Fig 1: Differential regulation of the BM niche by >52w and 3w AML. a Schematic overview of RNA seq data acquisition and analysis. b Plot of principal component analysis of gene expression in NH9FL, NH93w, NH910w and NH9>52w myeloid leukaemias, n = 3 in each group. c Flow cytometric gating strategy used to identify endothelial cells (EC), mesenchymal cells (MSC) and osteoblasts (OB) in the stroma (CD45−Ter119−). Leukaemic burden assessed by d %GFP in BM and e WBC count. Analysis of percentage ECs (top) and MSCs (bottom) in the stroma of central marrow of mice transplanted with f NH9+ AML blasts or g healthy LSKs of foetal (LSKFL) or >52w (LSK>52W) origin. Analysis of percentage OBs (bottom) and MSC (top) in the stroma of the compact bone of mice transplanted with h NH9+ AML blasts or i LSKFL or LSK>52W. d–i Graphs depict individual values with mean ± s.d. of mice transplanted with leukaemic blasts from NH93W (n = 13) or NH9>52W (n = 9) (only mice with >30% GFP in the bone marrow are included, and individual mice are indicated with different symbols on the graphs), and LSKFL(n = 4) or LSK>52w(n = 4) from healthy mice. NT not transplanted (n = 4). Significance determined by Student’s t-test, *p < 0.05, **p < 0.01
Fig 2: 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 3: Characterization of atherosclerotic plaques of VFEpoR mice.Control (EpoR-Cre) and VFEpoR mice were fed a Western diet and treated with low-density lipoprotein receptor antisense oligonucleotides (ASO) weekly for 12 weeks. (A) H&E staining and quantification of total lesion area and necrotic core area in the aortic root. Necrotic core regions are indicated by broken lines. Scale bar: 100 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) 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)–positive and erythrophagocytosis (Ter119+Mac2+) cell counts per section. Scale bar: 100 µm. (D) Representative 3D-rendered image from VFEpoR mice aortic root lesions of macrophage (anti-Mac2, green), smooth muscle cell (ACTA-2, magenta), and RBCs (anti-Ter119, red). Size: 332.80 µm × 332.80 µm × 7.50 µm. Calibration: XY:0.33 µm, Z:1.50 µm. Arrowheads show macrophage erythrophagocytosis. (E) TUNEL and immunofluorescence staining of macrophage (anti-Mac2) in aortic roots and quantification of TUNEL-positive cell counts per section. Scale bar: 75 µm. (F) Lipid peroxidation product 4-HNE staining, quantified as the percentage of total lesion area. 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. Unpaired 2-tailed t test or Mann–Whitney U test, *P < 0.05.
Fig 4: Liprox-1 alleviated accelerated atherosclerosis progression in Jak2VFMx-Cre clonal hematopoiesis.20% of Jak2VFMX-Cre (VFMX) mice were fed with Western diet together with Liprox-1 (10mg/kg, 3 times per week) or vehicle injection for 12 weeks. (A) Experiment timeline. (B) 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. (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. (D) Bar graph shows quantification of iron (II + III)–positive and erythrophagocytosis (Ter119+Mac2+) cell counts per section. Scale bar: 100 µm. (E) 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 (F) quantified as the ratio of total lesion area. Scale bar: 100 µm. (G) Lipid peroxidation product 4-HNE staining, quantified as the percentage of total lesion area. Scale bar: 100 µm. Unpaired 2-tailed t test. *P < 0.05, **P < 0.01, ***P < 0.001.
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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