Epigenetic mechanisms that regulate endothelial cell (EC) gene expression are now emerging. DNA methylation is the most stable epigenetic mark that confers persisting changes in gene expression. Not only is DNA methylation important in rendering cell identity by regulating cell type-specific gene expression throughout differentiation, but it is becoming clear that DNA methylation also plays a key role in maintaining EC homeostasis and in vascular disease development. Disturbed blood flow (d-flow) causes atherosclerosis while stable flow (s-flow) protects against it by differentially regulating gene expression in ECs. Recently, we and others have shown that flow-dependent gene expression and atherosclerosis development are regulated by mechanisms dependent on DNA methyltransferases (DNMT1 and 3A). D-flow upregulates DNMT expression both in vitro and in vivo which leads to genome-wide DNA methylation alterations and global gene expression changes in a DNMT-dependent manner. These studies revealed several mechanosensitive genes, such as HoxA5, Klf3, and Klf4, whose promoters were hypermethylated by d-flow, but rescued by DNMT inhibitors such as 5Aza-2-deoxycytidine. These findings provide new insight into the mechanism by which flow controls epigenomic DNA methylation patterns, which in turn alters endothelial gene expression, regulates vascular biology, and induces atherosclerosis.

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.

Atherosclerosis is a multifactorial disease that preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow). The mechanisms by which d-flow induces atherosclerosis involve changes in the transcriptome, methylome, proteome, and metabolome of multiple vascular cells, especially endothelial cells. Initially, we begin with the pathogenesis of atherosclerosis and the changes that occur at multiple levels owing to d-flow, especially in the endothelium. Also, there are a variety of strategies used for the global profiling of the genome, transcriptome, miRNAnome, DNA methylome, and metabolome that are important to define the biological and pathophysiological mechanisms of endothelial dysfunction and atherosclerosis. Finally, systems biology can be used to integrate these 'omics' datasets, especially those that derive data based on a single animal model, in order to better understand the pathophysiology of atherosclerosis development in a holistic manner and how this integrative approach could be used to identify novel molecular diagnostics and therapeutic targets to prevent or treat atherosclerosis.

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Background: Alternative macrophages exist in human atherosclerosis but their role in atherogenesis remains uncertain. Intraplaque hemorrhage (IPH) is an important stimulus driving alternative macrophage polarization. Intake of hemoglobin (Hb) by the hemoglobin: haptoglobin receptor CD163 leads to a distinct non-foam cell phenotype termed M(Hb). These cells demonstrate upregulation of CD163, lack of lipid retention, and anti-oxidative properties, characteristics considered ‘atheroprotective’. Here we reveal an unexpected but important pathogenic role for M(Hb) in atherosclerosis. Objectives: To determine the role of M(Hb) macrophages in human intraplaque angiogenesis and vascular permeability. Methods: Using human atherosclerotic samples, cultured cells, and a mouse model of IPH, we investigated the role of IPH on macrophage function with respect to angiogenesis and vascular permeability. Results: Within M(Hb) activation of hypoxia-inducible factor-1 alpha (HIF-1) via inhibition of prolyl hydroxylases promotes intraplaque angiogenesis and vascular permeability. In human carotid plaques, alternative CD163 positive macrophages were found to be highly associated with plaque vascularity and expressed high levels of HIF1- and vascular endothelial growth factor-A (VEGF-A). Supernatants from hemoglobin:haptoglobin differentiated M(Hb) macrophages increased endothelial permeability and led to internalization of the endothelial barrier protein vascular endothelial cadherin (VE-cadherin) via activation of VEGF receptor 2 (VEGFR2). Areas of plaque demonstrating high density CD163 high macrophage subsets showed irregular VE-cadherin immunostaining and diffuse perivascular collections of von Willebrand factor suggesting microvessel incompetence. Finally, in brachiocephalic plaques of one-year-old apoE -/- and apoE -/- CD163 -/- mice, CD163 deficiency significantly reduced plaque progression, lesion size, and intraplaque hemorrhage, but had little effect on lesions uncomplicated by hemorrhage. Conclusions: Our findings provide a novel non-lipid driven mechanism by which alterative M(Hb) macrophages promote plaque neoangiogenesis and microvessel incompetence via a HIF-1/VEGF-A-dependent pathway.