Cl-amidine treatment does not affect metabolic changes associated with high fat diet feeding.

A) 3D reconstruction of a NET in obese adipose tissue of HFD fed C57Bl6 mice imaged by immunofluorescence confocal microscopy. Co-localisation of the markers Ly6G (green), cathelicidin (red), and dapi (blue) is shown. Dotted circle shows a NET-releasing neutrophil between the dotted lines. B) Isolated neutrophils were incubated with medium, 100 nM PMA, or 100 nM PMA and 1 mM Cl-amidine (Cl-ami). NETosis was measured with the extracellular DNA dye Sytox green over a period of 4 hours (n = 7). C-G) Mice receiving a 60% HFD for ten weeks were treated with either Cl-amidine (n = 10; 10 mg/kg; s.c.; daily) or vehicle (PBS) (n = 10; s.c.; daily). C) The mice were weighted weekly. D) Prior (pre-HFD) and after 10 weeks of the HFD (Post-HFD) a glucose tolerance test was performed. The mice were fasted for 12 hours after which they received a subcutaneous injection of 1 mg glucose per g mice. Blood glucose levels were measured at different time points. E) Area under the curve was calculated from the GTT graphs Fig 1D. F) Plasma levels of cholesterol were measured by CHOD-PAP (Roche). G) Haematoxylin/eosin staining was performed on paraffin sections of EpAT and the diameter of 100 adipocytes per mice was calculated. Data is represented as mean ± SEM, *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001, n = 10 for each group unless stated otherwise.</p

Cl-amidine treatment does not affect metabolic changes associated with high fat diet feeding.

<p><b>A)</b> 3D reconstruction of a NET in obese adipose tissue of HFD fed C57Bl6 mice imaged by immunofluorescence confocal microscopy. Co-localisation of the markers Ly6G (green), cathelicidin (red), and dapi (blue) is shown. Dotted circle shows a NET-releasing neutrophil between the dotted lines. <b>B)</b> Isolated neutrophils were incubated with medium, 100 nM PMA, or 100 nM PMA and 1 mM Cl-amidine (Cl-ami). NETosis was measured with the extracellular DNA dye Sytox green over a period of 4 hours (n = 7). <b>C-G)</b> Mice receiving a 60% HFD for ten weeks were treated with either Cl-amidine (n = 10; 10 mg/kg; s.c.; daily) or vehicle (PBS) (n = 10; s.c.; daily). <b>C)</b> The mice were weighted weekly. <b>D)</b> Prior (pre-HFD) and after 10 weeks of the HFD (Post-HFD) a glucose tolerance test was performed. The mice were fasted for 12 hours after which they received a subcutaneous injection of 1 mg glucose per g mice. Blood glucose levels were measured at different time points. <b>E)</b> Area under the curve was calculated from the GTT graphs Fig 1D. <b>F)</b> Plasma levels of cholesterol were measured by CHOD-PAP (Roche). <b>G)</b> Haematoxylin/eosin staining was performed on paraffin sections of EpAT and the diameter of 100 adipocytes per mice was calculated. Data is represented as mean ± SEM, *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001, n = 10 for each group unless stated otherwise.</p

Simvastatin impairs neutrophil adhesion.

<p>Isolated human neutrophils were pre-treated with simvastatin (3 hours, 1 or 10 µM) and then activated with fMLP. Neutrophils were perfused over immobilized recombinant ICAM-1 or fibronectin at 1 dyne/cm² and the number of adherent cells was enumerated. n = 8–10 for flow chamber experiments (repetition of the experiment = 4). Statistical significance was tested using one way ANOVA with Newman-Keuls Multiple Comparison test.* indicates significant difference of fMLP treated samples compared to each other samples.</p

Effect of Cl-amidine treatment on immune cell accumulation and activation in adipose tissue and liver.

<p><b>A)</b> White adipose tissue was digested by 0.25 mg/mL Liberase to isolate the resident immune cells. Flow cytometry analysis was performed for leukocytes (CD45⁺), myeloid cells (CD45⁺/CD11b⁺), neutrophils (CD45⁺/CD11b⁺/Ly6G⁺), macrophages (CD45⁺/CD11b⁺/F4/80⁺) and expression of macrophage pro-inflammatory markers CD11c and MHCII on macrophages. <b>B)</b> Similar flow cytometry was performed on liver resident immune cells. Data is represented as mean ± SEM, n = 10 for each group unless stated otherwise.</p

Simvastatin does not affect neutrophil apoptosis.

<p>Apoptosis of neutrophis in presence or absence of simvastatin was measured using Annexin V with flow cytometry after 3h and 24 hours respectively (n = 6; repetition = 3). Statistical significance was tested using one way ANOVA with Newman-Keuls Multiple Comparison test. * indicates significant difference to the control group without neutrophils.</p

Simvastatin reduces LPS-induced acute lung injury by interference with neutrophil recruitment.

<p>Mice were challenged with LPS via inhalation and sacrificed 4 hours later. In addition, neutrophils were depleted by antibody injection or mice were treated with simvastatin (2 µg/g bodyweight) 12 hours and one hour before or one hour after LPS exposure as indicated. <b>A:</b> Quantification of alveolar (top), interstitial (middle), and intravascular neutrophils (bottom) in mice treated as indicated. <b>B:</b> FITC-dextran clearance (top), albumin concentration (middle), and elastase activity (bottom) in BAL fluids in mice treated as indicated (n = 8–10 for each bar). Statistical significance was tested using one way ANOVA with Newman-Keuls Multiple Comparison test. * indicates significant difference compared to LPS-treated animals.</p

Simvastatin blocks ROS formation in neutrophils.

<p>Isolated human neutrophils were pre-treated with Simvastatin (3 hours, 1 or 10 µM), stained with ROS-sensitive H2-DCFDA and then activated with fMLP. In addition, neutrophils were pre-treated with simvastatin as indicated and fluorescence was measured by flow cytometry FACS Canto (Becton Dickinson, San Jose, CA) every ten minutes following fMLP stimulation. n = 6 for each group; repetition = 4. Statistical significance was tested using one way ANOVA with Newman-Keuls Multiple Comparison test. * indicates significant difference in comparison to the control group at each different time point.</p

Vulnerability-Index of the lesions in the mouse models of atherosclerotic plaque destabilization.

<p>Vulnerability-Index (VI) was calculated by the relation between analyzed unstable (<i>U</i>) and stable (<i>S</i>) features of the plaque and corrected by the incidence of lesion formation (p, VI_c). Lesions not representing the type II phenotype were scored as zero. SD was not indicated for models with incidence of n = 1 of type II lesions. LCCA: left common coronary artery; LRA: left renal artery; CD: chow diet; HFD: high fat diet. (n = 3–8; p < 0.05, p < 0.01, p < 0.001 with 1-way ANOVA with Bonferroni’s Multiple Comparison test)</p><p>Vulnerability-Index of the lesions in the mouse models of atherosclerotic plaque destabilization.</p

Compositional features of the atherosclerotic lesions.

<p>Quantification of (<b>A</b>) macrophage (CD68+ area), (<b>B</b>) vascular smooth muscle cell (SMA+ area) and (<b>C</b>) total collagen (picrosirius red+ area) content. (<b>D</b>) Quantification of the incidence of intraplaque hemorrhage in Pearls’ Prussian blue stained sections. CD: chow diet; HFD: high fat diet; LCCA: left common coronary artery; LRA: left renal artery; IPH: intraplaque hemorrhages.</p

Summary of features of the plaque destabilization models.

<p>• low •• medium ••• high—n/a</p><p>LCCA: left common coronary artery; LRA: left renal artery; CD: chow diet; HFD: high fat diet; VI: Vulnerability-Index; VI_c: corrected Vulnerability-Index; FC: fibrous cap; NC: necrotic core; IPH: intraplaque hemorrhage.</p><p>Summary of features of the plaque destabilization models.</p