Vitamin D Deficiency and Exogenous Vitamin D Excess Similarly Increase Diffuse Atherosclerotic Calcification in Apolipoprotein E Knockout Mice

Background: Observational data associate lower levels of serum vitamin D with coronary artery calcification, cardiovascular events and mortality. However, there is little interventional evidence demonstrating that moderate vitamin D deficiency plays a causative role in cardiovascular disease. This study examined the cardiovascular effects of dietary vitamin D deficiency and of vitamin D receptor agonist (paricalcitol) administration in apolipoprotein E knockout mice. Methods: Mice were fed atherogenic diets with normal vitamin D content (1.5IU/kg) or without vitamin D. Paricalcitol, or matched vehicle, was administered 3× weekly by intraperitoneal injection. Following 20 weeks of these interventions cardiovascular phenotype was characterized by histological assessment of aortic sinus atheroma, soluble markers, blood pressure and echocardiography. To place the cardiovascular assessments in the context of intervention effects on bone, structural changes at the tibia were assessed by microtomography. Results: Vitamin D deficient diet induced significant reductions in plasma vitamin D (p<0.001), trabecular bone volume (p<0.01) and bone mineral density (p<0.005). These changes were accompanied by an increase in calcification density (number of calcifications per mm2) of von Kossa-stained aortic sinus atheroma (461 versus 200, p<0.01). Paricalcitol administration suppressed parathyroid hormone (p<0.001), elevated plasma calcium phosphate product (p<0.005) and induced an increase in calcification density (472 versus 200, p<0.005) similar to that seen with vitamin D deficiency. Atheroma burden, blood pressure, metabolic profile and measures of left ventricular hypertrophy were unaffected by the interventions. Conclusion: Vitamin D deficiency, as well as excess, increases atherosclerotic calcification. This phenotype is induced before other measures of cardiovascular pathology associated clinically with vitamin D deficiency. Thus, maintenance of an optimal range of vitamin D signalling may be important for prevention of atherosclerotic calcification

Effects of 20 weeks of vitamin D deficient diet and paricalcitol on 25(OH)D and bone structure.

<p>A, plasma 25(OH)D by intervention. B, trabecular bone volume by intervention. C, trabecular bone mineral density relative to that of mice fed a vitamin D replete diet. D, representative microCT images of trabecular bone after 20 weeks of interventions. Groups are labelled according to dietary vitamin D content. Mice that did not receive paricalcitol received matched vehicle. D+, vitamin D replete diet; D-, vitamin D deficient diet; D+/P, vitamin D replete diet with paricalcitol, D−/P, vitamin D deficient diet with paricalcitol. n = 7–8 per group, data presented as mean (SEM). *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. BMD, bone mineral density; TB, trabecular bone.</p

Vitamin D manipulation does not affect atheroma burden, cellularity or lipid cleft area.

<p>A, aortic sinus cross sectional atheroma burden. B, thoracic aorta en face atheroma burden. C, percentage of acellular atherosclerosis. D, percentage atheroma cross sectional area occupied by lipid clefts. n = 7–8 per group, data presented as mean (SEM).</p

Vitamin D manipulation does not affect blood pressure in ApoE^−/− mice.

<p>A, mean tail cuff blood pressure by week. B, time-averaged mean blood pressures. n = 4 per group, data presented as mean (SEM). D+, vitamin D replete diet; D-, vitamin D deficient diet; D+/P, vitamin D replete diet with paricalcitol, D−/P, vitamin D deficient diet with paricalcitol.</p

Patterns of atheromatous calcification in ApoE^−/− mice.

<p>A–D, representative atherosclerotic lesions demonstrating diffuse calcification (stained black by von Kossa method, arrows) from mice fed vitamin D replete and vitamin D deficient diets and coadministered vehicle (A and B respectively) or paricalcitol (C and D respectively). E–G, sequential sections stained with Miller’s elastin Van Gieson, von Kossa method and osteopontin-specific antibody respectively, demonstrating a necrotic area of lesion (E) containing a large calcification (F) with associated osteopontin staining (G) (arrows), taken from a vitamin D replete mouse administered paricalcitol.</p

Effects of vitamin D deficient diet and paricalcitol on metabolic profile.

<p>n = 7–8 per group, data are given as mean (SEM).</p>#<p>p<0.01 vs. D replete vehicle, †p<0.001 vs. D replete vehicle. NOx, nitric oxide; sVCAM, soluble vascular cell adhesion molecule.</p

Vitamin D deficiency and excess similarly increase atherosclerotic calcification.

<p>A, number of calcifications normalized to lesion area. B, percentage calcified lesion area due to calcifications <100 µm². C, number of very large calcifications (≥100 µm²). D, total calcified area due to diffuse and large calcifications. n = 7–8 per group, data presented as mean (SEM). *p<0.05, **p<0.01, ***p<0.005.</p

Vitamin D deficiency does not induce left ventricular hypertrophy in ApoE^−/− mice.

<p>A, heart weights by intervention. B and C, mean LV wall thickness and LV cross sectional area measured histologically. D and E, cardiomyocyte cross sectional area and transverse diameter. F, left ventricular ejection fraction. G, left ventricular fractional area change. H, cardiac index. n = 7–8 per group for histological analyses and weights, n = 5–6 per group for echocardiography. Data presented as mean (SEM).</p