Atherosclerosis 217 (2011) 427–432 Contents lists available at ScienceDirect Atherosclerosis jo ur nal homep age : www.elsevier.com/locate/atherosclerosis Imaging mass spectrometry-based histopathologic examination of atherosclerotic lesions Nobuhiro Zaima a,b,∗ , Takeshi Sasaki c,d , Hiroki Tanaka a,e , Xian Wu Cheng d , Kenji Onoue a , Takahiro Hayasaka a , Naoko Goto-Inoue a , Hirofumi Enomoto a , Naoki Unno e , Masafumi Kuzuya d , Mitsutoshi Setou a,∗∗ a Department of Cell Biology and Anatomy, Hamamatsu University School of Medicine, 1-20-1, Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan b Department of Applied Biological Chemistry, Kinki University, 3327-204, Naka-machi, Nara 631-8505, Japan c Department of Anatomy and Neuroscience, Hamamatsu University School of Medicine, Japan d Department of Geriatrics, Nagoya University Graduate School of Medicine, Japan e Department of Vascular Surgery, Hamamatsu University School of Medicine, Japan a r t i c l e i n f o Article history: Received 26 November 2010 Received in revised form 18 March 2011 Accepted 30 March 2011 Available online 9 April 2011 Keywords: ApoE-deficient mouse Lipids Smooth muscle cells Arachidonic acid Phosphatidylcholine Imaging mass spectrometry a b s t r a c t Aims: Imaging mass spectrometry (IMS) enables the visualization of individual molecules present on tissue sections. We attempted to identify and visualize specific markers for aortic atherosclerotic lesions. Methods and results: Atherosclerotic lesions were obtained from aortic roots of apolipoprotein E (ApoE)- deficient mice at 60 weeks of age and from femoral arteries of humans with peripheral artery occlusive disease. IMS was performed with a matrix-assisted laser desorption/ionization mass spectrometry time- of-flight (TOF)/TOF-type instrument. The molecular ions at m/z 671.6 and 673.6 were found to be specific molecules in the mouse and human lipid-rich regions. These molecules were assigned as cholesterol linoleate (CE 18:2) and cholesterol oleate (CE 18:1). In the case of the human samples, triacylglycerol was also localized in the lipid-rich regions. The distributions of the molecular ions at m/z 804.5 and 832.5 were the same as the distribution of both the mouse and the human SMCs. These molecules were assigned as phosphatidylcholine (PC) (diacyl 16:0/20:4) and PC (diacyl 18:0/20:4). The molecular ion at m/z 566.9 was localized in the mouse calcified regions, and the molecular ions at m/z 539.0 were localized in the human calcified regions. Conclusions: The IMS-based histopathologic examination (IbHE) revealed the characteristic peaks of lipid- rich regions, SMCs, and calcified regions in the atherosclerotic lesions. In addition, IbHE revealed the characteristic distribution of lipids in human atherosclerotic lesions. These data indicate that an IMS- based pathologic approach is of considerable value as a new histopathologic examination. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Atherosclerosis is a progressive and multigenic vascular disease that involves plaque formation in intimal areas that include var- ious cells and molecules. The development of an atherosclerotic plaque is reported to be closely associated with dyslipidemia and chronic inflammation [1]. The increased permeability of the vas- cular endothelium causes the intimal accumulation of oxidized low-density lipoprotein (LDL) [2]. Monocytes that are recruited ∗ Corresponding author at: Department of Applied Biological Chemistry, Kinki University, 3327-204, Naka-machi, Nara 631-8505, Japan. Tel.: +81 742 43 8067; fax: +81 742 43 8067. ∗∗ Corresponding author. Tel.: +81 53 435 2292; fax: +81 53 435 2292. E-mail addresses: zaimanobuhiro@gmail.com (N. Zaima), setou@hama- med.ac.jp (M. Setou). to the intima and subintima transform into macrophages and foam cells through the increased uptake of modified LDL, thereby leading to the formation of early atherosclerotic lesions (fatty streaks) [1]. Fatty streaks grow into atherosclerotic plaques follow- ing the accumulation of inflammatory cells and the evolution of a lipid core region that is surrounded by a cap of smooth mus- cle cells (SMCs) [3,4]. In the lipid-rich atherosclerotic plaques, such as macrophages or SMCs occurs, and these apoptotic cells form a necrotic core [5]. The persistence of these processes finally leads to vascular calcification and rupture of the plaque [6]. The development of atherosclerosis is accompanied by the progressive alteration of the vascular walls [7]. Therefore, it is important to examine the condition of the vascular walls to estimate the pathol- ogy of atherosclerosis. Conventional stainings such as oil staining or immunostaining are essential for examination of the patho- logic process of atherosclerosis. However, conventional stainings have 3 weaknesses. The first is that conventional oil staining can- 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.03.044