Atorvastatin inhibits hypercholesterolemia-induced calcification in the aortic valves via the Lrp5 receptor pathway

Abstract

Background: Calcific aortic valve disease is the most common indication for surgical valve replacement in the United States. The cellular mechanisms of valve calcification are not well understood. We have previously shown that cellular proliferation and osteoblastogenesis are important in the development of valvular heart disease. Lrp5, a known low-density receptor-related protein, plays an essential role in cellular proliferation and osteoblastogenesis via the beta-catenin signaling pathway. We hypothesize that Lrp5 also plays a role in aortic valve (AV) calcification in experimental hypercholesterolemia.

Methods and results: We examined the effects of cholesterol and atorvastatin in Watanabe rabbits (n=54). Group I (n=18) received a normal diet, group II (n=18) a 0.25% cholesterol diet, and group III (n=18) a 0.25% (w/w) cholesterol diet with atorvastatin for the development of calcification. The AVs were examined for cellular proliferation, Lrp5/beta-catenin, and bone matrix markers. Bone formation was assessed by micro-computed tomography, calcein injection, and osteopontin expression. Low-density lipoprotein with and without atorvastatin was also tested in AV myofibroblasts for cellular proliferation and regulation of the Lrp5/beta-catenin pathway. Our results demonstrate that the cholesterol diet induced complex bone formations in the calcified AVs with an increase in the Lrp5 receptors, osteopontin, and p42/44 expression. Atorvastatin reduced bone formation, cellular proliferation, and Lrp5/beta-catenin protein levels in the AVs. In vitro analysis confirmed the Lrp5/beta-catenin expression in myofibroblast cell proliferation.

Conclusions: Hypercholesterolemic AV calcification is attenuated by atorvastatin and is mediated in part by the Lrp5/beta-catenin pathway. This developmental pathway may be important in the signaling pathway of this disease.

Figure 1

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, the aortic valve leaflet is positioned on the left, with the aorta on the right. Arrow points to aortic valve in each figure. All frames 12.5× magnification. A, Hematoxylin and eosin stain. B, Masson trichrome stain. C, elastin Van Geison stain.

Figure 2

Proliferation marker and Lrp5/β-catenin protein expression. Left column, control diet; middle column, cholesterol diet; right column, cholesterol diet plus atorvastatin. Arrow points to aortic valve in each figure. A, α-actin immunostain. B, PCNA immunostain. C, p42/44 Western blot (1) and quantification (2). D, Lrp5 receptor immunostain. E, β-catenin immunostain.

Figure 3

Establishment of the bone-like phenotype in the calcified aortic valves. 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. Arrow points to aortic valve in each figure. All frames 12.5× magnification. B, Osteopontin Western blot and quantification. C, micro-computed tomography 2-dimensional reconstructed slices of each valve (3.4 mm horizontal field of view). D, Confocal microscopy of the calcein labeling in the mineralizing valve.

Figure 4

In vitro myofibroblast cell model of proliferation and osteopontin expression in the presence of LDL with and without atorvastatin. A, Western blot for Lrp5, βcatenin, osteopontin, and p42/44. B, Thymidine incorporation in the LDL treated cells with and without atorvastatin. *P<0.001.