Silencing hepatic PCSK9 via novel chimeric AAV8 mitigates the progression of atherosclerosis by inhibiting inflammation in ApoE−/− mice

Figures

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  Figure 1. Temporal changes in GFP expression in the liver and biodistribution of AAV8 and AAV8.P vectors in mouse tissues (A) Representative laser confocal microscopy images showing GFP expression in the liver at various time points (1, 2, 4 weeks and 3, 6, 12 months) following systemic injection of AAV8-GFP or AAV8.P-GFP vectors, scale bar indicates 100 μm. (B) Assessment of gene transduction efficiency in the liver over time following AAV8-GFP or AAV8.P-GFP injection (n = 4/group). Data are shown as means ± SEM. ∗∗p ≤ 0.01, ∗p ≤ 0.05 (two-way ANOVA with Bonferroni correction). (C) Western blot analysis of GFP expression levels in liver samples from AAV8 and AAV8.P groups at specified post-delivery time points (n = 3/group). Data are shown as means ± SEM. ∗∗p ≤ 0.01, ∗p ≤ 0.05 (two-way ANOVA with Bonferroni correction). (D) qPCR measurement of AAV8 and AAV8.P genome copies in liver tissues at multiple time intervals (1, 2, 4 weeks and 3, 6, 12 months) after injection. Data are shown as means ± SEM. ∗∗p ≤ 0.01, ∗p ≤ 0.05 (Unpaired t test). (E) Quantification of AAV genome copies in 1 μg of genomic DNA from heart, liver, spleen, lung, kidney, brain, and skeletal muscle at 4 weeks post-gene…

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  Figure 2. Targeted and robust hepatic gene transduction of GFP utilizing AAV8.P-GFP and AAV8.P-PCSK9 shRNA vectors (A) Representative images of GFP expression in various tissues obtained through laser confocal microscopy, assessed 4 weeks post systemic administration of AAV-GFP and AAV-PCSK9 shRNA vectors, scale bar indicates 100 μm. (B) Quantitative analysis of GFP mRNA expression performed via real-time PCR across different tissues at 4 weeks following injection of either AAV-GFP or AAV-PCSK9 shRNA vectors. n = 5/group. Data are shown as means ± SEM. Unpaired t test. (C) Detection of GFP expression through western blot analysis in assorted tissues 4 weeks subsequent to gene transfer. Abbreviations: Li, Liver; He, Heart; Sp, Spleen; Lu, Lung; Ki, Kidney; Br, Brain; Mu, Muscle. (D and E) Western blot assessment of GFP expression specifically in the liver 4 weeks following gene transfer. n = 4/group. Data are shown as means ± SEM. Unpaired t test.

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  Figure 3. Silencing hepatic PCSK9 leads to decreased serum levels of total cholesterol and triglycerides, without altering AST or ALT concentrations in C57BL/6J mice (A) Hepatic PCSK9 mRNA levels were quantified using real-time PCR 1-month post-administration of either AAV-GFP or AAV-PCSK9 shRNA, n = 4 for the AAV-GFP cohort and n = 7 for the AAV-PCSK9 shRNA cohort. Data are shown as means ± SEM. ∗∗p ≤ 0.01 (Unpaired t test). (B) Plasma PCSK9 concentrations were assessed through ELISA at 1 and 3 months following transduction with either AAV-GFP or AAV-PCSK9 shRNA, n = 5/group. Data are shown as means ± SEM. ∗∗p ≤ 0.01 (two-way ANOVA with Bonferroni correction). (C and D) Serum levels of total cholesterol and triglycerides were determined 1 month and 3 months subsequent to vector administration, n = 8/group. Data are shown as means ± SEM. ∗∗p ≤ 0.01 (two-way ANOVA with Bonferroni correction). (E) Serum ApoB concentrations (μg/mL) in C57BL/6J mice were measured 1 month after vector administration, n = 6/group. Data are shown as means ± SEM. ∗∗p ≤ 0.01 (Unpaired t test). (F and G) Immunoblot analysis was conducted for hepatic PCSK9, LDL-R, ApoE, and ApoB-100 1 month after vector…

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  Figure 4. PCSK9 silencing in ApoE−/− mice induces early changes in lipid profiles but lacks a sustained impact on circulating lipids (A) Hepatic mRNA levels of PCSK9 were quantified using real-time PCR in liver specimens collected 2 weeks post-administration, n = 4/group. Data are shown as means ± SEM. ∗∗p ≤ 0.01 (Unpaired t test). (B) Plasma levels of PCSK9 protein were evaluated via ELISA at baseline and at 2 weeks, 4 weeks, and 6 months following injection, n = 10 for AAV-GFP, n = 12 for AAV-PCSK9 shRNA. Data are shown as means ± SEM. ∗∗p ≤ 0.01 (two-way ANOVA with Bonferroni correction). (C and D) Serum concentrations of total cholesterol and triglycerides were monitored over time after the administration of AAV-PCSK9 shRNA or AAV-GFP, n = 12 or 15 per group. Data are shown as means ± SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01 (two-way ANOVA with Bonferroni correction). (E and F) Immunoblotting results depicting hepatic levels of PCSK9, LDL-R, and ApoB-100 proteins at 6 months and 2 weeks post-administration, with corresponding quantification shown on the right, n = 5 or 6/group. Data are shown as means ± SEM. ∗p ≤ 0.05, ∗∗p ≤ 0.01 (Unpaired t test). (G) Serum ApoB levels (μg/mL) were assessed in mice…

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  Figure 5. Silencing hepatic PCSK9 protects ApoE−/− mice from atherosclerosis development Twelve-week-old ApoE−/− male mice were injected with 2 × 1011 viral genomes (v.g.) of AAV-shPCSK9 or AAV-GFP and fed a chow diet for 6 months. (A) Representative Oil Red O-stained images of the en face arterial tree. (B–F) Quantification of the lesion areas in (A), n = 8/group. (G) Representative Oil Red O staining in aortic sinus cross-sections, scale bar indicates 400 μm. (H) Quantification of the lesion areas in (G), n = 7 or 8/group. Data are shown as means ± SEM. ∗p ≤ 0.05, ∗∗p < 0.01 (Unpaired t test).

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  Figure 6. Silencing PCSK9 improves plaque stability in aortic sinus lesions (A) Representative images of macrophages and smooth muscle cells by immunostaining with CD68 antibody (red) and α-smooth muscle actin antibody (red) in aortic sinus lesions, scale bar indicates 100 μm, plaque areas circled with yellow solid lines. (B) Quantification of macrophage-positive areas in (A), n = 4 or 5/group. (C) Quantification of smooth muscle cell-positive areas in (A). n = 4 or 5/group. Data are shown as means ± SEM. ∗p ≤ 0.05, ∗∗p < 0.01 (Unpaired t test).

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  Figure 7. Silencing PCSK9 attenuates atherosclerotic aorta and serum inflammatory monocyte infiltration and inflammatory gene expression in ApoE−/− mice (A) Representative images and quantification (B) of Ly6Chi-positive cells via aortic sinus lesions immunostaining, scale bar indicates 25 μm, n = 4 or 5/group. (C) Real-time PCR analysis of MCP-1, TNF-α, MIF, IL-1β, IL-6, and IL-10 mRNA expression in aortas from AAV-transduced ApoE−/−mice fed a chow diet for 6 months, n = 3 or 4/group. (D–F) Serum levels of MCP-1, MIF, and TNF-α from AAV-transduced ApoE−/−mice fed a chow diet for 6 months via ELISA, n = 7–12/group. Data are shown as means ± SEM. ∗p ≤ 0.05, ∗∗p < 0.01 (Unpaired t test).