Fish Swim Bladder‐Derived ECM Hydrogels Effectively Treat Myocardial Ischemic Injury through Immunomodulation and Angiogenesis

Figures

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  Figure 1. Schematic illustration of synthesis of Alg/dECM hydrogel and treatment for post‐MI cardiac repair. The cardiac function was improved due to the hydrogel treatment by improving mechanical support, inflammatory modulation and angiogenesis.

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  Figure 2. The protein profiles of fish swim bladder‐derived ECM were analyzed by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). A) The component of fish FSB‐extracellular matrix. B–E) The subtypes of ECM‐affiliated protein, collagen, glycoproteins, and proteoglycans.

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  Figure 3. Preparation and characterization of Alg@dECM hydrogel. A) SEM images of ALG hydrogel, BCM hydrogel, and Alg@dECM hydrogel. B) Rheology of alginate hydrogels of different molecular weight. C) Rheology of alginate hydrogels crosslinked by different types of crosslinker agents. D) Rheology of alginate hydrogels incorporation with different contents of dECM. E) Rheology of ALG, BCM, and Alg@dECM hydrogels over time. F) Angular frequency. G) Viscosity of ALG, BCM, and Alg@dECM hydrogels over time.

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  Figure 4. Mechanical stability and degradation of ALG@dECM hydrogel. A) Load cycle curve of compression 3000 cycles of the ALG@dECM hydrogel during 1–10, 1500–1510, and 2990–3000 cycles. B) Compress stress–strain curves of ALG, dECM, and ALG@dECM hydrogels. C) Schematic diagram of planar biaxial mechanical tests of ALG@dECM hydrogel after heart injection. D,E) Tension stress–strain curves of hydrogel‐embedded tissue in different axis. F) In vitro degradation of ALG, BCM, and Alg@dECM hydrogels in vitro assay. G) In vivo degradation of ALG, BCM, and Alg@dECM hydrogels in a mice subcutaneous implantation model (n = 5). All data in the figure are presented as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001).

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  Figure 5. ALG@dECM hydrogel promoted cells viability, migration, and tube formation. A) The F‐actin stained of cytoskeleton of H9C2 cells on different hydrogel. B) The OD values of H9C2 cells cultured for 2 d in ALG, dECM, and Alg@dECM hydrogels by CCK8 assay. C) Images and quantitative analysis of transwell assay for HUVECs at 24 h (n = 5). D) Images and quantitative analysis of tube formation of HUVECs (n = 5). G–J) Fish swim bladder dECM promoted the macrophages polarization into an anti‐inflammatory M2 phenotype (n = 3). G,H) Proportion of CD206+ and CD86+ expression cells in F4/80+ after RAW267.4 cells seeded on the hydrogels. I,J) Relative mRNA expression of TNF‐α, IL‐6, IL‐10, and Arg‐1 (n = 3). All data in the figure are presented as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001).

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  Figure 6. ALG@dECM hydrogel improved cardiac function in rat MI model. A) Schematic diagram of treatment HF in a rat MI model. B) Representative M‐mode echocardiographic images of heart function. C,E) EF values and its variation of left ventricular in different groups (Sham group, n = 5; Saline group, n = 5; ALG group, n = 8; BCM group, n = 8; ALG@dECM group, n = 8). D,F) LVAWds and its variation of left ventricular in different groups. G–J) Variation of LVAWs, FS, EDV, and ESV in different groups. All data in the figure are presented as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001).

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  Figure 7. Therapeutic effects of ALG@dECM hydrogel in rat MI model. A) H&E and Masson trichrome staining of harvested hearts from rats. B,E) Representative images and quantitative analysis of TUNEL staining in infarct area (n = 5). C,F) Representative images and quantitative analysis of WGA immunofluorescence staining in the border area (n = 5). D) Quantification of infarct area by Masson staining (Sham group, n = 5; Saline group, n = 5; ALG group, n = 8; BCM group, n = 8; ALG@dECM group, n = 8). All data in the figure are presented as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001).

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  Figure 8. The therapeutic mechanism of ALG@dECM hydrogel in rat MI model. A,D) Representative images and quantitative analysis of α‐SMA immunofluorescence staining in infarct area (n = 5). B,E) Representative images of CD86/CD206 immunofluorescence staining in infarct area and statistics of the proportion of M2 macrophages (n = 5). C,F) Representative images and quantitative analysis of CD68 immunofluorescence staining in infarct area (n = 5). All data in the figure are presented as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001).

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  Figure 9. Transcriptome RNA sequencing of MI rats in different groups at 7 d after hydrogel injection. A) Differentially expressed genes (DEGs) in each pairwise comparison. Red represents the number of upregulated genes, and blue represents the number of downregulated genes. B) GO enrichment scatterplot based on differential genes comparing ALG and dECM group. C) GO enrichment scatterplot based on differential genes comparing the groups (ALG vs Saline, BCM vs Saline, dECM vs Saline, and Saline vs Sham). D) KEGG enrichment barplot shows the pathway of interest in the DEGs when compared across multiple groups (ALG, BCM, dECM, Saline, Sham). The x‐axis represents the number of DEGs within a pathway, the y‐axis indicates the pathway names, and the color denotes the KEGG primary classification. E) Heatmap of Angptl4 family proteins across all samples. F) ANGPTL4 expression in H9C2 cells before and after dECM treatment by Elisa assay. G) The adsorption of ANGPTL4 in Alg and Alg/dECM hydrogel in vitro assay.