AGGF1-primed endothelial progenitor cells alleviate ischaemia-reperfusion injury in diabetic hearts

Figures

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  Fig. 1. EPCs isolation and characteristics. (A) Morphology of MNCs (cultured in EGM-2 and seeding on gelatin-coated dishes) isolated from C57BL/6J mice on day 1, 4, 7. Scale bar = 50 μm. (B) Representative images of Dil-acLDL (red) and FITC-UEA-1 (green) uptake. Blue, Hoechst staining of the nuclei, scale bar = 25 μm. (C) The immunofluorescence assay of cell surface profile of MNCs (CD31 and CD34), scale bar = 25 μm.

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  Fig. 2. AGGF1 reverses HG + HR impaired angiogenic functions of EPCs. (A) Proliferation assay of EPCs using CCK8. (B–C) Tube formation assay of EPCs using matrigel (8 h after cells seeding). (D–E) Transwell assay of EPCs (24 h after cells seeding). (F–G) Wound healing assay of EPCs (36 H after cells seeding). Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 4. ns, not significant.

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  Fig. 3. AGGF1-primed EPCs therapy markedly alleviates I/R injuries in mice hearts. (A) Overview of the T2DM model procedure. (B) IPGTT assay for C57BL/6 J mice after HFD + STZ, n = 6. (C) IPITT assay for C57BL/6 J mice after HFD + STZ, n = 6. (D–F) LDH, cTnT, and CK-MB activities in the serum (36 h post I/R). n = 6. Data are mean ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001.

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  Fig. 4. AGGF1-primed EPCs implantation suppresses oxidative damage, fibrosis and inflammation in diabetic I/R mice. (A) MDA content analysis of myocardial tissue. (B) SOD content assay of myocardial tissue. (C) Masson staining (Scale bar = 1000 μm), Sirius Red staining (Scale bar = 1000 μm) and immunohistochemistry assay (Scale bar = 50 μm) of cardiac sections from diabetic I/R mice. (D) Quantification of Masson staining and Sirius Red staining. (E) Representative images of mice hearts from sham controls, I/R mice, I/R + EPCs mice, and I/R + AGGF1-EPCs mice with immunohistochemical analysis of LY6G, MCP1, TNF-α, IL-1β, and IL-6. (F) Quantification data on inflammation markers LY6G, MCP1, TNF-α, IL-1β, and IL-6. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 6.

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  Fig. 5. AGGF1 improves the nuclear localization of Nrf2 and activation of downstream antioxidative molecules. (A–C) Western blot analysis for the n-Nrf2 (nuclear Nrf2) and its downstream antioxidative proteins (HO1, NQO1, CAT) under HG + HR. siNrf2: 5’-CGAGAAGUGUUUGACUUUATT-3’ (sense); 5’-UAAAGUCAAACACUUCUCGTT-3’ (antisense), EPCs were transfected with siRNAs (synthesized by RiboBio) using a transfection reagent (Santa Cruz Biotechnology, Dallas, TX). (D–G) Quantification data on n-Nrf2, CAT, HO1and NQO1 from images in (A–C). The grouping of gels/blots cropped from different parts of the same gel. Data are mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 3.

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  Fig. 6. siNrf2 blocked the protective effect of AGGF1 on EPCs. siNrf2 blocked the protective effect of AGGF1 on proliferation (A), angiogenesis (B–C), TEM (D–E), and migration (F–G). Data are mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 4.

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  Fig. 7. NRF2 is required for AGGF1-primed EPCs therapy. (A) Left ventricular ejection fraction (LVEF, n = 6). (B) Left ventricular fractional shortening (LVFS, n = 6). (C) Representative images of echocardiographic analysis for mice from Sham, I/R + PBS, I/R + EPCs, I/R + AGGF1-EPCs, I/R + AGGF1-EPCs-siNRF2. (D–E) Representative images for immunostaining with an anti-GFP antibody (EPCs homing evaluation) and anti-CD31 antibody (angiogenesis evaluation), n = 6. Data are mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001. ns, not significant.