Fig 1.
M3 inhibits chemokine activity in vitro.
The Boyden chamber migration assay was used to assess A. CCR2-, B. CCR5- and C. CX3CR1-directed cell migration in vitro. 293T cells were transfected with plasmids encoding CCR2, CCR5 or CX3CR1. D. Migration of human primary monocytes towards CCL2, CCL5, CX3CL1 and CXCL12 in Boyden chamber migration assay. Representative images of Calcein AM labelled monocytes migrated to the underside of transwell membranes with and without purified M3 protein (100 ng/ mL) are shown. Data are mean±SEM. *p<0.05, **p<0.01.
Fig 2.
Confirmation of gene transfer and inhibition of plasma chemokine activity by M3.
Two models of ‘rapid promotion’ or ‘slow progression’ of atherosclerosis were established in which a HFD or regular chow were fed and AdM3 or AdGFP were infused (n = 10–12/group). See “Materials and Methods” for details. Successful gene transfer and expression following adenoviral delivery was determined by Western immunoblotting. A. Culture media from Ad293 cells infected with AdM3 as well as plasma from mice infused with AdGFP and AdM3 were assessed from 4 to 12 weeks post adenoviral delivery following immunoprecipitation with anti-c-myc agarose beads. B. Chemokine activity was measured using the Boyden chamber migration assay. Calcein-AM labelled monocytes were allowed to migrate towards plasma from mice infused with AdGFP or AdM3 (n = 10–12/group). Data are mean±SEM. *p<0.05, ****p<0.0001.
Fig 3.
M3 reduces atherosclerotic plaque size when the rate of plaque development is less rapid.
Two models of ‘rapid promotion’ or ‘slow progression’ of atherosclerosis were established in which a HFD or regular chow were fed and AdM3 or AdGFP were infused (n = 10–12/group). See “Materials and Methods” for details. Images are representative pictures of aortic arches (upper panels) and Trichrome stained aortic sinus sections (lower panels). Quantification of total plaque area (μm2) was determined from 3 sections/mouse, spanning the entire aortic sinus for A. the ‘rapid promotion’ model and B. the ‘slow progression’ model. Data are mean±SEM. *p<0.05.
Fig 4.
M3 reduces plaque macrophage content and p65 activation when the rate of plaque development is more rapid.
Two models of ‘rapid promotion’ or ‘slow progression’ of atherosclerosis were established in which a HFD or regular chow were fed and AdM3 or AdGFP were infused (n = 10–12/group). See “Materials and Methods” for details. Upper panels are representative images of Mac-3+ macrophages (brown staining) in aortic sinus sections. Quantification of macrophage staining within plaques (μm2) for A. the ‘rapid promotion’ model and B. the ‘slow progression’ model. Phosphorylated p65 levels were measured in aortic arch samples for C. the ‘rapid promotion’ model and D. the ‘slow progression’ model. p65 mRNA levels were determined in aortic arch samples for E. the ‘rapid promotion’ model and F. the ‘slow progression’ model. Data expressed as mean±SEM. *p<0.05, **p<0.01, ****p<0.0001.
Fig 5.
M3 prevents a decrease in inflammatory monocytes in the rapid promotion model four weeks’ post-gene transfer.
Two models of ‘rapid promotion’ or ‘slow progression’ of atherosclerosis were established in which a HFD or regular chow were fed and AdM3 or AdGFP were infused (n = 7/group). See “Materials and Methods” for details. Circulating neutrophils (CD45hiCD115loLy6-C/Ghi) and monocytes (CD45hiCD115hi) were measured for Ly6-C/G by flow cytometry every week up to 4 weeks in A. the ‘rapid promotion’ model and every month up to 12 weeks in B. the ‘slow progression’ model.
Fig 6.
M3 increases plaque SMC content in the ‘slow progression’ atherosclerosis model.
Two models of ‘rapid promotion’ or ‘slow progression’ of atherosclerosis were established in which a HFD or regular chow were fed and AdM3 or AdGFP were infused (n = 10–12/group). See “Materials and Methods” for details. Images are representative sections of plaque α-actin+ SMCs (red staining) in aortic sinuses. SMC α-actin staining was quantified as a percentage of total aortic sinus plaque area and SMC α-actin mRNA levels were determined in aortic arch samples for the ‘rapid promotion’ model (A—B) and the ‘slow progression’ model (C—D). Data expressed as mean±SEM. *p<0.05.
Fig 7.
M3 reduces lipid deposition in descending aortas.
Two models of ‘rapid promotion’ or ‘slow progression’ of atherosclerosis were established in which a HFD or regular chow were fed and AdM3 or AdGFP were infused (n = 10–12/group). See “Materials and Methods” for details. Images are representative sections of Oil Red O stained descending thoracic aortas. Oil Red O staining was quantified as a percentage of total thoracic aorta area for the ‘rapid promotion’ model and the ‘slow progression’ model. Data are mean±SEM. *p<0.05.
Fig 8.
M3 regulation of chemokine levels in the plasma and tissue.
Two models of ‘rapid promotion’ or ‘slow progression’ of atherosclerosis were established in which a HFD or regular chow were fed and AdM3 or AdGFP were infused (n = 10–12/group). See “Materials and Methods” for details. CCL2, CCL5 and CX3CL1 protein levels were assessed in the liver (A-F) and CCL2 and CCL5 in the plasma (G-J) by ELISAs. Data are mean±SEM. *p<0.05, ****p<0.0001.
Fig 9.
Summary schematic comparing effects of broad-spectrum inhibition by M3 on atherosclerosis.
The effects of broad-spectrum chemokine inhibition by M3 was demonstrated via two models of atherosclerosis—‘rapid promotion’ and ‘slow progression’. In the rapid promotion model M3 inhibits chemokine activity, causing suppression of inflammatory monocytes, reducing adherence to the endothelium so they accumulate in the circulation rather than enter the plaque. This leads to a reduction in plaque macrophages and a suppression in lipid deposition in the descending thoracic aorta, which develops later, but not in the aortic sinus that would contain plaque of a more advanced stage. In the ‘rapid promotion’ model, aortic phosphorylated p65 was lower which may also have contributed to the reduction in plaque macrophages. In the more gradual slower progressive model, we saw an increase in plaque SMCs, a marker of improved plaque stability, with an overall reduction in atherosclerotic lesion area in the aortic sinus and descending thoracic aorta. Despite the decrease in lesion area, there was no effect on circulating monocytes or plaque macrophage content. This may be explained by the decline in M3 protein and activity at the later time points of this study and the overall lower levels of inflammation in this chow-fed model.