Background-The transition of a fatty streak into an atherosclerotic plaque is characterized by the appearance of focal and diffuse regions of cell death. We have investigated the distribution of apoptotic cell death and apoptosis-related proteins in early and advanced atherosclerotic lesions. Methods and Results-Human atherosclerotic plaques were studied by whole-mount carotid endarterectomy specimens (nϭ18). This approach allowed comparison of adaptive intimal thickenings, fatty streaks, and advanced atherosclerotic plaques of the same patient. The fatty streaks differed from adaptive intimal thickenings by the presence of BAX (PϽ0.01), a proapoptotic protein of the BCL-2 family. Both regions were composed mainly of smooth muscle cells (SMCs), and macrophage infiltration was low and not different. Apoptosis, as detected by DNA in situ end labeling (terminal deoxynucleotidyl transferase end labeling [TUNEL] and in situ nick translation) was not present in these regions. Apoptosis of SMCs and macrophages, however, was present in advanced atherosclerotic plaques that were present mainly in the carotid sinus. A dense infiltration of macrophages (5.8Ϯ3% surface area) was present in these advanced atherosclerotic plaques. Cytoplasmic remnants of apoptotic SMCs, enclosed by a cage of thickened basal lamina, were TUNEL negative and remained present in the plaques as matrix vesicles. Conclusions-We conclude that SMCs within human fatty streaks express BAX, which increases the susceptibility of these cells to undergo apoptosis. The localization of these susceptible SMCs in the deep layer of the fatty streaks could be important in our understanding of the transition of fatty streaks into atherosclerotic plaques, which are characterized by regions of cell death. Matrix vesicles are BAX-immunoreactive cytoplasmic remnants of fragmented SMCs that can calcify and may be considered the graves of SMCs that have died in the plaques. (Circulation. 1998;97:2307-2315.)

A nonocclusive silicone cuff placed around the rabbit carotid artery results in a diffuse intimal thickening. The early stages of this phenomenon were studied by light microscopy, immunohistochemistry, and electron microscopy. Neointimal formation appeared to be triphasic. The first phase started 2 hours after cuff placement, with vascular infiltration by polymorphonuclear leukocytes (PMNs). In the second phase, starting within 12 hours, 1.90±0J6% of the medial smooth muscle cells (SMCs) were replicating, as demonstrated by their immunoreactivity for proliferating cell nuclear antigen (PCNA). The third phase was characterized by the appearance, from day 3 onward, of subendothelial SMCs that were immunoreactive for or-SMC actin and vimentin. A few cells showed immunoreactivity for PCNA. During this phase all the PMNs disappeared, but SMC replication in the media was still present, as indicated by the presence of mitoses and the persisting immunoreactivity for PCNA (0.76±0.22% at day 7). In the third phase the number of subendothelial cells increased (104±15 SMC nuclei per section at day 7, of which 8.89±2.26% were PCNA-positive) and was associated with deposition of collagen type IV and flbronectin. At 14 days a complete, circular neointima was present and contained 2.13 ±0.28% replicating SMCs. The media showed 0.44±0.08% cell-cycling SMCs, which was still four times higher than normal. During the first week there was also a significantly higher PCNA activity in the media of sham-operated carotid arteries (no cuff present) than in nonsurgical ones. However, this did not lead to the formation of a neointima. We conclude that in the cuff system SMC replication in the media precedes the neointimal formation. The system can be used to study SMC replication, migration, and neointimal formation with minimal medial SMC damage. While the factors responsible for intimal development in humans are largely unknown, numerous methods of injury have been applied to produce intimal lesions in animals. These methods can be divided into two broad categories: those that use intraluminal (e.g., balloon denudation) and those that use perivascular manipulation. Examples of the latter are external electrical stimulation, 6 external compression, 7 stripping the adventitia from the arteries, 8 and positioning of a cuff around an artery. 9 -" Perivascular cuff placement is used to avoid direct injury to the vessel wall, particularly

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