Localization of apoptotic macrophages at the site of plaque rupture in sudden coronary death

Abstract

Although apoptosis is a well-recognized phenomenon in chronic atherosclerotic disease, its role in sudden coronary death, in particular, acute plaque rupture is unknown. Culprit lesions from 40 cases of sudden coronary death were evaluated. Cases were divided into two mechanisms of death: ruptured plaques with acute thrombosis (n = 25) and stable plaques with and without healed myocardial infarction (n = 15). Apoptotic cells were identified by staining of fragmented DNA and confirmed in select cases by gold conjugate labeling combined with ultrastructural analysis. Additional studies were performed to examine the expression and activation of two inducers of apoptosis, caspases-1 and -3. Ruptured plaques showed extensive macrophage infiltration of the fibrous cap, in particular at rupture sites contrary to stable lesions, which contained fewer inflammatory cells. Among the culprit lesions, the overall incidence of apoptosis in fibrous caps was significantly greater in ruptured plaques (P < 0.001) and was predominantly localized to the CD68-positive macrophages. Furthermore, apoptosis at plaque rupture sites was more frequent than in areas of intact fibrous cap (P = 0. 028). Plaque rupture sites demonstrated a strong immunoreactivity to caspase-1 within the apoptotic macrophages; staining for caspase-3 was weak. Immunoblot analysis of ruptured plaques demonstrated caspase-1 up-regulation and the presence of its active p20 subunit whereas stable lesions showed only the precursor; nonatherosclerotic control segments were negative for both precursor and active enzyme. These findings demonstrate extensive apoptosis of macrophages limited to the site of plaque rupture. The proteolytic cleavage of caspase-1 in ruptured plaques suggests activation of this apoptotic precursor. Whether macrophage apoptosis is essential to acute plaque rupture or is a response to the rupture itself remains to be determined.

Figure 1.

Cartoon diagram illustrating where measurements of cell type and apoptosis were performed. The color key at the bottom describes the various plaque constituents. The black-hatched regions represent areas where measurements were taken. Quantitatively, the size of the rupture site varied between cases such that cell counts were performed in areas ranging from 0.12 to 0.20 mm².

Figure 2.

Apoptosis in culprit plaques. A: Micrograph of a cross-section of an epicardial coronary artery shows a plaque rupture with an acute luminal thrombus (Th); note the thin fibrous cap, boxed area (H&E stain; original magnification, ×30). B: Serial section of A after DNA fragmentation staining by ISEL (see Materials and Methods). Numerous apoptotic cells (blue nuclear staining, arrowheads) are identified at the plaque rupture site (eosin counterstain; original magnification, ×150). The inset shows a high-power view illustrating the nuclear detail; note the fragmented nucleus (arrowhead; original magnification, ×1000). C: Micrograph of a cross-section of an epicardial coronary artery shows an eccentric stable plaque. The lesion is characterized by dense fibrous cap, boxed area, overlying a calcified region (H&E; original magnification, ×30). D: Serial section of C after DNA fragmentation staining, there is a paucity of apoptotic cells (arrowheads) relative to rupture site in B (eosin counterstain; original magnification, ×150). Abbreviations: L, lumen; NC, necrotic core.

Figure 3.

Immunoelectron microscopic characterization of apoptosis at the rupture sites. A: Ultrastructural examination of the macrophages (arrows) at the rupture site showed that the majority had nuclear changes suggestive of apoptosis. B: Example demonstrating varying stages of nuclear shrinkage with condensation of chromatin at the nuclear periphery. The presence of lipid globules and lack of contractile elements help identify the macrophages. ISEL was performed on ultra-thin sections with TdT and biotinylated nucleotides. Fragmented DNA was visualized using a 10-nm gold, streptavidin conjugate. C: High-power electron micrograph of the region indicated by the inset in B, note the gold particles are selectively localized on the condensed chromatin material.

Figure 4.

Identification of cells undergoing apoptosis in culprit plaques. Apoptotic cells were characterized using a combination of ISEL and specific antibodies for macrophages (KP-1/CD68; A and C) or SMCs (HHF-35; B and D). ISEL-positive nuclei were visualized with diaminobenzidine (dark-brown reaction product) and antibody staining was detected using an anti-mouse IgG conjugated with biotin and an avidin alkaline phosphatase-substrate system (red reaction product). The methyl green counterstain yields blue-green nuclei. A and B are photomicrographs of plaque rupture and C and D are stable plaques. A: CD68 and ISEL staining in plaque rupture, note the majority of apoptotic cells are macrophages (original magnification, ×150). Abbreviations: Th, thrombus; FC, fibrous cap. B: Serial section showing DNA fragmentation and HHF-35 staining in the fibrous cap in a ruptured plaque. Note that most of the apoptotic nuclei (upper left, rupture site) are negative for SMCs (original magnification, ×300). C: Stable plaque showing occasional apoptotic macrophages (arrowheads) in a region of the fibrous cap (original magnification, ×300). D: Serial section of C shows rare apoptotic SMCs (arrowheads) in the fibrous cap.

Figure 5.

Comparision of apoptosis relative to specific cell types in culprit plaques. Bar graphs represent the different cell populations and apoptotic index in the fibrous cap of ruptured and stable plaques. The cells were quantified after double-labeling experiments using serial frozen sections as shown in Figure 4 ▶ . Not all cells undergoing apoptosis could be identified by antibodies. The results are expressed as the mean ± SE of 21 ruptures and 11 stable plaques. A: Overall, cell populations expressed as a percentage of the total number of nuclei; note the predominance of macrophages and paucity of SMCs at the rupture site. B: Apoptotic cells as represented as a percentage of cell type; ∼45% of macrophages were recognized as apoptotic. C: Macrophage apoptosis compared with a remote region of the fibrous cap. Abbreviations: MΦs, macrophages; SMC, smooth muscle cells.

Figure 6.

Immunolocalization and biochemical activation of caspase-1 in plaque rupture. A: Immunohistochemical staining demonstrating caspase-1 at the site of plaque rupture (reddish-brown reaction product; original magnification, ×150). Note the intense immunoreactivity at the rupture site (a serial section from the case as shown in Figure 4 ▶ ). B: Stable plaque showing the localization of caspase-1 in an area of the fibrous cap (reddish-brown reaction product; original magnification, ×150). C: Representative immunoblot analysis of coronary segments showing extensive cleavage of caspase-1 in rupture; the stable plaques showed minimal cleavage. The analyses were repeated at least four times, yielding similar results.