Molecular Imaging of Atherosclerotic Plaques Using a Human Antibody Against the Extra-Domain B of Fibronectin by Christian M. Matter, Pia K. Schuler, Patrizia Alessi, Patricia Meier, Romeo Ricci, Dongming Zhang, Cornelia Halin, Patrizia Castellani, Luciano Zardi, Christoph K. Hofer, Matteo Montani, Dario Neri, and Thomas F. Lüscher Circulation Research Volume 95(12):1225-1233 December 10, 2004 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Characterization of miniantibody preparations. Figure 1. Characterization of miniantibody preparations. SDS-PAGE analysis in reducing and nonreducing conditions of SIP(L19) and SIP(HyHEL-10). A pure covalent homodimer is observed for both antibodies when running the gel in nonreducing conditions. Christian M. Matter et al. Circ Res. 2004;95:1225-1233 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Specific binding of 125I-labeled L19 to atherosclerotic plaques in ApoE−/− mice. a, Autoradiograms and fat stainings of longitudinally opened aortas 4 hours (4h) after injection of 125I-labeled SIP(L19) in normal wild-type (WT) and atherosclerotic ApoE−/− mice (n=6 each). Figure 2. Specific binding of 125I-labeled L19 to atherosclerotic plaques in ApoE−/− mice. a, Autoradiograms and fat stainings of longitudinally opened aortas 4 hours (4h) after injection of 125I-labeled SIP(L19) in normal wild-type (WT) and atherosclerotic ApoE−/− mice (n=6 each). The radioactivity uptake allowed even the identification of small plaques (arrows). Minimal antibody uptake in the normal aorta of WT mice comprises unspecific binding of the antibody. b, Time course of autoradiographic analysis of aortas after injection of SIP(L19) or SIP(HyHEL-10). A persistent uptake of radioactive SIP(L19) in plaques can be observed up to 3 days (3d) after intravenous administration. Plaque-to-normal vessel ratios increase over time and match fat staining profiles. In contrast, only a modest plaque uptake of SIP(HyHEL-10) can be seen at 4 hours but not at later time points. Vessel background with SIP(HyHEL-10) decreases more rapidly than with SIP(L19); n=6 each. c, Correlation between fat staining using Oil red O and autoradiography from 125I-labeled L19 in aortas of ApoE−/− mice. Aortas from atherosclerotic ApoE−/− mice (n=4) were harvested 24 hours (24h) after intravenous injection of 125I-labeled L19. Corresponding images obtained after autoradiography and fat staining were compared by tracing each positive area (given as percentage of total vessel area) using linear regression (r=0.89; P<0.0001; y=0.96×−0.05). Each data point (n=27) refers to a single plaque staining and its corresponding autoradiographic signal. Christian M. Matter et al. Circ Res. 2004;95:1225-1233 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. Microscopic analysis of plaque targeting using fluorescent antibodies. Figure 3. Microscopic analysis of plaque targeting using fluorescent antibodies. Microscopic fluorescence images of plaques, after intravenous injection of ApoE−/− mice with Cy5-labeled SIP(L19) [red] (b and e), are compared with phase contrast and von Willebrand staining (green) (a and d). The merged images, which include DAPI (blue) for nuclear staining, demonstrate a colocalization of L19 with endothelial cells (c and f, dotted arrow) and with plaque matrix adjacent to endothelial cells (f, solid arrow). In contrast, normal vessel sections from wild-type mice (g, phase contrast) injected with the same antibody do not exhibit any detectable fluorescence (h); n=3 mice. Christian M. Matter et al. Circ Res. 2004;95:1225-1233 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Near-infrared fluorescence imaging of atherosclerotic plaques in ApoE−/− mice. Figure 4. Near-infrared fluorescence imaging of atherosclerotic plaques in ApoE−/− mice. At 24 hours after injection of Cy7-labeled SIP(L19), plaques are macroscopically visualized using an infrared fluorescence imager (a; after removal of heart, lungs, and abdominal organs) and in isolated aortas (c). In contrast, mice injected with saline show only minimal autofluorescence (b, d, and e); fat staining of aortic plaques (c, f); arrow indicates abdominal aorta; n=4 for each group. Correlation between fat staining using Oil red O and fluorescence imaging from Cy7-labeled L19 in aortas of ApoE−/− mice (g). Corresponding fluorescence and fat staining images were compared by tracing each positive area (given as percentage of total vessel area) using linear regression; r=0.83; P<0.0005; y=0.80×−3.72. Each data point (n=13) refers to a single plaque staining and a fluorescence signal; n=3 mice. Christian M. Matter et al. Circ Res. 2004;95:1225-1233 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. Overexpression of fibronectin ED-B in murine and human atherosclerotic plaques. Figure 5. Overexpression of fibronectin ED-B in murine and human atherosclerotic plaques. Fibronectin ED-B expression using the L19 antibody staining (red) is virtually undetectable in the normal aorta of wild-type mice (a) but is highly expressed in aortic plaques of ApoE−/− mice (b). Similarly, fibronectin ED-B is only minimally present in normal human internal mammary arteries (c) but is abundantly expressed in human plaques from an abdominal aortic aneurysm (d), where it is found predominantly around vasa vasorum (arrow) and plaque matrix. Similarly, a cross-section through a human coronary artery (e, f) reveals faint ED-B staining in portions of the vessel that appears minimally diseased (e). In contrast, enhanced ED-B expression is found in atherosclerotic areas (f), around vasa vasorum (arrow), and plaque matrix, as well as in the diseased media. Light microscopy, longitudinal (mouse) and cross-sections (human); magnifications are ×100 (a and b; bar=200 μm) and ×40 (c through f; bar=500 μm) on the left and ×200 on the right (bar=100 μm); representative photomicrographs from n≥3 different samples for each condition. Christian M. Matter et al. Circ Res. 2004;95:1225-1233 Copyright © American Heart Association, Inc. All rights reserved.