Lowering plasma cholesterol levels halts progression of aortic valve disease in mice

Abstract

Background: Treatment of hyperlipidemia produces functional and structural improvements in atherosclerotic vessels. However, the effects of treating hyperlipidemia on the structure and function of the aortic valve have been controversial, and any effects could be confounded by pleiotropic effects of hypolipidemic treatment. The goal of this study was to determine whether reducing elevated plasma lipid levels with a "genetic switch" in Reversa mice (Ldlr-/-/Apob(100/100)/Mttp(fl/fl)/Mx1-Cre+/+) reduces oxidative stress, reduces pro-osteogenic signaling, and retards the progression of aortic valve disease.

Methods and results: After 6 months of hypercholesterolemia, Reversa mice exhibited increases in superoxide, lipid deposition, myofibroblast activation, calcium deposition, and pro-osteogenic protein expression in the aortic valve. Maximum aortic valve cusp separation, as judged by echocardiography, was not altered. During an additional 6 months of hypercholesterolemia, superoxide levels, valvular lipid deposition, and myofibroblast activation remained elevated. Furthermore, calcium deposition and pro-osteogenic gene expression became more pronounced, and the aortic cusp separation decreased from 0.85+/-0.04 to 0.70+/-0.04 mm (mean+/-SE; P<0.05). Rapid normalization of cholesterol levels at 6 months of age (by inducing expression of Cre recombinase) normalized aortic valve superoxide levels, decreased myofibroblast activation, reduced valvular calcium burden, suppressed pro-osteogenic signaling cascades, and prevented reductions in aortic valve cusp separation.

Conclusions: Collectively, these data indicate that reducing plasma lipid levels by genetic inactivation of the mttp gene in hypercholesterolemic mice with early aortic valve disease normalizes oxidative stress, reduces pro-osteogenic signaling, and halts the progression of aortic valve stenosis.

Figure 1

Superoxide in aortic valve before and after normalization of blood lipids. A) and B) 6 and 12 month control; C) and D) 6 and 12months hypercholesterolemia; E) 12 month “reversed”; F) mean data for all mice (8–12 per group). Note that superoxide levels were markedly increased in both 6 and 12 month hypercholesterolemic animals, and were completely normalized by reduction of blood lipids in the “reversed” animals. Arrows highlight areas of positive staining in aortic valve tissue. *, p < 0.05 versus the time-matched control group; #, p< 0.05 versus 12 month hypercholesterolemic group.

Figure 2

Lipid (red staining) in aortic valve before and after normalization of blood lipids. A) and B) 6 and 12 month control; C) and D) 6 and 12months hypercholesterolemia; E) 12 month “reversed”; F) mean data for all mice (8–12 per group). Valvular lipid deposition was significantly increased in hypercholesterolemic mice at both 6 and 12 months. Arrows indicate aortic valve tissue. Normalizing blood lipids produced significant reductions in valvular lipid in “reversed” animals. *, p < 0.05 versus the time-matched control group; #, p< 0.05 versus 12 month hypercholesterolemic group.

Figure 3

Macrophages (brown staining) in aortic valve before and after normalization of blood lipids. A) and B) 6 and 12 month control; C) and D) 6 and 12 months hypercholesterolemia; E) 12 month “reversed”; F) mean data for all animals studied (8–12 per group). Macrophage immunostaining was markedly increased in hypercholesterolemic mice at 6 and 12 months, and was almost completely eliminated by lowering blood lipid levels in “reversed” mice. Arrows indicate aortic valve tissue. *, p < 0.05 versus the time-matched control group; #, p< 0.05 versus 12 month hypercholesterolemic group.

Figure 4

Calcification (red staining) of aortic valve before and after normalization of lipid levels. A) and B) 6 and 12 month control; C) and D) 6 and 12 months hypercholesterolemia; E) 12 month “reversed”; F) mean data for all animals studied (8–12 per group). Calcium burden was significantly increased in hypercholesterolemic mice at 6 and 12 months. Normalizing blood lipids produced significant reductions in valvular calcium burden in “reversed” mice. Arrows indicate aortic valve tissue. *, p < 0.05 versus the time-matched control group; #, p< 0.05 versus 12 month hypercholesterolemic group.

Figure 5

Phospho-Smad2 immunofluorescence, alpha-smooth muscle actin immunofluorescence, and collagen (blue) staining and in the aortic valve before and after normalization of lipid levels. P-Smad2 levels were markedly increased in 6 and 12 month hypercholesterolemic mice. Normalizing blood lipids significantly reduced the amount of P-Smad2 immunofluorescence in “Reversed” mice. Alpha-smooth muscle actin immunofluorescence was increased in 6 and 12 month hypercholesterolemic mice, was virtually non-detectable in “Reversed” mice. Collagen deposition also progressively increased in hypercholesterolemic mice from 6 to 12 months, but was not reduced in “Reversed” mice. Arrows denote areas of aortic valve tissue when immunofluorescence is very faint.

Figure 6

Pro-calcific proteins in the aortic valve before and after normalization of lipid levels. P-Smad1/5/8 was markedly increased in 6 and 12 month hypercholesterolemic animals, and was substantially reduced in the “reversed” group. Immunofluorescence of the pro-osteogenic genes Msx2, CBFA1, and Osterix was increased in both 6 and 12 month hypercholesterolemic animals, and markedly reduced by normalizing blood lipid levels. Arrows denote aortic valve tissue when fluorescence is very faint, or point to valvular tissue when non-valvular tissue is present in the micrograph.

Figure 7

Aortic valve function examined by echocardiography (A) and MRI (B) before and after normalization of lipid levels. A) Leaflet separation distance derived from echocardiography during progression and regression of aortic valve disease from 6 to 12 months. Samples of echocardiographic images are provided in online supplementary Figure S3. B) Systolic short-axis magnetic resonance images acquired in the plane of the aortic valve, using a “white blood” pulse sequence. Blood velocity through the valve is sufficient to cause blood signal dephasing, rendering blood-filled voxels black (arrows) and providing contrast with valve tissue and surrounding cardiac structures Valve areas measured off-line by electronic planimetry: 6mo CTRL = 0.09 mm², 6mo Hchol = 1.0 mm², 12mo Hchol = 0.05 mm², “Reversed” = 1.19 mm²). Arrows point towards the aortic valve orifice.