The prevention and treatment of CVD remain an incontrovertible fact by reducing the CV risk factors such as increased lipid or systolic blood pressure promoting health by a change of an unhealthy lifestyle, e.g. lack of physical activity, poor-quality diet, overweight, smoking, as well as elevated blood cholesterol levels. Because a high blood cholesterol level is an independent cardiovascular risk factor, its lowering is a major goal of an anti-atherosclerotic therapy [
3,
29,
30]. Patients with an established CVD or with an increased risk of developing CVD, necessarily need to reduce their blood cholesterol levels by using medication, e.g. statins. However, nobody knows the consequences of varying cholesterol levels or statin treatment on plaque size and influence on the cellular composition of already existing plaques. Thus, and because rabbits are widely accepted for the study of human atherosclerosis [
31] since they are sensitive to a cholesterol diet and have similar features of lipoprotein metabolism, like humans but unlike rodents [
31], the purpose of our study, by using rabbits with pre-lesioned thoracic aortas, was to investigate the effects of a CEDrs with or without statin treatment on plaque size and/or cellular composition. We found that re-supplementation for 4 weeks resulted in a significant increase in plasma cholesterol levels, whereas afterward withdrawal of CED for 6 weeks reduced plasma cholesterol levels to almost the levels before starting the re-supplementation. In this context, it has most recently been shown, that in rabbits – after application of a 0.5% CED – a treatment with atorvastatin (2 mg/kg/day) for 10 weeks, did also not significantly reduce the absolute LDL-C and triglyceride levels in comparison with the control at distinct time points [
32]. However, the same study revealed that treatment of atorvastatin significantly reduced the lesion size by 68% [
32], whereas our data reveal that after CEDrs and withdrawal of CED (group III), plaque area is not significantly different from the group without re-supplementation (group II), despite an increased cholesterol content of the vessel wall; however, in our re-supplemental study (group IV), atorvastatin treatment did neither affect plaque size nor cholesterol content in comparison with the group without atorvastatin (group III) treatment. Thus, atorvastatin has different effects on inhibition of plaque development/progression depending on, whether the lesions already exist or just develop. Moreover, CEDrs (group III) did not significantly influence cell density, and atorvastatin treatment (group IV) did not as well. While the regression of advanced atherosclerotic plaques after normalization of pro-atherogenic factors in rodents and non-human mammals, as well as in patients, is differently discussed [
24,
33‐
35], the cellular reorganization of the plaque is suggested to lead to varying plaque stabilities [
16]. In our experiments, we have used the lowest dose as suggested by others [
36]. We assume that the atorvastatin dose was too low and / or the four-week application time was too short to see a more obvious effect on vessel morphology. In this context, the density of MΦ may also be an important factor for the regression of atherosclerosis, as demonstrated in rabbits [
11,
18,
37]. In respect thereof, we found characteristic signs of plaque remodelling as an increase of the presence of MΦ after CED induction (group I) or atorvastatin treatment after CEDrs (group IV), but not in group II (long-term withdrawal) or group III (short-term withdrawal after 4 weeks CEDrs). Additionally, we found a reduced number of apoptotic cells in atherosclerotic lesions of groups II and IV, which may evidence that MΦ are still necessary for the plaque remodelling after atorvastatin treatment [
11]. This is necessary to mention because in human atherosclerotic lesions apoptosis was abundantly found and contributed to the accumulation of gruel and plaque instability [
38] and macrophage death may be a promising pharmacological target in atherosclerosis [
39]. Moreover, we found similar densities of HLA-DR
+ cells in groups II, III and IV with a significant positive correlation between the density of Mac-1
+ MΦ and HLA-DR
+ cells, indicating that density of antigen-presenting cells (APC) is affected neither by CEDrs nor by atorvastatin treatment. However, APC localized in atherosclerotic plaques of the thoracic aortas of rabbits under test are suggested to be MΦ. As we also found the highest density of SOD2
+ cells in the plaques of group I (induction), this may indicate that oxidative stress in MΦ, possibly induced by oxidized low-density lipoprotein (oxLDL), may cause apoptosis and in terms of antigen presentation, attract more phagocytes from the blood stream [
40]. Since administration of atorvastatin induces plaque stabilization [
32] and an absence of TP53 accelerates atherosclerosis [
41], it is of interest, that we found a significant positive correlation between the density of Mac-1
+ MΦ and density of TP53
+ cells, which may be an indicator of improved plaque stabilization as found for lesions exclusively in group IV (with atorvastatin treatment). Accordingly, simvastatin was shown to stabilize or even regress established atheromatous lesions in rabbits at a dose rate of 7 mg/kg/day [
42], which was about 3-fold higher than the atorvastatin dose rate in our experiment. In this respect, 4 weeks of CEDrs and additional withdrawal for 6 weeks (Group III), resulted in a decreased density of MΦ and an increased density of α-actin
+ SMC compared with group I. Excess stimulation of SMCs by mitogens and secretion of the vessel wall components is assumed to contribute to plaque growth and might explain the lack of regression in some species under certain pathophysiological conditions [
43]. In agreement with our previous observations, after the withdrawal of CED for up to 1.5 years, neither regression nor progression of the lesion size was observed, whereas the atherosclerotic plaques respond by a decrease in the number of MΦ and an increase of SMCs [
24]. According to other publications, coronary plaque stabilization was found in parallel with a decrease of lipid and MΦ content, as well as impaired migration of SMCs in hypercholesterolemic miniature pigs [
11,
44]. Our data show, that the composition, but not the size of atherosclerotic plaques, respond to lowering of cholesterol levels, suggesting their reorganization to more stable plaques. It may be reasonable to assume that a complete cessation of fat consumption and statin treatment would not heal, reduce and/or stabilize the atherosclerotic lesion or arrest the further progression of the disease process, as we have shown after short-term statin treatment and CED interruption. In humans, this might be the case, e.g. if the patients abandon their treatment and discontinue a healthy lifestyle prior to reaching their therapeutic effect. However, this negative attitude can be more or less risky which depends on the length of time between the interruption phase and the re-establishment of the treatment as well as a healthy lifestyle. Nevertheless, it is unclear if these effects on the plaque morphology and quality occur in animals as well as in humans [
45]. Although, an increase of cholesterol concentration in the vessel wall (Gruppe IV) is suggested to be responsible for the activation of plaque MΦ leading to e.g. oxidative stress and apoptosis, as demonstrated in the present study. Atorvastatin treatment seems to activate conditions for plaque remodeling and to accelerate the stabilization compared with the group without atorvastatin treatment. In this context, in vulnerable plaques of humans, which present a thin fibroatheroma cap and a lipid core with macrophage infiltration, statins increased the fibrous cap thickness and reduced the lipid core, which may be interpreted as a process of plaque stabilization [
46].
We suggest, that either the dose of atorvastatin was too low and/or the application time of 4 weeks was too short. Moreover, high-intensity atorvastatin therapy, 80 mg daily for 24 months, has been demonstrated to promote coronary atheroma stabilization and regression [
47,
48]. Statin treatment was in direct association with changes in atheroma composition, reducing fibro-fatty components [
47]. However, atherosclerosis continues to progress [
49] or not retreats as happens in our study with rabbits. In this context, new therapeutic strategies must use a combination of treatments, e.g. dietary reduction of lipids and carbohydrate intake, together with statins and agents to inhibit cholesterol absorption (
e. g. ezetimibe) or proprotein convertase subtilisin/kexin type 9 (PCSK9) activity (
e. g. evolocumab) [
50]. Thus, it is noteworthy to mention, that recent investigations have shown a relationship between serum PCSK9 levels and cardiovascular events as e.g. myocardial infarction, revascularization, thrombotic stroke [
51]. PCSK9 released by endothelial and smooth muscle cells may have paracrine influence, on the expression of LDLR on macrophages and induce its differentiation in foam cells within atherosclerotic plaques [
52]. In patients with cardiovascular disease, PCSK9 blood concentrations were related to the frequent presence of necrotic cores in coronary plaques regardless of statin use [
53]. Unfortunately, the PCSK9 inhibition on plaque regression is unknown [
54]. In this regard, by using the experimental animal model of our study, it would be an interesting perspective to investigate the influence of PCSK9 on cholesterol levels and plaque morphology, as a potential promising target for new therapies, together with the reduction of dietary intake of lipids.
According to our results, long-term plaque reorganization suggests a predisposition to plaque stabilization due to normalized pro-atherogenic risk factors, like cholesterol levels. In contrast, cholesterol re-supplementation seems to induce a development of a more vulnerable plaque, but the re-withdrawal modifies the plaque to a more stable state. Consequently, the regression of atherosclerosis unfortunately depends on the unpredictable development of atherosclerotic lesion, which depends at the same time on the individual variability, as well as the history of the disease of each patient.
Therefore, further CEDrs studies – using rabbits with pre-lesioned thoracic aortas - with withdrawal period longer than 6 weeks are needed to investigate, whether the withdrawal time and/or the dose and/or time of atorvastatin treatment improve the plaque stability and/or definitely induce its regression partially or even completely. In order to define processes, which promote or reduce the development either of destabilizing or stabilizing plaque structures, further studies are needed, especially with regard to the development of atherosclerotic lesions in rabbits/patients with long-term dietary exogenous dyslipidemia, including prolongated withdrawal time and lipid-lowering drugs as well. According to our results, we suggest, that the period of time/concentration of statin treatment and/or regression of blood cholesterol levels may influence the curability of patients by modulating the grade of stability of atherosclerotic plaques; otherwise non-curable patients will land up at a point of no return.