Nuclear Receptor NR1D1 Regulates Abdominal Aortic Aneurysm Development by Targeting the Mitochondrial Tricarboxylic Acid Cycle Enzyme Aconitase-2

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  Figure 1.. NR1D1 is upregulated in human and murine aortic SMCs from AAA tissues. A, Relative mRNA expression of 49 NRs measured by Nanostring profiling in saline- or AngII-induced AAA mice (n=5 per group). The raw data were normalized to the mean expression levels of the housekeeping genes, and the normalized data were log2-transformed. The log2 (fold change) and P values were calculated, and multiple testing using the Benjamini-Hochberg method was applied to adjust the P values. With a Benjamini-Hochberg adjusted P<0.05 and |log2(fold change)|≥1 as the criteria, 3 genes (Nr1d1 [adjusted P=0.03], Nr4a1 [adjusted P=0.04], and Nr1f3 [adjusted P=0.04]) were identified as differentially expressed NRs in mouse AAA tissues. *Benjamini–Hochberg adjusted P<0.05. B, Top, Quantification of NR1D1 mRNA and NR1D1 protein expression measured by RT-qPCR and Western blot in human AAA and non-AAA segments (n=6 per group). Data were analyzed by unequal variance t test. *P<0.05; ***P<0.001. Bottom, NR1D1 protein expression assessed by Western blot in human AAA and non-AAA segments. C, Representative images of NR1D1 expression by immunofluorescence staining of human AAA and non-AAA segments and costaining…

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  Figure 2.. VSMC-specific NR1D1 deficiency represses AngII-induced AAA formation. A, Schematic protocol: ApoE−/−/Nr1d1flox/flox and ApoE−/−/Nr1d1ΔSMC mice were subcutaneously injected with saline or AngII by a mini osmotic pump for 28 days (n=30 per group). B, Representative images of the macroscopic features of AAA formation in indicated groups. C, Representative images of macroscopic features and HE staining of crossed-sections of aneurysm ruptures (arrow indicated). D, Survival curves in indicated groups (n=30 per group). Survival data were analyzed by the Kaplan-Meier method and compared using log-rank tests. *P<0.05. E, The incidence of AngII-induced AAA in indicated groups (n=30 per group). Data were analyzed by a Fisher exact test. **P<0.01; ***P<0.001. F, The incidence of AngII-induced aneurysm rupture in indicated groups (n=30 per group). Data were analyzed by a Fisher exact test. *P<0.05. G and H, Quantification of the maximal diameter of suprarenal abdominal aortas measured by a Digital Vernier Caliper and total aortic weight/BW in indicated groups (n=22–30 per group). Data were analyzed by 2-way ANOVA followed by the Bonferroni post hoc test. ***P<0.001. I, Representative images…

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  Figure 3.. NR1D1 perturbs mitochondrial metabolism by the TCA cycle in AAA. A, Heatmap of differentially expressed genes in ApoE−/−/Nr1d1flox/flox-AngII and ApoE−/−/Nr1d1ΔSMC-AngII groups. Each column represents an individual replicate, and each row represents an individual gene. Upregulated genes are shown in red, and downregulated genes are displayed in blue. n=3 for both groups. Differentially expressed genes were defined as genes with a Benjamini-Hochberg adjusted P value <0.05 and |log2(fold change)|≥1. B, Volcano plot reveals the magnitude and significance of altered genes in ApoE−/−/Nr1d1flox/flox-AngII and ApoE−/−/Nr1d1ΔSMC-AngII groups. Differentially expressed genes were defined as genes with a Benjamini-Hochberg adjusted P value <0.05 and |log2(fold change)|≥1. C, The top 10 Kyoto Encyclopedia Genes and Genomes (KEGG) pathways of differentially expressed genes. D, Heatmap of differential plasma metabolites in ApoE−/−/Nr1d1flox/flox-AngII and ApoE−/−/Nr1d1ΔSMC-AngII groups. n=7 for ApoE−/−/Nr1d1flox/flox-AngII, n=6 for ApoE−/−/Nr1d1ΔSMC-AngII. Differential metabolites in plasma were identified using variable important in projection (VIP) values of >1.2 in an orthogonal partial…

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  Figure 4.. Aco2 is the direct trans-repression target of NR1D1 in regulating mitochondrial function. A and B, Aco2 mRNA expression and ACO2 activity measured by RT-qPCR and ELISAs in human AAA and non-AAA segments (n=6 per group). Data were analyzed by Student t test. **P<0.01; ***P<0.001. C, Representative images of ACO2 expression by immunofluorescence staining of human AAA and non-AAA segments and costaining with the key smooth muscle cell–associated marker αSMA and DAPI. D, Left, ACO2 protein expression assessed by Western blot in human AAA and non-AAA segments. Right, Quantification of ACO2 protein expression measured by Western blot in indicated groups (n=6 per group). Data were analyzed by Student t test. **P<0.01. E and F, Aco2 mRNA expression and ACO2 activity measured by RT-qPCR and ELISAs in abdominal aortic segments from saline- and AngII-infused ApoE−/− mice (n=6 per group). Data were analyzed by Student t test. **P<0.01; ***P<0.001. G, Representative images of ACO2 expression by immunofluorescence staining in abdominal aortas from saline- and AngII-infused ApoE−/− mice. H, Left, ACO2 protein expression measured by Western blot in abdominal aortas from saline- and AngII-infused…

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  Figure 5.. VSMC-specific NR1D1 deficiency regulates the expression of mitochondria-related genes and inhibits mitochondrial ROS production. A, Summarized OCR tracings in MASMCs isolated from Nr1d1flox/flox and Nr1d1ΔSMC mice after stimulation with AngII for 48 hours (n=4 independent experiments). B through D, Basal, maximal, and ATP-coupled OCRs in indicated groups (n=4 independent experiments). Data were analyzed by Student t test. **P<0.01; ***P<0.001. E, mtDNA damage detection in MASMCs isolated from Nr1d1flox/flox and Nr1d1ΔSMC mice after stimulation with AngII for 48 hours (n=6 independent experiments). Data were analyzed by Student t test. *P<0.05. F and G, Relative mRNA expression of nuclear- and mitochondrial-encoded mitochondrial genes in MASMCs isolated from Nr1d1flox/flox and Nr1d1ΔSMC mice with AngII treatment for 48 hours, normalized to β-actin (n=6 independent experiments). Data were analyzed by Student t test. *P<0.05; **P<0.01; ***P<0.001. H and I, Heatmap of log2 (fold change) of nuclear- and mitochondrial-encoded mitochondrial genes in the abdominal aorta from AngII-infused ApoE−/−/Nr1d1flox/flox and ApoE−/−/Nr1d1ΔSMC mice (n=3 per group). Differentially expressed genes…

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  Figure 6.. NR1D1 deficiency results in early changes in mitochondrial function on day 3 after AngII infusion before a change in AAAs. Abdominal aortas from ApoE−/−/Nr1d1flox/flox and ApoE−/−/Nr1d1ΔSMC mice were harvested on the morning of day 3 after AngII infusion before a change in AAAs. A, Representative images of the macroscopic features and HE staining of suprarenal abdominal aortas in ApoE−/−/Nr1d1flox/flox and ApoE−/−/Nr1d1ΔSMC mice on day 3 after AngII infusion. B, Quantification of the maximal diameter of suprarenal abdominal aortas in indicated groups (n=10 per group). Data were analyzed by Student t test. C, Representative images of MMP activity and in situ DHE staining in indicated groups. D, Left, MMP-2 protein expression measured by Western blot and MMP activity measured by zymography in indicated groups. Right, Quantification of MMP-2 protein expression and MMP activity in indicated groups (n=6 per group). Data were analyzed by Student t test. **P<0.01; ***P<0.001. E, IL-6, TNF-α, and MCP-1 expression in abdominal aortas assessed by ELISAs in indicated groups (n=10 per group). Data were analyzed by Student t test. **P<0.01; ***P<0.001. F, IL-6, TNF-α, and MCP-1 expression…

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  Figure 7.. ACO2 silencing abolishes the protective effects of NR1D1 deficiency on mitochondrial function. A, Top, Quantification of Aco2 mRNA and ACO2 protein expression assessed by RT-qPCR and Western blot in MASMCs after transfection with SiCon and SiAco2-1, normalized to β-actin (n=6 independent experiments). Data were analyzed by Student t test. ***P<0.001. Bottom, ACO2 protein expression assessed by Western blot in indicated groups. B, Summarized OCR tracings in AngII-treated MASMCs isolated from Nr1d1flox/flox and Nr1d1ΔSMC mice pretreated with SiCon and SiAco2 (n=6 independent experiments). C, mtDNA damage assay in indicated groups (n=10 independent experiments). Data were analyzed by 2-way ANOVA followed by the Bonferroni post hoc test. *P<0.05; ***P<0.001. D, MitoTracker Red CMXRos staining was used to measure mitochondrial membrane potential for the assessment of early-stage apoptosis. E, Quantification of MitoTracker Red CMXRos staining (n=6 independent experiments). Data were analyzed by 2-way ANOVA followed by the Bonferroni post hoc test. *P<0.05; ***P<0.001. F, JC-1 staining was used to measure mitochondrial membrane potential for the assessment of early-stage apoptosis. G,…

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  Figure 8.. Supplementation with the ACO2 downstream metabolite αKG protects against AAA formation. A, Schematic protocol: saline- or AngII-infused ApoE−/−/Nr1d1flox/flox and ApoE−/−/Nr1d1ΔSMC mice were intraperitoneally injected with vehicle or DM-αKG (100 mg/kg/d) for 28 days. B, Representative images of the macroscopic features of AAA formation in indicated groups. C, The incidence of AAA formation in indicated groups (n=30 per group). Data were analyzed by a Fisher exact test. *P<0.05; **P<0.01; ***P<0.001. D, Quantification of the maximal diameter of suprarenal abdominal aortas measured using a Digital Vernier Caliper (n=22–30 per group). Data were analyzed by 2-way ANOVA followed by the Bonferroni post hoc test. **P<0.01; ***P<0.001. E, Quantification of total aortic weight/BW (n=22–30 per group). Data were analyzed by 2-way ANOVA followed by the Bonferroni post hoc test. ***P<0.001. F, Representative images of suprarenal abdominal aortas visualized by MUI using the B mode in indicated groups. G, Quantification of the maximal diameter of suprarenal abdominal aortas measured by MUI using the B mode in indicated groups (n=22–30 per group). Data were analyzed by 2-way ANOVA followed by…