(A) Representative images of atherosclerotic lesion burden of ovariectomized (OVX) LDLR^–/– mice fed a high-fat diet from 8 to 24 weeks of age, with or without exogenous estradiol (OVX+E2). Lipid-rich lesions were identified by en face Oil Red O staining in aortas from OVX (n = 10) and OVX+E2 groups (n = 12). Scale bar: 1 mm. (B) Quantification of data from A expressed as a percentage of aortic area. (C) Arterial stiffness (elastic modulus) determined by AFM; n = 4 per group. The arrowheads in B and C represent the median Oil Red O staining and elastic moduli of 6-month female LDLR^–/– mice on a high-fat diet without OVX (taken from Figure 2). (D) Blood cholesterol levels were measured after completion of the high-fat diet (n = 10 per condition). The arrow approximates the cholesterol level in C5BL/6 mice on a Western diet (71). (E) Aortic root sections of male and female LDLR^–/– mice on a high-fat diet from 8 to 24 weeks costained for CD68 (red) and MMP12 (green). The images were merged to show colocalization; see Supplemental Figure 2 for individual images. Closed and open arrowheads show MMP12 levels in CD68⁺ and CD68^– regions, respectively. Scale bar: 500 μm. (F) Quantification of MMP12 signal intensity in CD68⁺ regions from E (n = 5 per group). Graphs show box and whisker plots with Tukey’s whiskers; the horizontal lines of boxes represent the 25th percentile, the median, and the 75% percentile. Statistical significance for all panels was determined using Mann-Whitney tests.