Influence of serum cholesterol on atherogenesis and intimal hyperplasia after angioplasty: inhibition by amlodipine

Abstract

The objectives of the present study were to determine whether serum hypercholesterolemia (HC) promotes the development of spontaneous and angioplasty-induced lesions and whether amlodipine inhibits these lesions and cellular processes underlying their genesis. Rabbits were fed normal, 0.5%, or 2% cholesterol diets for 9 wk, which resulted in the development of increasing HC. After week one, balloon dilation of the abdominal aorta was performed while the thoracic aorta was not disturbed and monitored for the development of spontaneous lesions. Lesion size increased with the degree of HC and was accompanied by increased collagen synthesis and smooth muscle cell (SMC) proliferation at each site. Amlodipine (5 mg/kg p.o.) inhibited lesion size by 50% (P < 0.01) at both sites in cholesterol-fed animals but not at angioplasty sites in animals on a normal diet. Local collagen synthesis was inhibited at both sites by amlodipine in the diet animals. The increase in HC was accompanied by a 1.7-fold increase in basal Ca2+ uptake in SMCs in the thoracic aorta, which was not altered by amlodipine, nifedipine, Ni2+, or La3+, revealing an uninhibitable calcium leak during atherogenesis. In culture, cholesterol enrichment increased SMC proliferation, collagen synthesis, and the secretion of a soluble SMC mitogen, which were inhibited by amlodipine (10(-9) M). Finally, in SMC membranes, amlodipine uniquely restored the cholesterol-expanded membrane bilayer width without any effect on membrane fluidity. This study establishes a causal role between serum HC and the development of spontaneous and angioplasty-induced lesions and the ability of amlodipine to disrupt this action by a novel remodelling action on the SMC membrane.

Fig. 1

Size of spontaneous lesions (shaded bars) as well as angioplasty-induced intimal hyperplasia (open bars) increases with increasing blood cholesterol achieved by feeding animals a cholesterol diet supplemented with either 0.5% or 2% cholesterol. TC, total cholesterol; I/M ratio, intimal area-to-medial area ratio. ¶P < 0.001, 0.5% diet vs. control diet; §P < 0.05, 2% diet vs. 0.5% diet; *P < 0.01, 0.5% diet vs control diet; †P < 0.01, 2% diet vs. 0.5% diet. n ≥ 6 animals. Statistical analysis was by ANOVA.

Fig. 2

A: oral amlodipine (AML; shaded bars) suppresses the development of angioplasty-induced intimal hyperplasia in the abdominal aorta by ~40% compared with placebo (open bars) in hypercholesterolemic animals regardless of the magnitude of increase in serum cholesterol levels. Interestingly, AML had no effect on intimal hyperplasia in animals fed the normal (cholesterol free) diet. This finding suggests a dependence of the AML effect on enrichment of smooth muscle cells (SMCs) with cholesterol. B: oral AML inhibited the development of spontaneous lesions in the thoracic aorta by ~45% in hypercholesterolemic animals regardless of the magnitude of increase in serum cholesterol levels. *P < 0.05, AML treated vs. placebo. n = 3–6 animals. Statistical analysis was by ANOVA.

Fig. 3

A: collagen synthesis is increased at sites of spontaneous and angioplasty-induced lesions in cholesterol-fed (0.5%) rabbits, and oral AML (shaded bars) inhibited collagen synthesis at both sites. B: however, AML failed to significantly inhibit SMC proliferation at either site. C: hypercholesterolemia in rabbits on the 0.5% diet increased calcium uptake by 82% in the thoracic aorta (hatched bar), but neither the organic (amlodipine, nifedipine) nor inorganic (Ni²⁺, La³⁺) calcium channel blockers (CCBs; shaded bars) inhibited this increase, demonstrating the development of an uninhibitable calcium “leak” in the early atherogenic period. *P < 0.05, atherosclerosis vs. control. n = 7 animals. Statistical analysis was by unpaired Student’s t-test (A and B) and by ANOVA (C).

Fig. 4

A: in cultured SMCs, enrichment with exogenous cholesterol increased the free cholesterol (FC) content in the cell membrane by 2-fold. Incubation of the cells with AML (1 nM) had no effect on membrane cholesterol content in control cells or cells subjected to cholesterol enrichment. PL, phospholipid. *P < 0.01, cholesterol enriched vs. control. n = 5–7. B: cholesterol enrichment increased SMC collagen synthesis. Whereas AML had no effect on control SMCs, it abolished the cholesterol-induced increase in synthesis. *P < 0.01, cholesterol-enriched SMCs treated with AML vs. not treated. n = 4. C: cholesterol enrichment increased SMC proliferation by 3.6-fold. Whereas AML had no effect on control SMCs, it abolished the cholesterol-induced increase in cell proliferation. †P < 0.01, cholesterol enriched vs. control; *P < 0.05, cholesterol-enriched SMCs treated with AML vs. not treated. n = 6. D: culture medium (CM) conditioned with cholesterolenriched SMCs had twice the mitogenic activity on SMC proliferation as medium conditioned with growth-arrested control SMCs. AML treatment of cholesterol-enriched SMCs abolished the mitogenic activity in the conditioned medium. *P < 0.01, medium from cholesterol-enriched SMCs treated with AML vs. medium from cholesterol-enriched SMCs not treated with AML. n = 3. In C and D, cells were seeded at a density of 3.5 × 10³ cells/cm². Statistical analysis was by unpaired Student’s t-test (A–C) and by ANOVA (D).

Fig. 5

A: CCBs fail to alter membrane fluidity in FC/DMPC membrane vesicles. B: AML, but not felodipine, diltiazem, or verapamil, reduced membrane width. *P < 0.05, AML treated vs. control. n = 3. C: AML treatment reduced membrane width by 5.6% and phosphorus head group separation by 4.9% in native membranes isolated from SMCs freshly harvested from an atherosclerotic aorta. All CCBs were used at a concentration of 0.1 µM. Statistical analysis was by ANOVA (A and B).