The role of epigenetics in the endothelial cell shear stress response and atherosclerosis

Abstract

Currently in the field of vascular biology, the role of epigenetics in endothelial cell biology and vascular disease has attracted more in-depth study. Using both in vitro and in vivo models of blood flow, investigators have recently begun to reveal the underlying epigenetic regulation of endothelial gene expression. Recently, our group, along with two other independent groups, have demonstrated that blood flow controls endothelial gene expression by DNA methyltransferases (DNMT1 and 3A). Disturbed flow (d-flow), characterized by low and oscillating shear stress (OS), is pro-atherogenic and induces expression of DNMT1 both in vivo and in vitro. D-flow regulates genome-wide DNA methylation patterns in a DNMT-dependent manner. The DNMT inhibitor 5-Aza-2'deoxycytidine (5Aza) or DNMT1 siRNA reduces OS-induced endothelial inflammation. Moreover, 5Aza inhibits the development of atherosclerosis in ApoE(-/-) mice. Through a systems biological analysis of genome-wide DNA methylation patterns and gene expression data, we found 11 mechanosensitive genes which were suppressed by d-flow in vivo, experienced hypermethylation in their promoter region in response to d-flow, and were rescued by 5Aza treatment. Interestingly, among these mechanosensitive genes, the two transcription factors HoxA5 and Klf3 contain cAMP-response-elements (CRE), which may indicate that methylation of CRE sites could serve as a mechanosensitive master switch in gene expression. These findings provide new insight into the mechanism by which flow controls epigenetic DNA methylation patterns, which in turn alters endothelial gene expression, regulates vascular biology, and induces atherosclerosis. These novel findings have broad implications for understanding the biochemical mechanisms of atherogenesis and provide a basis for identifying potential therapeutic targets for atherosclerosis. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.

Keywords: Atherosclerosis; DNMT; Endothelial function; Epigenetic DNA methylation; Flow; Gene expression; Shear stress.

Figure 1. Flow regulates DNA methylation of Hox family

HoxA, HoxB, and HoxD gene family DNA methylation changes in endothelial cells due to disturbed flow are seen by reduced representation bisulfite sequencing (RRBS) in the UCSC Genome Browser from our mouse partial carotid ligation model (A). RRBS data in the UCSC Genome Browser showing 5Aza-reversible, d-flow-induced Hox promoter hypermethylation regions. Differentially methylated regions by shear stress are boxed in green (B). High methylation is denoted in yellow and low methylation is denoted in red.

Figure 1. Flow regulates DNA methylation of Hox family

HoxA, HoxB, and HoxD gene family DNA methylation changes in endothelial cells due to disturbed flow are seen by reduced representation bisulfite sequencing (RRBS) in the UCSC Genome Browser from our mouse partial carotid ligation model (A). RRBS data in the UCSC Genome Browser showing 5Aza-reversible, d-flow-induced Hox promoter hypermethylation regions. Differentially methylated regions by shear stress are boxed in green (B). High methylation is denoted in yellow and low methylation is denoted in red.

Figure 2. Flow- and differentiation-dependent methylation changes in the HoxA5 promoter

UCSC Genome Browser view of the RRBS data showing flow-dependent methylation changes in the HoxA5 promoter (A). The ENCODE consortium ChIP-Seq and Methyl450k array datasets for HUVEC show that the transcription factor CTCF and RNA Polymerase II are enriched at the HoxA5 promoter where there is a high density of CpG sites (B). Zoomed-in view of the red boxed area from panel A (C). UCSC Genome Browser screenshot of the ENCODE Methyl450k array datasets shows a comparison of the HoxA5 promoter methylation profile across cell types from the least differentiated (human embryonic stem cells) to the most endothelial-like (HUVECs). Highly methylated regions in orange, partially methylated regions in purple, and unmethylated regions in blue. (D).

Figure 2. Flow- and differentiation-dependent methylation changes in the HoxA5 promoter

UCSC Genome Browser view of the RRBS data showing flow-dependent methylation changes in the HoxA5 promoter (A). The ENCODE consortium ChIP-Seq and Methyl450k array datasets for HUVEC show that the transcription factor CTCF and RNA Polymerase II are enriched at the HoxA5 promoter where there is a high density of CpG sites (B). Zoomed-in view of the red boxed area from panel A (C). UCSC Genome Browser screenshot of the ENCODE Methyl450k array datasets shows a comparison of the HoxA5 promoter methylation profile across cell types from the least differentiated (human embryonic stem cells) to the most endothelial-like (HUVECs). Highly methylated regions in orange, partially methylated regions in purple, and unmethylated regions in blue. (D).