Histone and DNA Methylation–Mediated Epigenetic Downregulation of Endothelial Kruppel-Like Factor 2 by Low-Density Lipoprotein Cholesterol

Abstract

Objective-Low-density lipoprotein (LDL) cholesterol induces endothelial dysfunction and is a major modifiable risk factor for coronary heart disease. Endothelial Kruppel-like Factor 2 (KLF2) is a transcription factor that is vital to endotheliumdependent vascular homeostasis. The purpose of this study is to determine whether and how LDL affects endothelial KLF2 expression. Approach and Results-LDL downregulates KLF2 expression and promoter activity in endothelial cells. LDL-induced decrease in KLF2 parallels changes in endothelial KLF2 target genes thrombomodulin, endothelial NO synthase, and plasminogen activator inhibitor-1. Pharmacological inhibition of DNA methyltransferases or knockdown of DNA methyltransferase 1 prevents downregulation of endothelial KLF2 by LDL. LDL induces endothelial DNA methyltransferase 1 expression and DNA methyltransferase activity. LDL stimulates binding of the DNA methylCpG-binding protein-2 and histone methyltransferase enhancer of zeste homolog 2, whereas decreases binding of the KLF2 transcriptional activator myocyte enhancing factor-2, to the KLF2 promoter in endothelial cells. Knockdown of myocyte enhancing factor-2, or mutation of the myocyte enhancing factor-2 site in the KLF2 promoter, abrogates LDLinduced downregulation of endothelial KLF2 and thrombomodulin, and KLF2 promoter activity. Similarly, knockdown of enhancer of zeste homolog 2 negates LDL-induced downregulation of KLF2 and thrombomodulin in endothelial cells. for myocyte enhancing factor-2 (MEF2), a transcription factor which upregulates KLF2, 9,10,15 and the tumor suppressing transcription factor p53 which downregulates KLF2. 16 Although studies on epigenetic modulation of KLF2 expression are limited, emerging evidence suggests that histone modifiers, such as the histone methyltransferase enhancer of zeste homolog 2 (EZH2), the catalytic subunit of Polycomb repressive complex 2 which is implicated in tumorigenesis and is responsible for trimethylation of Histone 3 on lysine 27, play an important part in governing KLF2 expression. 17Cholesterol-rich lipid particles and hypercholesterolemia are associated with epigenetic changes in vitro and in experimental animal models as well as in humans. ApoE −/− mice fed a high-fat diet have aberrant DNA methylation patterns, including decreased global methylation in aorta and peripheral blood mononuclear cells, 18 and human atherosclerotic tissue samples display genomic hypomethylation.19 P66shc is one of the genes epigenetically modified by cholesterolrich proatherogenic particles, such as LDL.20 P66shc mediates endothelial oxidative stress and fatty streak formation in hypercholesterolemic mice. 21 However, p66shc is only one of many genes whose epigenetic modification by hypercholesterolemia may contribute to the development of vascular disease. Intrigued by the possibility that endothelial KLF2 might also be epigenetically regulated in a hypercholesterolemic environment, we asked whether LDL cholesterol changes endothelial KLF2 transcription, and investigated the rol...

The Complex Cardiac Atherosclerotic Disorder: The Elusive Role of Genetics and the New Consensus of Systems Biology Approach

“…But, soon it was realized that modern genomic technologies like SNP array, gene expression microarray and microRNA array were not sufficient to explain the genetic inclination in patients with CAD. Thus, GWAS showed that only 10.6% of people with atherosclerosis had a probable heritable genetic factor [10,11]. In the remaining cases epigenetic studies identified specific CAD risk loci with the DNA methylation status to play significant role [10,11].…”

Enzymatic Targets in Atherosclerosis

“…Both histone-and DNA-methylases were implicated in the LDL-induced repression of KLF2 transcription in endothelial cells. LDL-induced endothelial dysfunction effects were prevented by pharmacological inhibition or gene silencing of DNMT [258]. DNA methylation was also implicated in the reduced autophagy of THP-1 macrophages upon exposure to oxidized LDL (oxLDL) [259].…”

MiR-92a

“…Interestingly, a recent report showed that KLF2 is downregulated by LDL, and that the repression of KLF2 is mediated by both DNA and histone methylation, 8 suggesting that the oxLDL-induced repression of KLF2 is not solely mediated by miR-92a, but that other epigenetic phenomena also play an important role. Both endothelial KLF2 and KLF4 have been shown to be atheroprotective.…”

“…4 Inhibition of miR-92a also reduced the expression of the active form of nuclear factor-κB, phospho-p65, an important driver of inflammation, and promoted the expression of NOS3, 4 both targets of KLF2 and KLF4. 7 Interestingly, a recent report showed that KLF2 is downregulated by LDL, and that the repression of KLF2 is mediated by both DNA and histone methylation, 8 suggesting that the oxLDL-induced repression of KLF2 is not solely mediated by miR-92a, but that other epigenetic phenomena also play an important role. Both endothelial KLF2 and KLF4 have been shown to be atheroprotective.…”