Extracellular HMGB1 Contributes to the Chronic Cardiac Allograft Vasculopathy/Fibrosis by Modulating TGF-β1 Signaling

Figures

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  Figure 1. The effects of HMGB1 mAb on murine cardiac allograft survival and fibrosis. HMGB1 mAb or control mouse IgG (200 μg each time) was intraperitoneally (i.p.) injected into the allogeneic recipients, from the day before transplantation and then twice a week untill week 4 after transplantation. Allograft impulse was assessed by daily abdominal palpation and cardiac tissue samples were collected at indicated time after transplantation. (A) Therapeutic strategy about HMGB1 mAb. (B) The allograft survival time were evaluated in two groups (n=7/group). *: p<0.05, vs. the IgG group. (C) Fibrotic features of allografts from each experimental group were evaluated at week 8 post-transplant by Masson staining. Scale bar: 100 μm and 200 μm. (D) Quantitative analysis of cardiac fibrosis area by Masson staining. Values were shown as mean ± SEM (n=3/group). **: p<0.01. All data were representative of two or three independent experiments.

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  Figure 2. The expression of TGF-β1 in cardiac allografts. Cardiac tissue samples were collected at indicated time after transplantation. (A) Expression of TGF-β1 protein in allografts. (B) The relative quantity of TGF-β1 protein normalized to GAPDH. The data of (B) was shown as mean ± SEM (n=3/group). *: p<0.05; **: p<0.01, ***: p<0.001. (C) The localization of TGF-β1 in allografts was examined by immunohistochemistry staining. TGF-β1 was stained brown and the nuclei was counterstained blue. Upper image: scale bar: 50 μm; Lower image: enlarged view of the box above. The data were representative of three independent experiments.

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  Figure 3. HMGB1 blockade attenuated the synthesis and release of TGF-β1 in allografts. (A) Representative images showed the protein expression of TGF-β1 (brown arrows) by immunohistochemical analysis in cardiac allografts at week 8 post transplantation in two groups. Scale bar: 50 μm. (B) Concentration of TGF-β1 in recipients' sera and the allograft homogenate at week 8 post-transplant tested by ELISA. Values were expressed as means ± SEM (n = 4–8/group). **: p<0.01; ***: p<0.001, vs. the IgG group. (C) The protein expression of TGF-β1 by western blotting after HMGB1 mAb administration. (D) The relative quantity of TGF-β1 protein normalized to GAPDH. Data were shown as mean ± SEM. ***: p<0.001, vs. the IgG group. All data were representative of three independent experiments.

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  Figure 4. Extracellular HMGB1 promoted the release of TGF-β1 from fibroblasts. (A) The expression of TGF-β1 on myofibroblasts (α-SMA+) in the allograft at week 8 posttransplant and HMGB1 mAb treatment. α-SMA was labeled with green fluorescence, TGF-β1 was labeled with red color and nuclei were stained with DAPI in blue. Scale bar: 25 μm. (B) The protein expression of α-SMA by western blotting after HMGB1 mAb administration and (C) the relative quantity of α-SMA protein normalized to GAPDH in grafts. After 16 h of serum starvation, stimulation with rHMGB1 for 48 h at dose of 0 or 500 ng/ml in media containing 0.5% FBS, (D) western blotting analysis of α-SMA protein expression in cultured cardiac fibroblasts, (E) the relative quantity of α-SMA protein normalized to GAPDH, (F) free active and total TGF-β1 in the supernatants of fibroblasts after rHMGB1 stimulation detected by ELISA. Data were expressed as mean ± SEM (n=3/group) and representative of at least three separate experiments. *: p<0.05; **: p<0.01; ***: p<0.001, vs. negative control.

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  Figure 5. Extracellular HMGB1 enhanced macrophage released TGF-β1. (A) The expression of TGF-β1 on macrophages (F4/80+) in the allograft at week 8 posttransplant and HMGB1 mAb treatment. F4/80 was labeled with red color, TGF-β1 was labeled with green fluorescence and nuclei were stained with DAPI in blue. Scale bar: 25 μm. (B) After 16 h of serum starvation, BMMs were treated with recombinant HMGB1 at 500 ng/mL for 48h in the presence of 0.5% FBS. Free active and total TGF-β1 in the supernatants of BMMs were detected by ELISA. Data were expressed as means ± SEM (n = 3/group). **: p<0.01; ***: p<0.001, vs. negative control. All data were representative of three independent experiments.

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  Figure 6. HMGB1 induced phosphorylation of Smad2 and Smad3 in macrophages and fibroblasts. (A,C) The protein expression of p-Smad2 and p-Smad3 by western blot after HMGB1 Ab administration. (B,D) The relative quantity of p-Smad2, p-Smad3 protein normalized to total Smad2 and Smad3. Data were shown as mean ± SEM and represented of at least three separate experiments. *: p<0.05; **: p<0.01, ***: p<0.001, vs. the IgG group. All data were representative of three independent experiments. (E–H) After 16 h of serum starvation, cultured macrophages and fibroblasts were treated with rHMGB1 for 0, 10, 30 and 60 min in the presence of 0.5% FBS. The phosphorylation of Smad2 and Smad3 in BMMs (E,F) and fibroblasts (G,H) was observed with western blot. The relative quantity of p-Smad2 and p-Smad3 normalized to total Smad2 and Smad3, respectively. The data were shown as mean ± SEM (n = 3/group). **: p<0.01; ***: p<0.001, vs. negative control. All data were representative of three independent experiments. (I,J) ELISA to detect for free active and total TGF-β1 levels of BMMs (I) and fibroblasts (J) cultured supernatant. The data were shown as mean ± SEM (n = 3/group). *: p<0.05; **: p<0.01; ***: p<0.001,…