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Transcriptome profiling of plaque macrophages during atherosclerosis regression

ABSTRACT: We have carried out a systems biology investigation of regulators controlling arterial plaque macrophage transcriptional changes in response to lipid lowering, in the Reversa mouse model of plaque regression in which poly I:C injections induce recombination-mediated inactivation of Mttp in the liver and (when combined with a switch to chow diet) restore normal lipid levels in an Ldlr-/-Apob100/100 mouse (Lieu et al., 2003, Circulation, v.107(9), pp.1315-21). Twenty-four Reversa mice (12 males, 12 females) were maintained on Western diet for 16 weeks and given injections of either poly I:C or vehicle (saline) and then switched to normal chow. At seven days after the last injection, animals were sacrificed and CD68+ macrophages were isolated from the aortic root plaque using laser-capture microdissection. An additional two Ldlr-/- animals (male) were treated with poly I:C and CD68+ aortic root plaque macrophages were obtained, as a control group for effects of poly I:C independent of Mttp inactivation. RNA isolated from each of the 26 samples was reverse-transcribed, amplified, fragmented, labeled, and then hybridized to the Affymetrix Mouse Exon Array 1.0 ST GeneChip. Probe intensities were analyzed using transcript-level probesets for gene expression profiling, and for each mouse transcript represented on the microarray, a two-factor model (sex and treatment) was used for the analysis of the 24 samples from the Reversa mice, to test for a significant effect of the poly I:C (vs. saline) treatment.

ORGANISM(S): Mus musculus

PROVIDER: GSE52482 | GEO | 2014/12/04

SECONDARY ACCESSION(S): PRJNA229083

REPOSITORIES: GEO

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Project description:Purpose: The goals of this study is to evaluate the impact of LXRa phosphorylation at S196 in immune cells on atherosclerosi and obesity, on gene expression via RNA-seq. Lab Methods: Bone marrow from LXRa WT and S196A mice was transplanted into Ldlr-/- mice, which were fed a high fat, high cholesterol diet for 16 weeks prior to evaluation of atherosclerosis and obesity. Plaque CD68+ and T cells, and pWAT macrophages and T cells mRNA profiles of LXRa WT and S196A mice were generated by RNA-seq using Illumina HiSeq 2500 (plaque CD68+ samples) or HiSeq 4000 (Plaque T cells and pWAT FBC, FB and T cells samples). Bioinformatics Methods: Quality control and Illumina adapter removal was performed on the raw sequencing output, followed by mapping of each read pair to the GRCm38.96 transcriptome in order to generate the mRNA profile of each sample. Sample groups from different tissues as well as groups from wild-type or mutant samples were compared against each other for differential gene expression. Results: we analyzed genes differentially expressed in plaque LXRa S196A and found that LXRa S196A induced 365 and repressed 205 genes in CD68+ (LogFC > 0.6, p-value < 0.05), and induced 1,578 genes and repressed 2,392 genes in plaque T cells compared to LXRa WT (LogFC > 1, FDR < 0.05). In pWAT, LXRa S196A vs WT analysis showed that LXRa S196A repressed 419 genes and induced 379 genes in FBC, induced 182 genes and repressed 228 genes in FB, and 624 genes were induced and 622 genes were repressed in LXRa WT (LogFC > 1, FDR < 0.05). Conclusions: This study is the first detailed analysis of plaque and pWAT immune cells transcriptomes from LXRa S196A in bone marrow cells of LDLR-/- mice under high fat and high cholesterol diet, generated by RNA-seq. We demonstrated that LXRa S196A is largely affecting transcriptome of immune cells in atherosclerotic plaque and pWAT. The transcriptome remodeling observed is tissue and cell specific, and controls atherosclerosis and obesity progression.

Project description:Background: Somatic mutations in blood indicative of clonal hematopoiesis of indeterminate potential (CHIP), particularly in DNMT3A, TET2, and JAK2, are associated with an increased risk of hematologic malignancy, coronary artery disease, and all-cause mortality. However, whether CHIP is associated with increased risk of peripheral artery disease (PAD) remains unknown. In addition, chemotherapy frequently causes mutations in DNA Damage Repair (DDR) genes TP53 and PPM1D, and whether CHIP caused by somatic mutations in DDR genes results in increased risk of atherosclerosis is unclear. We sought to test whether CHIP, and CHIP caused by DDR genes, associates with incident peripheral artery disease (PAD) and atherosclerosis. Methods: We identified CHIP among 50,122 exome sequences in individuals from UK and Mass General Brigham Biobanks and tested CHIP status (N=2,851) with incident PAD and atherosclerosis across multiple arterial beds. To mimic the human scenario of clonal hematopoiesis and test whether the expansion of p53-deficient hematopoietic cells contributes to atherosclerosis, a competitive bone marrow transplantation (BMT) strategy was used to generate atherosclerosis-prone Ldlr-/- chimeric mice carrying 20% Trp53-/- hematopoietic cells (20% KO-BMT mice). We then evaluated aortic plaque burden and plaque macrophage accumulation 12 weeks after grafting. Results: CHIP associated with incident PAD (HR 1.7; P=2.2x10-5) and atherosclerosis in multiple beds (HR 1.3; P=9.7x10-5), with increased risk among individuals with DDR CHIP (HR 2.0; P=0.0084). Among atherosclerosis-prone Ldlr null mice, the p53 -/- 20% KO-BMT mice demonstrated increased aortic plaque size (p=0.013) and accumulation of p53-/- plaque macrophages (P<0.001), driven by an abundance of p53-deficient plaque macrophages. The expansion of p53-deficient cells did not affect the expression of the pro-inflammatory cytokines IL-6 and IL-1β in the atherosclerotic aortic wall. Conclusions: Our findings highlight the role of CHIP as a broad driver of atherosclerosis across the entire arterial system, with evidence of increased plaque among p53 -/- 20% KO-BMT mice via expansion of plaque macrophages. These observations provide new insight into the link between CHIP and cardiovascular disease, and lend human genetic support to the concept that post-cytotoxic chemotherapy patients may benefit from surveillance for atherosclerotic conditions in addition to therapy-related myeloid neoplasms.

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Transcriptome profiling of plaque macrophages during atherosclerosis regression

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