Analysis of gender and age-related patterns of lipid and calcium deposition in aortas and aortic valve leaflets of hyperlipidemic mice

Background. Efficient implementation of endothelial-protective and anti-atherosclerotic interventions in hyperlipidemic (including ApoE^–/– mice requires the identification of the optimal experimental parameters (sex and age) for the stable detection of aortic atherosclerosis as well as lipid and calcium deposition in the aortic valve.

Aim: To analyze gender and age-related patterns of lipid deposition in the aorta and lipid and calcific lesions of the aortic valve in ApoE^–/– mice.

Material and Methods. The study included 100 ApoE^–/– mice: 20 mice (10 males and 10 females) at each of the following ages: 1, 3, 6, 12, and 18 months. Aortas and left ventricle segments with the aortic valve (AV) were used as for histopathological analysis.

Results. Lipid deposition in the aorta of ApoE^–/– mice was first detected at 6 months of age, whilst the median proportion of lipid-positive aortic area was 0.90% (IQR: 0.00–1.44%) in males and 1.47% (IQR: 0.76–1.89%, p = 0.35) in females. By 12 months of age, median proportion of lipid-positive aortic area in the aorta increased to 29.80% (IQR: 19.22–33.42%) in males and 16.41% (IQR: 13.18–19.50%, p = 0.001) in females. Lipid deposition in the AV leaflets was detected starting from 1 month of age (median lipid-positive area: 0.76% [IQR: 0.14–1.54%] in males and 1.07% [IQR: 0.90–1.69%], p = 0.27 in females) and progressively increased (at 3 months: 2.95% [IQR: 1.11–7.64%] in males vs. 7.15% [IQR: 4.19–9.48%], p = 0.14 in females; at 6 months: 8.04% [IQR: 5.68–13.89%] in males vs. 5.70% [IQR: 4.63–10.21%], p = 0.31 in females; at 12 months: 15.58% [IQR: 12.27–21.44%] in males vs. 8.95% [IQR: 7.52–10.02%], p = 0.002 in females). Up to 6 months, only sporadic calcification foci were observed in ApoE^–/– mice. At 12 months, the median proportion of calcified AV leaflet area was 4.87% (IQR: 3.27–9.19%) in males and 5.76% (IQR: 2.96–9.26%, p = 0.91) in females. The increase in both the area and intensity of Alizarin Red staining correlated with the extent of lipid deposition in the aorta (r = 0.736 and r = 0.708, respectively).

Conclusion. Aortic lipid lesions in ApoE^–/– mice are consistently detectable after 6 months of age and stabilizes by 12 months. Lipid lesions in the AV leaflets of ApoE^–/– mice of both sexes is detectable as early as 1–3 months, progresses until 6 months, and stabilizes by 12 months. At 12 months, lipid lesions of both the aorta and AV are more pronounced in males than in females. Mineralization foci of AV leaflets begin to develop at approximately 6 months of age, with stabilization of calcification by 12 months. The strong correlation between lipid deposition in the aorta and calcium deposition in the AV leaflets suggests a pathogenetic parallelism of these processes within the circulatory system of ApoE^–/– mice. These findings indicate that ApoE^–/– mice can be optimally withdrawn from experiments between 9 and 12 months of age.

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