Inhibiting epigenetic enzymes to improve atherogenic macrophage functions Jan Van den Bossche a,⇑ , Annette E. Neele a , Marten A. Hoeksema a , Femke de Heij a , Marieke C.S. Boshuizen a , Saskia van der Velden a , Vincent C. de Boer b , Kris A. Reedquist c , Menno P.J. de Winther a a Department of Medical Biochemistry, Experimental Vascular Biology, Academic Medical Center, Amsterdam, The Netherlands b Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam, The Netherlands c Department of Rheumatology and Clinical Immunology, Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands article info Article history: Received 27 October 2014 Available online 18 November 2014 Keywords: Macrophages Inflammation Atherosclerosis Epigenetics Histone deacetylases (HDACs) Chromatin modifying enzymes (CME) abstract Macrophages determine the outcome of atherosclerosis by propagating inflammatory responses, foam cell formation and eventually necrotic core development. Yet, the pathways that regulate their atherogenic functions remain ill-defined. It is now apparent that chromatin remodeling chromatin modifying enzymes (CME) governs immune responses but it remains unclear to what extent they control atherogenic macrophage functions. We hypothesized that epigenetic mechanisms regulate atherogenic macrophage functions, thereby determining the outcome of atherosclerosis. Therefore, we designed a quantitative semi-high-throughput screening platform and studied whether the inhibition of CME can be applied to improve atherogenic macrophage activities. We found that broad spectrum inhibition of histone deacetylases (HDACs) and histone methyltransfer- ases (HMT) has both pro- and anti-inflammatory effects. The inhibition of HDACs increased histone acet- ylation and gene expression of the cholesterol efflux regulators ATP-binding cassette transporters ABCA1 and ABCG1, but left foam cell formation unaffected. HDAC inhibition altered macrophage metabolism towards enhanced glycolysis and oxidative phosphorylation and resulted in protection against apoptosis. Finally, we applied inhibitors against specific HDACs and found that HDAC3 inhibition phenocopies the atheroprotective effects of pan-HDAC inhibitors. Based on our data, we propose the inhibition of HDACs, and in particular HDAC3, in macrophages as a novel potential target to treat atherosclerosis. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Current atherosclerosis medication efficiently lowers plasma cholesterol levels, but reduces the risk for cardiovascular disease only partially. Therefore, alternative treatment strategies are needed [1]. During atherosclerosis initiation, accumulation of low-density lipoproteins (LDL) and their modifications (e.g. oxida- tion to oxLDL) in the arterial wall activate endothelial cells. Attracted monocytes adhere to and migrate into the vessel wall, where they differentiate into macrophages and can become lipid- loaded foam cells. Upon atherosclerosis progression, additional immune cells propagate chronic inflammation and proliferating smooth muscle cells enclose the lesion with a fibrous cap. Apopto- tic foam cells can be eliminated by neighboring macrophages through efferocytosis or can form a necrotic core. Additionally, macrophage-derived metalloproteases can induce thinning of the fibrous cap and when so-called ‘vulnerable’ plaques rupture this can cause myocardial infarction and stroke [2]. While macrophages clearly play a central role in different stages of atherogenesis, surprisingly little is known about the molecular mechanisms that regulate their phenotype within the plaque [3]. Macrophages display high heterogeneity and in response to the microenvironment adopt different polarization states [4]. Classi- cally activated (M1) macrophages, activated by Toll-like-receptor (TLR) triggers and Th1 cytokines, are pro-inflammatory and are therefore regarded as pro-atherogenic. Interleukin-4 (IL-4)/IL-13- induced alternatively activated (M2) macrophages are considered http://dx.doi.org/10.1016/j.bbrc.2014.11.029 0006-291X/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author at: Experimental Vascular Biology, Department of Medical Biochemistry, Academic Medical Center, Meibergdreef 9, 1105AZ Amsterdam, The Netherlands. E-mail address: j.vandenbossche@amc.uva.nl (J. Van den Bossche). Biochemical and Biophysical Research Communications 455 (2014) 396–402 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc