In the present review, we describe the causes and consequences of loss of vascular smooth muscle cells (VSMCs) or their function in advanced atherosclerotic plaques and discuss possible mechanisms such as cell death or senescence, and induction of autophagy to promote cell survival. We also highlight the potential use of pharmacological modulators of these processes to limit plaque progression and/or improve plaque stability. VSMCs play a pivotal role in atherogenesis. Loss of VSMCs via initiation of cell death leads to fibrous cap thinning and promotes necrotic core formation and calcification. VSMC apoptosis is induced by pro-inflammatory cytokines, oxidized low density lipoprotein, high levels of nitric oxide and mechanical injury. Apoptotic VSMCs are characterized by a thickened basal lamina surrounding the cytoplasmic remnants of the VSMC. Inefficient clearance of apoptotic VSMCs results in secondary necrosis and subsequent inflammation. A critical determinant in the VSMC stress response and phenotypic switching is autophagy, which is activated by various stimuli, including reactive oxygen and lipid species, cytokines, growth factors and metabolic stress. Successful autophagy stimulates VSMC survival, whereas reduced autophagy promotes age-related changes in the vasculature. Recently, an interesting link between autophagy and VSMC senescence has been uncovered. Defective VSMC autophagy accelerates not only the development of stress-induced premature senescence but also atherogenesis, albeit without worsening plaque stability. VSMC senescence in atherosclerosis is likely a result of replicative senescence and/or stress-induced premature senescence in response to DNA damaging and/or oxidative stress-inducing stimuli. The finding that VSMC senescence can promote atherosclerosis further illustrates that normal, adequate VSMC function is crucial in protecting the vessel wall against atherosclerosis.

An ideal animal model of atherosclerosis resembles human anatomy and pathophysiology and has the potential to be used in medical and pharmaceutical research to obtain results that can be extrapolated to human medicine. Moreover, it must be easy to acquire, can be maintained at a reasonable cost, is easy to handle and shares the topography of the lesions with humans. In general, animal models of atherosclerosis are based on accelerated plaque formation due to a cholesterol-rich/Western-type diet, manipulation of genes involved in the cholesterol metabolism, and the introduction of additional risk factors for atherosclerosis. Mouse and rabbit models have been mostly used, followed by pigs and non-human primates. Each of these models has its advantages and limitations. The mouse has become the predominant species to study experimental atherosclerosis because of its rapid reproduction, ease of genetic manipulation and its ability to monitor atherogenesis in a reasonable time frame. Both Apolipoprotein E deficient (ApoE-/-) and LDL-receptor (LDLr) knockout mice have been frequently used, but also ApoE/LDLr double-knockout, ApoE3-Leiden and PCSK9-AAV mice are valuable tools in atherosclerosis research. However, a great challenge was the development of a model in which intra-plaque microvessels, haemorrhages, spontaneous atherosclerotic plaque ruptures, myocardial infarction and sudden death occur consistently. These features are present in ApoE-/-Fbn1C1039G+/- mice, which can be used as a validated model in pre-clinical studies to evaluate novel plaque-stabilizing drugs.

Objective— Apoptotic cell death has been demonstrated in advanced human atherosclerotic plaques. Apoptotic cells (ACs) should be rapidly removed by macrophages, otherwise secondary necrosis occurs, which in turn elicits inflammatory responses and plaque progression. Therefore, we investigated the efficiency of phagocytosis of ACs by macrophages in atherosclerosis. Methods and Results— Human endarterectomy specimens and human tonsils were costained for CD68 (macrophages) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) (apoptosis). Free and phagocytized ACs were counted in both tissues. The ratio of free versus phagocytized AC was 19-times higher in human atherosclerotic plaques as compared with human tonsils, indicating a severe defect in clearance of AC. Impaired phagocytosis of AC was also detected in plaques from cholesterol-fed rabbits and did not further change with plaque progression. In vitro experiments with J774 or peritoneal mouse macrophages showed that several factors caused impaired phagocytosis of AC including cytoplasmic overload of macrophages with indigestible material (beads), free radical attack, and competitive inhibition among oxidized red blood cells, oxidized low-density lipoprotein and ACs for the same receptor(s) on the macrophage. Conclusion— Our data demonstrate that phagocytosis of ACs is impaired in atherosclerotic plaques, which is at least partly attributed to oxidative stress and cytoplasmic saturation with indigestible material.

Background— The formation of reactive oxygen species is a critical event in atherosclerosis because it promotes cell proliferation, hypertrophy, growth arrest, and/or apoptosis and oxidation of LDL. In the present study, we investigated whether reactive oxygen species-induced oxidative damage to DNA occurs in human atherosclerotic plaques and whether this is accompanied by the upregulation of DNA repair mechanisms. Methods and Results— We observed increased immunoreactivity against the oxidative DNA damage marker 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) in plaques of the carotid artery compared with the adjacent inner media and nonatherosclerotic mammary arteries. Strong 8-oxo-dG immunoreactivity was found in all cell types of the plaque including macrophages, smooth muscle cells, and endothelial cells. As shown by competitive ELISA, carotid plaques contained 160±29 8-oxo-dG residues/10 ⁵ dG versus 3±1 8-oxo-dG residues/10 ⁵ dG in mammary arteries. Single-cell gel electrophoresis showed elevated levels of DNA strand breaks in the plaque. The overall number of apoptotic nuclei was low (1% to 2%) and did not correlate with the amount of 8-oxo-dG immunoreactive cells (>90%). This suggests that initial damage to DNA occurs at a sublethal level. Several DNA repair systems that are involved in base excision repair (redox factor/AP endonuclease [Ref 1] and poly(ADP-ribose) polymerase 1 [PARP-1]) or nonspecific repair pathways (p53, DNA-dependent protein kinase) were upregulated, as shown by Western blotting and immunohistochemistry. Overexpression of DNA repair enzymes was associated with elevated levels of proliferating cell nuclear antigen. Conclusions— Our findings provide evidence that oxidative DNA damage and repair increase significantly in human atherosclerotic plaques.

Autophagy is a reparative, life-sustaining process by which cytoplasmic components are sequestered in double-membrane vesicles and degraded on fusion with lysosomal compartments. A growing body of evidence suggests that autophagy is stimulated in advanced atherosclerotic plaques by oxidized lipids, inflammation, and metabolic stress conditions. However, despite the increasing interest in autophagy in various pathophysiological situations such as neurodegeneration, cancer, and cardiac myopathies, the process remains an underestimated and overlooked phenomenon in atherosclerosis. As a consequence, its role in plaque formation and stability is poorly understood. Most likely, autophagy safeguards plaque cells against cellular distress, in particular oxidative injury, by degrading damaged intracellular material. In this way, autophagy is antiapoptotic and contributes to cellular recovery in an adverse environment. An interesting observation is that basal autophagy can be intensified by specific drugs. Excessively stimulated autophagic activity is capable of destroying major proportions of the cytosol, leading finally to type II programmed cell death that lacks several hallmarks of apoptosis or necrosis. Because atherosclerosis is an inflammatory disorder of the arterial intima, pharmacological approaches could be developed to stabilize vulnerable, rupture-prone lesions through selective induction of macrophage autophagic death.

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 (n=18). 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 ( P <0.01), 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 (5.8±3% 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.