Project description:We used single cell RNA-sequencing of aortic CD45+ cells, combined with immunohistologic, morphometric and flow cytometric analyses to define the changes in plaque immune cell composition, gene expression and function following miR-33 inhibition. We report that anti-miR-33 treatment of Ldlr–/– mice with advanced atherosclerosis reduced plaque burden and altered the plaque immune cell landscape by shifting the balance of pro- and anti-atherosclerotic macrophage and T cell subsets. By quantifying the kinetic processes that determine plaque macrophage burden, we found that anti-miR-33 reduced levels of circulating monocytes and splenic myeloid progenitors, decreased macrophage proliferation and retention, and promoted macrophage attrition by apoptosis and efferocytotic clearance. scRNA-sequencing of aortic arch plaques showed that anti-miR-33 reduced the frequency of MHCIIhi “inflammatory” and Trem2hi “metabolic” macrophages, but not tissue resident macrophages. Furthermore, anti-miR-33 led to derepression of distinct miR-33 target genes in the different macrophage subsets: in resident and Trem2hi macrophages, anti-miR-33 relieved repression of miR-33 target genes involved in lipid metabolism (e.g., Abca1, Ncoa1, Ncoa2, Crot), whereas in MHCIIhi macrophages, anti-miR-33 upregulated target genes involved in chromatin remodeling and transcriptional regulation. Anti-miR-33 also reduced the accumulation of aortic CD8+ and CD4+ Th1 cells, and increased levels of FoxP3+ regulatory T cells in plaques, consistent with an immune-dampening effect on plaque inflammation.
Project description:Inhibition of miR-33 results in increased cholesterol efflux and HDL-cholesterol levels in mice. In this study we examined the effect of miR-33 inhibition in a mouse model of atherosclerosis and observed significant reduction in atherosclerotic plaque size. At the end of the study, gene expression in macrophages from the atherosclerotic plaques was assessed. The results demonstrated a reduction in inflammatory gene expression and increased levels of mRNAs containing miR-33 binding sites. LDLR-/- mice on a Western diet with established atherosclerosis were switched to normal chow and treated with anti-miR-33, control anti-miR, or saline (n=4/group) for 4 weeks. Gene expression profiling was performed on macrophages in aortic plaques isolated at the end of the treatment period by laser capture microdissection.
Project description:Inhibition of miR-33 results in increased cholesterol efflux and HDL-cholesterol levels in mice. In this study we examined the effect of miR-33 inhibition in a mouse model of atherosclerosis and observed significant reduction in atherosclerotic plaque size. At the end of the study, gene expression in macrophages from the atherosclerotic plaques was assessed. The results demonstrated a reduction in inflammatory gene expression and increased levels of mRNAs containing miR-33 binding sites.
Project description:To investigate the expression profiles of miRNA in atherosclerotic plaques, the global features of miRNAs expression of three normal coronary artery tissues sample pools and three sample pools of advanced atherosclerosis plaques of coronary artery were studied using microarray technology,
Project description:Macrophages represent a major immune cell population in atherosclerotic plaques and play central role in the progression of this lipid-driven chronic inflammatory disease. Targeting immunometabolism is proposed as a strategy to revert aberrant macrophage activation to improve disease outcome. Here, we show ATP citrate lyase (Acly) to be activated in inflammatory macrophages and human atherosclerotic plaques. We demonstrate that myeloid Acly deficiency induces a stable plaque phenotype characterized by increased collagen deposition and fibrous cap thickness, along with a smaller necrotic core. In-depth functional, lipidomic, and transcriptional characterization indicate deregulated fatty acid and cholesterol biosynthesis and reduced liver X receptor (LXR) activation within the macrophages in vitro. This results in macrophages that are more prone to undergo apoptosis, whilst presenting increased phagocytosis of apoptotic cells. Together, our results indicate that targeting macrophage metabolism improves atherosclerosis outcome and we reveal Acly as a promising therapeutic target to stabilize atherosclerotic plaques.
Project description:We compared gene expression profiles between asymptomatic and symptomatic atherosclerotic plaques from the same patient. This was accomplished by analyzing carotid plaques from four patients with bilateral high-grade carotid artery stenoses one being symptomatic (TIA or stroke) and the other asymptomatic.
Project description:Macrophages represent a major immune cell population in atherosclerotic plaques and play central role in the progression of this lipid-driven chronic inflammatory disease. Targeting immunometabolism is proposed as a strategy to revert aberrant macrophage activation to improve disease outcome. Here, we show ATP citrate lyase (Acly) to be activated in inflammatory macrophages and human atherosclerotic plaques. We demonstrate that myeloid Acly deficiency induces a stable plaque phenotype characterized by increased collagen deposition and fibrous cap thickness, along with a smaller necrotic core. In-depth functional, lipidomic, and transcriptional characterization indicate deregulated fatty acid and cholesterol biosynthesis and reduced liver X receptor activation within the macrophages in vitro. This results in macrophages that are more prone to undergo apoptosis, whilst maintaining their capacity to phagocytose apoptotic cells. Together, our results indicate that targeting macrophage metabolism improves atherosclerosis outcome and we reveal Acly as a promising therapeutic target to stabilize atherosclerotic plaques.
Project description:In order to identify potential new biomarkers of atherosclerotic plaque composition we performed a large scale analysis of gene expression patterns in human atherosclerotic lesions. Whole genome expression analysis of 101 peripheral plaques identified a robust gene signature (1514 genes) dominated by inflammatory processes, and cholesterol metabolism and storage genes. Specific pathways enriched in this signature included activation of the Toll-like receptor signaling pathway, T-cell activation, cholesterol efflux, oxidative stress response, inflammatory cytokine production, vasoconstriction and lysosomal activity. Analysis of gene expression in plaque micro-dissected material revealed that the signature is strongly up-regulated in macrophage-rich regions and down-regulated in regions with high smooth muscle cell content. A smaller qPCR biomarker panel and inflammatory composite score (ICS) were developed to facilitate clinical translation of discoveries from gene expression profiling. We found that ICS correlates with histological features related to plaque vulnerability. In addition, ICS is able to separate groups of plaques obtained from symptomatic and asymptomatic patients undergoing carotid endarerectomy. In summary, we identified a robust mRNA biomarker panel associated with histo-pathological as well as clinical hallmarks of vulnerable atherosclerotic plaque. This panel may be used as a diagnostic and prognostic tool in clinical setting to evaluate novel anti-atherosclerotic therapies. 290 human peripheral plaques were excised using the FoxHollow silverhawk catheter and stored in RNAlater. RNA was extracted, amplified and hybridized to Affymetrix/Merck custom 1.0 arrays (GPL10687).
Project description:In order to identify potential new biomarkers of atherosclerotic plaque composition we performed a large scale analysis of gene expression patterns in human atherosclerotic lesions. Whole genome expression analysis of 101 peripheral plaques identified a robust gene signature (1514 genes) dominated by inflammatory processes, and cholesterol metabolism and storage genes. Specific pathways enriched in this signature included activation of the Toll-like receptor signaling pathway, T-cell activation, cholesterol efflux, oxidative stress response, inflammatory cytokine production, vasoconstriction and lysosomal activity. Analysis of gene expression in plaque micro-dissected material revealed that the signature is strongly up-regulated in macrophage-rich regions and down-regulated in regions with high smooth muscle cell content. A smaller qPCR biomarker panel and inflammatory composite score (ICS) were developed to facilitate clinical translation of discoveries from gene expression profiling. We found that ICS correlates with histological features related to plaque vulnerability. In addition, ICS is able to separate groups of plaques obtained from symptomatic and asymptomatic patients undergoing carotid endarerectomy. In summary, we identified a robust mRNA biomarker panel associated with histo-pathological as well as clinical hallmarks of vulnerable atherosclerotic plaque. This panel may be used as a diagnostic and prognostic tool in clinical setting to evaluate novel anti-atherosclerotic therapies. Total RNA from peripheral plaque (n=101) profiled in the Merck/Agilent 44k v1.1 against a reference pool of total RNA from 7 carotid plaques.
Project description:Genome wide Methylation profiling of DNA extracted from plaques of atherosclerotic plaque samples (n=14) and of controls (n=4). The Illumina Infinium HumanMethylation450 or HumanMethylation850 BeadChip was used representing potentially methylated CpG sites throughout the genome.