Flow-dependent epigenetic DNA methylation regulates endothelial gene expression and atherosclerosis
Jessilyn Dunn, … , I. King Jordan, Hanjoong Jo
Jessilyn Dunn, … , I. King Jordan, Hanjoong Jo
Published May 27, 2014
Citation Information: J Clin Invest. 2014;124(7):3187-3199. https://doi.org/10.1172/JCI74792.
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Flow-dependent epigenetic DNA methylation regulates endothelial gene expression and atherosclerosis

Abstract

In atherosclerosis, plaques preferentially develop in arterial regions of disturbed blood flow (d-flow), which alters endothelial gene expression and function. Here, we determined that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase–dependent (DNMT-dependent) manner. Induction of d-flow by partial carotid ligation surgery in a murine model induced DNMT1 in arterial endothelium. In cultured endothelial cells, DNMT1 was enhanced by oscillatory shear stress (OS), and reduction of DNMT with either the inhibitor 5-aza-2′-deoxycytidine (5Aza) or siRNA markedly reduced OS-induced endothelial inflammation. Moreover, administration of 5Aza reduced lesion formation in 2 mouse models of atherosclerosis. Using both reduced representation bisulfite sequencing (RRBS) and microarray, we determined that d-flow in the carotid artery resulted in hypermethylation within the promoters of 11 mechanosensitive genes and that 5Aza treatment restored normal methylation patterns. Of the identified genes, HoxA5 and Klf3 encode transcription factors that contain cAMP response elements, suggesting that the methylation status of these loci could serve as a mechanosensitive master switch in gene expression. Together, our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis.

Authors

Jessilyn Dunn, Haiwei Qiu, Soyeon Kim, Daudi Jjingo, Ryan Hoffman, Chan Woo Kim, Inhwan Jang, Dong Ju Son, Daniel Kim, Chenyi Pan, Yuhong Fan, I. King Jordan, Hanjoong Jo

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Figure 1

DNMT1 expression is induced by d-flow in endothelial cells in vivo and in vitro.

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DNMT1 expression is induced by d-flow in endothelial cells in vivo and i...
(A) Schematic diagram of the partial carotid ligation model, in which 3 of the 4 caudal branches of the left common carotid artery (LCA) are ligated, while the contralateral right common carotid artery (RCA) remains untouched as an internal control. Also depicted are the naturally, chronically flow-disturbed lesser curvature (LC) and the unidirectional-flow greater curvature (GC). (B) Validation of microarray results by qPCR was performed with endothelial-enriched total RNA obtained from the LCA and RCA at 0, 24, and 48 hours after ligation in C57BL/6 mice. DNMT1 mRNA levels were normalized to 18S. Data are shown as the mean ± SEM. *P < 0.05, n = 8 (0 hours), n = 9 (24 hours), n = 12 (48 hours). (C and D) LCA and RCA frozen sections 48 hours after ligation (scale bars: 50 μm) (C) and the LC and GC (en face preparation; scale bars: 100 μm) from C57BL/6 mice without ligation surgery (D) were stained with DNMT1 antibody (red). Nuclei are stained with DAPI (blue), and elastic laminae autofluoresce green. The lumen (L) is indicated. (E and F) DNMT1 expression is increased by oscillatory shear stress (OS) compared with laminar shear stress (LS). HUVECs exposed to LS (15 dyn/cm2) or OS (±5 dyn/cm2, at 1 Hz) for 24 hours were used for qPCR and Western blot analyses for DNMT1 using 18S and β-actin as respective internal controls. The bar graph (F) shows ImageJ Western blot quantification normalized to β-actin (n = 7 each, data are shown as the mean ± SEM. *P < 0.05; **P < 0.01).