Knockdown of angiopoietin-like 2 induces clearance of vascular endothelial senescent cells by apoptosis, promotes endothelial repair and slows atherogenesis in mice

Figures

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  Figure 1. Age-dependent increase of senescence-associated p21, Pai-1 and angptl2 expressions in the native endothelium parallels plaque growth in the aorta. (A) mRNA expression of indicated genes was quantified in the native aortic endothelium of 3-mo (n=4), 5-mo (n=4), 6-mo (n=4) and 12-mo (n=4) control ATX mice. The average level of gene expression in 3-mo ATX mice was arbitrarily set at 1. Plaque area was quantified from longitudinally open thoracic aortas of 3-mo (n=7), 5-mo (n=5), 6-mo (n=7), 9-mo (n=12) and 12-mo (n=4) ATX mice. Data are expressed as mean±SEM. *: p<0.0001 vs. 3-mo ATX mice. (B) Representative pictures of age-related increase in atherosclerotic plaque in 3-, 6-, 9- and 12-mo ATX mice.

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  Figure 2. Increased expression of senescence-associated p21, Pai-1 and angptl2 in the native aortic endothelium of 6-month old ATX compared to WT mice. mRNA expression of indicated genes was quantified in the native aortic endothelium of 6-mo WT and ATX mice (n=3). The average level of gene expression in 6-mo WT mice was arbitrarily set at 1. Data are expressed as mean±SEM. *: p<0.05 vs. WT mice.

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  Figure 3. Distribution of the AAV1-mCherry in the aortic wall and specificity of the AAV1-shAngptl2. (A) Immunofluorescence of AAV1-mCherry in frozen aortic sections of ATX mice at 6 months of age, 3 months post-infection: mCherry signal distributed throughout the vascular wall is shown in red and basal lamina in green; nuclei are shown in blue. At a higher magnification (40X), arrows show mCherry signal in the endothelium. A negative control (absence of primary antibody against mCherry) was performed (data not shown). (B) Neither cardiac nor liver Angptl2 and p21 mRNA expressions were affected by the AAV1-shAngptl2 in ATX mice, 3 months post-infection. Average gene expression level in shSCR mice was arbitrarily set at 1. Data are mean±SEM of n ATX mice. C) Cholesterol, triglycerides and glucose levels of ATX mice were not altered by the AAV1-shAngptl2, 3 months post-infection. Data are mean±SEM of n=7 ATX mice.

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  Figure 4. Down-regulation of vascular angptl2 is anti-atherogenic. (A) Representative images of longitudinally open thoracic aortas showing atherosclerotic plaque, and (B) Quantification of thoracic aorta atheroma plaque area in 3 mo control (CTL 3-mo, n=6), 6-mo control (CTL 6-mo, n=12), 6-mo AAV1-shScramble (+ shSCR, n=11) and 6-mo AAV1-shAngptl2 (+ shAngptl2, n=12) ATX mice revealing white atheroma plaques at 6-mo. Data are mean±SEM. *: p<0.0001 vs. CTL 6-mo and shSCR; $: p<0.0001 vs. CTL 3-mo.

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  Figure 5. Time-dependent decrease of senescent-associated Angptl2 and p21 mRNA expressions in the native endothelium of the aorta post-AAV1-shAngptl2 injection. Decrease of Angptl2 (A) and p21 (B) mRNA expression post-injections (1-week, n=5-6; 2-week, n=5-7; 4-week, n=4-7; 3-month, n=6-4) in the native endothelium freshly harvested from thoracic aortas of ATX mice treated with AAV1-shSCR or AAV1-shAngptl2. Average gene expression level in shSCR group was arbitrarily set at 1. Data are mean±SEM. *: p<0.05; **: p<0.01; ***: p<0.001 vs. shSCR group, at each time point.

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  Figure 6. Down-regulation of vascular angptl2 is senolytic and anti-atherogenic. (A) Decrease of mRNA expression of the indicated genes in the native aortic endothelium isolated from AAV1-shAngptl2-treated ATX mice, 3 months post-infection. Data are mean±SEM of n mice. *: p<0.05; **: p<0.01; ***: p<0.001 vs. shSCR group. (B) Decrease of mRNA expression (except for p21) in the de-endothelialized aortic wall (media) isolated from AAV1-shAngptl2-treated ATX mice, 3 months post-infection. Data are mean±SEM. *: p<0.05 and **p<0.01 vs. shSCR group.

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  Figure 7. Down-regulation of vascular angptl2 is senolytic by promoting apopotosis. (A) Induction of Angptl2+ senescent endothelial cells apoptosis 1-week post-injection of AAV1-shAngptl2 in aortas of ATX mice: Bax mRNA expression increased (left, p=0.005) while Bcl2 mRNA expression decreased (middle, p=0.003), increasing Bax/Bcl2 ratio (right). (B) Absence of apoptosis in cells from the media 1-week post-injection of AAV1-shAngptl2 in aortas of ATX mice. Data are mean±SEM of n ATX mice.

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  Figure 8. Down-regulation of vascular angptl2 is anti-atherogenic and promotes endothelial repair. (A) Increased expression of the progenitor marker Cd34 mRNA in the native aortic endothelium 4-week post-injection of shAngptl2 ATX mice. Data are mean±SEM. * : p<0.05 vs. shSCR group. The average gene expression levels in the shSCR group were arbitrarily set at 1. (B) Higher CD34 staining in the endothelium of 6-month old ATX mice 3 months post-injection of the AAV1-shAngptl2 compared to ATX mice injected with the scramble shRNA (shSCR). Data are mean±SEM. *: p<0.05 vs. shSCR. C) Representative images of CD34 immunostaining are shown.

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  Figure 9. Association of vascular ANGPTL2, senescence and inflammation in IMA from atherosclerotic patients undergoing coronary artery by-pass surgery. Linear correlations between (A) ANGPTL2 and p21 mRNA expression, (B) ANGPTL2 and TNF-α, IL-8 and IL-6 mRNA expression and (C) p21 and TNF-α, IL-8 and IL-6 mRNA expression in IMA segments (n=26). Non-parametric Spearman correlation test was applied. Patient’s characteristics are described in Table S2.

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  Figure 10. Schematic representation of the putative anti-atherogenic action of AAV1-shAngptl2. Elimination of angptl2+:p21+ senescent endothelial cells by apoptosis promotes endothelial repair. This leads to a reduced inflammatory profile, a lower immune cell adhesiveness and infiltration and therefore, a reduced atherogenicity.