title: Hemodynamic disturbed flow induces differential DNA methylation of endothelial Kruppel-Like Factor 4 promoter in vitro and in vivo.
abstract: BACKGROUND
  Hemodynamic disturbed flow (DF) is associated with susceptibility to atherosclerosis. Endothelial Kruppel-Like Factor 4 (KLF4) is an important anti-inflammatory atheroprotective transcription factor that is suppressed in regions of DF.
  
  
  OBJECTIVE
  The plasticity of epigenomic KLF4 transcriptional regulation by flow-mediated DNA methylation was investigated in vitro and in arterial tissue.
  
  
  RESULTS
  To recapitulate dominant flow characteristics of atheroprotected and atherosusceptible arteries, human aortic endothelial cells were subjected to pulsatile undisturbed flow or oscillatory DF containing a flow-reversing phase. Differential CpG site methylation was measured by methylation-specific polymerase chain reaction, bisulfite pyrosequencing, and restriction enzyme-polymerase chain reaction. 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 knockdown 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 myocyte enhancer factor-2 binding site in the KLF4 promoter near the transcription start site. DF also induced DNMT-sensitive propathological expression of downstream KLF4 transcription targets nitric oxide synthase 3, thrombomodulin, and monocyte chemoattractant protein-1. In support of the in vitro findings, swine aortic endothelium isolated from DF regions expressed significantly lower KLF4 and nitric oxide synthase 3, and bisulfite sequencing of KLF4 promoter identified a hypermethylated myocyte enhancer factor-2 binding site.
  
  
  CONCLUSIONS
  Hemodynamics influence endothelial KLF4 expression through DNMT enrichment/myocyte enhancer factor-2 inhibition mechanisms of KLF4 promoter CpG methylation with regional consequences for atherosusceptibility.
authors.affiliation: From the Department of Pathology & Laboratory Medicine and Institute for Medicine & Engineering (Y.-Z.J., J.M.J., M.E.M., L.-D.Z., P.F.D.) and Pharmacology Graduate Group (K.O.), Perelman School of Medicine, University of Pennsylvania, Philadelphia.
authors.forename: Yi-Zhou
authors.initials: YZ
authors.lastname: Jiang
authors.affiliation: From the Department of Pathology & Laboratory Medicine and Institute for Medicine & Engineering (Y.-Z.J., J.M.J., M.E.M., L.-D.Z., P.F.D.) and Pharmacology Graduate Group (K.O.), Perelman School of Medicine, University of Pennsylvania, Philadelphia.
authors.forename: Juan M
authors.initials: JM
authors.lastname: Jiménez
authors.affiliation: From the Department of Pathology & Laboratory Medicine and Institute for Medicine & Engineering (Y.-Z.J., J.M.J., M.E.M., L.-D.Z., P.F.D.) and Pharmacology Graduate Group (K.O.), Perelman School of Medicine, University of Pennsylvania, Philadelphia.
authors.forename: Kristy
authors.initials: K
authors.lastname: Ou
authors.affiliation: From the Department of Pathology & Laboratory Medicine and Institute for Medicine & Engineering (Y.-Z.J., J.M.J., M.E.M., L.-D.Z., P.F.D.) and Pharmacology Graduate Group (K.O.), Perelman School of Medicine, University of Pennsylvania, Philadelphia.
authors.forename: Margaret E
authors.initials: ME
authors.lastname: McCormick
authors.affiliation: From the Department of Pathology & Laboratory Medicine and Institute for Medicine & Engineering (Y.-Z.J., J.M.J., M.E.M., L.-D.Z., P.F.D.) and Pharmacology Graduate Group (K.O.), Perelman School of Medicine, University of Pennsylvania, Philadelphia.
authors.forename: Ling-Di
authors.initials: LD
authors.lastname: Zhang
authors.affiliation: From the Department of Pathology & Laboratory Medicine and Institute for Medicine & Engineering (Y.-Z.J., J.M.J., M.E.M., L.-D.Z., P.F.D.) and Pharmacology Graduate Group (K.O.), Perelman School of Medicine, University of Pennsylvania, Philadelphia. pfd@mail.med.upenn.edu.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Davies
mesh_terms: D000818:Animals; D050197:Atherosclerosis / Q000209:etiology*; D001775:Blood Circulation; D002478:Cells, Cultured; D018899:CpG Islands; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000502:physiology; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D004730:Endothelium, Vascular / Q000378:metabolism; D006439:Hemodynamics; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics*; D064326:MEF2 Transcription Factors / Q000235:genetics; D052250:Nitric Oxide Synthase Type III / Q000235:genetics; D011401:Promoter Regions, Genetic; D013552:Swine
chemical_list: C000717967:DNMT3A protein, human; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D064326:MEF2 Transcription Factors; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A
keywords: DNA methylation; atherosclerosis; epigenetics; hemodynamics
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