Growth differentiation factor 11 alleviates oxidative stress-induced senescence of endothelial progenitor cells via activating autophagy

Figures

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  Fig. 1. Rat bone marrow-derived CD34+VEGFR-2+ EPCs. (A) Analysis of CD34+VEGFR-2+ EPCs by flow cytometry. (B) CD34+VEGFR-2+ EPCs (arrows) in the mononuclear cells isolated from bone marrow. Immunostaining. Scale bar = 25 μm. (C) CD31 expression in CD34+VEGFR-2+ EPCs at 2 weeks after induction with VEGF. Immunostaining. Scale bar = 10 μm. (D, E) ALK-4 expression in EPCs demonstrated with immunostaining and Western blotting respectively. Scale bar = 25 μm. (F) The statistical result of ALK-4 expression. *P < 0.01 versus young group. n = 3

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  Fig. 2. Reduction of ROS production, lipofuscin accumulation and SA-β-gal expression in H2O2-induced cells after treatment with GDF11. (A) The levels of ROS. *P < 0.05 and **P < 0.01 versus control group, #P < 0.01 versus H2O2 group. n = 3. (B) Lipofuscin granules. Sudan black B staining. Scale bar = 10 μm. (C) Autofluorescence intensities of lipofuscin in the cells of young and old groups. Analysis by flow cytometry. The abscissa indicates the number of the cells, while the ordinate indicates autofluorescence intensity of lipofuscin. (D) The statistical result of autofluorescence intensities of lipofuscin. *P < 0.05 versus control group, #P < 0.05 versus H2O2 group. n = 4. (E) SA-β-gal-positive cells. SA-β-gal staining. Scale bar = 20 μm. (F) The statistical result of the numbers of SA-β-gal-positive cells. *P < 0.05 versus control group, #P < 0.01 versus H2O2 group, &P < 0.05 versus H2O2 + GDF11 group of young rats. †P < 0.05 versus H2O2 + GDF11 group. n = 3

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  Fig. 3. LC3 expression and autophagic structures in GDF11-treated cells. (A) LC3-positive puncta. Immunostaining. Scale bar = 10 μm. (B) The statistical result of LC3-positive puncta. *P < 0.05 versus control group, †P < 0.05 versus H2O2 group, #P < 0.05 versus young group. n = 3. (C, D) Western blotting of LC3 in the cells of young and old groups. (E, F) The statistical result of LC3 expression in the cells of young and old groups. *P < 0.05 and **P < 0.01 versus control group, #P < 0.01 versus H2O2 group. n = 3

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  Fig. 4. Representative autophagic ultrastructures in GDF11-treated cells. Arrows indicate the autophagosomes with double membranes or multilayer membranes. Arrowheads indicate the autolysosomes containing an autophagosome with single membrane. In H2O2 group, some mitochondria (M) and rough endoplasmic reticula (rER) are swollen. TEM images. N, nucleus. Scale bar = 0.5 μm

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  Fig. 5. Features and properties of the SAP. (A) The nanofibers assembled by SAP. AFM image. (B) The scaffolds formed from the SAP nanofibers. SEM image. Scale bar = 1 μm. (C) A cell spreading on the SAP nanofibers. Numerous filopodia protrude from the lamellipodium. SEM image. Scale bar = 2 μm. (D) The apoptotic cells (arrows) and necrotic cells (arrowheads) in the cells treated with hypoxia for 2 h. EB/AO staining. Scale bar = 25 μm. (E) The statistical result of the apoptotic cells and necrotic cells. *p < 0.05 versus control group. n = 6. (F) Viability of the hypoxic cells. CCK-8 assay. OD, optical density. *p < 0.001 versus control group. n = 6. (G) Concentration of VEGF in the supernate. *p < 0.01 versus normoxia group, #p < 0.01 versus control group. n = 6. (H) The profile of cumulative GDF11 released from the SAP hydrogel. n = 6

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  Fig. 6. The changes in cardiac function at 4 weeks after transplantation. (A) Representative echocardiograms of the LV free wall. (B) Ejection fractions (EF) of LV. (C) Fractional shortening (FS) of LV. *p < 0.05 versus control group, #p < 0.05 versus SAP group, †p < 0.05 versus SAP + GDF11 group, &p < 0.05 versus EPCs group, $p < 0.05 versus SAP + EPCs group. n = 6

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  Fig. 7. The changes in the cardiac structures at 4 weeks after transplantation. (A, B) The morphological changes of LV walls. The panels in A show the transverse sections of the ventricles at the widest part of the infarct region. The panels in B show the transverse sections of the infarct regions. Masson’s trichrome staining. Scale bar = 2 mm. (C) The statistical result of scar size. (D) The statistical result of the thickness of the LV wall at the infarct region. *p < 0.05 and **p < 0.01 versus control group, #p < 0.05 and ##p < 0.01 versus SAP group, †p < 0.05 and ††p < 0.01 versus SAP + GDF11 group, &p < 0.05 and &&p < 0.01 versus EPCs group, $p < 0.05 and $$p < 0.01 versus SAP + EPCs group. n = 6

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  Fig. 8. The microvessels at the peri-infarct region at 4 weeks after transplantation. (A) The microvessels. White dash lines indicate the epicardium. CD31 immunostaining. Scale bar = 50 μm. (B) The statistical result of the number of the microvessels. *p < 0.05 and **p < 0.01 versus control group, #p < 0.05 and ##p < 0.01 versus SAP group, †p < 0.01 versus SAP + GDF11 group, &p < 0.01 versus EPCs group, $p < 0.01 versus SAP + EPCs group. n = 6. (C) CD31-positive cells differentiated from the engrafted EPCs (GFP-positive cells) at infarct region. GFP+CD31+ cells (arrows) are located at the wall of the microvessels. In the panels of the third row, the large boxes are magnification of the small boxes. White dash lines indicate the epicardium. Immunostaing. Scale bar = 50 μm. n = 6

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  Fig. 9. The senescent cells in the transplanted cells at peri-infarct region at 4 weeks after transplantation. (A) GFP+SA-β-gal+ cells in the survived cells (GFP+ cells). Immunostaining and SA-β-gal staining. Arrows indicate GFP+SA-β-gal+ cells. Scale bar = 50 μm. (B) The statistical result of the number of GFP+SA-β-gal+ cells. **p < 0.01 versus EPCs group, ##p < 0.01 versus SAP + EPCs group. n = 6