ANGPTL8 deletion attenuates abdominal aortic aneurysm formation in ApoE−/− mice

Figures

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  Figure 1. ANGPTL8 expression is significantly up-regulated in human AAA and AngII-induced AAA of ApoE−/− mice (A) Immunohistochemical results of ANGPTL8 in human AAA tissue and control tissues. (B) Immunohistochemical results of ANGPTL8 in arterial tissues of saline and AngII-infused AAA in ApoE−/− mice. (C) Western blot results of ANGPTL8 in arterial tissues of saline and AngII-infused ApoE−/− mice. (D) Immunofluorescence analysis of ANGPTL8 (green), MAC-2, α‐SMA, and CD3 (red) and 4′,6‐diamidino‐2‐phenylindole (DAPI; blue for nuclei) in human AAA tissues. (E) Immunofluorescence analysis of ANGPTL8 (green), MAC-2, α‐SMA, and CD3 (red) and DAPI (blue for nuclei) in AngII-infused AAA in ApoE−/− mice. N=6/group, Data are presented as mean ± SEM, *P<0.05. Scale bars: 50 μm. (F) Circulating level of ANGPTL8 in AAA patients. N=40/group, Data are presented as mean ± SEM, ***P<0.01. (G) Circulating level of ANGPTL8 in AAA mice. N=6/group, Data are presented as mean ± SEM, *P<0.05.

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  Figure 2. Knockout of ANGPTL8 attenuated AngII-induced AAA in ApoE−/− mice (A) Representative morphology of abdominal aortic specimens in four groups of mice: the ApoE−/− + saline group, the ApoE−/− + AngII group, the ApoE−/−ANGPTL8−/− + saline group, and the ApoE−/−ANGPTL8−/− + AngII group. (B) Survival curve showing the survival of mice at week 4 after saline or AngII administration. (C) The incidence of AAA. (D) Blood pressure of mice in 4 weeks after saline or AngII administration. (E) Quantification of maximal aortic diameter after saline or AngII-infusion for 28 days. Data are presented as means ± SEM. *P<0.05. (F) Representative images from ultrasonography of abdominal aortas after saline or AngII-infusion for 28 days. (G) Representative images and quantitative analysis of Elastin staining of abdominal aortas in four groups of mice. Scale bars: 50 μm. Data are presented as means ± SEM. *P<0.05. (H) Histopathological analysis of representative abdominal aortas by H&E staining and quantification of aortic thickness. Scale bars: 50 μm. Data are presented as means ± SEM. *P<0.05. (I) Representative images and quantitative analysis of Masson’s Trichrome staining of abdominal aortas in…

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  Figure 3. Knockout of ANGPTL8 attenuated inflammation in AngII-induced AAA in ApoE−/− mice (A) Representative images of inflammatory factor IL-6, IL-1β, and MCP-1 staining of abdominal aortas in four groups of mice. Scale bars: 50 μm. (B) Quantitative analysis of inflammatory factor IL-6, IL-1β, and MCP-1 staining of abdominal aortas in four groups of mice. N=6/group. Data are presented as mean ± SEM. *P<0.05. (C) Western blot analysis the expression of IL-6, IL-1β, and TNF-α in RAW264.7. N=6/group. Data are presented as mean ± SEM. *P<0.05.

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  Figure 4. Knockout of ANGPTL8 attenuated MMPs in AngII-induced AAA in ApoE−/− mice (A) Representative photomicrographs and quantitative analysis of in-situ zymography of abdominal aortas in four groups of mice. Scale bars: 50 μm. (B) Representative images and quantitative analysis of MMP-2 and MMP-9 staining of abdominal aortas in four groups of mice. N=6/group. Data are presented as mean ± SEM. *P<0.05. Scale bars: 50 μm.

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  Figure 5. Knockout of ANGPTL8 attenuated apoptosis in AngII-induced AAA in ApoE−/− mice (A) TUNEL staining of the abdominal aortas in four groups of mice. Scale bars: 50 μm. Data are presented as means ± SEM. *P<0.05. (B) Representative images of α-SMA, cleaved-Caspase 3, and cleaved-PARP staining of abdominal aortas in four groups of mice. Scale bars: 50 μm. (C) Quantitative analysis of α-SMA, cleaved-Caspase 3, and cleaved-PARP staining of abdominal aortas in four groups of mice. N=6/group. Data are presented as mean ± SEM. *P<0.05. (D) TUNEL staining of HASMCs. Scale bars: 50 μm. N = 6/group. Data are presented as means ± SEM. *P<0.05. (E) Western blot analysis the expression of cleaved-PARP, cleaved-caspase 3, Bcl-2, and Bax in HASMCs. N=6/group. Data are presented as mean ± SEM. *P<0.05.

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  Figure 6. Inhibition of ANGPTL8 attenuated AngII-induced AAA in ApoE−/− mice (A) Representative morphology of abdominal aortic specimens in Control and sh-ANGPTL8 groups of mice. (B) Survival curve of Control and sh-ANGPTL8 groups of mice. (C) The incidence of AAA. (D) Representative images from ultrasonography and quantification of maximal aortic diameter after AngII-infusion for 28 days. Data are presented as means ± SEM. *P<0.05. (E) Representative images and quantitative analysis of H&E staining of abdominal aortas in Control and sh-ANGPTL8 groups of mice. Scale bars: 50 μm. Data are presented as means ± SEM. *P<0.05. (F) Representative images and quantitative analysis of Elastin staining of abdominal aortas in Control and sh-ANGPTL8 groups of mice. Scale bars: 50 μm. Data are presented as means ± SEM. *P<0.05.