Apocynin suppresses the progression of atherosclerosis in apoE-deficient mice by inactivation of macrophages Hiroyuki Kinoshita a,1 , Takeshi Matsumura a,⇑,1 , Norio Ishii a , Kazuki Fukuda a , Takafumi Senokuchi a , Hiroyuki Motoshima a , Tatsuya Kondo a , Kayo Taketa a , Shuji Kawasaki a , Satoko Hanatani a , Motohiro Takeya b , Takeshi Nishikawa a , Eiichi Araki a a Department of Metabolic Medicine, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556, Japan b Department of Cell Pathology, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556, Japan article info Article history: Received 3 January 2013 Available online 11 January 2013 Keywords: Apocynin Reactive oxygen species NADPH oxidase Atherosclerosis Macrophages abstract Production of reactive oxygen species (ROS) and other proinflammatory substances by macrophages plays an important role in atherogenesis. Apocynin (4-hydroxy-3-methoxy-acetophenone), which is well known as a NADPH oxidase inhibitor, has anti-inflammatory effects including suppression of the gener- ation of ROS. However, the suppressive effects of apocynin on the progression of atherosclerosis are not clearly understood. Thus, we investigated anti-atherosclerotic effects of apocynin using apolipoprotein E-deficient (apoE –/– ) mice in vivo and in mouse peritoneal macrophages in vitro. In atherosclerosis-prone apoE –/– mice, apocynin suppressed the progression of atherosclerosis, decreased 4-hydroxynonenal- positive area in atherosclerotic lesions, and mRNA expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) in aorta. In mouse peritoneal macrophages, apocynin suppressed the Ox-LDL-induced ROS generation, mRNA expression of MCP-1, IL-6 and granulocyte/macrophage colony-stimulating factor, and cell proliferation. Moreover, immunohistochemical studies revealed that apocynin decreased the number of proliferating cell nuclear antigen-positive macrophages in atheroscle- rotic lesions of apoE –/– mice. These results suggested that apocynin suppressed the formation of athero- sclerotic lesions, at least in part, by inactivation of macrophages. Therefore, apocynin may be a potential therapeutic material to prevent the progression of atherosclerosis. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction The progression of atherosclerosis and the stability of atheroscle- rotic plaques decide cardiovascular disease outcome. The develop- ment of atherosclerosis depends on a fragile balance between proinflammatory and oxidative stimuli on one phase, and anti- inflammatory and anti-oxidative defense mechanisms on the other phase [1,2]. Therefore, the control of reactive oxygen species (ROS) is one of the beneficial steps for the prevention of cardiovascular disease. Apocynin (4-hydroxy-3-methoxy-acetophenone) is a constitu- ent of the Himalayan herb Picrorhiza kurroa Royle (Scrophularia- ceae) that is well known as a traditional Indian medicine (Ayurveda). It has been reported that apocynin has several anti- inflammatory effects such as the suppression of neutrophil oxida- tive burst, neutrophil-mediated oxidative tissue damage [3,4], monocyte adhesion to endothelial cells [5], chemotaxis of poly- morphonuclear granulocyte [6], peroxynitrite production by mac- rophages [7] and cyclooxygenase-2 expression in human monocytes [8]. The underlying mechanism for these biological ef- fects of apocynin is explained by the inhibition of NADPH oxidases, superoxide O À 2 generating enzymes [3,9]. NADPH oxidases play critical roles in the regulation of inflam- matory processes by promoting oxidation of lipids and proteins, which ultimately leads to tissue damages and organ failure [10]. NADPH oxidases are expressed in all vascular cells, including endo- thelial cells (ECs), smooth muscle cells (SMCs), fibroblasts, and monocytes/macrophages [10,11]. Notably, NADPH oxidases also plays a key role in the development and progression of atheroscle- rotic lesions [12]. Interestingly, apocynin has been reported to have both NADPH oxidase-dependent and NADPH oxidase-independent anti-oxidative effects [13]. It is well known that atherosclerosis is a chronic inflammatory disease, and macrophages and macrophage-derived foam cells play pivotal roles in the progression of atherosclerosis by expressing inflammatory cytokines and chemokines [1]. Since apocynin has both anti-inflammatory and anti-oxidative effects, apocynin may suppress the progression of atherosclerosis via inactivation of macrophages. However, the suppressive effects of apocynin on 0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2013.01.014 ⇑ Corresponding author. Address: Department of Metabolic Medicine, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan. Fax: +81 96 366 8397. E-mail address: takeshim@gpo.kumamoto-u.ac.jp (T. Matsumura). 1 These authors contributed equally to this work. Biochemical and Biophysical Research Communications 431 (2013) 124–130 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc