Protective role of Pannexin1 in lymphatic endothelial cells in the progression of atherosclerosis in female mice

Figures

• 

  Fig 1. Timeline for initiation and progression of atherosclerosis. (A) For the initiation study, Panx1 deletion was induced at 6 weeks of age by tamoxifen injection for consecutive 4 days. When mice reached 10 weeks of age, blood was taken from female and male Panx1LECdelApoe-/- and control Panx1fl/flApoe-/- mice and they were placed on a HCD for 6 weeks. At 16 weeks of age, mice were killed and blood and different tissues were collected. (B) For the progression study, blood was sampled at 6 weeks of age and atherosclerosis was induced in control Panx1fl/flApoe-/- and Panx1LECdelApoe-/- mice by feeding them with a HCD for 10 weeks. Four weeks after the start of the HCD, Panx1 deletion was induced with tamoxifen injections. At the end of the 10 weeks HCD period, mice were killed and blood and tissues were collected.

• 

  Fig 2. Adventitial lymphatic vessel number is similar in male and female Panx1LECdelApoe-/- and control mice. (A) Representative images of LYVE1 immunofluorescent staining (red) in aortic roots. Nuclei were stained with DAPI (blue). Scale bar represents 50 μm. (B) The lymphatic vessel number in the aortic roots was counted in female control Panx1fl/flApoe-/- mice (white), female Panx1LECdelApoe-/- mice (grey), male control Panx1fl/flApoe-/- mice (light blue) and male Panx1LECdelApoe-/- mice (dark blue). Mean ± SEM, N = 9–10.

• 

  Fig 3. Males have higher serum levels of TG and FFA after 6 weeks of HCD. Body weight (A), serum cholesterol (B), LDL (C), HDL (D), TG (E) and FFA (F) levels of female control Panx1fl/flApoe-/- mice (white), female Panx1LECdelApoe-/- mice (grey), male control Panx1fl/flApoe-/- mice (light blue) and male Panx1LECdelApoe-/- mice (dark blue) before and after 6 weeks of HCD. Mean ± SEM, N = 8–10, **P≤0.01, ***P≤0.001, ****P≤0.0001.

• 

  Fig 4. Females have larger atherosclerotic plaques in aortic roots after 6 weeks of HCD. Sudan-IV staining (A) and quantification of atherosclerotic plaque burden in aortic roots (B) and thoraco-abdominal aortas (C) of female control Panx1fl/flApoe-/- mice (white), female Panx1LECdelApoe-/- mice (grey), male control Panx1fl/flApoe-/- mice (light blue) and male Panx1LECdelApoe-/- mice (dark blue) after 6 weeks of HCD. Scale bar represents 200 μm. Mean ± SEM, N = 9–10, *P≤0.05.

• 

  Fig 5. Reduced atherosclerotic plaque stability in male mice after 6 weeks of HCD. Representative images and quantification of (immuno-) stainings for collagen (A,B; in blue), α-SMA (C,D; in green), Sudan-IV (E,F; in red) and CD68 (G,H; in brown) performed on aortic roots of female control Panx1fl/flApoe-/- mice (white), female Panx1LECdelApoe-/- mice (grey), male control Panx1fl/flApoe-/- mice (light blue) and male Panx1LECdelApoe-/- mice (dark blue) after 6 weeks of HCD. Scale bars represent 100 μm for A and E, and 200 μm for C and G. Mean ± SEM, N = 9–10, **P≤0.01, ***P≤0.001, ****P≤0.0001.

• 

  Fig 6. Increased advanced atherosclerotic plaque burden in female Panx1LECdelApoe-/- mice. Sudan-IV staining (A) and quantification of atherosclerotic lesion extent in the aortic roots (B) of female control Panx1fl/flApoe-/- mice (white), female Panx1LECdelApoe-/- mice (grey), male control Panx1fl/flApoe-/- mice (light blue) and male Panx1LECdelApoe-/- mice (dark blue) after 10 weeks of HCD. Scale bar represents 200 μm. Mean ± SEM, N = 8–9, **P≤0.01.

• 

  Fig 7. Panx1 deletion in LECs does not affect serum lipid levels and advanced atherosclerotic plaque burden in males. Body weight (A), serum cholesterol (B), LDL (C), HDL (D), TG (E) and FFA (F) levels of male control Panx1fl/flApoe-/- mice (light blue) and male Panx1LECdelApoe-/- mice (dark blue) after 10 weeks of HCD. Sudan-IV staining (E) and quantification of atherosclerotic lesion burden in thoraco-abdominal aortas (F) of male Panx1fl/flApoe-/- mice (light blue) and male Panx1LECdelApoe-/- mice (dark blue) after 10 weeks of HCD. Mean ± SEM, N = 9, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

• 

  Fig 8. Panx1 deletion in LECs does not affect advanced plaque stability in males. Quantification of (immuno-)stainings for collagen (A), α-SMA (B), Sudan-IV (C), CD68 (D) and CD3 (E) in aortic roots of male Panx1fl/flApoe-/- mice (light blue) and male Panx1LECdelApoe-/- mice (dark blue) after 10 weeks of HCD. Mean ± SEM, N = 9.

• 

  Fig 9. Panx1 deletion in LECs enhances advanced atherosclerosis in aortic roots of female mice. Body weight (A), serum cholesterol (B), LDL (C), HDL (D), TG (E) and FFA (F) levels of female control Panx1fl/flApoe-/- mice (white) and female Panx1LECdelApoe-/- mice (grey) after 10 weeks of HCD. Sudan-IV staining (E) and quantification of atherosclerotic lesion burden in thoraco-abdominal aortas (F) of male Panx1fl/flApoe-/- mice (white) and male Panx1LECdelApoe-/- mice (grey) after 10 weeks of HCD. Mean ± SEM, N = 8–9, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

• 

  Fig 10. Panx1 deletion in LECs results in T cell enrichment in advanced atherosclerotic lesions of female mice. Quantification of (immuno-)stainings for collagen (A), α-SMA (B), Sudan-IV (C), CD68 (D) and CD3 (E) in aortic roots of female Panx1fl/flApoe-/- mice (white) and female Panx1LECdelApoe-/- mice (grey) after 10 weeks of HCD. Mean ± SEM, N = 8–9. VCAM-1 expression (F) in Panx1-expressing (white) and Panx1-deficient (grey) LECs isolated from LNs of female mice. Mean ± SEM, n = 4. *P≤0.05, **P≤0.01.