Figure 1.
Immunohistochemistry of FKN and CX3CR1 in atherosclerotic lesions of human carotid arteries.
Representative images of human atherosclerotic plaque in different stages of lesion development (I–VI). Staining was performed with antibodies against FKN, CX3CR1, the macrophage marker CD68, and α-smooth muscle actin (SMA). Bars, 200 µm. Asterisks indicate the luminal side of the vessel.
Figure 2.
FKN positive structures in an advanced atherosclerotic lesion.
Image of an advanced atherosclerotic plaque (VI) isolated by CEA from the human carotid artery. Insets magnify FKN positive structures within the plaque (upper row). Bottom row shows consecutive stainings for smooth muscle cells (SMA), endothelium and neovessels (Factor VIII), and macrophages (CD68). Upper scale bar: 200 µm, lower scale bar: 40 µm. Asterisk indicates the luminal side of the vessel.
Figure 3.
Double immunofluorescence analysis of FKN and CX3CR1 expressing cells.
(A) Percent distribution of FKN (green) and CX3CR1 (red) single positive and double positive (red-green stripes) cells in early (I–III) and advanced (V–VI) plaque. n = 4 carotids per group. (B) Quantitative analysis of the number of FKN and CX3CR1 positive cells in human carotid plaque. The correlation between the co-incidence of both markers was calculated by linear regression analysis. Upper panel: early plaque (I–III), r = 0.44, p<0.01. Lower panel: advanced plaque (V–VI), r = 0.74, p<0.0001. (C) A representative section from an atherosclerotic lesion (V–VI) was stained for FKN (green), CX3CR1 (red), and DAPI (blue). Bars, 100 µm. Asterisks indicate the luminal side of the vessel.
Figure 4.
Quantitative analysis of FKN expression and FKN serum levels.
(A) PCR analysis of FKN expression in atherosclerotic plaques of different AHA stages. Graph indicates relative expression ratio as calculated from the real-time PCR efficiencies and the crossing point deviation of FKN versus control. Mean FKN expression per gram plaque tissue did not differ significantly between AHA stages I–VI. (B) Serum levels of sFKN in 10 patients with moderate (50–69%), 83 with advanced (70–89%), and 44 with subocclusive (90–99%) carotid artery stenosis. Patients with subocclusive stenosis showed significantly higher levels of sFKN. **p<0.001 for 50–69% vs. 90–99% (0.44±0.04 ng/ml vs. 0.88±0.14 ng/ml). ***p<0.0001 for 70–89% vs. 90–99% (0.43±0.02 ng/ml vs. 0.88±0.14 ng/ml). p = ns for 50–69% vs. 70–89%.
Figure 5.
Monocyte and WEHI 274.1 recruitment to intact and injured atherosclerotic carotid artery lesions.
(A) Monocyte adhesion to atherosclerotic endothelium (indicated ‘before injury’) and to the injured atherosclerotic vascular wall (indicated ‘after injury’) in ApoE−/− mice (B) WEHI 274.1 adhesion to inflamed atherosclerotic endothelium and to the injured atherosclerotic vascular wall in ApoE−/− mice. Adhesion of monocytes and WEHI 274.1 was significantly increased following injury. n = 4–8, *p<0.05, **p<0.001.
Figure 6.
FKN and CX3CR1 mediate WEHI 274.1 recruitment to injured atherosclerotic carotid arteries.
(A) WEHI 274.1 adhesion to mechanically injured atherosclerotic carotids was studied over 30 minutes in the presence of an IgG control antibody (black bars) or a function blocking anti-FKN antibody (white bars). (B) Representative intravital microscopic images from mouse carotid arteries at baseline (left) and 15 minutes (right) after injury of the atherosclerotic vascular wall, pretreated with either an anti-FKN antibody (upper row) or an isotype IgG control antibody (lower row). WEHI 274.1 cells were stained with DCF (green). Bars, 50 µm. n = 4–8, *p<0.05, **p<0.01. (C) WEHI 274.1 adhesion is dependent on FKN-CX3CR1 interactions. WEHI 274.1 were transfected with shRNA-encoding plasmids to silence CX3CR1 expression (grey and white bars) or transfected with a plasmid encoding a scrambled control shRNA (black bars). Prior to transfusion the animals were pretreated either with a rabbit IgG control antibody (grey bars) or a function blocking anti-FKN antibody (white bars). WEHI 274.1 adhesion to injured atherosclerotic carotids was analyzed over 30 minutes.