Rivaroxaban, a Direct Oral Factor Xa Inhibitor, Attenuates Atherosclerosis by Alleviating Factor Xa–PAR2-Mediated Autophagy Suppression

Figure 1. Pharmacologic Effects of Polyethylene Glycol 400–Aggregated RIV Administration on Plasma Concentration, Plasma Factor Xa Levels, and PT in Mice (A) Plasma concentration of rivaroxaban (RIV) in response to 60- or 120-mg/kg/d administration of RIV. ∗P < 0.05, †P < 0.01 (n = 5). (B) Quantitative analysis of plasma factor Xa activity in mice administered with the indicated dosage of RIV. ∗P < 0.05, †P < 0.01 (n = 5). (C) Quantitative analysis of prothrombin time in mice administered with the indicated dosage of RIV. ∗P < 0.05 (n = 8). (D) Quantitative analysis of activated partial thromboplastin time in mice administered with the indicated dosage of RIV (n = 8). All data are expressed as dot plots, with lines indicating the mean ± standard error of the mean.

Figure 2. Administration of RIV Markedly Reduces Atherosclerotic Plaques in HFD-Fed ApoE–/– Mice (A) Quantitative analysis of plasma factor Xa activity in apolipoprotein E–deficient (ApoE–/–) mice with or without a high-fat diet (HFD) and rivaroxaban (RIV) 120 mg/kg/d. ∗P < 0.05 (n = 7). (B) (Top) Representative images of the aorta en face stained with oil red O. (Bottom) Quantitative analysis of the area of atherosclerotic plaques in the aorta. ∗P < 0.05 (n = 10). (C) (Top) Representative pictures of Mallory-Azan–stained aortic root plaques. (Bottom) Quantitative analysis of the area of aortic root plaques. ∗P < 0.05 (n = 10). All data are expressed as dot plots, with lines indicating the mean ± standard error of the mean.

Figure 3. The Effects of Factor Xa on Macrophage Autophagy Both In Vivo and In Vitro (A)(Left) Representative transmission electron microscopic images of autophagosomes (arrows and insets) in macrophages derived from the atheroma of high-fat diet (HFD)-fed apolipoprotein E–deficient (ApoE–/–) mice with or without rivaroxaban (RIV) administration. (Right) Quantitative analysis of the number of autophagosomes in macrophages residing in the atheroma with or without RIV. ∗P < 0.05 (n = 10). (B) (Left) Representative transmission electron microscopic images of autophagosomes in RAW264.7 macrophages with and without the administration of indicated compounds. (Right) Quantitative analysis of the number of autophagosomes with and without the administration of indicated compounds. ∗P < 0.05 (n = 10). (C) A representative immunoelectron microscopic image of autophagosomes stained with anti-LC3 antibody in 7KC-treated RAW264.7 macrophages. (D) (Left) Representative immunoblot images of mammalian target of rapamycin (mTOR), phospho-mTOR (pmTOR) (Ser2448), p62, LC3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (Right) Densitometric analysis of immunoblots. ∗P < 0.05 (n = 3). All data are…

Figure 4. The Effects of Factor Xa on NLRP3 Inflammasomes of Macrophages Both In Vivo and In Vitro (A)(Left) Representative immunoblot images of NLR family pyrin domain containing 3 (NLRP3), caspase-1, interleukin (IL)-1β, and GAPDH. (Right) Densitometric analysis of immunoblots. ∗P < 0.05 (n = 3). All data are expressed as mean ± standard error of the mean. (B) Immunohistochemical images of NLRP3 inflammasome markers in RAW264.7 macrophages with and without the administration of indicated compounds. (C) RAW264.7 macrophages with and without the administration of indicated compounds were analyzed for IL-1β secretion in the supernatants by enzyme-linked immunosorbent assay. ∗P < 0.05 (n = 6). Data are expressed as dot plots, with the lines indicating the mean ± standard error of the mean. (D) (Left) Representative images of NLRP3 immunostaining in atheromas derived from high-fat diet–fed apolipoprotein E knockout (KO) mice with or without the rivaroxaban administration. (Right) Quantitative analysis of the number of NLRP3 staining intensity in immunohistochemical samples. ∗P < 0.05 (n = 9). Data are expressed as dot plots, with the lines indicating the mean ± standard error of the mean. WT…

Figure 5. The Role of Protease-Activated Receptor 2–Mediated Signaling in Regulating Autophagy and NLRP3 Inflammasome Both on PAR2 Knockout and WT Mice–Derived Macrophages (A) (Top) Representative immunoblot images of LC3, phospho–mammalian target of rapamycin (p-mTOR), mammalian target of rapamycin (mTOR), NLR family pyrin domain containing 3 (NLRP3), interleukin (IL)-1β, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (Bottom) Densitometric analysis of immunoblots. ∗P < 0.05 (n = 3). All data are expressed as mean ± standard error of the mean. (B) Macrophages taken from both protease-activated receptor 2 (PAR2) knockout (KO) and wild-type (WT) mice with and without the administration of indicated compounds were analyzed for IL-1β secretion in the supernatants by enzyme-linked immunosorbent assay. ∗P < 0.05 (n = 6). Data are expressed as dot plots, with the lines indicating the mean ± standard error of the mean.

Figure 6. The Inflammasome Is Degraded by TRIM20-Mediated Precision Autophagy in 7-Ketocholesterol–Treated RAW264.7 Macrophages (A) Lysates from RAW264.7 macrophages treated with or without 7-ketocholesterol (7KC) were subjected to immunoprecipitation with the anti-TRIM20 antibody. The immunoprecipitants and 5% inputs were immunoblotted with the anti-ULK1, anti-Beclin1, and anti-ATG16L1 antibodies. (B) Lysates from RAW264.7 macrophages treated with or without 7KC were subjected to immunoprecipitation with the anti–NLR family pyrin domain containing 3 (NLRP3) antibody. The immunoprecipitants and 5% inputs were immunoblotted with the anti-TRIM20 antibody. (C) Representative immunoblot images of p-ULK1(Ser317), p-ULK1(Ser757), and total ULK1. (D) The representative images of in situ proximity ligation assays. Green dots indicate the NLRP3-TRIM20 interaction in RAW264.7 macrophages. (E) A schematic representation of precision autophagy. NLRP3 inflammasome is degraded by precision autophagy through the TRIM20-mediated mechanism. IP = immunoprecipitation.

Figure 7. The Role of Both PAR2 and Autophagy in the Progression of Atherosclerosis in a HFD-fed Apo–/– Knockout Mice (A) A schematic of the experimental design for evaluating the effect of factor Xa–PAR2-mediated autophagy regulation on atherosclerosis. ApoE–/– mice, ApoE–/–/protease-activated receptor 2 (PAR2) knockout (PAR2–/–) bigenic mice, or wild-type mice were fed a high-fat diet (HFD) for 20 weeks with or without treatment with rivaroxaban (RIV) or chloroquine (CQ) (an autophagy inhibitor). The mice were sacrificed for analyses after 20 weeks. (B) (Top) Representative images of the aorta en face derived from HFD-fed ApoE–/– mice, ApoE–/–/PAR2–/– bigenic mice, or wild-type mice with and without the administration of RIV and CQ stained with oil red O. (Bottom) Quantification of aortic en face plaque area in HFD-fed ApoE–/– mice or ApoE–/–/PAR2–/– bigenic mice, as shown in the representative images. ∗P < 0.05 versus ApoE–/– + HFD + Riv(+). †P < 0.05 versus ApoE–/–;PAR2–/– + HFD (n = 10). Data are expressed as dot plots, with lines indicating the mean ± standard error of the mean. ApoE-/- = apolipoprotein E; HFD = high-fat diet; PAR2 = protease-activated receptor-2.