Fig 1: VFEpoR RBCs, red pulp macrophages, and splenic CD11b+ cells show increased ROS and lipid peroxidation.Control and VFEpoR mice were fed a Western diet and treated with LDLR ASO and EPO (3 times per week) for 12 weeks. (A) ROS in RBCs was assessed by H2 DCFDA staining and analyzed by flow cytometry as MFI. (B) Immunoblot and quantification of NOX2 expression in RBC lysates, which was normalized to ß-actin. (C) RBC lipid peroxidation was assessed by C11 BODIPY staining and quantified by flow cytometry as MFI. (D) Splenic red pulp macrophages, CD11b+ cells, neutrophils, and Ly6Chi and Ly6Clo monocytes were stained with C11 BODIPY for lipid peroxidation and analyzed by flow cytometry. (E) Assessment of externalized phosphatidylserine level by annexin V staining in RBCs through flow cytometry. (F) Interaction network and clustering of upregulated proteins in VFEpoR RBCs. Bar graphs show relative abundance protein level of EIF2AK1 (also known as heme-regulated inhibitor, HRI) and EIF2A (eukaryotic translation initiation factor 2A). The lines between proteins represent interactions among proteins with confidence level; dashed line is the lowest and thick line is the highest confidence. (G) Proteins involved in cellular oxidant stress were analyzed and quantifications of relative abundance of glutamate cysteine ligase catalytic subunit (GCLC), glutathione transferase theta 1 (GSTT1), glutathione peroxidase 1 (GPX1) are shown. (H) The level of reduced and oxidized glutathione ratio in RBCs. Unpaired 2-tailed t test, *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2: Erythroid parameters and platelets in blood from atherogenic and control mice. (A) Number of circulating RBC from ApoE+/+ and ApoE-/- mice fed either with a CD or with a WD for the last 13 weeks (n = 13–6, from 6–2 independent experiments). One-way ANOVA with Bonferroni correction was used to compare all pairs of columns between groups. (B) Hematocrit, hemoglobin, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) determined in blood from ApoE+/+ mice with a CD and ApoE-/- fed with a WD for the last 13 weeks. Two-tailed Student's t-tests were used for comparisons between two groups (n = 7–7, from 3 independent experiments). (C) Epo mRNA levels in kidney and circulating Epo in mice described in B (n = 10–12, from 4–5 independent experiments). (D) Hepcidin mRNA levels in liver (n = 7–9, from 4 independent experiments) and circulating hepcidin in mice described in B (n = 7–10, from 4 independent experiments). (E,F) Number of circulating CD41+ events (E) and leukocytes (F) from mice described in B (n = 5–9, from 2–4 independent experiments). (A–F) Data show the mean ± SEM. (A,B) *p < 0.05, **p < 0.01.
Fig 3: Increased lesion area, plaque instability, and iron deposition in VFEpoR mice after EPO injection.Control and VFEpoR mice were fed a Western diet and treated with LDLR ASO and EPO (3 times per week) for 12 weeks. (A) H&E staining of aortic root sections and quantification of absolute lesion and necrotic core area. Necrotic core regions indicated by broken lines. Scale bar: 200 µm. (B) Aortic root sections were stained with Masson’s trichrome staining for fibrous cap (red, outlined by broken lines) and collagen (blue) content area, and then quantified as the ratio of total lesion area. Scale bar: 100 µm. (C) Iron (Perl’s blue) and redox-active iron deposition (Perl’s blue + DAB), IHC staining of RBCs (anti-Ter119) and macrophages (anti-Mac2) in aortic roots. Bar graph shows quantification of iron (II + III)–positive and erythrophagocytosis (Ter119+Mac2+) cell counts per section. Scale bar: 100 µm. (D) TUNEL and immunofluorescence staining of macrophage (anti-Mac2) in aortic roots and quantification of TUNEL-positive cell counts per section. Scale bar: 50 µm. (E) Lipid peroxidation product 4-HNE staining, quantified as the percentage of total lesion area. Scale bar: 100 µm. (F) Immunofluorescence staining of TfR1 and macrophage (anti-Mac2), and quantification of TfR1 and macrophage costaining cell counts per section. Scale bar: 50 µm. (G) Aortic root sections were immunostained for Mac2 and quantified as absolute Mac2-positive area. Scale bar: 250 µm. (H) Lesions were stained for citrullinated histones (H3Cit) and activated neutrophils using myeloperoxidase (MPO); the overlap H3cit and MPO (NETs) cell counts were quantified. Scale bar: 50 µm. Unpaired 2-tailed t test or Mann-Whitney U test, *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4: Aortic endothelial permeability was increased in VFEpoR mice.Control and VFEpoR mice were fed a Western diet (WD) and treated with LDLR ASO and EPO (3 times per week) for 6 or 12 weeks. (A) FITC dextran (green, 70 kDa) and nuclei (blue, DAPI) in aortic root cross-section, which were probed for endothelial permeability after 12 weeks of a WD. Scale bar: 50 µL. (B) Aortic arch and descending aorta were probed for endothelial permeability using Evans blue intravital staining after 6 or 12 weeks of a WD. The bar graph shows quantification of Evans blue extravasation normalized by the tissue weight. (C) En face scanning electron microscopy showing the luminal surface of the aortic arches; the bar graph shows numbers of the field that had RBC attachment or no RBC attachment (n = 3 per group); ?2 test. (D) Ldlr–/– mice were fed a WD for 2 weeks and then transfused with packed and PKH26-labeled 80–100 µL control or VFEpoR RBCs once per week for another 5 weeks, in total 7 weeks of WD. Representative 3D-rendered image from RBC-transfused mice aortic root lesions staining of macrophages (anti-Mac2, green) and RBCs (PKH26, red), and quantification of infiltrated RBC counts and erythrophagocytic macrophage counts in the lesions. Size: 332.80 µm × 332.80 µm × 7.50 µm. Calibration: XY:0.65 µm, Z:1.50 µm. Unpaired 2-tailed t test. *P < 0.05, **P < 0.01.
Fig 5: Liprox-1 reverses VFEpoR-induced atherosclerosis progression.Control and VFEpoR mice were fed a Western diet and treated with LDLR ASO and EPO for 12 weeks, with Liprox-1 (10 mg/kg, 3 times per week) or vehicle injection for the last 10 weeks. (A) Experiment timeline. (B) Representative C11 BODIPY staining histogram and quantification of C11 BODIPY CD11b+ cells as the percentage of total aortic CD11b+ cells. (C) H&E-stained images of aortic root sections. Necrotic core regions indicated by broken lines and quantification of total lesion area and necrotic core area are shown. Scale bar: 200 µm. (D) Aortic root sections were stained with Masson’s trichrome staining for fibrous cap (red, outlined by broken lines) and collagen (blue) content area, and then quantified as the ratio of total lesion area. Scale bar: 100 µm. (E) 4-HNE IHC in the aortic root cross sections. Scale bar: 100 µm. (F) Representative images of Perl’s blue plus DAB staining, IHC staining of RBCs (anti-Ter119), and macrophages (anti-Mac2) in aortic roots. Bar graph shows quantification of iron (II + III)–positive and erythrophagocytosis (Ter119+Mac2+) cell counts per section. Scale bar: 100 µm. (G) Immunofluorescence staining of TfR1 and macrophage (anti-Mac2), and quantification of TfR1 and macrophage costaining cell counts per section. Scale bar: 50 µm. (H) Evans blue intravital staining of arches and quantification of Evans blue extravasation. (I) L-selectin and CD11a expression levels in neutrophils and monocytes from peripheral blood assessed by flow cytometry. (J) VFEpoR mice were fed a Western diet and injected with LDLR ASO and EPO for 3 weeks, then divided into two groups, treated with Isotype or anti–Gr-1 mAb twice per week for another 4 weeks. In total 7 weeks WD and EPO injection. Evans blue intravital staining of arches and descending aorta, and quantification of extravasation. One-way ANOVA (B–D) or unpaired 2-tailed t test (E–J). *P < 0.05, **P < 0.01, ***P < 0.001.
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