RESUMO
AIMS: We conducted a genome-wide analysis to identify differentially methylated genes in atherosclerotic lesions. METHODS: DNA methylation at promoters, exons and introns was identified by massive parallel sequencing. Gene expression was analysed by microarrays, qPCR, immunohistochemistry and western blots. RESULTS: Globally, hypomethylation of chromosomal DNA predominates in atherosclerotic plaques and two-thirds of genes showing over 2.5-fold differential in DNA methylation are up-regulated in comparison to healthy mammary arteries. The imprinted chromatin locus 14q32 was identified for the first time as an extensively hypomethylated area in atherosclerosis with highly induced expression of miR127, -136, -410, -431, -432, -433 and capillary formation-associated gene RTL1. The top 100 list of hypomethylated promoters exhibited over 1000-fold enrichment for miRNAs, many of which mapped to locus 14q32. Unexpectedly, also gene body hypermethylation was found to correlate with stimulated mRNA expression. CONCLUSION: Significant changes in genomic methylation were identified in atherosclerotic lesions. The most prominent gene cluster activated via hypomethylation was detected at imprinted chromosomal locus 14q32 with several clustered miRNAs that were up-regulated. These results suggest that epigenetic changes are involved in atherogenesis and may offer new potential therapeutic targets for vascular diseases.
Assuntos
Metilação de DNA/genética , MicroRNAs/genética , Placa Aterosclerótica/genética , Idoso , Estudos de Casos e Controles , Cromossomos Humanos Par 14/genética , Regulação para Baixo/genética , Éxons/genética , Feminino , Ontologia Genética , Loci Gênicos/genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , MicroRNAs/metabolismo , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Regulação para Cima/genéticaRESUMO
AIMS: Statins have beneficial vascular effects beyond their cholesterol-lowering action. Since macrophages play a central role in atherogenesis, we characterized the effects of simvastatin on gene expression profile of human peripheral blood monocyte (HPBM)-macrophages. METHODS AND RESULTS: Gene expression profile was studied using Affymetrix gene chip analysis. Lentiviral gene transfer of Kruppel-like factor 2 (KLF-2) was used to further study its role in macrophages. Simvastatin treatment lead to downregulation of many pro-inflammatory genes including several chemokines [e.g. monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory proteins-1alpha and beta, interleukin-2 receptor-beta], members of the tumour necrosis factor family (e.g. lymphotoxin beta), vascular cell adhesion molecule-1, and tissue factor (TF). Simvastatin also modulated the expression of several transcription factors essential for inflammation: NF-kappaB relA/p65 subunit and ets-1 were downregulated, and an atheroprotective transcription factor KLF-2 was upregulated. The effects of simvastatin on MCP-1 and TF could be mimicked by KLF-2 overexpression using lentiviral gene transfer. CONCLUSION: Simvastatin has a strong anti-inflammatory effect on HPBM cells including upregulation of the atheroprotective factor KLF-2. This may partly explain the beneficial effects of statins on cardiovascular diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Citocinas/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Macrófagos/efeitos dos fármacos , Sinvastatina/farmacologia , Linhagem Celular , Células Cultivadas , Quimiocina CCL2/metabolismo , Citocinas/genética , Perfilação da Expressão Gênica/métodos , Técnicas de Transferência de Genes , Humanos , Fatores de Transcrição Kruppel-Like/genética , Macrófagos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Prenilação de Proteína , Proteínas Proto-Oncogênicas c-ets/metabolismo , RNA Mensageiro/metabolismo , Tromboplastina/metabolismo , Fatores de Tempo , Fator de Transcrição RelA/metabolismo , Fatores de Necrose Tumoral/metabolismo , Regulação para CimaRESUMO
No mouse model is currently available where the induction of type 2 diabetes on an atherosclerotic background could be achieved without significant concomitant changes in plasma lipid levels. We crossbred 2 genetically modified mouse strains to achieve a model expressing both atherosclerosis and characteristics of type 2 diabetes. For atherosclerotic background we used low-density lipoprotein receptor-deficient mice synthetizing only apolipoprotein B100 (LDLR(-/-) ApoB(100/100)). Diabetic background was obtained from transgenic mice overexpressing insulin-like growth factor-II (IGF-II) in pancreatic beta cells. Thorough phenotypic characterization was performed in 6- and 15-month-old mice on both normal and high-fat Western diet. Results indicated that IGF-II transgenic LDLR(-/-)ApoB(100/100) mice demonstrated insulin resistance, hyperglycemia, and mild hyperinsulinemia compared with hypercholesterolemic LDLR(-/-)ApoB(100/100) controls. In addition, old IGF-II/LDLR(-/-)ApoB(100/100) mice displayed significantly increased lesion calcification, which was more related to insulin resistance than glucose levels, and significantly higher baseline expression in aorta of several genes related to calcification and inflammation. Lipid levels of IGF-II/LDLR(-/-)ApoB(100/100) mice did not differ from LDLR(-/-)ApoB(100/100) controls at any time. In conclusion, type 2 diabetic factors induce increased calcification and lesion progression without any lipid changes in a new mouse model of diabetic macroangiopathy.