Atorvastatin inhibits hypercholesterolemia-induced cellular proliferation and bone matrix production in the rabbit aortic valve

Abstract

Background: Despite the common occurrence of aortic stenosis, the cellular causes of the disorder are unknown, in part because of the absence of experimental models. We hypothesized that atherosclerosis and early bone matrix expression in the aortic valve occurs secondary to experimental hypercholesterolemia and that treatment with atorvastatin modifies this transformation.

Methods and results: To test this hypothesis, we developed an experimental hypercholesterolemic rabbit model. New Zealand White rabbits (n=48) were studied: group 1 (n=16), normal diet; group 2 (n=16), 1% (wt/wt) cholesterol diet; and group 3 (n=16), 1% (wt/wt) cholesterol diet plus atorvastatin (3 mg/kg per day). The aortic valves were examined with hematoxylin and eosin stain, Masson trichrome, macrophage (RAM 11), proliferation cell nuclear antigen (PCNA), and osteopontin immunostains. Cholesterol and highly sensitive C-reactive protein (hsCRP) serum levels were obtained by standard assays. Computerized morphometry and digital image analysis were performed for quantifying PCNA (% area). Electron microscopy and immunogold labeling were performed for osteopontin. Semiquantitative RT-PCR was performed for the osteoblast bone markers [alkaline phosphatase, osteopontin, and osteoblast lineage-specific transcription factor (Cbfa-1)]. There was an increase in cholesterol, hsCRP, PCNA, RAM 11, and osteopontin and osteoblast gene markers (alkaline phosphatase, osteopontin, and Cbfa-1) in the cholesterol-fed rabbits compared with control rabbits. All markers except hsCRP were reduced by atorvastatin.

Conclusions: These findings of increased macrophages, PCNA levels, and bone matrix proteins in the aortic valve during experimental hypercholesterolemia provide evidence of a proliferative atherosclerosis-like process in the aortic valve associated with the transformation to an osteoblast-like phenotype that is inhibited by atorvastatin.

Figures

Figure 1

Serum cholesterol and hsCRP levels. A, Total serum cholesterol levels in each treatment group. B, Total hsCRP levels in serum of each treatment group. Chol indicates cholesterol-fed; Atorv, atorvastatin treatment. *P<0.001.

Figure 2

Light microscopy of rabbit aortic valves and aorta. Left column, control diet; middle column, cholesterol diet; right column, cholesterol diet plus atorvastatin. In each panel, aortic valve leaflet is positioned on the left, with aorta on the right. All frames ×12.5 magnification. A, Hematoxylin and eosin stain; B, Masson trichrome stain; C, macrophage, RAM 11; D, PCNA stain.

Figure 3

Quantification of PCNA in each treatment group. *P<0.001.

Figure 4

Evidence for osteopontin production in aortic valve. Left column, control diet; middle column, cholesterol diet; right column, cholesterol diet plus atorvastatin. A, Osteopontin immunostain light microscopy, aortic valve on the left and aorta on the right. All frames ×12.5 magnification. B, Osteopontin immunogold electron microscopy (magnification, 150 000).

Figure 5

Semiquantitative RT-PCR. A, RT-PCR with the total RNA from aortic valves for alkaline phosphatase (AlkPhos) (475bp), results normalized to GAPDH (451 bp). B, RT-PCR with the total RNA from aortic valves for osteopontin (OP) (347bp), results normalized to GAPDH (451 bp). C, RT-PCR with the total RNA from the aortic valves for Cbfa-1 (289bp), results normalized to GAPDH (451 bp). Cont indicates control; Chol, cholesterol-fed; Atorv, atorvastatin treatment.