ANGPTL2 inhibits macrophage pyroptosis and alleviates rheumatoid arthritis progression by regulating mitophagy via IGFBP5

Figures

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  Schematic diagram of the mechanism of ANGPTL2 in the treatment of rheumatoid arthritis.

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  Fig. 1. ANGPTL2 deficiency exacerbates pyroptosis and joint damage in a mouse model of rheumatoid arthritis. A Representative macroscopic images showing paw swelling in WT and Angptl2–/– CIA mice. B Assessment of arthritis progression, including body weight changes, paw thickness, and clinical arthritis scores over time. C Representative 3D μCT reconstruction images of ankle joints. Quantification of talus bone volume (mm³) (D) and the ratio of bone volume to tissue volume (BV/TV, %) (E) in ankle joints. F Representative H&E-stained sections of ankle joints showing inflammation and structural damage. G Representative TRAP-stained sections of ankle joints showing osteoclast distribution and activity. H Representative ALP-stained sections of ankle joints indicating osteoblast activity and bone formation in periarticular regions. I Dual immunofluorescence (IF) staining of ankle joint sections using anti-F4/80 and antibodies against NLRP3, Caspase-1, GSDMD, or IL-1β in CIA mice. The average IF signals for total NLRP3, Caspase-1, GSDMD, and IL-1β expression, and co-localization with F4/80+ macrophages were determined. T: tibia; TAL: talus; ST: synovial tissue. J Western blot analysis showing…

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  Fig. 2. Inflammatory stimulation reduces ANGPTL2 expression and induces pyroptotic signaling in macrophages in vitro. A qPCR analysis of Angptl2, Nlrp3, Caspase-1, Gsdmd, and Il-1β expression in RAW264.7 cells stimulated with LPS alone or LPS + ATP. B ELISA quantification of IL-1β secretion in culture supernatants from RAW264.7 cells stimulated with LPS alone or LPS + ATP. C Time-course qPCR analysis of Angptl2, Nlrp3, Caspase-1, Gsdmd, and Il-1β mRNA expression in RAW264.7 cells primed with LPS for 0, 3, 6, 12, and 24 h, followed by 1-h ATP stimulation prior to RNA extraction. D Corresponding time-course Western blot analysis of ANGPTL2, NLRP3, GSDMD, Caspase-1, and IL-1β expression in RAW264.7 cells under identical conditions. E qPCR analysis of Angptl2, Nlrp3, Caspase-1, Gsdmd, and Il-1β expression in BMDMs stimulated with LPS alone or LPS + ATP. F ELISA quantification of IL-1β secretion in culture supernatants from BMDMs stimulated with LPS alone or LPS + ATP. G Time-course qPCR analysis of Angptl2, Nlrp3, Caspase-1, Gsdmd, and Il-1β mRNA expression in BMDMs primed with LPS for 0, 3, 6, 12, and 24 h, followed by 1-h ATP stimulation prior to RNA extraction. H Corresponding time-course…

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  Fig. 3. Knockdown of ANGPTL2 enhances LPS-induced pyroptosis in macrophages. qPCR analysis of Angptl2, Nlrp3, Caspase-1, Gsdmd, and Il-1β mRNA expression in RAW264.7 cells (A) and BMDMs (B) following siAngptl2 or siNC transfection, with or without LPS + ATP stimulation. Western blot analysis of ANGPTL2, NLRP3, GSDMD, Caspase-1, and IL-1β protein expression in RAW264.7 cells (C) and BMDMs (D) under identical conditions. ELISA measurement of IL-1β levels in culture supernatants of RAW264.7 cells (E) and BMDMs (F). Cell death assessment using LDH release assay in RAW264.7 cells (G) and BMDMs (H) under identical conditions. Representative TEM images showing morphological features of pyroptotic cell death in RAW264.7 cells (I) and BMDMs (J). Representative flow cytometry plots showing Annexin V and PI staining of RAW264.7 cells (K) and BMDMs (L) under identical conditions. Quantification of Annexin V+PI+ populations in RAW264.7 cells (M) and BMDMs (N). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 in the indicated groups.

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  Fig. 4. Transcriptomic profiling reveals suppression of mitophagy and activation of inflammatory signaling pathways in ANGPTL2-deficient macrophages. A Volcano plot of DEGs comparing Angptl2–/– and NC BMDMs following LPS + ATP stimulation. Genes with significant upregulation (yellow) or downregulation (blue) are highlighted based on thresholds of adjusted p < 0.05 and |log2(fold change)| ≥2. Non-significant genes are shown in gray. B Heatmap showing hierarchical clustering of DEGs between Angptl2–/– and NC BMDMs following LPS + ATP stimulation. Each column represents an individual sample, and each row represents a gene. Color gradient indicates Z-score normalized expression levels (red: upregulated, green: downregulated). C GSEA plot showing enrichment of the gene set “negative regulation of autophagy of mitochondrion” between groups. D GO enrichment dot plot for DEGs. Enriched terms are classified by Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). Dot size reflects gene count, color indicates adjusted p-value, and X-axis denotes gene ratio. E KEGG pathway enrichment map for DEGs in KO-LPS vs. NC-LPS. Dot size reflects gene count, color indicates adjusted…

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  Fig. 5. ANGPTL2 deficiency exacerbates mitochondrial damage and impairs mitophagy in macrophages. A Western blot analysis of mitophagy-related proteins (PINK1, PARKIN, LC3B, and p62) in joint tissues from WT and Angptl2–/– mice under basal and CIA-induced conditions. B Western blot analysis of mitophagy markers in RAW264.7 cells and BMDMs transfected with siAngptl2 or siNC, with or without LPS + ATP stimulation. Flow cytometry analysis of mitochondrial membrane potential using JC-1 staining in RAW264.7 cells (C) and BMDMs (D). The ratio of red (aggregates) to green (monomers) fluorescence (R/G) was calculated to assess mitochondrial integrity. Intracellular ROS levels measured by flow cytometry in RAW264.7 cells (E) and BMDMs (F) under the indicated conditions. G Flow cytometry analysis of mito-Keima-loaded RAW264.7 cells to assess mitophagic flux under different conditions. H Representative confocal microscopy images showing colocalization of LC3B (red) and mitochondria (MitoTracker Green) in BMDMs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 in the indicated groups.

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  Fig. 6. Recombinant ANGPTL2 restores mitochondrial function and suppresses pyroptosis via mitophagy in BMDMs. A Western blot analysis of ANGPTL2 expression in BMDMs following treatment with recombinant mouse ANGPTL2 protein (rMmANGPTL2). B qPCR analysis of Nlrp3, Caspase-1, Gsdmd, and Il-1β mRNA expression in BMDMs stimulated with LPS + ATP and treated with increasing concentrations of rMmANGPTL2 (0, 250, 500, 750, 1000 ng/mL). C Flow cytometry analysis of mitochondrial membrane potential using JC-1 staining in BMDMs under NC, LPS + ATP, or LPS + ATP + rMmANGPTL2 treatment conditions. D Flow cytometry analysis of intracellular ROS levels in BMDMs under NC, LPS + ATP, or LPS + ATP + rMmANGPTL2 treatment conditions. qPCR (E) and Western blot (F) analysis of mitophagy-related markers in BMDMs following LPS + ATP stimulation with or without rMmANGPTL2 and the autophagy inhibitor 3-MA. qPCR (G) and Western blot (H) analysis of pyroptosis-associated markers in BMDMs under the same treatment conditions. I Flow cytometry analysis of mito-Keima-loaded RAW264.7 cells to assess mitophagic flux under the same treatment conditions. J Representative confocal microscopy images showing LC3B and…

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  Fig. 7. ANGPTL2 modulates mitophagy via an IGFBP5-dependent mechanism. A Heatmap showing the expression correlation between ANGPTL2 and insulin-like growth factor (IGF) family members, including IGF1, IGF2, and IGFBP proteins. qPCR (B) and Western blot (C) analysis of IGFBP5 expression in BMDMs following Igfbp5 knockdown and LPS + ATP stimulation. D Western blot analysis of mitophagy-related proteins (PINK1, PARKIN, LC3B, p62) and NLRP3 in BMDMs after Igfbp5 knockdown with LPS + ATP treatment. qPCR (E) and Western blot (F) analysis of IGFBP5 expression in BMDMs following Angptl2 knockdown and LPS + ATP stimulation. qPCR (G) and Western blot (H) analysis of IGFBP5 expression in BMDMs treated with rMmANGPTL2 under LPS + ATP stimulation. I–J Co-immunoprecipitation (Co-IP) assay to evaluate protein-protein interaction between ANGPTL2 and IGFBP5 in RAW264.7 cells. K 3D model of structured ANGPTL2 interfaces with IGFBP5 predicted by HDOCK. L Western blot analysis of mitophagy-related proteins in BMDMs treated with LPS + ATP and rMmANGPTL2, with or without Igfbp5 knockdown. *p < 0.05, **p < 0.01, ***p <0.001, ****p <0.0001 in the indicated groups.

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  Fig. 8. Intra-articular delivery of ANGPTL2 alleviates rheumatoid arthritis by restoring mitophagy and suppressing pyroptosis. A Representative images of mouse paws from control, CIA, and AAV-Angptl2-treated groups showing macroscopic joint swelling. B Assessment of arthritis progression, including body weight changes, paw thickness, and clinical arthritis scores over time. C Representative 3D μCT reconstruction images of ankle joints. D Representative H&E-stained sections of ankle joints illustrating synovial inflammation and tissue architecture. E Representative TRAP-stained sections showing osteoclast distribution and activity. F Representative ALP-stained sections indicating osteoblast activity and bone formation. G Representative dual immunofluorescence images of ankle joint sections stained for F4/80 and either NLRP3, GSDMD, Caspase-1, or IL-1β. The average IF signals for total NLRP3, GSDMD, Caspase-1, and IL-1β expression, and co-localization with F4/80+ macrophages were determined. T: tibia; TAL: talus; ST: Synovial Tissue. H Western blot analysis of ANGPTL2 protein levels in joint tissues from each group. I Western blot analysis of pyroptosis and inflammasome-related proteins…