Objective: The use of animal models of aortic stenosis (AS) remains essential to further elucidate its patho-physiology 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 D 3 supplementation in this model. Methods: HFD and subcutaneous vitamin D 3 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) 2 D 3 and cholesterol), histology and immunohistochem-istry (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) 2 D 3 were significantly increased relative to the control group. HFD and vitamin D 3 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 D 3 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. Please cite this article as: Colleville B, Perzo N, Avinée G, Dumesnil A, Ziegler F, Billoir P, Eltchaninoff H, Richard V, Durand E. Impact of high-fat diet and vitamin D 3 supplementation on aortic stenosis establishment in waved-2 epidermal growth factor receptor mutant mice.