Summary schematic comparing effects of broad-spectrum inhibition by M3 on atherosclerosis.

<p>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.</p

M3 reduces lipid deposition in descending aortas.

<p>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 (<i>n</i> = 10–12/group). See “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173224#sec002" target="_blank">Materials and Methods</a>” 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. *<i>p</i><0.05.</p

M3 inhibits chemokine activity <i>in vitro</i>.

<p>The Boyden chamber migration assay was used to assess <b>A.</b> CCR2-, <b>B.</b> CCR5- and <b>C.</b> CX₃CR1-directed cell migration <i>in vitro</i>. 293T cells were transfected with plasmids encoding CCR2, CCR5 or CX₃CR1. <b>D.</b> 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. *<i>p</i><0.05, **<i>p</i><0.01.</p

M3 reduces plaque macrophage content and p65 activation when the rate of plaque development is more rapid.

<p>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 (<i>n</i> = 10–12/group). See “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173224#sec002" target="_blank">Materials and Methods</a>” 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 <b>A.</b> the ‘rapid promotion’ model and <b>B.</b> the ‘slow progression’ model. Phosphorylated p65 levels were measured in aortic arch samples for <b>C.</b> the ‘rapid promotion’ model and <b>D.</b> the ‘slow progression’ model. p65 mRNA levels were determined in aortic arch samples for <b>E.</b> the ‘rapid promotion’ model and <b>F.</b> the ‘slow progression’ model. Data expressed as mean±SEM. *<i>p</i><0.05, **<i>p</i><0.01, ****<i>p</i><0.0001.</p

M3 reduces atherosclerotic plaque size when the rate of plaque development is less rapid.

<p>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 (<i>n</i> = 10–12/group). See “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173224#sec002" target="_blank">Materials and Methods</a>” 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 <b>A.</b> the ‘rapid promotion’ model and <b>B.</b> the ‘slow progression’ model. Data are mean±SEM. *<i>p</i><0.05.</p

M3 prevents a decrease in inflammatory monocytes in the rapid promotion model four weeks’ post-gene transfer.

<p>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 (<i>n</i> = 7/group). See “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173224#sec002" target="_blank">Materials and Methods</a>” for details. Circulating neutrophils (CD45^hiCD115^loLy6-C/G^hi) and monocytes (CD45^hiCD115^hi) were measured for Ly6-C/G by flow cytometry every week up to 4 weeks in <b>A.</b> the ‘rapid promotion’ model and every month up to 12 weeks in <b>B.</b> the ‘slow progression’ model.</p

Evaluation of synthetic vascular grafts in a mouse carotid grafting model

<div><p>Current animal models for the evaluation of synthetic grafts are lacking many of the molecular tools and transgenic studies available to other branches of biology. A mouse model of vascular grafting would allow for the study of molecular mechanisms of graft failure, including in the context of clinically relevant disease states. In this study, we comprehensively characterise a sutureless grafting model which facilitates the evaluation of synthetic grafts in the mouse carotid artery. Using conduits electrospun from polycaprolactone (PCL) we show the gradual development of a significant neointima within 28 days, found to be greatest at the anastomoses. Histological analysis showed temporal increases in smooth muscle cell and collagen content within the neointima, demonstrating its maturation. Endothelialisation of the PCL grafts, assessed by scanning electron microscopy (SEM) analysis and CD31 staining, was near complete within 28 days, together replicating two critical aspects of graft performance. To further demonstrate the potential of this mouse model, we used longitudinal non-invasive tracking of bone-marrow mononuclear cells from a transgenic mouse strain with a dual reporter construct encoding both luciferase and green fluorescent protein (GFP). This enabled characterisation of mononuclear cell homing and engraftment to PCL using bioluminescence imaging and histological staining over time (7, 14 and 28 days). We observed peak luminescence at 7 days post-graft implantation that persisted until sacrifice at 28 days. Collectively, we have established and characterised a high-throughput model of grafting that allows for the evaluation of key clinical drivers of graft performance.</p></div

Cell tracking.

<p>A: Schematic of experimental design. Bone marrow mono-nuclear cells are isolated from FVB-L2G mice and injected into FVB-N mice that received that carotid grafting. B: Representative IVIS images of carotid grafted mice with BM-MNC injection. Warm colours denote higher signal and cold colours denote lower signal. C: Representative images of cross sections with eGFP staining in green and nucleus in blue. Scale bar = 100 μm. D: Quantification of eGFP positive cells cells. Data expressed as mean ± SEM and analysed using one-way ANOVA, n = 3 animals/timepoint.</p

Neointimal area.

<p>A: Distribution of NH throughout the graft. Neointimal area represented as a percentage of total luminal area as defined by the inner graft wall. Data expressed as mean ± SEM and analysed using two-way ANOVA, n = 7 animals/timepoint. B: Representative images of H&E staining of cross sections. Black dotted lines indicate the graft wall. Scale bar = 100 μm.</p

Endothelialisation.

<p>A: Scanning electron microscopy images of the luminal side of the graft. Scale bar = 500 μm. Inset scale bar = 50 μm. B: Quantification of CD31 coverage represented as a percentage of total luminal circumference. Data expressed as mean ± SEM and the mid section of the grafts were analysed using one-way ANOVA, n = 7 animals/timepoint. C: Representative images of cross sections with CD31 staining in red, nucleus in blue. White dotted lines indicate the graft wall. Scale bar = 100 μm.</p