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

The Multifunctional Ca²⁺/Calmodulin-Dependent Kinase IIδ (CaMKIIδ) Regulates Arteriogenesis in a Mouse Model of Flow-Mediated Remodeling

<div><p>Objective</p><p>Sustained hemodynamic stress mediated by high blood flow promotes arteriogenesis, the outward remodeling of existing arteries. Here, we examined whether Ca²⁺/calmodulin-dependent kinase II (CaMKII) regulates arteriogenesis.</p><p>Methods and Results</p><p>Ligation of the left common carotid led to an increase in vessel diameter and perimeter of internal and external elastic lamina in the contralateral, right common carotid. Deletion of CaMKIIδ (CaMKIIδ−/−) abolished this outward remodeling. Carotid ligation increased CaMKII expression and was associated with oxidative activation of CaMKII in the adventitia and endothelium. Remodeling was abrogated in a knock-in model in which oxidative activation of CaMKII is abolished. Early after ligation, matrix metalloproteinase 9 (MMP9) was robustly expressed in the adventitia of right carotid arteries of WT but not CaMKIIδ−/− mice. MMP9 mainly colocalized with adventitial macrophages. In contrast, we did not observe an effect of CaMKIIδ deficiency on other proposed mediators of arteriogenesis such as expression of adhesion molecules or smooth muscle proliferation. Transplantation of WT bone marrow into CaMKIIδ−/− mice normalized flow-mediated remodeling.</p><p>Conclusion</p><p>CaMKIIδ is activated by oxidation under high blood flow conditions and is required for flow-mediated remodeling through a mechanism that includes increased MMP9 expression in bone marrow-derived cells invading the arterial wall.</p></div

CaMKII is upregulated and activated in arteriogenesis.

<p>(A) CaMKII activity in right carotid arteries from WT and CaMKIIδ−/− mice 14 days after left carotid ligation. (B) Immunolabeling for total CaMKII (green; SM–actin red; nuclei blue) in WT and CaMKIIδ−/− right carotid artery sections on day 14 after ligation. Arrow, adventitia; arrowhead, endothelium. (C) Fold change in mRNA expression of CaMKIIδ and γ in right carotids isolated from WT and CaMKIIδ−/− mice by quantitative RT-PCR. (D) Immunolabeling for oxidized (ox-CaMKII, green, left panel) and phosphorylated CaMKII (p-CaMKII, green, right panel) in WT right carotid artery sections (SM–actin red; nuclei blue). Arrowheads indicate single cells with p-CaMKII labeling. Scale bar = 30 µm.</p

CaMKIIδ is required for flow-mediated remodeling.

<p>(A) Diagram of experimental approach. Arteriogenesis is induced in the right common carotid artery (CCA) after left CCA ligation. (B) Representative H&E-stained right carotid arteries of WT and CaMKIIδ−/− mice at baseline (day 0) and day 28 post-left carotid ligation. Scale bar = 200 µm. (C) Quantification of the perimeter of the internal (IEL) and external elastic lamina (EEL) (n = 6 for day 0 and n = 10 for days 14 and 28). (D) Ultrasound cross-sectional images of the right common carotid artery 28 days after left carotid ligation. The insets demonstrate color Doppler flow in the right carotid. No flow was detected in the left common carotid. (E) Quantification of the anterior-posterior diameter of right common carotid arteries of WT and CaMKIIδ−/− mice (n = 10 per genotype, experiments are independent of (B) and (C)). *p<0.05 compared to baseline; **p<0.05 compared to WT.</p

Transplantation of WT bone marrow cells into CaMKIIδ−/− mice restores arteriogenesis.

<p>(A) Quantification of the anterior-posterior diameter of right carotid arteries of WT or CaMKIIδ−/− mice transplanted with WT BM (n = 6 for WT and n = 10 for CaMKIIδ−/− mice, *p<0.05 compared to day 0). (B) Representative H&E-stained WT and CaMKIIδ−/− right carotid arteries after transplantation of WT bone marrow. Carotid artery ligations were performed 8 weeks after BM transplantation. Scale bar = 200 µm (C) Quantification of the perimeter of the IEL and EEL (n = 6 for WT and n = 10 for CaMKIIδ−/− mice).</p