Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide expression profiling of laser micro-dissected macrophages from ruptured and stable atherosclerotic human plaques

ABSTRACT: Objective: Resident macrophages play an important role in atheromatous plaque rupture. The macrophage gene expression signature associated with plaque rupture is incompletely defined due to the complex cellular heterogeneity in the plaque. We aimed to characterise differential gene expression in resident plaque macrophages from ruptured and stable human atheromatous lesions. A cell-specific approach has the potential to address the question of gene expression differences between particular cell types in stable and unstable plaques with greater precision than approaches based on the study of whole plaques. Using laser micro-dissection, we isolated total RNA from macrophage-rich regions of stable and ruptured human atheromatous plaques derived from carotid endarterectomy samples which were comprehensively characterized using clinical, radiological and histological criteria, and carried out genome-wide gene expression profiling using microarrays. Results: The profiles were characteristic of activated macrophages. At a false discovery rate of 10%, 914 genes were differentially expressed between stable and ruptured plaques. The findings were confirmed in fourteen further stable and ruptured samples for a subset of eleven genes with the highest expression differences (p<0.05). Pathway analysis revealed that components of the PPAR/Adipocytokine signaling pathway were the most significantly upregulated in ruptured compared to stable plaques (p=5.4x10-7). Two key components of the pathway, fatty-acid binding-protein 4 (FABP4) and leptin, showed nine-fold (p=0.0086) and five-fold (p=0.0012) greater expression respectively in macrophages from ruptured plaques. Conclusions: We found differences in gene expression signatures between macrophages isolated from stable and ruptured human atheromatous plaques. Our findings indicate the involvement of FABP4 and leptin in the progression of atherosclerosis and plaque rupture, and suggest that down-regulation of PPAR/adipocytokine signaling within plaques may have therapeutic potential. Methods: We performed genome-wide expression analyses of isolated macrophage-rich regions of stable and ruptured human atherosclerotic plaques. Plaques present in carotid endarterectomy specimens were designated as stable or ruptured using clinical, radiological and histopathological criteria. Macrophage-rich regions were excised from 5 ruptured and 6 stable plaques by laser micro-dissection. Total RNA were subjected to two cycles of linear amplification. Transcriptional profiling was performed using Affymetrix HG-U133 plus 2.0 GeneChip arrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Kelvin Lee

PROVIDER: E-GEOD-41571 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Objective: Resident macrophages play an important role in atheromatous plaque rupture. The macrophage gene expression signature associated with plaque rupture is incompletely defined due to the complex cellular heterogeneity in the plaque. We aimed to characterise differential gene expression in resident plaque macrophages from ruptured and stable human atheromatous lesions. A cell-specific approach has the potential to address the question of gene expression differences between particular cell types in stable and unstable plaques with greater precision than approaches based on the study of whole plaques. Using laser micro-dissection, we isolated total RNA from macrophage-rich regions of stable and ruptured human atheromatous plaques derived from carotid endarterectomy samples which were comprehensively characterized using clinical, radiological and histological criteria, and carried out genome-wide gene expression profiling using microarrays. Results: The profiles were characteristic of activated macrophages. At a false discovery rate of 10%, 914 genes were differentially expressed between stable and ruptured plaques. The findings were confirmed in fourteen further stable and ruptured samples for a subset of eleven genes with the highest expression differences (p<0.05). Pathway analysis revealed that components of the PPAR/Adipocytokine signaling pathway were the most significantly upregulated in ruptured compared to stable plaques (p=5.4x10-7). Two key components of the pathway, fatty-acid binding-protein 4 (FABP4) and leptin, showed nine-fold (p=0.0086) and five-fold (p=0.0012) greater expression respectively in macrophages from ruptured plaques. Conclusions: We found differences in gene expression signatures between macrophages isolated from stable and ruptured human atheromatous plaques. Our findings indicate the involvement of FABP4 and leptin in the progression of atherosclerosis and plaque rupture, and suggest that down-regulation of PPAR/adipocytokine signaling within plaques may have therapeutic potential.

Project description:Cardiovascular disease is the leading cause of death in developed countries and there is compelling evidence that the majority of these fatalities are secondary to rupture of unstable atherosclerotic plaques. However, the mechanisms that control plaque stability are poorly understood, although it is widely believed that plaques having a decreased ratio of cells positive for smooth muscle cell (SMC) markers such as ACTA2 relative to macrophage markers are more likely to rupture. Herein we employMyh11-CreERT2 ROSA floxed STOP eYFP Apoe-/- mice to trace SMC lineage and show that traditional methods for detecting SMCs based on immunostaining for SMC markers like ACTA2 are unable to detect 82% of SMC-derived cells within advanced atherosclerotic lesions. Moreover, we show that these SMC-derived cells within advanced lesions exhibit multiple phenotypes including activation of multiple markers of macrophages, mesenchymal stem cells (MSC), and myofibroblasts (MF). Importantly, we show that SMC-derived macrophage like cells are phagocytic based on electron microscope YFP immunolabeling. In addition, results of single cell epigenetic assays developed in our lab shows that nearly 20% of macrophage-like cells within human lesions are of SMC not myeloid origin. These phenotypic transitions appear to be critical in lesion pathogenesis, in that we show that SMC-specific conditional knockout (KO) of the pluripotency factor, Krüppel-like factor 4 (KLF4), resulted in marked reductions in lesion size, increases in multiple indices of plaque stability, and reduced numbers of SMC-derived MSC- and macrophage-like cells. Finally, we show that cholesterol loading of cultured SMC is associated with activation of multiple markers of macrophages and MSC, secretion of pro-inflammatory cytokines, and increased phagocytosis, all of which are Klf4 dependent. Taken together, results indicate that the contribution of SMCs within atherosclerotic plaques has been greatly underestimated, and that loss of Klf4 within SMC result in major changes in SMC phenotype and function that play a major role in lesion pathogenesis. Examination of KLF4 transcription factor in an atherosclerosis setting

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Genome-wide expression profiling of laser micro-dissected macrophages from ruptured and stable atherosclerotic human plaques

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