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

Characteristics of mouse arterial thrombi induced by FeCl₃ injury or wire denudation in mice.

<p>(A) Immunohistological images of platelet aggregate area (red) in arterial thrombi (n = 3/group) and control stainings. Bars, 100μm. Control (isotype) or secondary antibody alone. (B) Comparison of leukocyte recruitment to the mouse carotid artery 3h after FeCl₃ exposure or wire denudation (n = 3/group). Representative images show immunohistochemical staining for leukocytes (CD45, green) and their subsets, as distinguished by expression of neutrophil elastase (NE, red) for neutrophils and CD68 (red) for blood monocytes. Nuclei were counterstained with Hoechst (including controls). Bars, 10μm. Control (isotype) or secondary antibody alone. (C) Association between number of leukocytes and thrombus age (n = 3/group). Mean ± SD. (D) Quantification of monocyte and neutrophil subsets within mouse thrombi 3h after FeCl₃ exposure (n = 3/group). Mean ± SD.</p

List of antibodies used for immunohistochemistry.

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

Patients’ baseline characteristics.

<p>Patients’ baseline characteristics.</p

NETs in arterial thrombi of mice and humans.

<p>(A) Representative illustration of NETs stained for NE and DNA (DAPI) in the early phase of arterial thrombosis. Human and mouse thrombi showed comparable morphology after 3, 6 or 12h. Extracellular DNA originates from NE+ neutrophils. Bars, 10μm. Arrows, nuclei; arrowheads, NET fibers. (B) Quantification of NETs per 100 neutrophils in human thrombi (<12h) (n = 10) and experimental thrombosis (FeCl₃) (3–6h) (n = 5). Dots represent individual experiments; lines indicate mean values for each group. (C) Association between thrombus age and number of NETs in mice and humans.</p

Characteristics of human arterial thrombi.

<p>(A) Pie chart shows the distribution of thrombus age. 50 out of 81 patients described the precise onset of AMI symptoms, which allowed the calculation of thrombus age following its removal during PCI. The majority of human thrombi (with precise onset of symptoms) was younger than 24h. (B) Leukocyte accumulation in human thrombi. Representative images of HE staining (n = 3). Bars, 200μm (top image) and 50μm (bottom image). (C) Immunohistochemical visualization of leukocytes (CD45, green, n = 3), neutrophils (NE, red, n = 81) and monocytes (CD14, green, n = 11). Nuclei are counterstained with Hoechst (including controls). Control (isotype) or secondary antibody alone. Bars, 10μm. (D) The graph shows the quantification of monocytes (n = 11) and neutrophils (n = 81) in human thrombi. Results are shown as mean ± SD. (E) Correlation between human thrombi younger than 12h and the number of leukocytes (n = 33).</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

Cl-amidine reduces myocardial ischemia-reperfusion injury.

<p>(A) Representative masson-trichrome stainings of myocardial sections from mice 7 days after myocardial ischemia-reperfusion injury treated with vehicle (left) or Cl-amidine (right). Mice treated with Cl-amidine show a decrease in fibrotic tissue compared to vehicle. Bars 2mm. (B) Infarct size 7 days after myocardial ischemia-reperfusion injury in mice treated with vehicle (n = 10) and Cl-amidine (n = 7). (C) Myocardial function was evaluated by measuring ejection fraction (in %) and (D) cardiac output (in μl/min) 7 days after myocardial injury in mice treated with vehicle (n = 5) and Cl-amidine (n = 6). Dots represent individual experiments, lines indicate mean values for each group.</p