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

Abstract

Investigating atherosclerosis and endothelial dysfunction has mainly become established in genetically modified ApoE^-/- or LDL-R^-/- mice transgenic models. A new AAV-PCSK9DY^DY mouse model with no genetic modification has now been reported as an alternative atherosclerosis model. Here, we aimed to employ this AAV-PCSK9^DY mouse model to quantify the mechanical stiffness of the endothelial surface, an accepted hallmark for endothelial dysfunction and forerunner for atherosclerosis. Ten-week-old male C57BL/6 N mice were injected with AAV-PCSK9^DY (0.5, 1 or 5 × 10¹¹ VG) or saline as controls and fed with Western diet (1.25% cholesterol) for 3 months. Total cholesterol (TC) and triglycerides (TG) were measured after 6 and 12 weeks. Aortic sections were used for atomic force microscopy (AFM) measurements or histological analysis using Oil-Red-O staining. Mechanical properties of in situ endothelial cells derived from ex vivo aorta preparations were quantified using AFM-based nanoindentation. Compared to controls, an increase in plasma TC and TG and extent of atherosclerosis was demonstrated in all groups of mice in a viral load-dependent manner. Cortical stiffness of controls was 1.305 pN/nm and increased (10%) in response to viral load (≥ 0.5 × 10¹¹ VG) and positively correlated with the aortic plaque content and plasma TC and TG. For the first time, we show changes in the mechanical properties of the endothelial surface and thus the development of endothelial dysfunction in the AAV-PCSK9^DY mouse model. Our results demonstrate that this model is highly suitable and represents a good alternative to the commonly used transgenic mouse models for studying atherosclerosis and other vascular pathologies.

Keywords: AAV-PCSK9DY mouse model; Atherosclerosis; Atomic force microscopy; Cortical stiffness; Endothelial dysfunction.

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 PCSK9^DY 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