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Noninvasive Imaging of Atherosclerotic Lesions in Apolipoprotein E–Deficient and Low-Density-Lipoprotein Receptor–Deficient Mice with Annexin A5

¹ Division of Cardiology, University of California, Irvine School of Medicine, Irvine, California; ² Division of Cardiovascular Diseases, University of California, San Diego, La Jolla, California; ³ Department of Cardiology, Maastricht University Hospital, Maastricht, The Netherlands; ⁴ Division of Biochemistry, Maastricht University, Maastricht, The Netherlands; ⁵ Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan; ⁶ Armed Forces Institute of Pathology, Washington, District of Columbia; and ⁷ Department of Pathology, University of California, Irvine School of Medicine, Irvine, California

View larger version (8K): [in a new window]   FIGURE 1.  Schematic presentation of experimental protocol. Three hours after annexin A5 injection, in vivo micro-SPECT was acquired for approximately 1 h and, immediately after SPECT acquisition, in vivo micro-CT was acquired for 15 min. After the in vivo imaging study, animals were sacrificed and aortas were explanted, and ex vivo planar imaging was acquired for 30 min.

View larger version (45K): [in a new window]   FIGURE 2.  In vivo and ex vivo images of control mice (A) and apoE–/– mice without cholesterol diet (B) and with cholesterol diet (C). For all images, left panel represents transverse images, middle panel represents sagittal images, and right panel represents ex vivo images (A–C). Top panel shows micro-CT, middle panel shows micro-SPECT, and bottom panel shows fusion images. (A) No obvious annexin A5 uptake was seen on either in vivo or ex vivo images of control animals. (B) Distinct uptake was observed in the arch on in vivo images and in the arch and abdominal aorta on ex vivo image. (C) Distinct uptake and calcification were observed in the arch on the in vivo images; annexin uptake was seen in whole aorta on the ex vivo image. (D) Quantitative uptake was highest in cholesterol-fed apoE–/– mice, followed by chow-fed apoE–/– and control mice in lesions at arch, thoracic, or abdominal level. Ch = cholesterol fed.

View larger version (44K): [in a new window]   FIGURE 3.  In vivo and ex vivo images in control mice (A) and LDLR–/– mice without cholesterol diet (B) and with cholesterol diet (C). For all images, left panel represents transverse images, middle panel represents sagittal images, and right panel represents ex vivo images (A–C). Top panel shows micro-CT, middle panel shows micro-SPECT, and bottom panel shows fusion images. (A) No obvious annexin A5 uptake was seen on either in vivo or ex vivo images of control animals. (B) Significant uptake was observed in the arch on in vivo images and in the arch and abdominal aorta on ex vivo image. (C) Distinct uptake was observed in the arch on in vivo images and distinct uptake was seen in whole aorta on ex vivo image. However, uptake in any area was lower compared with that of apoE–/– mice. (D) Quantitative uptake was highest in cholesterol-fed LDLR–/– mice, followed by chow-fed LDLR–/– and control mice in lesions at arch, thoracic, or abdominal level. Ch = cholesterol fed.

View larger version (39K): [in a new window]   FIGURE 4.  Histopathologic characterization of atherosclerotic lesions including Movat's pentachrome staining (x100) (A), Mac-3 antibody staining (x400) (B), -actin staining (x400) (C), and TUNEL staining (x400) (D) in control, chow-fed LDLR–/–, cholesterol-fed LDLR–/–, chow-fed apoE–/–, and cholesterol-fed apoE–/– mice. (B) Prevalence of Mac-3–positive cells was significantly higher in cholesterol- and chow-fed apoE–/– and cholesterol-fed LDLR–/– mice than that in chow-fed LDLR–/– mice. (C) Prevalence of SMC, detected by -actin, was significantly lower compared with macrophages. -Actin–positive cells were predominantly located in fibrous caps and were relatively more commonly observed in cholesterol- and chow-fed apoE–/– mice than in cholesterol- or chow-fed LDLR–/– mice. (D) TUNEL-positive nuclei in core region were more frequently observed in apoE–/– mice than in LDLR–/– mice and more so in cholesterol-fed animals (arrows). TUNEL-positive nuclei were not detected in chow-fed LDLR–/– mice. Ch = cholesterol fed.

View larger version (14K): [in a new window]   FIGURE 5.  Quantitative histologic analysis and correlation with radiotracer uptake. (A) Percentage prevalence of cellular components in various groups. Percentage immunostaining field areas for macrophages are highest in cholesterol-fed apoE–/– mice, followed by chow-fed apoE–/–, cholesterol-fed LDLR–/–, and chow-fed LDLR–/– mice. Similar trends are seen for apoptotic nuclei. Correlation of annexin A5 uptake is evident with macrophages (C) and apoptosis (D), but no significant correlation is seen with SMCs (B). Ch = cholesterol fed.

View larger version (107K): [in a new window]   FIGURE 6.  Localization of annexin A5 spontaneous atherosclerotic lesions. Biotinylated annexin A5 was injected intravenously into apoE–/– mice and traced histochemically in arterial wall. (A) Advanced atherosclerotic lesion in femoral artery (Movat's pentachrome, x100). Area outlined by black box in A is magnified in B–D. (B) Annexin uptake (black arrowhead, apoptotic; black arrows, nonapoptotic) was localized in macrophages identified by Mac-3 antibody staining as shown in C (x400). Inset in B shows that some nuclei were apoptotic (black reaction product, pink arrows) and localized in same area (x1,000, blue-green nuclear counterstain; interference contrast microscopy). (D) Annexin A5 uptake was not associated with SMCs.