Increased dietary intake of vitamin A promotes aortic valve calcification in vivo

Abstract

Objective: Calcific aortic valve disease (CAVD) is a major public health problem with no effective treatment available other than surgery. We previously showed that mature heart valves calcify in response to retinoic acid (RA) treatment through downregulation of the SRY transcription factor Sox9. In this study, we investigated the effects of excess vitamin A and its metabolite RA on heart valve structure and function in vivo and examined the molecular mechanisms of RA signaling during the calcification process in vitro.

Methods and results: Using a combination of approaches, we defined calcific aortic valve disease pathogenesis in mice fed 200 IU/g and 20 IU/g of retinyl palmitate for 12 months at molecular, cellular, and functional levels. We show that mice fed excess vitamin A develop aortic valve stenosis and leaflet calcification associated with increased expression of osteogenic genes and decreased expression of cartilaginous markers. Using a pharmacological approach, we show that RA-mediated Sox9 repression and calcification is regulated by classical RA signaling and requires both RA and retinoid X receptors.

Conclusions: Our studies demonstrate that excess vitamin A dietary intake promotes heart valve calcification in vivo. Therefore suggesting that hypervitaminosis A could serve as a new risk factor of calcific aortic valve disease in the human population.

Figure 1. Excess dietary vitamin A promotes heart valve calcification phenotypes in mice

C57BL/6J wild type mice were fed either a control diet containing 20 IU/g retinyl palmitate or an excess vitamin A diet containing 200 IU/g over a period of 12 months. (A, B) von Kossa reactivity indicates calcific nodule formation (arrows) in aortic valves from mice fed control (A), or an excess vitamin A (B) diet. (C) Quantification of von Kossa as a percentage of total area defined by Alcian blue staining (n=3). (D) qPCR to show fold changes in gene expression in aortic valves from mice fed an excess vitamin A diet compared to controls (n=3). (E–H) Echocardiography and Doppler flow analysis to show aortic valve peak pressure gradient (E) and peak velocity (F) in mice fed excess vitamin A. (G, H) Representative Doppler profiles of aortic outflow velocity for mice subjected to control (G) and excess vitamin A (H) diets (n=6). Blue line indicates measurements taken. * p<0.05 over control.

Figure 2. RARb,g isoforms are highly expressed in mouse and chicken aortic and mitral valves

Quantitative real-time PCR to show approximate absolute transcript numbers of various RAR and RXR isoforms in postnatal mouse (A), and embryonic day 10 chicken (B) aortic (AoV) and mitral (MV) valves.

Figure 3. ATRA-mediated heart valve calcification requires RAR and RXR activity

cE10 mitral valve explants were treated for 48 hours with indicated agonists, with or without a 4 hour antagonist pre-treatment. (A) Diagram depicting selectivity of RAR and RXR agonists and antagonists. (B–C) qPCR to show changes in RARβ expression (B, C) as a fold change with agonist treatments (B), and as a percent change following pre-treatment with RAR (LE540) or RXR (PA452) pan-antagonists compared to ATRA and 9-cis agonists alone. (D) qPCR analysis to show percentage changes in Sox9 expression in agonist treatment alone, and pre-treatment with antagonists. * p<0.05 over vehicle; # p<0.05 over agonist.

Figure 4. Retinoic acid treatment promotes calcific nodule formation in cE10 mitral valve explants

Compared to vehicle controls (A), von Kossa reactivity is increased following treatment with ATRA (B) and 9-cis RA (E). von Kossa reactivity is attenuated when co-treated with pharmacological antagonists (C,D,F,G,H). (I) Quantification of von Kossa reactivity normalized to area, as indicated by Alcian blue counterstain. * p<0.05 over vehicle; # p<0.05 over agonist.

Figure 5. Treatment with specific RAR isoform agonists promotes expression of osteogenic genes at the expense of cartilaginous markers

(A) qPCR to show fold changes in RARb expression following treatment of cE10 or postnatal mouse aortic valve explants with RARa- (AM580) and RARb,g,-specific (Adapalene) agonists relative to vehicle controls. (B, C) qPCR to show changes in osteogenic (Spp1) and cartilaginous (Sox9, Col2a1) gene expression following treatments. * p<0.05 over vehicle controls.