Modified bFGF targeting connective tissue growth factor in the injured microenvironment improved cardiac repair after chronic myocardial ischemia

Figures

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  Scheme 1. Schematic illustration of responsive release of targeting CFBP-bFGF improved cardiac repair after chronic myocardial ischemia in rats.

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  Fig. 1. Preparation and properties of CFBP-bFGF. (A) SDS-PAGE of purified bFGF and CFBP-bFGF; (B) Western blot of purified bFGF and CFBP-bFGF; (C) The biological activity assay of bFGF and CFBP-bFGF on HSFS cells proliferation, n = 5; (D) The biological assay of bFGF and CFBP-bFGF on H9c2 cells proliferation, n = 5; (E) Cardiomyocytes protection assay on OGD/R model of H9c2 cells, n = 5; (F) Tube formation assay in vitro, n = 5; (G) Statistics of the number of tubules formed in vitro. Scale bar = 100 μm. Data were expressed as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

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  Fig. 2. Evaluation of the targeting efficiency of CFBP-bFGF in ischemic myocardium. (A) Western blot analysis of CTGF expression in ischemic myocardium at 1d, 3d, 1w and 2w after MI; (B)The content of bFGF in ischemic myocardium and serum was determined by ELISA at 3d, 1w and 2w after MI; (C) Western blot analysis of bFGF in ischemic myocardium at 3d, 1w and 2w after MI; (D) The statistical analysis of Western blot statistical analysis of bFGF at 3d after MI; (E) The statistical analysis of Western blot statistical analysis of bFGF at 1w after MI; (F) The statistical analysis of Western blot statistical analysis of bFGF at 2w after MI. Data were expressed as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, n = 5.

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  Fig. 3. Colocalization of CFBP-bFGF and CTGF in ischemic myocardial tissue. (A) Immunofluorescence staining of CTGF (red) and bFGF (green) in ischemic myocardium at 1w after MI. Yellow staining indicates colocalization; (B)The colocalization analysis of CTGF and bFGF in PBS group; (C) The colocalization analysis of CTGF and bFGF in bFGF group; (D) The colocalization analysis of CTGF and bFGF in CFBP-bFGF group; (E) Immunofluorescence staining of CTGF (red) and bFGF (green) in ischemic myocardium 2w after MI. Yellow staining indicates colocalization; (F) The colocalization analysis of CTGF and bFGF in the PBS group; (G) The colocalization analysis of CTGF and bFGF in the bFGF group; (H) The colocalization analysis of CTGF and bFGF in CFBP-bFGF group. Scale bar = 100 μm, n = 3.

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  Fig. 4. The evaluation of cardiac function after CFBP-bFGF or bFGF intravenous administration. (A) H&E staining panoramic scan representation of each group; (B) H&E staining of myocardial infarction; (C) Masson staining panoramic scan representation of each group; (D) Masson staining of infarcted myocardium; (E) Statistical analysis of infarct size; (F) Statistical analysis of LV wall thickness. Data represent mean ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 5.

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  Fig. 5. Evaluation of blood vessel regeneration, cardiocyte survival, and apoptosis after CFBP-bFGF or bFGF by intravenous administration. (A) Immunofluorescence staining of α-SMA, vWF, cTnT, and Tunel staining in ischemic myocardium; (B) Statistical analysis of the number of arterioles labeled by α-SMA; (C) Statistical analysis of the number of capillaries labeled by vWF; (D) Statistical analysis of the positive area of myocardium labeled by cTnT; (E) Statistical analysis of the number of TUNEL-positive cells. Scale bar = 100 μm. Data were expressed as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, n = 6.

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  Fig. 6. Transcriptome analysis to determine the mechanism of CFBP-bFGF mediated ischemic myocardial repair. (A) Gene ontology (GO) analysis after continuous injection for one month; (B) KEGG analysis; (C)Differential gene expression in the bFGF group and CFBP-bFGF group; (D) Significantly different gene profiles associated with ischemic myocardial regeneration and repair, n = 3; (E) qPCR validation of mRNA expression trends of key genes, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, n = 3.

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  Fig. 7. The administration of CFBP-bFGF can alleviate MI-induced cardiac fibrosis and alleviate adverse cardiac remodeling. (A) Immunofluorescence staining and statistical analysis of collagenⅠ; (B) Immunofluorescence staining and statistical analysis of Vimentin; (C)Western blot results of TGF-β pathway and Smad7, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 6.