[18F]-Sodium fluoride imaging of calcification in a novel murine model of calcific aortic valve disease

33 Objectives: Calcific aortic valve disease (CAVD), the major cause of aortic stenosis (AS), is characterized by fibro-calcific remodeling of the aortic valve leaflets. In the absence of an effective medical therapy for CAVD, therapeutic options remain limited to aortic valve replacement for advanced, symptomatic disease. Several therapeutic agents which were found to be effective in animal models have failed in clinical trials. This failure may be related gaps in knowledge regarding the CAVD pathophysiology, lack of representative animal models for human disease, and lack of appropriate tools for tracking therapeutic effectiveness in vivo. Indeed, preclinical studies of CAVD are often performed in hyperlipidemic or hyperphosphatemic animal models, which are confounded by the presence of atherosclerosis or non-physiologic mineral levels. Fluorine-18 sodium fluoride ([18F]-NaF) can be used for positron emission tomography (PET) imaging of the calcification process by leveraging the rapid exchange of F-18 with hydroxyl groups of hydroxyapatite. Discoidin, CUB and LCCL Domain Containing 2 (DCBLD2) is a neuropilin-like protein which regulates vascular remodeling and growth factor signaling. Here, using [18F]-NaF to detect the calcification process in vivo, we investigated whether DCBLD2 regulates valvular remodeling. Methods: Dcbld2-/- mice at 3-4 months (young) and 9-12 months of age (old) and age-matched wild-type animals (WT) were used for this study. The presence of AS was evaluated in old female Dcbld2-/- mice (n = 12) and age-match wild type (WT) animals (n = 3) by echo Doppler, using an aortic valve jet velocity of >2.5 m/s to define AS. Young (n = 3) and old (n = 3) Dcbld2-/- mice and age-matched WT animals were injected intra-venously with 16.3 ± 4.9 MBq of [18F]-NaF. Animals were kept under anesthesia for 1 h and blood samples were collected. After 1 h, tissue samples were collected to evaluate [18F]-NaF biodistribution. The whole aorta, including the aortic valve, was carefully dissected for quantitative autoradiography. The presence of valvular calcification was investigated in Dcbld2-/- mice by Alizarin red staining of aortic valve tissue sections and compared with age-matched WT animals. Results: No AS was present in WT Mice, but 42% (5/12) of Dcbld2-/- mice had hemodynamically significant AS. Young and old ESDN-/- animals injected with [18F]-NaF showed similar activities in blood and all harvested tissues with the exception of bone (49.9 ± 5.9 vs 30.0 ± 5.0 % injected dose (ID)/g, for young and old mice, respectively, p < 0.05). A similar biodistribution profile was observed in age-matched WT animals. Autoradiography analysis showed a trend toward higher uptake of [18F]-NaF uptake in aortic valves of old Dcbld2-/- mice compared to young animals (ratio to age-matched WT: 1.59 ± 0.10 vs 1.17 ± 0.26, p = 0.057). Of note, while considerable heterogeneity of aortic valve [18F]-NaF signal was noted in young Dcbld2-/- mice, potentially reflecting differences in the calcification process in young animals, in old Dcbld2-/- mice the uptake was consistently high (range of ratio to aged-matched WT: 1.50-1.69). Additionally, foci of elevated [18F]-NaF signal was noted in the aortae of old Dcbld2-/- mice, reflecting concomitant vascular calcification. The presence of aortic valve microcalcifications was confirmed in 67% (8/12) of old Dcbld2-/- mice, while none of the tested young Dcbld2-/- or WT mice revealed any calcification on histological analysis. Conclusions: DCBLD2 regulates aortic valve calcification, with its deficiency leading to spontaneous CAVD and hemodynamically significant AS. The extent of valvular and vascular calcification in Dcbld2-/- mice may be detected by [18F]-NaF-based molecular imaging. Furthermore, [18F]-NaF autoradiography appears more sensitive than Alizarin red staining for detection of microcalcifications. This unique model of AS will be of value in developing novel therapeutic and imaging agents for CAVD.