Molecular Imaging of Apoptosis in Atherosclerosis by Targeting Cell Membrane Phospholipid Asymmetry

Abstract

Background: Apoptosis in atherosclerotic lesions contributes to plaque vulnerability by lipid core enlargement and fibrous cap attenuation. Apoptosis is associated with exteriorization of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell membrane. Although PS-avid radiolabeled annexin-V has been employed for molecular imaging of high-risk plaques, PE-targeted imaging in atherosclerosis has not been studied.

Objectives: This study sought to evaluate the feasibility of molecular imaging with PE-avid radiolabeled duramycin in experimental atherosclerotic lesions in a rabbit model and compare duramycin targeting with radiolabeled annexin-V.

Methods: Of the 27 rabbits, 21 were fed high-cholesterol, high-fat diet for 16 weeks. Nine of the 21 rabbits received ^99mTc-duramycin (test group), 6 received ^99mTc-linear duramycin (duramycin without PE-binding capability, negative radiotracer control group), and 6 received ^99mTc-annexin-V for radionuclide imaging. The remaining normal chow-fed 6 animals (disease control group) received ^99mTc-duramycin. In vivo microSPECT/microCT imaging was performed, and the aortas were explanted for ex vivo imaging and for histological characterization of atherosclerosis.

Results: A significantly higher duramycin uptake was observed in the test group compared with that of disease control and negative radiotracer control animals; duramycin uptake was also significantly higher than the annexin-V uptake. Quantitative duramycin uptake, represented as the square root of percent injected dose per cm (√ID/cm) of abdominal aorta was >2-fold higher in atherosclerotic lesions in test group (0.08 ± 0.01%) than in comparable regions of disease control animals (0.039 ± 0.0061%, p = 3.70·10^-8). Mean annexin uptake (0.060 ± 0.010%) was significantly lower than duramycin (p = 0.001). Duramycin uptake corresponded to the lesion severity and macrophage burden. The radiation burden to the kidneys was substantially lower with duramycin (0.49% ID/g) than annexin (5.48% ID/g; p = 4.00·10^-4).

Conclusions: Radiolabeled duramycin localizes in lipid-rich areas with high concentration of apoptotic macrophages in the experimental atherosclerosis model. Duramycin uptake in atherosclerotic lesions was significantly greater than annexin-V uptake and produced significantly lower radiation burden to nontarget organs.

Keywords: annexin-V; cell death; duramycin; radionuclide imaging; vulnerable plaques.

FIGURE 1. Experimental Protocol and Timeline

Various experimental groups and a timeline of experimental protocol.

FIGURE 2. In Vivo Images of Atherosclerotic Lesions With Radiolabeled Duramycin

In vivo images of the abdominal aorta of the atherosclerotic animal (left) and the disease control animal (right) injected with 99mTc-duramycin (test tracer) at 0 (top) and 4 h (bottom). Please note radiotracer uptake in the abdominal aorta of the atherosclerotic animal (red arrows, left) compared with the disease control animal (red arrows, right) at 4 h. Please refer to Video 1 for 3D video visualization of duramycin uptake at 4 h in abdominal aorta of atherosclerotic rabbit and Video 2 for lack of uptake in abdominal aorta of control animal.

FIGURE 3. Harvested Aortas and Ex Vivo Images

Ex vivo images of the explanted aortas with ^99mTc-duramycin (test tracer [A]) and ^99mTc-annexin-V (positive control tracer [B]), compared with ^99mTc-linear duramycin (negative radiotracer control [C]) for atherosclerotic animals, and ^99mTc-duramycin (test tracer [D]) in disease control animal. Ex vivo images are adjacent to their corresponding gross picture. Please note that radiolabeled duramycin targeting is visually noninferior to annexin-V images.

FIGURE 4. Radiation Burden for the Nontarget Organs

Quantitative %ID/g uptake in nontarget organs for ^99mTc-labeled duramycin and annexin-V administered to atherosclerotic animals. Significant differences (*) are given in comparison with ^99mTc-duramycin uptake. ID = injected dose.

FIGURE 5. Histopathologic Characterization of Thoracic and Abdominal Aorta Samples

(A and B) Histopathological sections from the thoracic and abdominal aorta of a diseased animal, respectively. The top rows show low (x10) magnification, and the bottom rows show the microphotographs at high (×40) magnification. The left 2 columns of both A and B are stained with hematoxylin and eosin, whereas the right 2 columns are immunohistochemically stained with RAM-11 (macrophages, column 3) and HHF-35 (smooth-muscle cells, column 4). Pathological intimal thickening is seen in the thoracic region (A) compared with fibroatheromatous lesion (B) seen in the abdominal aorta. Staining is intense for the smooth-muscle cells in the medial layer and scattered in the neointima in the thoracic region (A). Abundant inflammation is evident in the thick neointima carrying large necrotic core in the abdominal aorta specimen (B). The duramycin uptake of the thoracic aorta segment was low at 0.002269 %ID/cm and 3-fold higher in the abdominal aorta segment of the same animal at 0.006824 %ID/cm. (C) The correlation of the RAM-11-stained aortic cross-sectional area with the respective piece-by-piece %ID/g uptake of 99mTc-duramycin. ID = injected dose.

CENTRAL ILLUSTRATION. Molecular Imaging of Phospholipid Asymmetry During Apoptosis

The phospholipids in the cell membrane lipid bilayer are asymmetrically distributed with phosphatidylserine (PS) and phosphatidylethanolamine (PE) in the inner tab and phosphatidylcholine (PC) and sphingomyelin (SM) on the outer. This asymmetric distribution of phospholipids across the membrane bilayer is maintained by enzymes such as translocase and flippase. During apoptotic cell death, as a result of enzymatic alteration, normal phospholipid asymmetry within the cell membrane is lost, resulting in exteriorization of PS and PE. Activation of scrambalase exaggerates the random distribution. The exposure of PS acts as an “eat-me signal” for macrophages to have the apoptotic bodies removed. An endogenous protein, annexin-V possesses nanomolar affinity for externalized PS. Radiolabeled annexin-V has previously been used for noninvasive imaging of apoptotic cell death. Although effective for apoptosis imaging, annexin-V shows substantial off-target organ radiation burden. Like PS, apoptotic cell membranes also express PE. Even in larger quantities, PE can be targeted by an antibiotic-duramycin radiolabeled with technetium-99m. The current study compares the 2 probes targeted at the altered cell membranes. The study not only demonstrates the efficacy of imaging with duramycin but also its favorable off-target organ profile.