Cardiac and vascular changes in elderly atherosclerotic mice: the influence of gender

Abstract

Background: Although advanced age is considered a risk factor for several diseases, the impact of gender on age-associated cardiovascular diseases, such as atherosclerotic processes and valvular diseases, remains not completely clarified. The present study was designed to assess aortic valve morphology and function and vascular damage in elderly using the apolipoprotein E knockout (ApoE KO) mouse. Our hypothesis was that advanced age-related cardiovascular changes are aggravated in atherosclerotic male mice.

Methods: The grade (0 to 4) of aortic regurgitation was evaluated through angiography. In addition, vascular lipid deposition and senescence were evaluated through histochemical analyses in aged male and female ApoE KO mice, and the results were compared to wild-type C57BL/6J (C57) mice.

Results: Aortic regurgitation was observed in 92% of the male ApoE KO mice and 100% of the male C57 mice. Comparatively, in age-matched female ApoE KO and C57 mice, aortic regurgitation was observed in a proportion of 58% and 53%, respectively. Histological analysis of the aorta showed an outward (positive) remodeling in ApoE KO mice (female: 1.86 ± 0.15; male: 1.89 ± 0.68) using C57 groups as reference values. Histochemical evaluation of the aorta showed lipid deposition and vascular senescence only in the ApoE KO group, which were more pronounced in male mice.

Conclusion: The data show that male gender contributes to the progression of aortic regurgitation and that hypercholesterolemia and male gender additively contribute to the occurrence of lipid deposition and vascular senescence in elderly mice.

Figure 1

Influence of gender on aortic regurgitation in aged normocholesterolemic and hypercholesterolemic mice. Top panel: typical angiographies showing aortic regurgitation in the male (right image) but not in the female (left image). Bottom panel: scatter plot showing the severity of aortic regurgitation (grades: 0 = none, 1 = light, 2 = mild, 3 = moderate, 4 = severe) in males compared with females in both C57 and ApoE KO groups. Horizontal bars represent the median for each group. p values were calculated using the Fisher's exact test and indicate statistical significance between proportions. The number of animals per group is shown in parentheses.

Figure 2

Typical photomicrographs of the aortic valves of male and female mice from the normocholesterolemic C57 and hypercholesterolemic ApoE KO groups. Squares indicate the maximum thickness of each valve. Arrows indicate valvar lesions in males of both C57 and ApoE KO groups as indicated by hemosiderin deposition. The sections were stained with both Oil-Red-O and hematoxylin. Magnification: 400×.

Figure 3

Typical photomicrographs of aortic valves on a longitudinal axis from a male aged hypercholesterolemic ApoE KO and a normocholesterolemic C57 mouse. Samples were stained with hematoxylin-eosin (HE), Masson's trichrome (MT), and von Kossa (VK). Black arrows indicate diffuse acellularity and myxoid thickening of the spongy layer (HE, MT and VK) in the ApoE KO mouse. Red arrows indicated valvular fibrosis with dense collagen (MT) mainly in the ApoE KO mouse. Magnification: 250×.

Figure 4

Typical angiography analysis of the internal diameter (parallel red lines) of the aorta divided into four segments: A) ascendant aorta - left common carotid; B) left common carotid - descendant thoracic portion of aorta; C) descendant thoracic portion of aorta - right renal artery; D) descendant thoracic portion of aorta - bifurcation of the common iliac artery. For a better visualization, A and B images were performed at an angle of 45° whereas C and D were at 0°.

Figure 5

Top panel: typical photomicrographs of cross sections of ascending aorta showing an augmented vessel diameter in the hypercholesterolemic ApoE KO compared with the normocholesterolemic C57 mouse. The sections were stained with both oil-red-O and hematoxylin (magnification: 400×). Bottom panel: Bar graphs showing average vessel (white bars) and lumen (black bars) cross-sectional areas of C57 (n = 6) and ApoE KO (n = 6) mice. Values are mean ± SEM. Numbers in parentheses indicate a positive remodeling ratio (>1.05). *p < 0.05 compared with vessel area of C57 groups.

Figure 6

Morphological analysis of aortas in both genders of normocholesterolemic C57 and hypercholesterolemic ApoE KO mice. (A) typical micrographs showing the severity of calcium deposition (Von Kossa staining; magnification: 250×) and en face images of (B) lipid deposition (Oil-Red-O staining) and (C) vascular senescence (X-gal, pH 6.0), in ApoE KO mice mainly in the male but not in either male or female C57 mice.