Circ Cardiovasc Imaging Development of Receptor for Advanced Glycation End Products–Directed Imaging of Atherosclerotic Plaque in a Murine Model of Spontaneous AtherosclerosisCLINICAL PERSPECTIVE by Yared Tekabe, Qing Li, Rosa Rosario, Marija Sedlar, Stan Majewski, Barry I. Hudson, Andrew J. Einstein, Ann Marie Schmidt, and Lynne L. Johnson Circ Cardiovasc Imaging Volume 1(3):212-219 November 19, 2008 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. A, Planar γ images of the anteroposterior (AP) and lateral (LAT) views of a 20-week apoE−/− mouse on Western-type diet with atherosclerotic lesion 4 hours after intravenous injection of 99mTc-labeled anti-RAGE F(ab′)2, corresponding to the location of the atherosclerotic lesions shown on the photograph. Figure 1. A, Planar γ images of the anteroposterior (AP) and lateral (LAT) views of a 20-week apoE−/− mouse on Western-type diet with atherosclerotic lesion 4 hours after intravenous injection of 99mTc-labeled anti-RAGE F(ab′)2, corresponding to the location of the atherosclerotic lesions shown on the photograph. B, Planar γ images of the anteroposterior and lateral views of a 20-week apoE−/− mouse on Western-type diet with atherosclerotic lesion 4 hours after intravenous injection of 99mTc-labeled nonimmune rabbit IgG F(ab′)2 show no tracer uptake in the thorax, although the in situ dissection of the aortic arch showed extensive atherosclerotic plaque. C, Anteroposterior planar γ image of a wild-type control C57BL/6 mouse 4 hours after intravenous injection of 99mTc-labeled anti-RAGE F(ab′)2 and gross examination of the aorta revealed no lesion. Yared Tekabe et al. Circ Cardiovasc Imaging. 2008;1:212-219 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. A, Bar graph shows uptake of radiotracer in thoracic organs expressed as % ID/g in apoE−/− mice injected with 99mTc anti-RAGE F(ab′)2. Figure 2. A, Bar graph shows uptake of radiotracer in thoracic organs expressed as % ID/g in apoE−/− mice injected with 99mTc anti-RAGE F(ab′)2. There is significantly higher uptake in the proximal aorta as compared with the uptake in the heart or the lungs. B, Graph shows uptake of radiotracer in the proximal aorta expressed as % ID/g for apoE−/− mice injected with 99mTc anti-RAGE F(ab′)2 (striped bar), apoE−/− mice injected with 99mTc-labeled nonimmune IgG (black bar), and C57BL/6 mice injected with 99mTc anti-RAGE F(ab′)2 (gray bar). Yared Tekabe et al. Circ Cardiovasc Imaging. 2008;1:212-219 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. Biodistribution of 99mTc-labeled anti-RAGE F(ab′)2 (open bar) and nonimmune IgG F(ab′)2 (black bar) 4 hours after intravenous administration of the radiotracer in nontarget organs of 20-week apoE−/− mice. Figure 3. Biodistribution of 99mTc-labeled anti-RAGE F(ab′)2 (open bar) and nonimmune IgG F(ab′)2 (black bar) 4 hours after intravenous administration of the radiotracer in nontarget organs of 20-week apoE−/− mice. Yared Tekabe et al. Circ Cardiovasc Imaging. 2008;1:212-219 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Epifluorescent micrographs of 5-μm-thick paraffin section of the aortic sinus from an apoE−/− mouse injected with 99mTc and rhodamine-labeled anti-RAGE F(ab′)2 (A) and immunohistochemical stained adjacent sections of the aortic sinus with anti-RAGE IgG (B). Figure 4. Epifluorescent micrographs of 5-μm-thick paraffin section of the aortic sinus from an apoE−/− mouse injected with 99mTc and rhodamine-labeled anti-RAGE F(ab′)2 (A) and immunohistochemical stained adjacent sections of the aortic sinus with anti-RAGE IgG (B). Fluorescence in the aorta corresponds to localization of RAGE-specific staining in the lesion. (Magnification ×100.)‏ Yared Tekabe et al. Circ Cardiovasc Imaging. 2008;1:212-219 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. A, Immunohistochemical characterization of atherosclerotic lesions in 20-week apoE−/− mouse. Figure 5. A, Immunohistochemical characterization of atherosclerotic lesions in 20-week apoE−/− mouse. Serial sections were stained for endothelial cells (FVIII), SMCs (α-actin), macrophages (Mac-2), and RAGE. The chromagen stains brown. (Magnification ×400.) B, Aortic tissue subjected to immunofluorescence staining to detect RAGE antigen (A, D, and G). Sites of prominent RAGE expression were confirmed to be predominantly macrophages based on colocalization of RAGE expression with Mac-2 (E) in the merged image (F). Colocalization of RAGE with endothelial cells (B) was seen in the merged image (C). RAGE was also expressed in smooth muscle cells, based on colocalization of smooth muscle actin (H) in the merged image (I). Areas in yellow represent colocalization. (Magnification ×100.)‏ Yared Tekabe et al. Circ Cardiovasc Imaging. 2008;1:212-219 Copyright © American Heart Association, Inc. All rights reserved.