Monocyte and WEHI 274.1 recruitment to intact and injured atherosclerotic carotid artery lesions.

<p>(<b>A</b>) Monocyte adhesion to atherosclerotic endothelium (indicated ‘before injury’) and to the injured atherosclerotic vascular wall (indicated ‘after injury’) in ApoE^−/− mice (<b>B</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.</p

FKN and CX3CR1 mediate WEHI 274.1 recruitment to injured atherosclerotic carotid arteries.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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.</p

Double immunofluorescence analysis of FKN and CX3CR1 expressing cells.

<p>(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.</p

Quantitative analysis of FKN expression and FKN serum levels.

<p>(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%.</p

FKN positive structures in an advanced atherosclerotic lesion.

<p>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.</p

Immunohistochemistry of FKN and CX3CR1 in atherosclerotic lesions of human carotid arteries.

<p>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.</p

Histological comparison of arterial thrombi in mice and men and the influence of Cl-amidine on thrombus formation

<div><p>Aims</p><p>Medical treatment of arterial thrombosis is mainly directed against platelets and coagulation factors, and can lead to bleeding complications. Novel antithrombotic therapies targeting immune cells and neutrophil extracellular traps (NETs) are currently being investigated in animals. We addressed whether immune cell composition of arterial thrombi induced in mouse models of thrombosis resemble those of human patients with acute myocardial infarction (AMI).</p><p>Methods and results</p><p>In a prospective cohort study of patients suffering from AMI, 81 human arterial thrombi were harvested during percutaneous coronary intervention and subjected to detailed histological analysis. In mice, arterial thrombi were induced using two distinct experimental models, ferric chloride (FeCl₃) and wire injury of the carotid artery. We found that murine arterial thrombi induced by FeCl₃ were highly concordant with human coronary thrombi regarding their immune cell composition, with neutrophils being the most abundant cell type, as well as the presence of NETs and coagulation factors. Pharmacological treatment of mice with the protein arginine deiminase (PAD)-inhibitor Cl-amidine abrogated NET formation, reduced arterial thrombosis and limited injury in a model of myocardial infarction.</p><p>Conclusions</p><p>Neutrophils are a hallmark of arterial thrombi in patients suffering from acute myocardial infarction and in mouse models of arterial thrombosis. Inhibition of PAD could represent an interesting strategy for the treatment of arterial thrombosis to reduce neutrophil-associated tissue damage and improve functional outcome.</p></div

Accumulation of fibrinogen/fibrin in human and mouse arterial thrombi.

<p>(A) Representative immunohistochemical staining of mouse and human thrombi for fibrinogen/fibrin (red) and control stainings. Nuclei were counterstained with Hoechst (including controls). Bars: 50μm (top left and right), 200μm (bottom left and right), 300μm (top and bottom middle). (B) Fibrinogen/fibrin-covered area in the thrombus (human thrombi n = 6, mouse thrombi n = 3). Data are shown as mean ± SD.</p

List of antibodies used for immunohistochemistry.

<p>List of antibodies used for immunohistochemistry.</p

Cl-amidine inhibits arterial thrombosis in mice.

<p>(A) Representative intravital microscopy images 5, 10 and 20min after FeCl₃ injury in mice treated with Cl-amidine or vehicle. Platelets were labeled in vivo (green). Bars, 200μm. (B) Time until occlusion (left) and duration of vessel occlusion (right) after FeCl₃ exposure in mice treated with vehicle (n = 8) or Cl-amidine (n = 8). (C) Left: Representative histological images (Ly6G in red, cit H3 in green, DAPI in blue) of NETs in mice treated with vehicle or Cl-amidine (n = 5/group). Bars, 5μm. Arrowhead, NET fiber. Middle: Quantification of NETs per 100 neutrophils (n = 5/group). Right: Quantification of leukocytes (left axis) and neutrophils (right axis) in murine arterial thrombi.</p