Loss of soluble guanylyl cyclase in platelets contributes to atherosclerotic plaque formation and vascular inflammation

Figures

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  Extended Data Fig. 1 |. sGC-Expression in Pf4-Cre+Gucy1b1LoxP/LoxP compared to Pf4-Cre+Gucy1b1+/LoxP mice. a. Expression of sGC-β1 in platelets, peripheral blood mononuclear cells (PBMC), aorta, and lung. Representative of three independently performed Western blots on different samples. B+C. Gucy transcript expression analysis in megakaryocytes of Pf4-Cre+Gucy1b1+/LoxP and Pf4-Cre+Gucy1b1LoxP/LoxP mice. b. Gucy1a1 and Gucy1b1. C. Gucy1a2 and Gucy1b2. Each symbol represents one independent animal (n = 6 for control group, n = 5 for Pf4-Cre+Gucy1b1LoxP/LoxP). Two-sided unpaired t-test. Data are mean and s.e.m.

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  Extended Data Fig. 2 |. Platelet aggregation in control and platelet sGC knockout (Pf4-Cre+Gucy1b1LoxP/LoxP) mice. Platelet aggregation in control and platelet sGC knockout (Pf4-Cre+Gucy1b1LoxP/LoxP) mice after stimulation with adenosine diphosphate (ADP, a) the platelet-activated receptor 4 agonist PAR4-AP (b), the thromboxane analog U46619 (c), and collagen (d). Each experiment was performed in the presence of DMSO (vehicle) or the nitric oxide donor sodium nitroprusside (SNP). Each symbol represents one independent animal (n = 6). Aggregation tracings represent the mean values of investigated animals per genotype. Two-sided unpaired t-test (except A, vehicle and D, vehicle: two-sided Mann-Whitney U-test). Data are mean and s.e.m.

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  Extended Data Fig. 3 |. Pf4-Cre+Gucy1b1LoxP/LoxPLdlr−/− compared to Pf4-Cre+Gucy1b1+/LoxPLdlr−/− mice after Western diet (see Fig. 1e). a. Serum cholesterol levels (n = 11). b. Platelet count (n = 11). c. Body weight (n = 11) at baseline (left) and after ten weeks (right). d. Blood leukocyte numbers and subsets (n = 11 for controls, n = 10 for Pf4-Cre+Gucy1b1LoxP/LoxPLdlr−/−). Each symbol represents one independent animal. Two-sided unpaired t-test (except C 10 weeks: two-sided Mann-Whitney U-test). One outlier was removed in the analysis of D according to the ROUT method. Data are mean and s.e.m.

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  Extended Data Fig. 4 |. Leukocyte adhesion to atherosclerotic plaques of Pf4-Cre+Gucy1b1LoxP/LoxPLdlr−/− (n = 13) compared to Pf4-Cre+Gucy1b1+/LoxPLdlr−/− mice (n = 11) after Western diet assessed by fluorescence intravital microscopy. a. Ly6Chigh monocytes. b. Neutrophils. Each symbol represents one independent animal. Two-sided unpaired t-test. Data are mean and s.e.m.

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  Extended Data Fig. 5 |. Quantification of monocyte adhesion after preincubation of either EC or monocytes with supernatant of activated platelets from Pf4-Cre + Gucy1b1LoxP/LoxP mice. Each symbol represents one paired sample (derived from n = 8 independent animals). Two-sided paired t-test. Data are mean and s.e.m. Abbreviations: EC, endothelial cells; rfu, relative fluorescence units.

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  Extended Data Fig. 6 |. Vcam1 expression of wild-type endothelial cells after incubation with supernatant of activated Pf4-Cre+Gucy1b1+/LoxP or Pf4-Cre+Gucy1b1LoxP/LoxP platelets. Each symbol represents one independent animal (n = 6). Data are mean and s.e.m. Two-sided paired t-test.

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  Extended Data Fig. 7 |. Cytokine profiling. a-h. Cytokine profiling assay results for cytokines detected at a mean relative intensity ≥1 (in addition to Angiopoietin-1). I. Replication attempt for RBP4 release using ELISA. Each symbol represents one independent animal (A-H: n = 8; I: n = 5). Two-sided unpaired t-test (except B and G: Two-sided Mann-Whitney U-test). Data are mean and s.e.m. Abbreviations: au, arbitrary units; CXCL5, C-X-C Motif Chemokine Ligand 5; IGFPB2, Insulin Like Growth Factor Binding Protein 2; RBP4, Retinol Binding Protein 4; REG3G, Regenerating islet-derived protein 3 gamma.

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  Extended Data Fig. 8 |. ngiopoietin-1 (ANGPT1) release by control or Pf4-Cre+Irag1LoxP/LoxP platelets after activation by shaking. A Each symbol represents one independent animal (n = 6). Two-sided unpaired t-test. Data are mean and s.e.m.

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  Extended Data Fig. 9 |. Adenosine diphosphate-induced platelet aggregation in Pf4-Cre+Gucy1b1+/LoxP and Pf4-Cre+Gucy1b1LoxP/LoxP platelets after pretreatment with vehicle or BAY-747. Each symbol represents one independent animal (n = 3). Two-sided unpaired t-test. Data are mean and s.e.m. Abbreviation: ADP, adenosine diphosphate.

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  Extended Data Fig. 10 |. Characteristics of Ldlr−/− mice receiving 0 ppm compared to Ldlr−/− mice receiving 150 ppm BAY-747 after ten weeks of Western diet. a. Serum cholesterol levels (n = 12). b. Platelet count (n = 9 in 0 ppm, n = 8 in 150 ppm group). c. Blood leukocytes (n = 9 in 0 ppm, n = 8 in 150 ppm group). d. Body weight after (n = 12). e. Plasma concentrations of the soluble guanylyl cyclase stimulator BAY-747 in treated mice. f. Blood leukocyte numbers and subsets. Each symbol represents one independent animal (n = 12). Two-sided unpaired t-test. Two outliers were removed in the analysis of F: neutrophils (150 ppm group; n = 10) and Ly6Chigh monocytes (150 ppm group; n = 11) according to the ROUT method. Data are mean and s.e.m.

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  Fig. 1 |. Atherosclerotic plaque formation and vascular inflammation in mice lacking platelet sGC. a–c, Atherosclerotic plaque formation as assessed by aortic root histology (a), aortic en face ORO staining (b), and monocyte and macrophage content (c) in 12 (b: 8) Pf4-Cre+Gucy1b1LoxP/LoxPLdlr−/− mice compared to 14 (b: 9) Pf4-Cre+Gucy1b1+/LoxPLdlr−/− mice that were fed a Western diet for 10 weeks. Two-sided unpaired t-test. d, Leukocyte adhesion to atherosclerotic plaques in n = 13 Pf4-Cre+Gucy1b1LoxP/LoxPLdlr−/− mice compared to n = 11 Pf4-Cr e+Gucy1b1+/LoxPLdlr−/− mice that were fed a Western diet for 6 weeks to induce atherosclerotic plaque formation. Two-sided unpaired t-test. e, Quantification of vascular inflammation by flow cytometry analysis of aortic cell suspensions of Pf4-Cre+Gucy1b1LoxP/LoxPLdlr−/− mice compared to Pf4-Cre+Gucy1b1+/LoxPLdlr−/− mice (n = 11 per group). Two-sided Mann–Whitney U-test. Each symbol represents one independent animal. Data are the mean ± s.e.m.

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  Fig. 2 |. Influence of platelet sGC on leukocyte adhesion in vitro. a,b, WT monocyte (a) and neutrophil (b) adhesion to WT ECs after incubation with supernatant of activated platelets isolated from either Pf4-Cre+Gucy1b1LoxP/LoxP or Pf4-Cre+Gucy1b1+/LoxP mice. Each symbol represents 1 independent animal (n = 8 per group). Two-sided unpaired t-test. Data are the mean ± s.e.m. c, Quantification of neutrophil adhesion after preincubation of either ECs or neutrophils with supernatant of activated platelets from Pf4-Cre+Gucy1b1LoxP/LoxP mice in comparison to non-preincubation conditions. Each symbol represents 1 paired sample (each derived from n = 8 independent animals). Repeated measures one-way ANOVA with Tukey test for multiple testing. Data are the mean ± s.e.m.