Atorvastatin inhibits calcification and enhances nitric oxide synthase production in the hypercholesterolaemic aortic valve

Abstract

Objective: To study in a rabbit model the expression of endothelial nitric oxide synthase (eNOS) in association with the development of calcification of the aortic valve, and to assess the effects of atorvastatin on eNOS expression, nitrite concentration, and aortic valve calcification.

Methods: Rabbits (n = 48) were treated for three months: 16, forming a control group, were fed a normal diet; 16 were fed a 0.5% (wt/wt) high cholesterol diet; and 16 were fed a 0.5% (wt/wt) cholesterol diet plus atorvastatin (2.5 mg/kg/day). The aortic valves were examined with eNOS immunostains and western blotting. Cholesterol and high sensitivity C reactive protein (hsCRP) concentrations were determined by standard assays. Serum nitrite concentrations were measured with a nitric oxide analyser. eNOS was localised by electron microscopy and immunogold labelling. Calcification in the aortic valve was evaluated by micro-computed tomography (CT).

Results: Cholesterol, hsCRP, and aortic valve calcification were increased in the cholesterol fed compared with control animals. Atorvastatin inhibited calcification in the aortic valve as assessed by micro-CT. eNOS protein concentrations were unchanged in the control and cholesterol groups but increased in the atorvastatin treated group. Serum nitrite concentrations were decreased in the hypercholesterolaemic animals and increased in the group treated with atorvastatin.

Conclusion: These data provide evidence that chronic experimental hypercholesterolaemia produces bone mineralisation in the aortic valve, which is inhibited by atorvastatin.

Figures

Figure 1

Evidence for endothelial nitric oxide synthase (eNOS) localisation and calcification in the aortic valve. Left column, control diet; middle column, cholesterol (Chol) diet; right column, cholesterol diet plus atorvastatin (Atorv). In each panel, the aortic valve leaflet is positioned on the left, with the aorta on the right. (A) eNOS immunostaining (all frames 4× magnification). (B) eNOS immunostaining (all frames 20× magnification). (C) eNOS immunogold electron microscopy (low magnification, 25 000×) with areas of mineralisation (arrows). (D) eNOS immunogold electron microscopy (high magnification, 125 000×). (E) Western blot analysis of eNOS protein expression and the control.

Figure 2

Micro-computed tomography of the aortic valves in the treatment groups indicating an area of calcification in the cholesterol treated aortic valve. Aortic valves and aortas from the (A) control group; (B) hypercholesterolaemic group (the arrow shows early stages of calcification); and (C) hypercholesterolaemic, atorvastatin treated group.