Hyperlipidemic ApoE^-/- mice as a model for studying lipid and calcium deposition in the aortic valve in preclinical studies

Aim. To perform a pathomorphological and semi-quantitative assessment of lipid and calcific lesions of aortic valve leaflets in 12-month-old male and female ApoE^–/– mice in order to substantiate the feasibility of using this model in preclinical studies of preventive therapy for calcific aortic stenosis.

Materials and Methods. The study included 12-month-old male and female ApoE^–/– mice (n = 20). After euthanasia, complexes of the aortic arch with a fragment of the left ventricle containing the aortic valve and the descending aorta were isolated. For macroscopic evaluation of lipid and calcific lesions, whole-mount specimens were stained with 1% Oil Red O and 2% Alizarin Red. For detailed analysis, serial cryosections of aortic valve leaflets were prepared and stained with Oil Red O, Alizarin Red, and hematoxylin and eosin. Visualization was performed using light and fluorescence microscopy. Semi-quantitative analysis of the area of lipid and calcific lesions was carried out using ImageJ software.

Results. Macroscopic evaluation did not reveal lipid or calcific lesions in aortic valve leaflets. However, pathomorphological analysis of cryosections demonstrated multiple lipid inclusions of various sizes, sometimes forming extended clusters, as well as heterogeneous microcalcifications located in both superficial and deeper layers of the valve leaflets. Semi-quantitative analysis showed significantly more pronounced lipid lesions in male ApoE^–/– mice compared with females (p = 0.002), whereas calcification demonstrated comparable values between the groups with a tendency toward higher levels in females.

Conclusion. In 12-monthold ApoE^–/– mice, lipid and calcific lesions develop in aortic valve leaflets and morphologically resemble early changes described in the initial stages of calcific aortic stenosis in humans. Detection of these lesions requires pathomorphological analysis of cryosections using lipophilic and calcium-specific dyes in combination with fluorescence microscopy. The obtained results support the use of ApoE^–/– mice as an experimental model for studying early stages of aortic valve calcification and for conducting preclinical studies of preventive interventions.

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