Effect of cholesterol re-supplementation and atorvastatin on plaque composition in the thoracic aorta of New Zealand white rabbits

Abstract

Background: Effects of re-supplementation of a cholesterol-enriched diet (CEDrs) on size, cholesterol content and morphology of already existing plaques are not known to date.

Methods: A group of rabbits received standard chow (SC) for 6 weeks ("negative control"; for plasma lipid measurements only). Group I-IV received 2% CED (induction) for 6 weeks; thereafter, groups II-IV have been fed a SC (= cholesterol withdrawal) for 68 weeks. Afterwards, feeding of groups II-IV was continued as follows: Group II - 10 weeks SC, group III - 4 weeks 0.5% CED (~re-supplementation), afterwards 6 weeks SC (~withdrawal again); group IV - 4 weeks 0.5% CED (re-supplementation) + atorvastatin (2.5 mg/kg body weight/day), afterwards 6 weeks SC (~withdrawal again) + atorvastatin. Plasma lipids, but also plaque size, morphology and cholesterol contents of thoracic aortas were quantified.

Results: After CEDrs, plasma cholesterol levels were increased. However, after withdrawal of CEDrs, plasma cholesterol levels decreased, whereas the cholesterol content of the thoracic aorta was increased in comparison with the group without CEDrs. Plaque size remained unaffected. Atorvastatin application did not change plasma cholesterol level, cholesterol content of the thoracic aorta and plaque size in comparison with the group without drug treatment. However, atorvastatin treatment increased the density of macrophages (MΦ) compared with the group without treatment, with a significant correlation between densities of MΦ (Mac-1⁺) and apoptotic (TUNEL⁺; TP53⁺), antigen-presenting (HLA-DR⁺) or oxidatively stressed (SOD2⁺) cells.

Conclusions: In rabbits with already existing plaques, CEDrs affects plaque morphology and cellular composition, but not plaque size. Despite missing effects on plasma cholesterol levels, cholesterol content of the thoracic aorta and size of already existing atherosclerotic plaques, atorvastatin treatment transforms the already existing lesions to a more active form, which may accelerate the remodelling to a more stable plaque.

Keywords: Apoptosis; Atherosclerosis; Atorvastatin; Hypercholesterolemic rabbits; Macrophage; Re-supplementation.

Fig. 1

Time schedule of the dietary interventions used in this study. Cholesterol-enriched diet (CED); CED re-supplementation (CEDrs) and control group after feeding for six week with standard chow. Black arrow, “6 weeks”, indicates the end point of the CED induction period. Black arrow, “74 weeks” indicates the end point of the 68 weeks withdrawal and the initiation of the 4 weeks CEDrs. Black arrow, “78 weeks”, indicates the end of 4 weeks CEDrs with or without atorvastatin treatment. Black arrow, “84 weeks”, indicates the endpoint of the study

Fig. 2

Plasma cholesterol (a), triglyceride (b) and (c) phospholipid levels (in mg/dl) of rabbits of negative control group (neg. control), as well as groups I, II, III, and IV. Black arrows indicate the CEDrs point. Data are expressed as means + SEM; *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001 vs. group I (6 weeks CED, [6w]); ^#p ≤ 0.05, ^##p ≤ 0.01, ^###p ≤ 0.001 vs group II at the corresponding time point; ^§p ≤ 0.05, ^§§p ≤ 0.01, ^§§§p ≤ 0.001 vs 78 weeks (78w); n = 5–8

Fig. 3

Cholesterol content in proximal segment of the thoracic aorta (a) of groups under test. Plaque area [mm²] in the thoracic aorta (b) and cell density [n/mm²] in atherosclerotic plaques (c) of the experimental groups. Data are expressed as means + SEM; *p < 0.05, **p ≤ 0.01 vs. group I (6 weeks CED, [6w]); ^§p ≤ 0.05 vs control; n = 5–8

Fig. 4

Effect of the CEDrs (at 84 weeks of the experiment) on density of (a) MΦ (Mac-1⁺), (b) apoptotic cells (TUNEL⁺), (c) antigen presenting cells (HLA-DR⁺), (d) oxidatively stressed cells (SOD2⁺), (e) apoptotic cells (TP53⁺) and (f) smooth muscle cells (α-actin⁺) in atherosclerotic plaques of thoracic aortas compared with group I (after 6 weeks induction with CED). Data are expressed as Mean + SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. group I; ^##P < 0.01 vs group II; ^§P < 0.05 vs group III. n = 5–8

Fig. 5

Representative microphotographs of atheromatous lesions of aortic wall sections of groups under test. Black arrows indicate Mac-1 MΦ, TUNEL+, HLA-DR+, SOD2+ or TP53+ immunoreactivity and TUNEL-positive cells. Magnification: × 200