Fig 1.
APN derived from PVAT protects mice from atherosclerosis.
A, HE staining of arteria carotis sections from mice after perivascular collar placement for 12 weeks. B, Quantitative analysis of intimal surface area in arteria carotis of each groups (n = 6 per group). *P<0.05 versus sham. C, Analysis of intima/media ratio in arteriacarotis with each groups (n = 6 per group). D, Bar graph shows quantification of lumen stenosis in arteria carotis with each groups (n = 6 per group). *P<0.05 versus sham. E, HE staining of arteria carotis sections from ApoE-/- mice transplanted with WT or APN-/- PVAT 12 weeks after atherosclerosis established. F, Quantitative analysis of intimal surface area in arteria carotis with WT or APN-/- PVAT (n = 6 per group). *P<0.05 versus (WT) PVAT. G, Bar graph shows quantification of lumen stenosis in arteria carotis with WT or APN-/- PVAT (n = 6 per group). *P<0.05 versus (WT) PVAT. H, HE staining of atherosclerotic plaques from the indicated mice. I, plaque disruption rate of he indicated mice (n = 6 per group). *P<0.05 versus surgery control.
Fig 2.
APN secreted by PVAT aggravates autophagy in plaque.
A, Representative western blot of LC3 expression in arteria carotis transplanted with WT or APN-/- PVAT 4 weeks after atherosclerosis (n = 6 per group). B, Quantitative analysis of LC3 protein expression in various groups. *P<0.05 versus (WT) PVAT. C, Immunofluorescence of p62, another marker of autophagy, in the arteria carotis with WT or APN-/- PVAT (n = 6 per group). D, Histogram shows p62 positive cells per 100 cells. *P<0.05 versus (WT) PVAT.
Fig 3.
APN exacerbates macrophage autophagy in vitro.
A and B respectively show the representative western blot and quantitative analysis of LC3 protein level in VSMC and macrophage stimulated with or without APN (5 μg/ml). n = 6 per group. *P<0.05 versus macrophage without APN. C and D respectively show the western blot and quantitative analysis of P62 and Beclin 1 protein level in macrophage treated with or without APN (5 μg/ml). n = 6 per group. *P<0.05 versus macrophage without APN.
Fig 4.
APN induces autophagy in macrophage through Akt-FOXO3a pathway.
A, Western blot shows the protein level of p-Akt, Akt, p-FOXO3a, FOXO3a in macrophages stimulated with phosphate buffered saline, APN, Akt agonist (740Y-P) or with APN in combination with 740Y-P. B, Western blot shows the protein level of PTEN, p-mTOR, mTOR in macrophages stimulated with phosphate buffered saline, APN, Akt agonist (740Y-P) or with APN in combination with 740Y-P. C, Quantitative analysis of p-Akt/Akt ratio, p-FOXO3a/FOXO3a ratio and p-mTOR/mTOR ratio in macrophages stimulated with phosphate buffered saline, APN, and APN in combination with 740Y-P, respectively. n = 6 per group. *P<0.05 versus macrophage treated with saline. D, Quantification of the optical density of PTEN in each groups. n = 6 per group. *P<0.05 versus macrophage treated with saline.