Role of HIF-1α-Activated IL-22/IL-22R1/Bmi1 Signaling Modulates the Self-Renewal of Cardiac Stem Cells in Acute Myocardial Ischemia

Figures

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  Fig. 1. Hypoxia/ischemia increased the expression of IL-22/IL-22R1 in PCCs and hearts. (A) The enhancement of IL-22 production was presented in the ischemic rat hearts in comparison with non-ischemic controls by ELISA. (B) Higher IL-22R1 and HIF-1α expression was observed in the tissue samples of the peri-infarcted region than in controls in a time-dependent manner by western blotting. (C) AMI induced an increase in IL-22R1+ expression in the peri-infarcted area (yellow square) via IHC analysis. (D-E) Compared with controls without hypoxia, IL-22R1 expression was observed in a dose-dependent (D, higher at 3% O2 level) and time-dependent (E, higher at 8 h) manner in hypoxic PCCs by western blotting. n = 5 in (A), (C), and (D). n = 4 in (B) and (E). Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control. Bar = 50 μm

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  Fig. 2. HIF-1α activation regulated the upregulation of IL-22R1 expression in vitro and in vivo. (A) 2-ME2 inhibited the upregulation of IL-22R1 in a dose-dependent manner after AMI by western blotting. (B) In HIF-1α−/− mice, AMI was not able to increase IL-22R1 expression compared with the littermate (HIF-1α-NL) heart samples. (C) Both HIF-1α and IL-22R1 upregulation required hypoxia-induced HIF-1α activation, which was demonstrated by 2-ME2 (1 µM) or HIF-1α knockdown using LV-HIF-1α-sh in PCCs. (D) Administration of 2-ME2 inhibited HIF-1α-induced IL-22R1 expression. However, 2-ME2 did not block LV-IL-22R1-induced IL-22R1 overexpression. (E-F) Treatment of LV-HIF-1α-sh with hypoxia and LV-HIF-1α, but not LV-HIF-2α-sh with hypoxia and LV-HIF-2α, significantly modulated IL-22R1 expression in PCCs. (G) Schematic representation of a putative HIF-1α-binding site (HRE) in the IL-22R1 promoter sequence from − 826 to − 821 (5′-ACGTG-3′). (H) The recruitment and direct binding of HIF-1α to the IL-22R1 promoter were demonstrated in PCCs subjected to 4 h of hypoxia but not in PCCs subjected to 4 h of normoxia or PCCs treated with LV-HIF-1α-sh with 4 h of hypoxia by the chromatin…

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  Fig. 3. IL-22 administration attenuated left ventricular (LV) dysfunction, reduced infarct size, and diminished fibrosis and apoptotic cell death via IL-22/IL-22R1 signaling activation after AMI. (A) Representative images of the gross view and T–M stain of mice hearts revealed a larger infarct size and thinner infarct wall thickness in IL-22R1−/− mice than in IL-22R1-NL mice 3 days after AMI. (B-D) Representative images of the gross view and T–M stain of rats’ heart revealed that IL-22 (5 and 50 µg/kg)-treated groups had lower (C) infarct volumes and higher (D) infarct wall thickness than the control groups 28 days after AMI. IL-22-mediated decrease in infarct volumes and increase in thickness of infarct wall were inhibited with rIL-22BP-Fc pretreatment 28 days after AMI. (E-F) M-mode echocardiography of rat hearts revealed that the IL-22 (5 and 50 µg/kg)-treated groups had significantly decreased (E) LVESD–LVEDD and increased (F) EF–FS after IL-22 injection compared with the control groups 28 days after AMI. IL-22-induced lower LVESD–LVEDD and higher FS–EF were blocked with rIL-22BP-Fc pre-treatment 28 days after AMI. (G) T–M staining from (B) revealed significant attenuation of…

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  Fig. 4. HIF-1α-activated IL-22/IL-22R1 pathway stimulated the proliferation and self-renewal of CSCs in vitro. (A) Representative immunocytochemical (ICC) images of CSCs and IL-22R1+ CSCs colocalized with the progenitor cell markers (upper panel, CD105) and the proliferation markers (lower panel, BrdU and Ki-67). (B) Representative images of CSCs under normoxic and hypoxic conditions (1%, 3%, and 5% O2 level) for 8 h. (C) Hypoxia with 3% O2 for 8 h promoted a higher frequency of sphere formation (also in 5% O2 level), sphere size (the average sphere size was obtained of five fields per sample), and self-renewal potential than ordinary O2 level (21%). In contrast, the proliferative effect on the sphere formation by hypoxia was abolished by LV-HIF-1α-sh or LV-IL-22R1-sh (left panel) administration into CSCs and IL-22R1−/− mice (right panel). (D) Hypoxia with 3% O2 for 8 h induced more enhancement of BrdU incorporation and Ki-67 immunostaining in CSCs than in normal cells. However, a significant increase in BrdU incorporation and Ki-67 immunostaining was blocked by the administration of LV-HIF-1α-sh or LV-IL-22R1-sh (left panel) in CSCs and IL-22R1−/− mice (right panel). (E) After 8 h of…

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  Fig. 5. Systemic activation of the IL-22/IL-22R1 cascade enhanced eCSC proliferation after AMI in the ischemic myocardium. After AMI, IHC analysis revealed that compared with sham control, a significant increase in the cell numbers of (A) CD105+/BrdU+ and (B) CD105+/Ki-67+ CSCs was observed with a peak at 3 days in a time-dependent manner. (C) CD105+/Nkx2.5+ CSCs, which was blocked using the rIL-22BP-Fc injection (10 µg/kg, i.p., was administered 4 h before the AMI), in the ischemic heart 3 days after AMI. n = 3 mice for each group, and the images were representative of 6 images for each group. (A)-(B) represented images after 3 days of AMI; (C) represented an image after 3 days of AMI with a subcutaneous injection of mouse recombinant IL-22 (50 µg/kg, s.c.) on 1 day after AMI. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control. Bar = 50 μm

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  Fig. 6. Bmi1 enhanced CSCs activation via the IL-22/IL-22R1 signaling pathway. (A) Upregulated and downregulated gene analyses of the differentially expressed genes (DEGs) with screening criteria of |log2FC| ≥ 2 and p-value ≤ 0.05. (B-C) The heatmap after hierarchical clustering of samples treated with 8 h of hypoxia-IL-22 (H22) versus normoxia-IL-22 (N22) based on the mRNA expression data obtained from RNA microarray analysis. The heatmap revealed the fold change of DEGs (log2FC of DEGs and p-value ≤ 0.05) across the two treatment groups of (B) cardiac progenitor genes and (C) cardiac progenitor cell-related transcription factors (TFs). (D) The mRNA expression of Bmi1 was significantly increased after treating CSCs with mouse 100 µg/ml IL-22 by RTq-PCR (n = 3 in each experimental group). (E) Pharmacological IL-22 treatment (50 µg/kg) increased the cell numbers of CD105+/Bmi1+ eCSCs, which was blocked using the rIL-22BP-Fc injection (10 µg/kg, i.p., was administered 4 h before the AMI), in the ischemic heart 3 days after AMI. The representative image after 3 days of AMI with a subcutaneous injection of mouse recombinant IL-22 (50 µg/kg, s.c.) on 1 day after AMI. n = 3 mice for each…

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  Fig. 7. Bmi1 promoted CSC-mediated cardiomyocyte repair after AMI. (A) Representative images of primary and secondary CSCs from Bmi1-NL and Bmi1−/− mice after IL-22 administration (100 ng/ml) before 3% O2 hypoxia for 8 h. (B-D) IL-22 treatment significantly increased the frequency of (B) cardiac sphere formation, (C) sphere size (the average sphere size was obtained of five fields per sample), and (D) self-renewal potential in CSCs derived from Bmi1-NL, which were inhibited via rIL-22BP-Fc treatment (1 µg/mL). However, these effects of IL-22 treatment were abolished in CSCs derived from Bmi1−/− mice. The trophic factors produced by CSCs, such as (E) stromal cell-derived factor 1 (SDF-1), (F) hepatocyte growth factor (HGF), and (G) insulin-like growth factor 1 (IGF-1), were assayed in CSCs derived from Bmi1-NL and Bmi1−/− mice after hypoxia with IL-22 treatment by RTq-PCR. n = 3 in each experimental group. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control. Bar = 50 μm