Phosphoglycerate Mutase 5 Is Important Mediator for Instigating Arterial Lipid Accumulation and Aggravating Atherosclerosis

Figures

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  Figure 1. Histological Staining and p-RIPK3 Protein Expression in Coronary Artery Samples From the Patients (A) Graphical representation of a human experiment. (B) Pathological staining of human coronary arteries at different stages of the atherosclerotic lesion. (C) Quantification of necrotic core area and its percentage in human coronary arteries (n = 3-4). (D) Immunofluorescence staining of p-RIPK3, CD68, and α-SMA in the atherosclerotic lesion of human coronary artery. (E) Representative images of p-RIPK3 protein expression in human coronary arteries by IHC staining. (F) The correlation analysis of p-RIPK3 expression and necrotic core area percentage in humans (n = 10). The zoom (1 or 2) fields represent enlarged images of the boxes in corresponding low-power field sections. Data are shown as mean ± SEM. Statistical analysis among 3 groups was performed using 1-way ANOVA with a Sidak post hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. α-SMA = alpha smooth muscle actin; ANOVA = analysis of variance; DAPI = 4′,6-diamidino-2-phenylindole; IF = immunofluorescence; IHC = immunohistochemistry; HE = hematoxylin & eosin; Masson = Masson trichome; p-RIPK3 = phosphorylated receptor-interacting…

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  Figure 2. Inhibition of RIPK3 Phosphorylation Alleviates Atherosclerotic Lesions in ApoE−/− Mice and ox-LDL–Exposed Macrophages (A-F) WT-C57 and ApoE−/− mice were fed normal diet and high-fat diet (40% fat, containing 1.25% cholesterol) for 24 weeks, respectively. ApoE−/− mice were treated with intraperitoneal injection of GSK-872 during the last 8 weeks. (F-I) Bone marrow–derived macrophages (BMDMs) were exposed to oxidized low-density lipoprotein (ox-LDL) for 24 hours with or without z-VAD (for the last 10 hours) and GSK-872 treatment (24 hours). (A) A graphical representation of an animal experiment. (B) Representative images and quantitative analysis of Oil Red O staining in the aortas of mice (n = 3). (C and D) Pathological staining (HE and Masson) and quantitative analysis of lesion area and fibrosis content in the aortas of mice (n = 3). (E) Immunofluorescence staining of p-RIPK3, F4/80, and α-SMA in the atherosclerotic lesion of mouse. (F and G) Representative immunoblots and quantitative analysis of p-RIPK3 and RIPK3 proteins in the aortas of mice (n = 6) and BMDMs (n = 5-6). (H and I) Representative images and quantitative analysis of Oil Red O staining in BMDMs (n = 3). Data are…

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  Figure 3. PGAM5 Expression Levels Within Macrophages in Atherosclerotic Lesions Are Suppressed by an RIPK3 Inhibitor (A and B) The human coronary artery samples. (A) Immunofluorescence staining of PGAM5, CD68, and α-SMA in the atherosclerotic lesion of human coronary artery. (B) IHC staining of PGAM5 protein in human coronary arteries. (C-G) WT-C57 and ApoE−/− mice were fed normal diet and high-fat diet (40% fat, containing 1.25% cholesterol) for 24 weeks, respectively. ApoE−/− mice were treated with intraperitoneal injection of GSK-872 during the last 8 weeks. (C) Immunofluorescence staining of PGAM5, F4/80, and α-SMA in the atherosclerotic lesion of mouse. (D and E) IHC staining and quantitative analysis of PGAM5 protein in the aortas of mice (n = 3). (F and G) Representative immunoblots and quantitative analysis of PGAM5 protein in the aortas of mice (n = 7). (H-L) BMDM or THP-1–derived macrophages were exposed to ox-LDL for 24 hours with or without z-VAD (for the last 10 hours) and GSK-872 (24 hours) treatment. (H-K) Representative immunoblots and quantitative analysis of PGAM5 protein in BMDMs (n = 9) and THP-1–derived macrophages (n = 3). (L) Immunofluorescence staining of PGAM5…

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  Figure 4. Overexpression of PGAM5 Aggravates Lipid Accumulation and Atherosclerotic Lesions in the Aortas of ApoE−/− Mice (A-L) ApoE−/− mice were fed a high-fat diet for 24 weeks and treated with GSK-872 during the last 8 weeks. ApoE−/− mice treated with GSK-872 were transduced with plasmid expressing the Pgam5 gene or empty vector using lentiviral vector at the 20th week. (A) A graphical representation of an animal experiment. (B) IHC staining of PGAM5 protein in the aortas of mice. (C) Representative immunoblots of PGAM5 protein in the aortas of mice. (D and E) Quantitative analysis of PGAM5 IHC staining and immunoblotting (n = 3-4). (F and G) Representative images and quantitative analysis of Oil Red O staining in the aortas of mice (n = 3). (H-J) Pathological staining (HE and Masson) and quantitative analysis of lesion area and fibrosis content in the aortas of mice (n = 3). (K and L) Representative immunoblots and quantitative analysis of p-RIPK3 and RIPK3 proteins in the aortas of mice (n = 5). Data are shown as mean ± SEM. Statistical analysis among 4 groups was performed using a 1-way ANOVA with a Sidak post hoc test. ns, P > 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations…

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  Figure 5. Lipid Accumulation in BMDMs Was Reduced by Pgam5 Gene Silencing and Aggravated by PGAM5 Overexpression (A-G) BMDMs were transduced with plasmid expressing PGAM5 gene or empty vector using lentiviral. (A) A representative image of lentiviral-transducted BMDMs. (B and C) Representative images and quantitative analysis of Oil Red O staining in BMDMs (n = 4). (D-F) Representative immunoblots and quantitative analysis of PGAM5 (n = 6) and p-RIPK3/RIPK3 (n = 3) proteins in BMDMs. (G) Immunofluorescence staining of PGAM5 and RIPK3 proteins in BMDMs. (H-N) BMDMs were transfected with siRNA (si-NC or si-Pgam5) for 72 hours plus ox-LDL exposure during the last 24 hours. (H) A representative image of siRNA-transfected BMDMs. (I and J) Representative images and quantitative analysis of Oil Red O staining in BMDMs (n = 4). (K-M) Representative immunoblots and quantitative analysis of PGAM5 (n = 6) and p-RIPK3/RIPK3 (n = 3) proteins in BMDMs. (N) Immunofluorescence staining of PGAM5 and RIPK3 proteins in BMDMs. Data are shown as mean ± SEM. Statistical analysis among 2 groups was performed using a 2-tailed unpaired Student’s t-test. ns, P > 0.05, ∗P < 0.05, ∗∗P < 0.01. si-NC = negative control…

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  Figure 6. PGAM5 Regulates the Expression Levels of the ANGPTL3 Gene and Protein in Macrophages (A-F) BMDMs were transfected with siRNA (si-NC or si-Pgam5) for 72 hours plus ox-LDL exposure during the last 24 hours. (A) The heatmap showing differentially expressed genes in BMDMs (n = 3). (B and C) The Gene Ontology (GO) analysis of differentially expressed genes in BMDMs. (D) The expression levels of genes in BMDMs (n = 3). (E and F) Representative immunoblots and quantitative analysis of ANGPTL3 protein in BMDMs (n = 12). (G-I) BMDMs were administrated with si-Pgam5 transfection with or without ANGPTL3 ASO treatment for 72 hours in the presence of ox-LDL exposure for the last 24 hours. (G and H) Representative immunoblots and quantitative analysis of PGAM5 and ANGPTL3 proteins in BMDMs (n = 6). (I) Representative images of Oil Red O staining in BMDMs. (J-L) BMDMs were administrated with si-Pgam5 transfection for 72 hours in the presence of ox-LDL exposure for the last 24 hours with or without ANGPTL3 mAb treatment for the last 24 hours. (J and K) Representative immunoblots and quantitative analysis of PGAM5 (n = 8) and ANGPTL3 (n = 9) proteins in BMDMs. (L) Representative images of Oil Red…

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  Figure 7. PGAM5 Mediates Cellular Communication Between Macrophages and ECs or SMCs (A-E) ECs were cultured with mixed medium (at a ratio of 1:1) of EGM2 and the CM obtained from BMDMs. ECs cultured in EGM2 medium for 36 hours was considered as the control group. (A) The coculture flowchart of ECs and the CM. (B) EC activity (n = 8). (C) Representative immunoblots of HMGB1, NF-κB p65, and TNF-α in ECs. (D-E) Quantitative analysis of HMGB1, NF-κB p65, and TNF-α (n = 5-9) proteins in ECs. (F-L) SMCs were cultured with mixed medium (at a ratio of 1:1) of DMEM and the CM obtained from BMDMs. SMCs cultured in DMEM medium was considered as the control group. (F) The coculture flowchart of SMCs and the CM. (G) SMC activity (n = 8). (H and I) SMC migration (n = 3). (J) Representative immunoblots of HMGB1, NF-κB p65, and TNF-α in SMCs. (K and L) Quantitative analysis of HMGB1, NF-κB p65, and TNF-α (n = 5-9) proteins in SMCs. Data are shown as mean ± SEM. Statistical analysis among 3 groups was performed using 1-way ANOVA with a Sidak post hoc test. Statistical analysis among 2 groups was performed using a 2-tailed unpaired Student t-test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. CM = conditioned…

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  Figure 8. The Expression Levels of PGAM5 and ANGPTL3 Protein in Macrophages Are Associated With the Severity of the Atherosclerotic Lesion in Humans and Mice (A to E, G and H, and J and K) WT-C57 and ApoE−/− mice were fed normal diet or high-fat diet (40% fat, containing 1.25% cholesterol) for 12 or 24 weeks, respectively. (F) WT-C57 and ApoE−/− mice were fed normal diet and high-fat diet (40% fat, containing 1.25% cholesterol) for 24 weeks, respectively. ApoE−/− mice were treated with intraperitoneal injection of GSK-872 during the last 8 weeks. (I, L-N) The human coronary artery samples. (A to C) Pathological staining (HE and Masson) and quantitative analysis of lesion area and the percentage of necrotic core area in the aortas of mice (n = 5). (D) The correlation analysis of lesion area and the percentage of necrotic core area in ApoE−/− mice (n = 10). (E and F) Serum ANGPTL3 levels (n = 4-5). (G) IHC staining of PGAM5 protein in the aortas of mice. (H and I) The correlation analysis of PGAM5 protein expression and the percentage of necrotic core area in ApoE−/− mice (n = 8) and humans (n = 10). (J) Immunofluorescence staining of F4/80 and ANGPTL3 proteins in the aortas of mice. (K) The…