AFM-based nanoindentation indicates an impaired cortical stiffness in the AAV-PCSK9DY atherosclerosis mouse model

Fig. 1. Growth of WD-fed mice in dependency of the PCSK9 viral load. A development of body weight within the 12-week treatment period; means ± SDs (n = 5–6), 2-way ANOVA followed by Tukey’s multiple comparisons test [time: F = 277, P < 0.0001, treatment: F = 2.9, P = 0.068, interaction (time × treatment): F = 216, P < 0.0001]; B gain in body weight (1-way ANOVA [F = 5.568, P = 0.0065) followed by Tukey’s multiple comparisons test); C mass distribution after 12 weeks (Kruskal Wallis Test (fat mass P = 0.0028; free body fluid P = 0.0906; lean mass P = 0.359) followed by Dunn’s multiple comparisons test was calculated as Gaussian distribution of the values was not given; the median is depicted in box blots; the box extends from the 25th to 75th percentiles and the whiskers go down to the smallest value and up to the largest; n = 5–6; *P < 0.05 vs 0

Fig. 2. Plasma concentrations of cholesterol (A) and triglycerides (B) after 6 and 12 weeks in WD-fed mice in dependency of the PCSK9DY viral load. Plasma cholesterol and triglycerides were calculated using by using 2-way ANOVA considering the factors time and viral load (cholesterol F = 17.75, P < 0.0001; TG F = 11.36, P = 0.0002) followed by Sidak’s multiple comparisons test. n = 5–6; the median is depicted in box blots; the box extends from the 25th to 75th percentiles and the whiskers go down to the smallest value and up to the largest. *P < 0.05 vs 0; †P = 0.05 vs. 0.5 × 1011

Fig. 3. Histologic evaluation of aortic root of WD-fed mice in dependency of the PCSK9.DY viral load. A depicts exemplary aortic segments of each treatment group upon Oil Red O staining (the brownish colorations marked with an arrow are blood residues in the samples and are evaluated as artifacts; scale bar: 100 µm); B quantitative evaluation of plaque content; C quantitative evaluation of fat content. A Kruskal Wallis test (plaque content P = 0.0044; fat content P = 0.0017) followed by Dunn’s multiple comparisons test was calculated; correlation analyses were performed by 2-tailed Pearson test; the median is depicted in box blots; the box extends from the 25th to 75th percentiles and the whiskers go down to the smallest value and up to the largest

Fig. 4. Cortical stiffness in WD-fed C57BL/6 N mice which were treated with AAV-PCSK9 (0, 0.5, 1 or, 5 × 10.11 VG). A Stiffness values were calculated in 44–62 cells of each mouse from force-distance curves in a blinded manner by using the PUNIAS 3D version 1.0 release 1.8 (http://punias.voila.net/); to detect statistical differences between groups, a Kruskal Wallis test (P < 0.0001) was calculated followed by Dunn’s multiple comparisons test; B evaluation considering aortic stiffness in individual mice; statistical differences between groups were detected by 1-way ANOVA testing (F = 12.24, P = 0.0001) followed by Tukey’s multiple comparisons test; values are depicted as box plots showing 25th and 75th percentile (box) and maximum/minimum values (whiskers). The horizontal black line depicts the median in each group; C–E Correlation analysis between cortical stiffness and cholesterol, triglycerides, or plaque content. Correlations between 2 factors were calculated by Pearson