Dead or alive: gene expression profiles of advanced atherosclerotic plaques from autopsy and surgery

Since inclusion of atherosclerotic tissues from different sources is often indispensable to study the full atherogenic spectrum, we investigated to what extent the expression profiles of advanced, stable atherosclerotic lesions obtained during autopsy and surgery are comparable. The gene expression profiles of human carotids with advanced atherosclerosis obtained at autopsy and at vascular surgery were studied by microarray analysis. Expression analysis was performed both at the single gene (Rosetta, Gene Ontology) and at the pathway level using Ingenuity and Gene Set Enrichment Analysis. In addition, mRNA and protein expression levels were validated using quantitative (q) RT-PCR and immunohistochemistry on unrelated advanced carotid lesions from autopsy and surgery. Microarray analysis indicated that the 97.2% of genes showed similar expression levels in advanced atherosclerotic lesions from autopsy and surgery. While the expression data revealed no differences in common atherosclerotic related pathways such as lipid metabolism and inflammation, the differentially expressed genes were mainly involved in basal cell metabolism and hypoxia driven pathways. qRT-PCR confirmed the differential expression of hypoxia-driven genes VEGF-A (2.3-fold upward arrow), glucose transporter (GLUT)-1 (2.5-fold upward arrow), GLUT3 (8.3-fold upward arrow), and hexokinase 1 (2.4-fold upward arrow) in autopsy vs. surgical specimens. Immunohistochemistry revealed that the transcriptional differences in these hypoxia-related genes were not reflected at the protein level. The gene expression profiles of advanced atherosclerotic lesions from autopsy and surgery are largely similar. However, >500 genes, mostly involved in basal cell metabolism and hypoxia were differentially expressed at mRNA, but not at the protein leve

Hypoxia, hypoxia-inducible transcription factor, and macrophages in human atherosclerotic plaques are correlated with intraplaque angiogenesis

We sought to examine the presence of hypoxia in human carotid atherosclerosis and its association with hypoxia-inducible transcription factor (HIF) and intraplaque angiogenesis. Atherosclerotic plaques develop intraplaque angiogenesis, which is a typical feature of hypoxic tissue and expression of HIF. To examine the presence of hypoxia in atherosclerotic plaques, the hypoxia marker pimonidazole was infused before carotid endarterectomy in 7 symptomatic patients. Also, the messenger ribonucleic acid (mRNA) and protein expression of HIF1 alpha, HIF2 alpha, HIF-responsive genes (vascular endothelial growth factor [VEGF], glucose transporter [GLUT]1, GLUT3, hexokinase [HK]1, and HK2), and microvessel density were determined in a larger series of nondiseased and atherosclerotic carotid arteries with microarray, quantitative reverse transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. Pimonidazole immunohistochemistry demonstrated the presence of hypoxia, especially within the macrophage-rich center of the lesions. Hypoxia correlated with the presence of a thrombus, angiogenesis, and expression of CD68, HIF, and VEGF. The mRNA and protein expression of HIF, its target genes, and microvessel density increased from early to stable lesions, but no changes were observed between stable and ruptured lesions. This is the first study directly demonstrating hypoxia in advanced human atherosclerosis and its correlation with the presence of macrophages and the expression of HIF and VEGF. Also, the HIF pathway was associated with lesion progression and angiogenesis, suggesting its involvement in the response to hypoxia and the regulation of human intraplaque angiogenesi

Identification of a novel microRNA. A,

<p>Schematic representation of the conservation of a novel microRNA, miR-3615376 among different species. <b>B, C,</b> Real-time quantitative PCR analysis of miR-3615376 expression in different mouse tissues. <b>D</b>, Expression pattern of miR-3615376 in the DCM and HCM libraries.</p

Hypertrophic cardiomyopathy-specific microRNA expression pattern.

<p>From the most differentially regulated microRNAs in the DCM and HCM libraries, 8 are specific for primates and conserved between primates (<b>A</b>) and 11 are highly conserved amongst vertebrates (<b>B</b>). C<b>,</b> From a total of 332 microRNAs detected in the HCM library, 29 were HCM-specifc, from which 10 had been previously described in profiling studies and 19 remain to be described. <b>D</b>, Relative abundance of HCM-specific microRNAs that were detected, or not, in previous profiling studies. MicroRNAs that are significantly expressed between HCM and DCM libraries are depicted in purple.</p

RNA content in the non-failing and failing human heart. A

<p>, Composition of global libraries, <b>B</b>, non-failing, <b>C</b>, hypertrophic cardiomyopathy (HCM), and <b>D</b>, dilated cardiomyopathy (DCM). <b>E</b>, Correlation of number of reads with cycle threshold (Ct) values obtained by real time quantitative PCR analysis of expression of 4 different microRNAs (miR-199a, miR-199b, miR-143 and miR-145). Data pairs are represented individually, independent of the miRNA analyzed. <b>F</b>. Real time quantitative PCR analysis of cardiac stress markers (nppa and nppb) in DCM and HCM samples.</p

Distinctive Expression of Chemokines and Transforming Growth Factor-β Signaling in Human Arterial Endothelium during Atherosclerosis

Knowledge about the in vivo role of endothelium in chronic human atherosclerosis has mostly been derived by insights from mouse models. Therefore, we set out to establish by microarray analyses the gene expression profiles of endothelium from human large arteries, as isolated by laser microbeam microdissection, having focal atherosclerosis of the early or the advanced stage. Within individual arteries, the endothelial transcriptomes of the lesional and unaffected sides were compared pairwise, thus limiting genetic and environmental confounders. Specific endothelial signature gene sets were identified with changed expression levels in either early (n = 718) or advanced atherosclerosis (n = 403), relative to their paired plaque-free controls. Gene set enrichment analysis identified distinct sets of chemokines and differential enrichments of nuclear factor-κB-, p53-, and transforming growth factor-β-related genes in advanced plaques. Immunohistochemistry validated the discriminative value of corresponding endothelial protein expression between early (fractalkine/CX3CL1, IP10/CCL10, TBX18) or advanced (BAX, NFKB2) stages of atherosclerosis and versus their plaque-free controls. The functional involvement of transforming growth factor-β signaling in directing its downstream gene repertoire was substantiated by a consistent detection of activated SMAD2 in advanced lesions. Thus, we identified truly common, local molecular denominators of pathological changes to vascular endothelium, with a marked distinction of endothelial phenotype between early and advanced plaques