Radiation‐induced AXL upregulation in CCR2 + monocytes promotes cardiac fibrosis

Figures

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  FIGURE 1. Temporal progression of cardiac injury and fibrosis following whole‐thorax irradiation. (A) Schematic diagram of the experimental design and tissue collection from Sham mice and irradiated mice at IR_D7, IR_D30, and IR_D60 after whole‐thorax irradiation. (B) Representative Masson's trichrome and Sirius Red staining of cardiac tissues from the Sham, IR_D7, IR_D30, and IR_D60 groups. Scale bar, 50 µm. (C) Representative immunohistochemical staining of Collagen I in cardiac tissues at the indicated time points. Boxed regions in the upper panels are shown at higher magnification below. Scale bar, 100 µm in the upper panels. (D) Representative immunohistochemical staining of α‐SMA in cardiac tissues at the indicated time points. Boxed regions in the upper panels are shown at higher magnification below. Scale bar, 100 µm in the upper panels. (E–H) Quantification of the Masson's trichrome‐positive area (E), Sirius Red‐positive area (F), Collagen I‐positive area (G), and α‐SMA‐positive area (H), expressed as percentages of the analyzed myocardial interstitial area. (I) Representative H&E staining of cardiac tissues from the Sham, IR_D7, IR_D30, and IR_D60 groups. Scale bar, 50 µm. (J, M)…

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  FIGURE 2. Transcriptomic and single‐cell analyses reveal increased cardiac monocyte accumulation following irradiation. (A) Schematic of the bulk RNA‐sequencing experiment using cardiac tissues collected at baseline (IR_D0) and at IR_D7 and IR_D30 after irradiation (n = 3 independent biological samples per group). (B) Cell‐proportion deconvolution of bulk cardiac RNA‐sequencing data from the IR_D0, IR_D7, and IR_D30 groups. (C) UMAP visualization of monocyte, macrophage, and dendritic‐cell subsets identified in the public radiation‐associated cardiac single‐cell RNA‐sequencing dataset, with their distributions at d0, d7, and d35 shown on the right. (D) Relative proportions of Dc2, Dc1, Macro1, Macro2, and Monocyte populations at d0, d7, and d35. (E) Schematic of lineage tracing in Lyz2CreERT2; Rosa26tdTomato mice following whole‐thorax irradiation. (F) Representative immunofluorescence staining of RFP and CCR2 in cardiac tissues at IR_D7 and IR_D30. Scale bar, 50 µm. (G) Quantification of RFP‐positive cells among total cardiac cells. (H) Quantification of CCR2+RFP+ cells among RFP+ cells. Data in panels G and H are presented as mean ± SEM. n = 6 mice per group. Statistical significance was…

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  FIGURE 3. Anti‐CCR2 treatment attenuates radiation‐induced cardiac fibrosis. (A) Schematic diagram of the anti‐CCR2 treatment schedule following whole‐thorax irradiation. Mice received anti‐CCR2 antibody or isotype control from IR_D3 to IR_D6. Peripheral blood was collected at IR_D7, and cardiac tissues were collected at IR_D30. (B) Representative flow‐cytometry plots of circulating CCR2+Ly6Chi monocytes in peripheral blood from isotype‐control and anti‐CCR2‐treated mice. (C) Quantification of circulating CCR2+Ly6Chi monocytes among CD45+ leukocytes. Data are shown as box‐and‐whisker plots with individual data points. (D, E) Representative Masson's trichrome (D) and Sirius Red (E) staining of cardiac tissues from isotype‐control and anti‐CCR2‐treated mice. (F, G) Representative immunohistochemical staining of Collagen I (F) and α‐SMA (G). Boxed regions are shown at higher magnification on the right. (H–K) Quantification of Masson's trichrome‐positive area (H), Sirius Red‐positive area (I), Collagen I‐positive area (J), and α‐SMA‐positive area (K). Scale bars, 50 µm. n = 6 mice per group. Data in panels H–K are presented as mean ± SEM. Statistical significance in panels C and H–K was…

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  FIGURE 4. AXL expression is enriched in cardiac monocytes during radiation‐induced cardiac remodeling. (A) Venn diagram showing the overlap among the top 10 significantly upregulated genes, ranked by adjusted P value, from the IR_D30 versus IR_D0, IR_D30 versus IR_D7, and IR_D7 versus IR_D0 comparisons. (B) Axl expression in bulk cardiac RNA‐sequencing data from the IR_D0, IR_D7, and IR_D30 groups. (C) Violin plots showing Axl expression across the major cardiac immune‐cell populations, including the mononuclear phagocyte system (MPS), NKT cells, Neutrophils, and B cells, in the public radiation‐associated single‐cell RNA‐sequencing dataset (PRJNA1088304). (D) Violin plots showing Axl expression across Dc2, Dc1, Macro1, Macro2, and Monocyte populations within the MPS compartment. (E) UMAP visualization of monocyte subclusters and their distributions at d0, d7, and d35 in the public dataset. (F) Feature plots showing Axl expression in monocytes at d0, d7, and d35. (G) Violin plots showing monocyte Axl expression across the indicated time points. (H) Representative immunofluorescence staining of AXL and CCR2 in cardiac tissues at IR_D7 and IR_D30. Scale bar, 50 µm. n = 6 mice per group. (I)…

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  FIGURE 5. Myeloid Axl deletion and pharmacological AXL inhibition attenuate radiation‐induced cardiac fibrosis. (A) Schematic diagram of the generation of tamoxifen‐inducible myeloid Axl conditional‐knockout mice. (B) Schematic diagram of the experimental design. Control and Axl‐cKO mice underwent whole‐thorax irradiation, and cardiac tissues were collected at IR_D30. (C) Representative western blot analysis of AXL protein in flow‐sorted cardiac CCR2+Ly6Chi monocytes from Control and Axl‐cKO mice at IR_D30. GAPDH served as the loading control. (D) Representative immunofluorescence staining of AXL and RFP in cardiac tissues from Control and Axl‐cKO mice at IR_D30. Green, AXL; red, RFP; blue, DAPI. (E) Relative mRNA expression of Acta2, Col1a1, and Col3a1 in cardiac tissues from Control and Axl‐cKO mice at IR_D30, as determined by quantitative real‐time PCR (qPCR). (F, G) Representative Masson's trichrome (F) and Sirius Red (G) staining of cardiac tissues from Control and Axl‐cKO mice at IR_D30. (H, I) Representative immunohistochemical staining of Collagen I (H) and α‐SMA (I) in cardiac tissues from Control and Axl‐cKO mice. Boxed regions are shown at higher magnification on the right. (JM)…

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  FIGURE 6. AXL regulates MEK/ERK‐MAPK signaling in cardiac CCR2+Ly6Chi monocytes. (A) Schematic diagram of bulk RNA‐sequencing analysis of irradiated Control and Axl‐cKO hearts (n = 3 independent biological samples per group). (B) Volcano plot showing differentially expressed genes between Axl‐cKO and Control groups. (C) KEGG pathway‐enrichment analysis of upregulated and downregulated genes in Axl‐cKO hearts. (D) GSEA showing negative enrichment of the MAPK signaling gene set in Axl‐cKO versus Control cardiac transcriptomes. (E) Violin plots showing Mapk1, Map2k1, Nras, and Kras expression in monocytes across different time points in the cardiac single‐cell dataset. (F) Western blot analysis of pAXL, AXL, pMEK, MEK, pERK, ERK, and GAPDH in flow‐sorted cardiac CCR2+Ly6Chi monocytes from Control and Axl‐cKO mice. (G) Quantification of pMEK/MEK and pERK/ERK protein levels in panel F. (H) Western blot analysis of pAXL, AXL, pMEK, MEK, pERK, ERK and GAPDH in cardiac CCR2+Ly6Chi monocytes isolated at IR_D30 from irradiated mice treated systemically with vehicle or R428. (I) Quantification of pAXL/AXL, pMEK/MEK, and pERK/ERK protein levels in panel H. GAPDH served as the loading control. For…

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  FIGURE 7. AXL–MEK/ERK signaling in cardiac CCR2+Ly6Chi monocytes promotes fibroblast activation. (A) Schematic diagram of the monocyte‐conditioned medium experiments. Cardiac CCR2+Ly6Chi monocytes were isolated from Sham or irradiated hearts under the indicated genetic conditions or treated ex vivo with inhibitors, and the collected conditioned medium was applied to primary cardiac fibroblasts. (B) Western blotting of COL1A1, α‐SMA, and GAPDH in cardiac fibroblasts cultured alone or with conditioned medium from Sham or IR_D30 monocytes. (C) Quantification of COL1A1 and α‐SMA protein levels in panel B. (D) Western blotting of COL1A1, α‐SMA, and GAPDH in cardiac fibroblasts cultured alone or with conditioned medium from Control or Axl‐cKO monocytes. (E) Quantification of COL1A1 and α‐SMA protein levels in panel D. (F) Western blotting of pAXL, AXL, pMEK, MEK, pERK, ERK, and GAPDH in flow‐sorted IR_D30 cardiac CCR2+Ly6Chi monocytes treated ex vivo with vehicle or R428. (G) Quantification of pAXL/AXL, pMEK/MEK and pERK/ERK protein levels in panel F. (H) Western blotting of COL1A1, α‐SMA, and GAPDH in cardiac fibroblasts cultured alone or with conditioned medium from Sham monocytes or IR_D30…