ...[more]

PMID: 26197235

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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:Mipu1 (myocardial ischemic preconditioning upregulated protein 1) is a novel N-terminal Kruppel-associated box (KRAB)/C2H2 zinc finger superfamily protein, that displays a powerful effect in protecting H9c2 cells from oxidative stress-induced cell apoptosis. The present study aims to investigate the effect of Mipu1 overexpression on oxidized low-density lipoprotein (oxLDL)-induced foam cell formation, cell apoptosis, and its possible mechanisms. New Zealand healthy rabbits were used to establish atherosclerosis model, and serum levels of triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol were detected by an automatic biochemical analyzer. Sudan IV staining was used to detect atherosclerotic lesions. The RAW264.7 macrophage cell line was selected as the experimental material. Oil red O staining, high-performance liquid chromatography, and Dil-labeled lipoprotein were used to detect cholesterol accumulation qualitatively and quantitatively, respectively. Flow cytometry was used to determine cell apoptosis. Real-time quantitative polymerase chain reaction (PCR) was used to detect the mRNA expression of the main proteins that are associated with the transport of cholesterol, such as ABCA1, ABCG1, SR-BI, and CD36. Western blot analysis was used to detect the protein expression of Mipu1. There were atherosclerotic lesions in the high-fat diet group with Sudan IV staining. High-fat diet decreased Mipu1 expression and increased CD36 expression significantly at the 10th week compared with standard-diet rabbits. Mipu1 overexpression decreased oxLDL-induced cholesterol accumulation, oxLDL uptake, cell apoptosis, and cleaved caspase-3. Mipu1 overexpression inhibited the oxLDL-induced CD36 mRNA and protein expression, but it did not significantly inhibit the mRNA expression of ABCA1, ABCG1, and SR-BI. Mipu1 overexpression inhibits oxLDL-induced foam cell formation and cell apoptosis. Mipu1 overexpression reduces the lipid intake of macrophages and might be associated with the downregulation of CD36 expression in the presence of oxLDL.

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Foam Cell Formation In Vivo Converts Macrophages to a Pro-Fibrotic Phenotype.

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Foam Cell Formation In Vivo Converts Macrophages to a Pro-Fibrotic Phenotype.

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