Objective:The use of animal models of aortic stenosis (AS) remains essential to further elucidate its pathophysiology and to evaluate new therapeutic strategies.The waved-2 mouse AS model has been proposed;data have indicated that while aortic regurgitation (AR) is effectively induced,development of AS is rare.We aimed to evaluate the effect of high-fat diet (HFD) and vitamin D3 supplementation in this model.Methods:HFD and subcutaneous vitamin D3 injections were initiated at the age of 6 weeks until the age of 6 (n =16,6-month treatment group) and 9 (n =11,9-month treatment group) months.Twelve waved-2 mice without supplementation were used as control.Echocardiography was performed at 3,6 and 9 months.Blood serum analysis (calcium,1,25(OH)2D3 and cholesterol),histology and immunohistochemistry (CD-31,CD-68 and osteopontin) were evaluated at the end of the experiment (6 or 9 months).Results:Total cholesterol and 1,25(OH)2D3 were significantly increased relative to the control group.HFD and vitamin D3 supplementation did result in improvements to the model,since AS was only detected in 6 (15.3％) mice (2 in the 3 groups) and AR was developed in the remaining animals.Echocardiographic parameters,fibrosis,thickness,inflammation and valvular calcification,were not significantly different between the 6-month treatment and control groups.Similar results were also observed in the 9-month treatment group.Conclusion:These results suggest that HFD and vitamin D3 supplementation have no effect in the waved-2 mouse model.This model essentially mimics AR and rarely AS.Further studies are needed to find a reliable animal model of AS.