Hypoxia, Hypoxia-Inducible Transcription Factor, and Macrophages in Human Atherosclerotic Plaques Are Correlated With Intraplaque Angiogenesis Judith C. Sluimer, Jean-Marie Gasc, Job L. van Wanroij, ...Natasja Kisters, Mathijs Groeneweg, Maarten D. Sollewijn Gelpke, Jack P. Cleutjens, Luc H. van den Akker, Pierre Corvol, Bradly G. Wouters, Mat J. Daemen, Ann-Pascale J. Bijnens Hypoxia was directly demonstrated in advanced atherosclerotic lesions of symptomatic patients with the use of pimonidazole administration before carotid endarterectomy. Hypoxia correlated with the presence of macrophages, angiogenesis, and a thrombus, and the expression of hypoxia-inducible transcription factor (HIF) and vascular endothelial growth factor. Also, the HIF pathway and microvessel density were associated with lesion progression and angiogenesis, suggesting the involvement of the HIF pathway in the response to hypoxia and the regulation of human intraplaque angiogenesis.
Although aberrant reactivation of embryonic gene programs is intricately linked to pathological heart disease, the transcription factors driving these gene programs remain ill-defined. Here we report ...that increased calcineurin/Nfat signalling and decreased miR-25 expression integrate to re-express the basic helix-loop-helix (bHLH) transcription factor dHAND (also known as Hand2) in the diseased human and mouse myocardium. In line, mutant mice overexpressing Hand2 in otherwise healthy heart muscle cells developed a phenotype of pathological hypertrophy. Conversely, conditional gene-targeted Hand2 mice demonstrated a marked resistance to pressure-overload-induced hypertrophy, fibrosis, ventricular dysfunction and induction of a fetal gene program. Furthermore, in vivo inhibition of miR-25 by a specific antagomir evoked spontaneous cardiac dysfunction and sensitized the murine myocardium to heart failure in a Hand2-dependent manner. Our results reveal that signalling cascades integrate with microRNAs to induce the expression of the bHLH transcription factor Hand2 in the postnatal mammalian myocardium with impact on embryonic gene programs in heart failure.
MicroRNAs (miRNAs) are a class of non-coding RNAs of ∼22 nucleotides in length, and constitute a novel class of gene regulators by imperfect base-pairing to the 3'UTR of protein encoding messenger ...RNAs. Growing evidence indicates that miRNAs are implicated in several pathological processes in myocardial disease. The past years, we have witnessed several profiling attempts using high-density oligonucleotide array-based approaches to identify the complete miRNA content (miRNOME) in the healthy and diseased mammalian heart. These efforts have demonstrated that the failing heart displays differential expression of several dozens of miRNAs. While the total number of experimentally validated human miRNAs is roughly two thousand, the number of expressed miRNAs in the human myocardium remains elusive. Our objective was to perform an unbiased assay to identify the miRNOME of the human heart, both under physiological and pathophysiological conditions. We used deep sequencing and bioinformatics to annotate and quantify microRNA expression in healthy and diseased human heart (heart failure secondary to hypertrophic or dilated cardiomyopathy). Our results indicate that the human heart expresses >800 miRNAs, the majority of which not being annotated nor described so far and some of which being unique to primate species. Furthermore, >250 miRNAs show differential and etiology-dependent expression in human dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM). The human cardiac miRNOME still possesses a large number of miRNAs that remain virtually unexplored. The current study provides a starting point for a more comprehensive understanding of the role of miRNAs in regulating human heart disease.
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 level.
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.
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