Hemodynamic Disturbed Flow Induces Differential DNA Methylation of Endothelial Kruppel-Like Factor 4 (KLF4) Promoter In Vitro and In Vivo
Rationale: Hemodynamic disturbed flow is associated with susceptibility to atherosclerosis. Endothelial KLF4 is an important anti-inflammatory atheroprotective transcription factor that is suppressed in regions of disturbed flow.
Objective: The plasticity of epigenomic KLF4 transcriptional regulation by flow-mediated DNA methylation was investigated in vitro and in arterial tissue.
Methods and Results: To recapitulate dominant flow characteristics of atheroprotected and atherosusceptible arteries, human aortic endothelial cells (HAEC) were subjected to pulsatile undisturbed flow (UF) or oscillatory disturbed flow (DF) containing a flow-reversing phase. Differential CpG site methylation was measured by methylation specific PCR, bisulfite pyrosequencing and restriction enzyme-PCR. The methylation profiles of endothelium from disturbed and undisturbed flow sites of adult swine aortas were also investigated. In vitro, DF increased DNA methylation of CpG islands within the KLF4 promoter that significantly contributed to suppression of KLF4 transcription; the effects were mitigated by DNA methyltransferase (DNMT) inhibitors and knock-down of DNMT3A. Contributory mechanisms included DF-induced increase of DNMT3A protein (1.7 fold), DNMT3A enrichment (11-fold) on the KLF4 promoter, and competitive blocking of a MEF2 binding site in the KLF4 promoter near the TSS. DF also induced DNMT-sensitive pro-pathological expression of downstream KLF4 transcription targets NOS3, thrombomodulin (THBD) and MCP-1. In support of the in vitro findings, swine aortic endothelium isolated from DF regions expressed significantly lower KLF4 and NOS3, and bisulfite sequencing of KLF4 promoter identified a hypermethylated MEF2 binding site.
Conclusions: Hemodynamics influence endothelial KLF4 expression through DNMT enrichment/MEF2 inhibition mechanisms of KLF4 promoter CpG methylation with regional consequences for atherosusceptibility.
- Received March 7, 2014.
- Revision received April 21, 2014.
- Accepted April 22, 2014.