FSP27 in adipocytes promotes macrophage migration through the CCL2/CCR2 axis. (a) Relative mRNA expression of Ccl2 in the aneurysmal tissues of Fsp27^fl/fl and Fsp27^AKO mice fed with an HFD and then infused with Ang Ⅱ (n = 6). (b) Immunohistochemical staining of CCL2 and quantitative analysis of positive staining signals in the abdominal aortic sections from Fsp27^fl/fl and Fsp27^AKO as treated in (a). Scale bar, 50 μm. (c and d) Gene expression levels of Fsp27 (c) and Ccl2 (d) determined by qPCR in 3T3-L1 adipocytes expressing negative control (shNC) or Fsp27 shRNA (shFsp27). (e) Secreted CCL2 in the supernatant from shNC and shFSP27 3T3-L1 adipocytes after 24 h culture (n = 4). (f) RAW264.7 cells were allowed to migrate towards the conditioned medium from shNC and shFsp27 3T3-L1 adipocytes for 24 h, followed by staining with crystal violet. Three fields from each chamber were counted and averaged. Scale bar, 50 μm (n = 4). (g) Representative images of F4/80⁺ (red) stained macrophages of PVAT sections from WT and Fsp27^−/− mice fed with an HFD. F4/80⁺ (red) stained macrophages are denoted by arrowheads. Scale bar, 50 μm. Quantification of F4/80⁺ macrophages per field is shown on the right (n = 4). (h and i) Gene expression of Fsp27 (h) and Ccl2 (i) determined by qPCR in 3T3-L1 adipocytes expressing an empty vector (Vector) or Fsp27-overexpression vector (Fsp27-OE) (n = 3). (j) Secreted CCL2 in the supernatant from 3T3-L1 adipocytes overexpressing an empty vector or Fsp27 after 24 h culture (n = 4). (k) RAW264.7 cells were allowed to migrate towards the conditioned medium from 3T3-L1 adipocytes overexpressing an empty vector or Fsp27 for 24 h in the presence or absence of RS102895 (5 μmol/L). Three fields from each chamber were counted and averaged (n = 4). Scale bar, 50 μm. (l and m) The concentration of CCL2 was determined by ELISA (l) and the capacity of the conditioned medium to stimulate RAW264.7 cell migration was assessed by a transwell assay (m). PVAT was isolated from both WT and Fsp27^−/− mice that had been fed with an HFD for three months, and conditioned medium from the PVAT was collected. Data were expressed as means ± SEM. P values were calculated by Student’s t-test (a−m) and two-way ANOVA with Bonferroni test (k). ns, not significant.