S100A9 Aggravates Cardiac Fibrosis by TLR4/PGC‐1α Mediated Macrophage‐Myofibroblast Crosstalk

Figures

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  FIGURE 1. S100A9 promotes macrophage‐to‐myofibroblast transition (MMT) after MI and exacerbates myocardial fibrosis. (A–B) UMAP plots showing the single‐cell transcriptomic landscape of cardiac macrophages from Sham mice and mice at Day 7 post–MIR. n = 9. (C–D) Masson staining showing cardiac fibrosis at Day 7 after MIR, with corresponding quantitative analysis. n = 5. (E–F) t‐SNE plots showing MMT levels in cardiac macrophages from Sham and Day‐7 MIR mice. n = 9. G. Detection of α‐SMA and endogenously red‐fluorescent macrophages in heart tissue by immunofluorescence. n = 3. (H–I) Immunofluorescence staining of α‐SMA and CD68 in heart tissue, with relative quantification. n = 5. (J–K) WGA staining showing cardiomyocyte cross‐sectional area with normalized quantification. n = 5. (L) Heart weight/body weight ratios and correlative analyses in each group. n = 5. (M–O) Echocardiographic assessment of cardiac function, including LVPWs, LVEF, and FS, with quantitative analysis. n = 5. (P–Q) Western blot analysis of protein expression in mouse heart tissue with normalized quantification. n = 5. ns, not significant; *, p < 0.05; **, p < 0.01.

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  FIGURE 2. Recombinant S100A9 protein promotes MMT and exacerbates myocardial fibrosis. WT and S100a9 MKO mice received intravenous rS100A9 after MIR and were sacrificed on Day 7 post‐MIR. (A–B) Masson staining showing cardiac fibrosis in mice, with corresponding quantitative analysis. n = 5. (C–D) WGA staining showing cardiomyocyte cross‐sectional area with normalized quantification. n = 5. (E) Heart weight/body weight ratios and quantitative analyses in each experimental group. n = 5. (F–H) Echocardiographic assessment of cardiac function, including LVPWs, LVEF, and FS, with quantitative analysis. n = 5. (I–J) Immunofluorescence staining of α‐SMA and CD68 in heart tissue, with relative quantification. n = 5. (K–L) Western blot analysis of protein expression in mouse heart tissue with normalized quantification. n = 5. WT mice received intravenous injections of S100A9 NTAB or isotype control after MIR and were sacrificed on Day 7 post‐MIR. (M–N) Masson staining showing cardiac fibrosis in mice, with corresponding quantitative analysis. n = 5. (O–P) WGA staining showing cardiomyocyte cross‐sectional area with normalized quantification. n = 5. (Q) Heart weight/body weight ratios and…

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  FIGURE 3. S100A9 promotes fibroblast migration, activation, and migrasome release. (A–B) Crystal violet staining showing fibroblast migration, with quantitative analysis. n = 5. (C–E) Co‐immunofluorescence staining of α‐SMA and Col1α1 to assess fibroblast activation, with corresponding fluorescence quantification. n = 5. (F–G) Western blot analysis of Integrin α5 expression in fibroblasts following rS100A9 stimulation, with normalized quantification. n = 5. (H) qPCR measurement of mtDNA levels in cardiac fibroblasts after rS100A9 stimulation. n = 5. (I–K) Western blot analysis of mitochondrial oxidative phosphorylation (OXPHOS) complex proteins, with normalized quantification. n = 5. (L–M) Flow cytometry analysis of ROS levels in cardiac fibroblasts following rS100A9 stimulation, with quantitative assessment. n = 5. (N–O) Flow cytometry analysis of MMP in rS100A9‐treated fibroblasts, with quantitative assessment. n = 5. (P–R) ECAR, OCR, and ATP levels in rS100A9‐stimulated fibroblasts. n = 5. (S) Fluorescence images showing α‐SMA and S100A9 expression in fibroblasts at different time points following rS100A9 stimulation. n = 5. (T–U) WGA staining showing migrasome level and corresponding…

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  FIGURE 4. S100A9 induces mitochondrial dysfunction and migrasome release in fibroblasts via the TLR4/PGC‐1α pathway. (A–B) Western blot analysis of protein expression in mouse cardiac fibroblasts stimulated with rS100A9, with normalized quantification. n = 5. (C–D) Western blot analysis of protein expression in cardiac fibroblasts overexpressing PGC‐1α and stimulated with rS100A9, with normalized quantification. n = 5. (E–F) Flow cytometry analysis showing macrophages co‐expressing α‐SMA and F4/80, with quantitative assessment. n = 5. (G–H) Western blot analysis of PGC‐1α expression in mouse cardiac fibroblasts, with normalized quantification. n = 5. (I–J) Flow cytometry measurement of ROS levels in cardiac fibroblasts after rS100A9 stimulation, with quantitative analysis. n = 5. (K–L) Flow cytometry analysis of MMP in cardiac fibroblasts stimulated with rS100A9 and pharmacological inhibitor, with quantitative assessment. n = 5. (M–O) ECAR, OCR, and ATP levels in rS100A9‐stimulated fibroblasts following treatment with a TLR4 inhibitor or a RAGE inhibitor. n = 5. (P–Q) WGA staining evaluating migrasome release level in cardiac fibroblasts, with quantitative analysis. n = 5. (R–S) Western…

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  FIGURE 5. S100A9‐induced migrasome release from fibroblasts promotes MMT. (A) Schematic diagram of the co‐culture system of macrophages and cardiac fibroblasts. n = 5. (B–C) Flow cytometry analysis showing macrophages co‐expressing α‐SMA and F4/80, with quantitative assessment. n = 5. (D–E) Western blot analysis of protein expression in macrophage co‐cultured with cardiac fibroblasts treated with rS100A9 or not, with normalized quantification. n = 5. (F–G) WGA Staining showing migrasome level in cardiac fibroblasts treated with rS100A9 and SAR407899, with corresponding quantitative analysis. n = 5. (H) Schematic diagram of an alternative co‐culture experiment of macrophages and cardiac fibroblasts. n = 5. (I–J) Flow cytometry analysis of macrophages co‐expressing α‐SMA and F4/80 which co‐cultured with fibroblasts in each group, with quantitative assessment. n = 5. (K–L) Western blot analysis of protein expression in cardiac fibroblasts, with normalized quantification. n = 5. (M–N) Western blot analysis of α‐SMA and Fibronection expression in macrophage co‐cultured with cardiac fibroblasts with Tspan4‐gene knock‐down and rS100A9 treatment. n = 5. (O–P) Flow cytometry analysis of macrophages…

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  FIGURE 6. Fibroblast‐derived migrasomes promote MMT through the integrin α5/Src signaling pathway. (A–B) Masson staining showing cardiac fibrosis in mice at Day 7 post‐MIR, with corresponding quantitative analysis. n = 5. (C–D) WGA staining showing cardiomyocyte cross‐sectional area with normalized quantification. n = 5. (E) Heart weight/body weight ratios and correlative analyses in each experimental group. n = 5. (F–H) Echocardiographic assessment of cardiac function, including LVPWs, LVEF, and FS, with quantitative analysis. n = 5. (I–J) Immunofluorescence staining of α‐SMA and CD68 in heart tissue, with relative quantification. n = 5. (K–L) Western blot analysis of protein expression in mouse heart tissue with normalized quantification. n = 5. (M) Survival rates of mice following MIR and treatment with integrin α5 inhibitor over a 28‐day period. n = 15. (N–O) Masson staining showing cardiac fibrosis in mice at day 28 post‐MIR, with corresponding quantitative analysis. n = 5. (P) Heart weight/body weight ratios and correlative analyses in each experimental group. n = 5. (Q–S) Echocardiographic assessment of cardiac function, including LVPWs, LVEF, and FS, with quantitative analysis. n =…

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  FIGURE 7. Pharmacological inhibition of S100a9 attenuates MMT and protects against cardiac fibrosis. (A) Survival rates of mice within 7‐days following MIR, with Tasquinimod administered at different post‐operative time points. n = 13. (B–C) Masson staining showing cardiac fibrosis in mice at Day 7 post‐MIR and treated with tasquinimod, with corresponding quantitative analysis. n = 5. (D–E) WGA staining showing cardiomyocyte cross‐sectional area with normalized quantification. n = 5. (F) Heart weight/body weight ratios and correlative analyses in each experimental group. n = 5. (G–I) Echocardiographic assessment of cardiac function, including LVPWs, LVEF, and FS, with quantitative analysis. n = 5. (J–K) Immunofluorescence staining of α‐SMA and CD68 in heart tissue, with relative quantification. n = 5. (L–M) Western blot analysis of protein expression in mouse heart tissue with normalized quantification. n = 5. (N) Serum S100A9 levels in patients with coronary reperfusion after myocardial infarction compared with those in healthy subjects. n = 8. (O–P) Monocytes isolated from the peripheral blood of the subjects were co‐cultured with S100A9‐stimulated human fibroblasts, and the expression…