In the present study, inhibitors of caspase-8 and -9 were used to differentiate between involvement of the death receptor and mitochondrial pathways of apoptosis. IL = interleukin; ROS = reactive oxygen species.

Annexin A5 uptake in 19 animals treated with broad and specific caspase inhibitors was compared with 6 animals each of treatment (Rx), disease, and radiotracer control groups.

The images were obtained in both control (nonatherosclerotic) and atherosclerotic (athero) rabbits with or without caspase (Casp) inhibitor therapy. The top panel demonstrates planar gamma images, and the bottom panel presents micro-single photon emission computed tomography (CT) images superimposed on a morphologic background of micro-CT images. (A) Lack of annexin uptake in the region of the abdominal aorta as denoted by arrows in the normal rabbit with no atherosclerotic lesions. The annexin metabolism and excretion results in variation burden to liver (L), spleen (S), and kidney (K). (B) Compared with the negative control, significant radiotracer accumulation can be seen in the abdominal aorta (shown by arrows in front of vertebral column activity) of the animal with experimental atherosclerotic lesions. The animals receiving (C) nonselective caspase inhibitor or (D) selective caspase-1 inhibitor demonstrate total abrogation of annexin uptake. (E and F) The bottom panel reveals superimposed nuclear and CT images after treatment with selective caspase-3 inhibitor and caspase-8 inhibitor. The images are present in the set of 2 projections, transverse and sagittal. Whereas the caspase-3 inhibitor completely prevented annexin A5 uptake (arrows pointing to aortic area in front of vertebral uptake), caspase-8 inhibition did not affect annexin uptake or apoptosis. In caspase-8 inhibitor-treated animals, vivid uptake of radiotracer is seen in the abdominal aorta.

The left panel demonstrates the uptake of annexin A5 in abdominal aortas. Disease control animals (C) with no atherosclerotic lesions are represented by an open bar and the remaining atherosclerotic animals by solid bars. Atherosclerotic animals imaged with mutant annexin were used as tracer controls (gray bar), and the remaining atherosclerotic animals were either untreated (black bar) or treated with various caspase inhibitors (colored bars). Broad and caspase-3 inhibition bars are shown in pink, caspase-8 and -9 inhibitors differentiating between mitochondrial and extramitochondrial pathways are in blue, and capase-1–specific inhibition is represented by a green solid bar. The statistical significance of caspase inhibition, in comparison with the disease control and untreated atherosclerotic animals, is shown above and below the bars, respectively. The uptake is significantly higher in the abdominal aortas of untreated atherosclerotic animals compared with that in the disease and tracer control groups. Treatment with a broad caspase inhibitor or selective caspase-1, -9, or -3 inhibitor significantly reduced the apoptotic activity as represented by lower annexin uptake. On the other hand, selective caspase-8 inhibition did not affect apoptosis. The right panel compares histologic characterization of atherosclerotic lesions in atherosclerotic untreated animals and atherosclerotic animals receiving the broad caspase inhibitor. Movat pentachrome (M5Ch), smooth muscle cell (-actin), and macrophage (Ram-11) staining (x200) in untreated atherosclerotic animals (left) and broad caspase inhibitor-treated animals (right) demonstrate similar cholesterol crystal-rich necrotic core, foam cell-rich, and smooth muscle-deficient lesions. The morphologic characteristics of the lesions in untreated and caspase-treated atherosclerosis are unchanged. However, terminal deoxyribonucleotide transferase-mediated nick-end labeling (TUNEL) reveals evidence of marked apoptotic nuclei in untreated atherosclerotic animals but marked resolution in the atherosclerotic lesions in caspase-inhibited animals.

Movat pentachrome (M5Ch), smooth muscle cell (-actin), macrophage (Ram-11), and terminal deoxyribonucleotide transferase-mediated nick-end labeling (TUNEL) staining (x200) in representative sections of aorta from nonatherosclerotic control animals, untreated atherosclerotic (Athero) animals, and those treated with selective caspase (Casp)-1, -3, and -8 inhibitors are shown. The pentachrome stains demonstrate atherosclerotic lesions with large necrotic cores, and the immunostains show intense macrophage infiltration and low smooth muscle cell content in all treated and untreated animals. Therefore, caspase treatment did not change the morphologic characteristics of the atherosclerotic lesions. The TUNEL staining showed evidence of marked apoptotic nuclei in untreated atherosclerotic animals, but treatment with caspase-1 and -3 resulted in decreased apoptotic nuclei in neointima; treatment with caspase-8 did not affect the number of TUNEL-positive nuclei.