Fig 2: CCR5 inhibition results in loss of enhanced recruitment of patrolling monocytes, Mafb+AIM+ macrophage accumulation and AT acceleration in MT4-MMP-null BMT Ldlr–/– mice. a Scheme depicts the experimental design of CCR5 blocking strategy by Maraviroc (MRV) administration as described in M&M. b Representative orthogonal XZ view of confocal microscopy images of whole mount-stained aortic arch from Ldlr–/– mice transplanted with MT4-MMP+/+ or MT4-MMP–/– BM cells, fed the HFD for 3 days and treated with MRV or vehicle. Samples were stained for CD115 (red) and Ly6C (green); elastin autofluorescence (green) and nuclei (Hoechst, blue) are also shown. Scale bar, 20 µm. The bar graph (right) shows the quantification of the number of patrolling monocytes (CD115+Ly6C–) in the aorta lumen; n = 7 vehicle and n = 8 MRV mice per genotype in two independent experiments. c Bar graphs show the quantification of the number of Mac3+ cells (left), Mac3+Mafb+ cells (middle), and Mac3+Mafb+AIM+ cells (right) in the plaques of BM-transplanted Ldlr–/– mice fed a HFD for 1 week and treated with Maraviroc or vehicle. n = 8 mice per genotype and condition in two independent experiments. d Representative microscopy images of transverse sections of aortic sinus from BM-transplanted Ldlr–/– mice fed a HFD for 8 weeks and treated with Maraviroc or vehicle; sections were labeled for Mac3 (red) and Hoechst (blue; nuclei) (upper, scale bar, 50 µm) and graph shows the quantification of the number of Mac3+ cells in the plaque (lower). n = 7 vehicle and n = 8 MRV BM-transplanted mice per genotype in two independent experiments. e Graph showing the lesion area of aortas stained with Oil Red from BM-transplanted Ldlr–/– mice fed a HFD for 8 week and treated with MRV or vehicle. n = 7 and n = 8 mice for Vehicle and MRV mice per genotype in two independent experiments. f Stary scoring (I-VI) of transverse aortic H&E-stained sections from BM-transplanted Ldlr–/– mice fed a HFD for 8 weeks and treated with Maraviroc or vehicle, shown as a percentage of all mice for each score. n = 7 vehicle and n = 8 MRV BM-transplanted mice per genotype in two independent experiments. Data were tested by one-way ANOVA followed by Bonferroni’s post test in b–e and by ?2-test for a trend in f. Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005

Fig 3: LOX transgenesis did not affect the systemic and local inflammatory response after carotid artery ligation. (a) Number of circulating white cells in wild-type (WT) and TgLOX mice after carotid artery ligation. (b) Count of the number of circulating monocytes (MID: Mid-sized cells; a subgroup of white blood cells that consists primarily of monocytes), lymphocytes (LYMF) and granulocytes (GRAN) in these animals. Data are expressed as mean ± SEM (*p < 0.001 vs. WT or TgLOX mice at time 0; Two-way ANOVA; n = 6). (c) Mac3 immunostaining in hematoxylin counterstained cross-sections of ligated carotid arteries from wild-type and TgLOX mice 3 weeks after ligation (n = 3). Bar: 20 µm.

Fig 4: Effect of bone marrow transplantation on the atherosclerosis lesion area in ApoE -/- and BubR1 L/L-ApoE -/- mice. A, Photomicrographs of the en face evaluation of ApoE -/-?ApoE -/-, BubR1 L/L-ApoE -/-?ApoE -/-, ApoE -/-?BubR1 L/L-ApoE -/- and BubR1 L/L-ApoE -/-?BubR1 L/L-ApoE -/- mouse aortas stained with Zudan III, and quantitative analysis of atherosclerosis of the total aorta after 12 weeks of a HCD. Data are presented as the mean±SE; *P<0.05, Student t test, n=6 in each group. B, Atherosclerosis lesion area of mice in the aortic root lesion, stained with EVG. Data are presented as the mean±SE; *P<0.05, Student t test; n=6 in each group. C, Necrotic core lesion area of mice in the aortic root lesion stained with HE. Data are presented as the mean±SE; *P<0.05, Student t test; n=6 in each group. D, Mac3-positive absolute cell count in the aortic root lesion area of mice. Data are presented as the mean±SE; *P<0.05, Student t test; n=6 in each group. ApoE-/- indicates apolipoprotein E-deficient; BubR1, Budding uninhibited by benzimidazole-related 1; EVG, elastic van Gieson; HCD, high-cholesterol diet; HE, Hematoxylin-Eosin.

Fig 5: Mice under RSS develop vulnerable lesion phenotypes. A, Representative images of hematoxylin-eosin staining with necrotic cores outlined by dotted line and quantification of areas of necrosis. B, Representative images of Masson's trichrome staining, which stains collagen in blue, and quantification of collagen-positive areas. C, Mac-3 immunostaining (brown) and quantification of Mac3-positive areas. DS indicates day shift; N.S., not significant; and RSS, rotating shift schedules. Bar=200 µm. N=11. *P<0.05, **P<0.01, t test.