AREG+ regulatory T cells mediating myocardial repair and neovascularization after myocardial infarction

Figures

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  Fig. 1. Changes in AREG expression after myocardial infarction. A The relative mRNA level of AREG at day 1,day 3, day 5, day 7 and day 14 after AMI or Sham operation. n = 6 per group. *P < 0.05 vs. Sham. B AREG protein levels in the infarcted myocardium were quantified by Western blotting at Sham, day 1,day 3, day 5, day 7 and day 14 after AMI. C Summary data of B. n = 6 per group. *P < 0.05 vs. Sham. D The proportions of Tregs in the hearts at day 1, day 3, day 7 and day 14 after AMI or Sham operation. n = 6 per group. *P < 0.05 vs. Sham. E The proportions of AREG+ Tregs in the hearts at day 1, day 3, day 7 and day 14 after AMI or Sham operation. n = 6 per group. *P < 0.05 vs. Sham. F The proportions of Tregs in the hearts in Ctrl and Anti CD25 group. n = 6 per group. *P < 0.05 vs. Ctrl. G Relative mRNA level of AREG in Sham, lgG and Anti CD25 group. n = 6 per group. *P < 0.05 vs. Sham, #P < 0.05 vs. lgG. H AREG protein levels in the infarcted myocardium were quantified by Western blotting in Sham, lgG and Anti CD25 group respectively. I Summary data of H. n = 6 per group. *P < 0.05 vs. Sham, #P < 0.05 vs. lgG. J The proportions of Tregs in the hearts lgG and IL-2/JES6-1. n = 6 per…

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  Fig. 2. Effect of Tregs on cardiac function and neovascularization after AMI. B LVEF, LVFS, LVDV, and LVSV at day 28 post-AMI by echocardiography in sham, Ctrl, lgG, anti CD25 and IL2/JES6 group. n = 6 per group. *P < 0.05 vs. Sham, #P < 0.05 vs. Ctrl. B Representative Masson’s trichrome and HE staining of day 28 post-AMI heart sections from 6 independent experiments. Scale bars, 2 mm. C Quantification of infarct size on day 28 post- AMI hearts. n = 6 per group. *P < 0.05 vs. Sham, #P < 0.05 vs. Ctrl. D Immunostaining with an anti-CD31 or anti-αSMA antibody on day 28 post-AMI heart sections. Scale bars, 50 mm. E Quantification of CD31 on day 28 post-AMI hearts. n = 6 per group. *P < 0.05 vs. Sham, #P < 0.05 vs. Ctrl. F Quantification of α-SMA on day 28 post-AMI hearts. n = 6 per group. *P < 0.05 vs. Sham, #P < 0.05 vs. Ctrl. G and H Representative flow cytometry pseudo color plot and percentages of FoxP3+ gated in CD45+CD25+CD4+ cells (Tregs). I LVEF, LVFS, LVDV, and LVSV at day 28 post-AMI by echocardiography in Ctrl, Treg, anti CD25 and anti CD25 + Treg group. n = 6 per group. *P < 0.05 vs. Ctrl, #P < 0.05 vs. Treg. J Representative Masson’s trichrome and HE staining of day 28 post-AMI…

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  Fig. 3. Effect of AREG+ Tregs on cardiac function and neovascularization after AMI. A The relative mRNA level of AREG in oe-NC and oe-AREG group. n = 6 per group. *P < 0.05 vs. oe-NC. B AREG protein levels in the infarcted myocardium were quantified by Western blotting at oe-NC and oe-AREG group. C Summary data of B. n = 6 per group. *P < 0.05 vs. oe-NC. D LVEF, LVFS, LVEF, LVFS, LVDV, and LVSV at day 28 post-AMI by echocardiography in Ctrl, oe-NC-Treg, oe-AREG-Treg group. n = 6 per group. *P < 0.05 vs. Ctrl, #P < 0.05 vs. oe-NC-Treg. E Representative Masson’s trichrome and HE staining of day 28 post-AMI heart sections from 6 independent experiments. Scale bars, 2 mm. F Quantification of infarct size on day 28 post-AMI hearts. n = 6 per group. n = 6 per group. *P < 0.05 vs. Ctrl, #P < 0.05 vs. oe-NC-Treg. G Immunostaining with an anti-CD31 or anti-αSMA antibody on day 28 post-AMI heart sections. cale bars, 50 mm. H Quantification of CD31 on day 28 post-AMI hearts. n = 6 per group. *P < 0.05 vs. Ctrl, #P < 0.05 vs. oe-NC-Treg. I Quantification of α-SMA on day 28 post-AMI hearts.n = 6 per group,*P < 0.05 vs. Ctrl, #P < 0.05 vs. oe-NC-Treg

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  Fig. 4. AREG is an important factor in the promotion of CMECs proliferation, migration and tube formation by Tregs. A Representative EdU staining images of CMECs, quantification of EdU-positive cells is shown on the right. n = 3 per group. *P< 0.05vs Nor,#P< 0.05 vs Ctrl. B Flow cytometry analysis of the cell cycle in CEMCs in the Nor, Ctrl, oe-NC, oe-AREG groups. The proportion of cells in G0/G1, S, and G2/M phases is displayed in the accompanying bar graph. n = 3 per group. *P< 0.05 vs Nor,#P< 0.05 vs Ctrl. C Representative migration assay results (crystal violet staining) with corresponding quantification of fold change in migrated cells.*P< 0.05 vs Nor,#P< 0.05 vs Ctrl. D and E Tube formation assay illustrating the effects of AREG overexpression on angiogenesis, with quantification of the number of junctions, meshs and total tuble length.*P< 0.05 vs Nor,#P< 0.05 vs Ctrl. F Expression of AREG, VEGF-A, and FGF in CMECs co-cultured with Tregs under the specified conditions.*P< 0.05 vs Nor,#P< 0.05 vs Ctrl. G Representative EdU staining images of CMECs in the Nor, Ctrl, sh-NC, sh-AREG groups, and quantification of EdU-positive cells is shown on the right. n = 3 per group. *P< 0.05 vs…

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  Fig. 5. AREG target FoxM1 to regulate CMECs. A Heatmap of RNA-sequencing analysis showed that gene expression was significantly upregulated in CMECs of the AREG-overexpression Tregs (AREGgroup) compared with the Treg (Nor) group. For all statistical plots, the data are presented as the mean ± SEM. *p < 0.05 between the two indicated groups by 2-tailed Student’s t-test. B The relative mRNA level of Fen1, Myb12, Mcm10, Gins2, FoxM1 in Nor and Treg group. n = 3 per group.*P< 0.05 vs Nor. C FoxM1 protein levels in CMECs were quantified by Western blotting in Nor or Treg group. n = 3 per group.*P< 0.05 vs Nor. D The relative mRNA level of FoxM1 in Sham and AMI group. n = 3 per group.*P< 0.05 vs Nor. E FoxM1 protein levels in CMECs were quantified by Western blotting in Sham or AMI group. n= 3 per group.*P< 0.05 vs Nor. F Co-immunoprecipitation of AREG and FoxM1 in CMECs. G Immunofluorescence staining of FoxM1 in CMECs, showing nuclear localization. n = 3 per group.*P< 0.05 vs Nor.Scale bars, 50 mm. H qRT-PCR analysis of FoxM1 expression in CMECs transfected with si-NC and si-FoxM1. n = 3 per group.*P< 0.05 vs si-NC. I Western blot analysis of FoxM1 expression in CMECs transfected with si-NC…

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  Fig. 6. AREG⁺Tregs enhance neovascularization post-AMI through FoxM1. A qRT-PCR analysis of FoxM1 mRNA levels in the Sham, Ctrl, Vector, and siFoxM1 groups, with quantification showing a significant reduction in the siFoxM1 group. n = 6 per group.*P< 0.05 vs Sham,#P< 0.05 vs Ctrl. B Western blot analysis of FoxM1 expression in the Ctrl and siFoxM1 groups, with corresponding quantification of FoxM1 protein levels. n = 6 per group.*P< 0.05 vs Ctrl. C LVEF, LVFS, LVDV, and LVSV at day 28 post-AMI by echocardiography in Ctrl and siFoxM1 groups.n = 6 per group.*P< 0.05 vs Ctrl. D Representative HE and Masson staining of heart sections from Ctrl and siFoxM1 groups, showing increased infarct size in the siFoxM1 group, with quantification of infarct size percentage. n = 6 per group.*P< 0.05 vs Ctrl. E Immunofluorescence staining of CD31 and α-SMA in heart sections, indicating reduced capillary and arteriole density in the siFoxM1 group, with corresponding quantification of the number of vessels per mm². n = 6 per group.*P< 0.05 vs Ctrl. F LVEF, LVFS, LVDV, and LVSV at day 28 by echocardiography in AMI+anti-CD25, AMI+anti-CD25+AREG+Treg and AMI+anti-CD25+AREG+Treg+siFoxM1 group.n = 6 per…

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  Fig. 7. AREG-expressing Tregs promote angiogenesis following acute myocardial infarction by releasing AREG, VEGF, and FGF, hence improving cardiac functionality