Dapagliflozin promotes angiogenesis in hindlimb ischemia mice by inducing M2 macrophage polarization

Figures

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  FIGURE 1. Dapagliflflozin improves blood flow recovery and reduces necrosis in ischemic limbs following HLI. (A) Schematic of schedule of the whole experiment. (B) Representative Laser Doppler perfusion imaging showed dynamic changes in blood perfusion in limb ischemia at days 0, 3, 7, 14, and 21 after HLI. (C) Quantitative analysis of blood flow is expressed as a ratio of left to right limb perfusion. (D) Representative images of ischemic limbs in mice at 5 days postoperatively. (E) A semi-quantitative analysis of the ischemic limb necrosis score. (F) A semi-quantitative analysis of the limb motor function score in mice. NS indicates no significance, n = 12. Statistical comparisons were made using a two-way ANOVA, followed by Sidak’s multiple comparisons post-tests (C) or an unpaired t-test (E, F), *p < 0.05, **p < 0.01.

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  FIGURE 2. Histological evaluation of angiogenesis and fibrosis in mouse gastrocnemius muscle. (A, B) Representative images (A) and quantification (B) of anti-CD31 immunohistochemical staining of mice gastrocnemius muscle 21 days after HLI. Scale bar = 100 μm. (C, D) Representative anti-α smooth muscle actin (α-SMA) immunohistochemical staining (C) of gastrocnemius muscle sections and quantitative analysis (D) of the α-SMA + arteriole density. Scale bar = 250 μm. (E, F) Masson-trichrome (Masson) staining (E) and quantitative analysis (F) for identification of fibrotic area (blue) in gastrocnemius muscle tissue. Scale bar = 250 μm. NS indicates no significance, n = 6–10, Multiple group comparisons were made using one-way ANOVA, followed by Bonferroni post hoc test, *p < 0.05, **p < 0.01.

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  FIGURE 3. The effect of dapagliflozin on the polarization state of macrophages. (A) Representative images of CD206 and DAPI staining in the ischemic gastrocnemius muscle. (B) Quantitative analysis of the M2 macrophages was calculated as the ratio of CD206+ cells and DAPI + cells. Scale bar = 200 μm. (C) Representative images of iNOS and DAPI staining in the ischemic gastrocnemius muscle. (D) Quantitative analysis of the M1 macrophages was calculated as the ratio of iNOS + cells and DAPI + cells. Scale bar = 200 μm. (E, F, and (G) Representative Western blotting images (E) and summary densitometry graph illustrating expression of Arg-1 (F) and iNOS (G) in the ischemic gastrocnemius muscle at 21 days postoperatively. (H–J) Representative Western blotting images (H) and summary densitometry graph illustrating expression of Arg-1 (I) and iNOS (J) in mouse bone marrow-derived macrophages (BMDM) stimulated with or without LPS in vitro. n = 6–10 (A–D); n = 6–10 (E–J), Statistical comparisons were made using Mann–Whitney test (C) or an unpaired t-test (D,F,G,I and J), *p < 0.05, **p < 0.01.

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  FIGURE 4. The effect of dapagliflozin on macrophage M2 polarization may be achieved through the NF-κB pathway. (A–C) The protein levels of IκB, phosphorylated- IκB, NF-κB, and phosphorylated-NF-κB in the ischemic gastrocnemius muscle were examined using Western blotting. (A) Representative Western blotting images and (B, C) summary densitometry graphs were shown. (D) Representative western blots demonstrating the expression levels of IL-6, IL-1β, and TNF-α. The quantitative results of IL-6 (E), IL-1β (F), and TNF-α (G) are shown in the histogram. NS indicates no significance, n = 6, Multiple group comparisons were made using one-way ANOVA, followed by Bonferroni post hoc test, *p < 0.05, **p < 0.01.

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  FIGURE 5. Promotion of HUVECs migration, proliferation, and tube formation by dapagliflozin-treated macrophage CM. Representative images (A) of the scratch assay in vitro and quantitative analysis (B) of the migration area were presented. Scale bar = 500 µm. Representative images (C) of the Transwell assay and quantitative analysis (D) of the migrated cells were presented to show the HUVECs’ migration ability. Scale bar = 500 µm. (E) Representative images of EdU staining showing the effects of the macrophage-CM on the proliferation of HUVECs. Quantification results (F) of EdU staining were calculated as the ratio of EdU + cells and DAPI + cells. Scale bar = 200 µm. Representative images (G) of the HUVECs tube formation test. The number of branches (H) and total length (I) of the tubule structure were quantified. Scale bar = 500 µm n = 5 (A–B); n = 6 (C–I), Statistical comparisons were made using unpaired t-test, *p < 0.05, **p < 0.01.

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  FIGURE 6. The schematic diagram of the major study findings shows how dapagliflozin promoted angiogenesis in mice with hindlimb ischemia. Dapagliflozin may inhibit the NF-κB signaling pathway of macrophages in ischemic muscle, thereby inhibiting the excessive secretion of inflammatory factors by macrophages and promoting the transformation of M2-type macrophages. Afterward, M2 macrophages promote ischemic tissue repair and promote angiogenesis.