Fig 1.
Clopidogrel and ticagrelor equally inhibit platelet aggregation at 25 mg/kg/day and 180 mg/kg/day, respectively.
Abbreviations: CTL, control; CLO, clopidogrel; TIC, ticagrelor; A.U., arbitrary units; RPI, platelet reactivity index; BP, blood pressure; N = 4–6 per group for (A–F), 26 for (G), and 11–14 for (H–I); Error bars, means ± SD, statistical analyses performed using ANOVA with Fisher’s multiple comparison; NS, not statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001 (See also S1 Fig). (A) Platelet aggregation assay using a hematology blood analyzer. (B) Platelet reactivity index (RPI) using flow cytometry. (C) Bleeding assay using tail blood loss measurement. (D) Systolic blood pressure measured. (E) Mean blood pressure calculated. (F) Diastolic blood pressure measured. (G) Body weights at 16th week of treatment with a high-fat diet (HFD). (H) Serum cholesterol measured at 16th week of treatment with a HFD. (I) Serum triglyceride measured at the 16th week of treatment with a HFD.
Fig 2.
Ticagrelor exhibits superior protection against atherosclerosis compared to clopidogrel in the Ldlr-/-Apobec1-/- hypercholesterolemic mice on a high-fat diet (HFD).
Abbreviations: CTL, control; CLO, clopidogrel; TIC, ticagrelor; H&E, hematoxylin & eosin; ORO, oil red o; Size bars, 300 μm; N = 12–15 per group; Error bars, means ± SD, statistical analyses performed using ANOVA with Fisher’s multiple comparison; NS, not statistically significant; *, P < 0.05; **, P < 0.01; ****, P < 0.001 (See also S2 Fig). Both en face (A) and cross-sectional (B) analyses show that TIC-treated animals were better protected against atherosclerosis than are CLO- or CTL-treated animals. CLO-treated animals exhibited less atherosclerosis than CTL-treated animals (B).
Fig 3.
Immunohistochemical (IHC) analysis of the aortae of TIC-, CLO-, and CTL-treated animals.
Abbreviations: CTL, control; CLO, clopidogrel; TIC, ticagrelor; A.U., arbitrary units; F4/80, F4/80 antigen also known as adhesion G protein-coupled receptor E1 (EMR1); MΦ, macrophages; α-SMA, alpha smooth muscle cell actin; VSMC, vascular smooth muscle cells; S100A4, S100 calcium-binding protein A4; FB, fibroblasts; TGFβ1, transforming growth factor β1; 4-HNE, 4-hydroxynonenal; P-IRE1, phosphorylated inositol-requiring enzyme 1; Size bars, 300 μm; N = 13–18, 6–7, 13–18, 6–7, 11–17, and 6–7 per group for (A), (B), (C), (D), (E), and (F), respectively; Error bars, means ± SD, statistical analyses performed using ANOVA with Fisher’s multiple comparison; NS, not statistically significant; *, P < 0.05; **, P < 0.01. (See also S3 Fig) (A) Decreased MΦ infiltration to the atherosclerotic intima of CLO- and TIC-treated compared to CTL-treated Ldlr-/-Apobec1-/- mice placed on a HFD for 16 weeks as assessed by IHC staining using anti-F4/80 antibody. (B) No significant VSMCs migration to the intima of CTL-, CLO-, and TIC-treated mice. (C) Decreased fibroblasts in the intima of CLO-treated mice in comparison with CTL- and TIC-treated mice. (D) Decreased TGFβ1 in the intima of CLO-treated mice in comparison with CTL- and TIC-treated mice. (E) Increased 4-HNE lipid peroxidation marker in CLO-treated mouse aortae compared with CTL- and TIC-treated aortae. (F) Increased P-IRE1 ER stress marker in CLO-treated mouse aortae compared with TIC-treated aortae.
Fig 4.
Serum chemo/cytokine profiles of CTL-, CLO-, and TIC-treated Ldlr-/-Apobec1-/- mice placed on a HFD.
Abbreviations: CTL, control; CLO, clopidogrel; TIC, ticagrelor; IL-6, interleukin 6; CCL4, C-C motif chemokine ligand 4 (also known as macrophage inflammatory protein 1β or MIP-1β); CXCL10, C-X-C motif chemokine ligand 10; TNFα, tumor necrosis factor alpha; N = 9–16, 13–16, 9–17, 15–16 per group, for (A), (B), (C), and (D), respectively; Error bars, means ± SD, statistical analyses performed using ANOVA with Fisher’s multiple comparison; NS, not statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001 (See also S4 Fig). (A) Decreased serum IL-6 levels in CLO- and TIC-treated mice compared with CTL-treated mice. (B) Decreased serum CCL4 levels in TIC-treated mice compared with CTL- and CLO-treated mice. (C) Increased serum CXCL10 levels in CLO-treated mice compared with TIC-treated mice. (D) Increased serum TNFα levels in CLO-treated mice compared with CTL- and TIC-treated mice.
Fig 5.
Ticagrelor treatment, but not clopidogrel treatment, induces the praroxonase-1 (PON1) message, increases tissue and serum PON1 protein, and mitigates atherosclerosis.
Abbreviations: CTL, control; CLO, clopidogrel; TIC, ticagrelor; RNA-Seq, RNA sequencing using next generation sequencing; PON1, paraoxonase 1; A.U., arbitrary unit; N = 5 per group; Error bars, means ± SD, statistical analyses performed using one-way ANOVA test with Fisher’s multiple comparisons; NS, not statistically significant; *, P < 0.05; **, P < 0.01 (See also S5 Fig). (A) Experimental protocol to explore the mechanism(s) by which TIC decreases atherosclerosis more than does CLO. (B) Comparison of differentially expressed genes of the CLO/CTL group with those of the TIC/CTL group. Fifty-six genes were concordantly and differentially expressed in both CLO/CTL and TIC/CTL groups, of which 48 genes were concordantly upregulated and 8 concordantly downregulated in both groups. (C) List of genes that were concordantly and differentially expressed in both CLO/CTL and TIC/CTL groups. These are the genes that could explain the reduction of atherosclerosis by both CLO and TIC. Among these genes, only PON1 has been associated with the amelioration of atherosclerosis. (D) RT-qPCR confirming induction of the PON1 message by TIC compared with CTL and CLO. (E) Increased PON1 activity in the sera of TIC-treated Ldlr-/-Apobec1-/- mice placed on a HFD compared with mice treated with CLO or CTL. (F) Increased tissue expression of PON1 in the atherosclerotic intima of TIC-treated mice compared with CTL-treated mice. (G) At the dose that inhibits platelets equally, TIC decreases atherosclerosis and associated inflammation more robustly than does CLO through its ability to induce PON1.
Fig 6.
Biological phenotypes of CTL-, CLO-, and TIC-treated mice.
Abbreviations: CTL, control; CLO, clopidogrel; TIC, ticagrelor; Orange, the results that could explain why TIC protects against atherosclerosis more than does CLO; Green, the results that could explain why TIC and CLO both protect against atherosclerosis, compared with CTL; Blue, the results that raise concerns on the possible negative vascular effects of CLO.