Osteoprotegerin Inhibits Aortic Valve Calcification and Preserves Valve Function in Hypercholesterolemic Mice

<div><p>Background</p><p>There are no rigorously confirmed effective medical therapies for calcific aortic stenosis. Hypercholesterolemic <i>Ldlr</i>^−/−<i>Apob</i>^100/100 mice develop calcific aortic stenosis and valvular cardiomyopathy in old age. Osteoprotegerin (OPG) modulates calcification in bone and blood vessels, but its effect on valve calcification and valve function is not known.</p><p>Objectives</p><p>To determine the impact of pharmacologic treatment with OPG upon aortic valve calcification and valve function in aortic stenosis-prone hypercholesterolemic <i>Ldlr</i>^−/−<i>Apob</i>^100/100 mice.</p><p>Methods</p><p>Young <i>Ldlr</i>^−/−<i>Apob</i>^100/100 mice (age 2 months) were fed a Western diet and received exogenous OPG or vehicle (N = 12 each) 3 times per week, until age 8 months. After echocardiographic evaluation of valve function, the aortic valve was evaluated histologically. Older <i>Ldlr</i>^−/−<i>Apob</i>^100/100 mice were fed a Western diet beginning at age 2 months. OPG or vehicle (N = 12 each) was administered from 6 to 12 months of age, followed by echocardiographic evaluation of valve function, followed by histologic evaluation.</p><p>Results</p><p>In Young <i>Ldlr</i>^−/−<i>Apob</i>^100/100 mice, OPG significantly attenuated osteogenic transformation in the aortic valve, but did not affect lipid accumulation. In Older <i>Ldlr</i>^−/−<i>Apob</i>^100/100 mice, OPG attenuated accumulation of the osteoblast-specific matrix protein osteocalcin by ∼80%, and attenuated aortic valve calcification by ∼ 70%. OPG also attenuated impairment of aortic valve function.</p><p>Conclusions</p><p>OPG attenuates pro-calcific processes in the aortic valve, and protects against impairment of aortic valve function in hypercholesterolemic aortic stenosis-prone <i>Ldlr</i>^−/−<i>Apob</i>^100/100 mice.</p></div

Morphometric and metabolic parameters.

<p><b>Veh-LA:</b> vehicle-treated Ldlr^−/−Apob^100/100 mice; <b>OPG-LA</b>: osteoprotegerin-treated Ldlr^−/−Apob^100/100 mice.</p>*<p>p<0.05 vs. Veh-LA;</p>†<p>p<0.05 vs. Young mice.</p

Immunostaining for monocyte chemo-attractant protein-1 (MCP-1) in Older LA mice.

<p>OPG decreased levels of MCP-1 in the aortic valve. N = 11 (Veh) and N = 6 (OPG). *p<0.05 for Veh vs. OPG.</p

Calcification in the aortic valve.

<p>Alizarin Red staining in a valve from an Older vehicle-treated mouse (<b>A,C</b>) demonstrates bright red staining, indicating valve calcification (arrows). Valve cusps are thickened in an Older OPG-treated mouse, but are minimally calcified (<b>B,D</b>). Dashed borders contain valve cusps, with care taken to exclude the aortic annulus (aa). Group data for valve calcification in Young mice (E) and Older mice (F). *p<0.05 Veh vs. OPG, N = 12.</p

Aortic valve function.

<p><b>A,B</b>: M-mode echocardiograms from Older mice depict aortic valve systolic dimension (arrows). Vertical white bar = 1 mm. <b>C</b>: Aortic systolic valve dimension in Young Mice studied at age 8 mo. N = 12, p = NS. <b>D</b>: Aortic valve systolic valve dimension in Older LA mice before (Pre-Rx, age 6 mo.) and after (Post-Rx, age 12 mo.) treatment with VEH or OPG, N = 12. *p<0.05 for VEH <i>vs</i>. OPG. † p<0.05 for 12 mo. vs. 6 mo. for comparisons within each treatment group.</p

Immunostaining for osteocalcin in the aortic valve in Older mice.

<p>In vehicle-treated mice (<b>A,C</b>), osteocalcin (dark brown) is abundant near the cusp base (arrows). There is only scant staining in the valve of an OPG-treated mouse (<b>B,D</b>). N = 4. *p<0.05 Veh vs. OPG.</p