IDO1 improves postischemic neovascularization in aged mice by boosting endothelial NAD+ de novo synthesis and curbing endothelial senescence

Figures

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  Fig. 1. The expression of the endothelial NAD + de novo synthesis enzyme IDO1 is reduced after lower limb ischemia in aged mice. (A) Scheme depicting the experiment for RNA-seq and NAD+-target metabolomics of young and aged mice following HLI. (B) Diagram showing the pathway of NAD+ synthesis and consuming. (C) Volcano plot of transcriptional alterations in hindlimb muscle ECs 7 days after HLI. (D) RNA-seq analysis of NAD+ metabolism related enzymes in hindlimb muscle ECs 7 days after HLI. Young group was set as 1. Two-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. (E) Heatmap of the NAD+ related metabolites in hindlimb muscle ECs 7 days after HLI. Young group was set as 1. Two-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns: not significant. (F) Western blot analysis and quantification of IDO1 in hindlimb muscle ECs 7 days after HLI. Young group was set as 1. Unpaired two-tail t-test, n = 3 per group, data are mean ± SEM, ∗∗∗P < 0.001. (G) Violin plots of log-transformed gene expression of Ido1 in cell populations of hindlimb muscle from…

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  Fig. 2. Ido1 deficiency leads to ECs senescence and impairs neovascularization in the ischemic hindlimb muscle of young mice. (A) Design showing the experiment for ECs senescence and vasculogenic phenotypes in Ido1−/− mice. (B) Mass spectrometry analysis of NAD+ related metabolites in hindlimb muscle ECs 7 days after HLI. WT group was set as 1. Two-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗∗∗P < 0.0001, ns: not significant. (C) Western blot analysis and quantification of P16 in hindlimb muscle ECs 7 days after HLI. WT group was set as 1. Unpaired two-tail t-test, n = 3 per group, data are mean ± SEM, ∗∗∗∗P < 0.0001. (D–F) RT-qPCR quantification of p16 (D), p21 (E) and Lmnb1 (F) in hindlimb muscle ECs 7 days after HLI. WT group was set as 1. Unpaired two-tail t-test, n = 3 per group, data are mean ± SEM, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. (G, H) ROS levels (G) and NO levels (H) quantification in hindlimb muscle ECs 7 days after HLI. Data of WT group in G was set as 1. Unpaired two-tail t-test, n = 3 per group, data are mean ± SEM, ∗P < 0.05, ∗∗∗∗P < 0.0001. (I) Immunofluorescence staining for CD31, α-SMA and KI67 in ischemic adductor…

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  Fig. 3. IL-17A/F represses ECs Ido1 transactivation and NAD+ biosynthesis, and facilitates ECs senescence in the ischemic hindlimb muscle of young mice. A) Schematic diagram for testing methods was indicated. (B) Bar plot of the most prominent categories by KEGG pathway (left) and GO analysis (right). (C) Western blot analysis and quantification of IDO1 in hindlimb muscle ECs 7 days after HLI. PBS group was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗P < 0.01, ∗∗∗P < 0.001. (D) RT-qPCR quantification of Ido1 in hindlimb muscle ECs 7 days after HLI. PBS group was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗P < 0.05. (E) Western blot analysis and quantification of IDO1 and P16 in hindlimb muscle ECs 7 days after HLI. Con group was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗P < 0.01, ∗∗∗∗P < 0.0001, ns: not significant. (F–I) RT-qPCR quantification of Ido1 (F), p16 (G), p21 (H) and Lmnb1 (I) in hindlimb muscle ECs 7 days after HLI. Con group was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group,…

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  Fig. 4. IL-17A/F counteracts Endothelial Ido1 overexpression-induced neovascularization under conditions of lower limb ischemia. (A) Scheme showing the experiment for rescuing the IL-17A/F-treated young mice by endothelial Ido1 overexpression. (B and C) Representative laser Doppler images (B) of hindlimbs of mice and quantification (C) of relative perfusion recovery as the blood flow ratio of the left limb to that of the right limb in AAV-NC + PBS, AAV-NC + IL-17A/F, AAV-Ido1 + PBS and AAV-Ido1 + IL-17A/F mice before and after HLI at the indicated time points. The areas under the curves of relative perfusion are shown, Two-way ANOVA with Tukey multiple comparison test, n = 6 per group, data are mean ± SEM, ∗AAV-NC + IL-17A/F vs. AAV-NC + PBS; #AAV-Ido1 + IL-17A/F vs. AAV-NC+ IL-17A/F, #/∗P < 0.05, ###P < 0.001, ∗∗∗∗/####P < 0.0001. (D) Representative micro-CT images of ischemic hindlimbs 21 days after HLI. Scale bar, 3 mm. (E) Quantification of micro-CT analysis of arterial vasculature in ischemic hindlimbs. Two-way ANOVA with Tukey multiple comparison test, n = 6 per group, data are mean ± SEM, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns: not significant. (F) Representative…

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  Fig. 5. IDO1 protein alleviates ECs senescence, and boosts ECs migration, tube formation and proliferation in vitro. (A) Schematic diagram for HUVECs treated with H2O2 for 24 h and incubated with or without IDO1 for 24 h. (B and C) Cell senescence was determined using SA-β-Gal staining, and the SA-β-Gal positive cells were quantified. Scale bar, 75 μm. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗∗∗P < 0.0001, ns: not significant. (D) P16 levels were measured using western blot analysis, and the mean gray value was quantified. Con group was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗∗∗P < 0.0001, ns: not significant. (E–G) P16 (E), P21 (F) and LMNB1 (G) levels were analyzed using RT-qPCR analysis. Con group was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.001, ns: not significant. (H–J) ROS (H), NAD+ (I) and NO (J) levels were quantified. Data of Con group in H was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns: not…

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  Fig. 6. IDO1 protein enhances the neovascularization in aged mice following lower limb ischemia. (A) Scheme illustrating the experiment of aged mice administrated with IDO1 protein. (B) Mass spectrometry analysis of NAD+ related metabolites in hindlimb muscle ECs 7 days after HLI. Aged + PBS group was set as 1. Two-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns: not significant. (C) Western blot analysis and quantification of P16 in hindlimb muscle ECs 7 days after HLI. Aged + PBS group was set as 1. Unpaired two-tail t-test, n = 3 per group, data are mean ± SEM, ∗∗∗P < 0.001. (D–F) RT-qPCR quantification of p16 (D), p21 (E), Lmnb1 (F) in hindlimb muscle ECs 7 days after HLI. Aged + PBS group was set as 1. Unpaired two-tail t-test, n = 3 per group, data are mean ± SEM, ∗P < 0.05, ∗∗P < 0.01. (G and H) ROS (G) and NO (H) levels quantification in hindlimb muscle ECs 7 days after HLI. Data of Aged + PBS group in G was set as 1. Unpaired two-tail t-test, n = 3 per group, data are mean ± SEM, ∗∗P < 0.001, ∗∗∗P < 0.001. (I) Immunofluorescence staining for CD31, α-SMA and KI67 in ischemic adductor muscles 7 days…

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  Fig. 7. Plasma IDO1 levels are lower in elderly PAD patients and correlate with PAD severity, onset risk, and cardiovascular outcome. (A) Schematic diagram for the process of sample collection and correlation analysis between plasma IDO1 levels and clinical indicators. (B) ELISA analysis of IDO1 levels in plasma from young and aged mic at 7 days following HLI. Unpaired two-tail t-test, n = 6 per group, data are mean ± SEM, ∗∗∗∗P < 0.0001. (C) ELISA analysis of IDO1 levels in plasma from young <65 years: and elderly ≥65 years: patients with PAD. Unpaired two-tail t-test, n = 30 per group, data are mean ± SEM, ∗∗∗∗P < 0.0001. (D–K) Spearman correlation between plasma concentration of IDO1 and ABI (D), HDL (E), PCSK9 (F), NLR (G), hs-CRP (H), Fibrinogen (I), D-dimer (J) and NT-proBNP (K). n = 30 per group. (L) Schematic diagram for the changes in plasma IDO1 levels and clinical indicators between young and elderly PAD patients.