Table 1.
Statin treatment decreases total cholesterol without affecting HDL-C in western-diet fed apoE-/- mice.
Figure 1.
Statin treatment decreases CD68+ cell and cholesteryl ester contents in atherosclerotic plaques.
ApoE-/- mice (n = 10 per group) were fed a high fat diet (western diet) after which they were either maintained on the western diet, or switched to a western diet that included atorvastatin or rosuvastatin. A) Macrophage content was assessed by CD68 staining, and B) cholesteryl ester content was assessed by oil red-O staining. The symbol * indicates statistical significance, p<0.05.
Figure 2.
Statin treatment does not significantly affect plaque area likely due to the increase in collagen content.
A) Plaque size was assessed by morphometric analysis after hematoxylin and eosin staining of serial sections (n = 10 per group). B) Collagen levels were measured using Sirius red staining under polarizing light microscopy (n = 10 per group). The symbol * indicates statistical significance, p<0.05.
Figure 3.
Statin treatment promotes Ly-6Chi and Ly-6Clo monocyte emigration from atherosclerotic plaques in a CCR7-dependent manner and represses MCP-1 expression.
The number of beads remaining in the plaque 4 weeks post-statin treatment that corresponded to; A) CCR2 (Ly-6Chi) monocytes and B) CX3CR1 (Ly-6Clo) highly positive monocytes are shown (n = 8 per group). C) Relative CCR7 mRNA expression levels as a function of statin treatment were determined by RT-PCR (n = 9 per group). D) CD68+ cell content in plaques from CCR7-/-apoE-/- double knockout mice treated with statin were determined by morphometric analysis of the immunostained areas (n = 7 per group). The symbol * indicates statistical significance, p<0.05. E) MCP-1 expression in plaques as a function of statin treatment. Sections of aortic roots from baseline, atorvastatin and rosuvastatin treated mice were stained with a biotinlyated mouse MCP-1 antibody, and visualized using chromogenic and fluorescent reaction products. Brightfield images of MCP-1 immunostaining of the whole root (top row) and confocal fluorescent images of the selected areas (inserts, bottom row) are shown and demonstrate reduced levels of MCP-1 protein in plaques of statin treated mice compared to baseline controls.
Figure 4.
Statins Induce SREBP and CCR7 expression.
RAW264.7 macrophage cells were treated with 5 μM statins for the times indicated, and expression of SREBP-1a (A), SREBP-1c (B), SREBP-2 (C), and CCR7 (D) were determined by RT-PCR. The experiments were repeated 3 times with similar results. The symbol * indicates statistical significance, p<0.05.
Figure 5.
Statins regulate CCR7 expression through modulating SREBP-2.
A) Luciferase assays were performed in RAW264.7 cells co-transfected with expression plasmids for SREBP-2 and a series of CCR7 reporter constructs in the absence (DMSO) and presence of rosuvastatin treatment. Relative luciferase units (RLU) normalized to β-galactosidase activity are shown. B) The consensus SRE-binding site in the -0.32 kb CCR7-luciferase reporter was mutated as indicated. COS7 cells were co-transfected with an empty vector (pCDNA3) or mature SREBP-2, along with either the wild type or mutated SRE-reporter construct and RLU measured as above. C) RAW264.7 cells were transfected with an SREBP-2 expression plasmid or control empty vector control, and expression of CCR7 was determined by RT-PCR. D) SREBP-2 and E) CCR7 mRNA expression was measured by RT-PCR after siRNA knockdown with a scrambled siRNA control or a specific siRNA against SREBP-2 in the absence and presence of 5 µM rosuvastatin. The symbol * indicates statistical significance, p<0.05.
Figure 6.
Statins induce acetylation of histones H3 and H4, with the dismissal of HDACs and recruitment of SREBP-2 and p300 to CCR7.
Luciferase assays were performed in RAW264.7 cells co-transfected with HDAC expression plasmids, the SREBP-2 expression plasmid, and the CCR7 promoter-luciferase construct. B) RAW 264.7 cells were treated with 5 µM rosuvastatin and the recruitment of acetylated histone H3 and H4 at the promoter region of the CCR7 gene was assessed by ChIP assays (C). RAW264.7 cells were mock treated or treated with 5 µM rosuvastatin. ChIP assays of the CCR7 promoter were performed using antibodies against HDAC6, HDAC7, SREBP-2 and p300 in RAW264.7 cells either mock treated or treated with rosuvastatin. % Input values represent the mean ± SEM.