Exendin-4 attenuates atherosclerosis progression via controlling hematopoietic stem/progenitor cell proliferation

Figures

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  Figure 1. Ex-4 treatment attenuates atherosclerotic plaque progression in hypercholesterolemic LDLr−/− mice. LDLr−/− mice were placed on HFD for 12 weeks. After 6 weeks, mice received saline or Ex-4 treatment using minipump till the end of the experiment. (A) Glucose tolerance test. n = 4–18. *P < 0.05 and **P < 0.01 for HFD vs. HFD + Ex-4. (B and C) Plasma levels of cholesterol and triglyceride. n = 7–11. (D and E) Absolute and differential white blood cell count. n = 6–12. (F) White blood cells were stained for CD11b and F4/80 and analyzed by FACS. n = 18–22. (G–L) Histology and immunohistochemistry of plaque in the aorta. (G) H&E analysis of plaque size. n = 17 for each group. (H and I) The numbers of α-SMA+ and CD68+; cells in plaque. n = 8–10. (J–L) Representative images of H&E, α-SMA, and CD68 staining.

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  Figure 2. Ex-4 treatment inhibits HSPC proliferation in hypercholesterolemic LDLr−/− mice. LDLr−/− mice were placed on HFD for 12 weeks. After 6 weeks, mice received saline or Ex-4 treatment using minipump till the end of the experiment. (A–D) Frequencies of HSPCs, CMPs, GMPs, and MEPs in BMCs. n = 21–34. (E) A representative flow of FACS analysis for HSPCs, CMPs, GMPs, and MEPs. (F) Percentages of BrdU-incorporating HSPCs in the HSPC population. n = 8–12. (G) Representative FACS analysis for BrdU-incorporating HSPCs.

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  Figure 3. GLP-1r expression in HSPCs. LDLr−/− mice were placed on HFD for 12 weeks. After 6 weeks, mice received saline or Ex-4 treatment using minipump till the end of the experiment. (A) BMCs were stained with HSPC surface markers and anti-mouse RAGE for FACS analysis. n = 9–21. (B) Western blotting for GLP-1r and GAPDH expression in livers. n = 4–6. (C) HSPCs were sorted out by FACS and subjected to capillary western blotting for GLP-1r and GAPDH expression. n = 3.

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  Figure 4. GLP-1r deficiency impaires glucose tolerance and white blood cell reconstitution. (A) Western blotting for GLP-1r expression in wild-type and GLP-1r−/− pancreas and HSPCs. n = 4. (B and C) Lipid and glucose tolerance in wild-type and GLP-1r−/− mice. n = 5–10. *P < 0.05. (D) Glucose tolerance in wild-type and GLP-1r−/− mice on HFD with or without Ex-4 treatment. n = 9. (E–H) LDLr−/− recipients were lethally irradiated and then injected with wild-type or GLP-1r−/− BMCs. After 4 weeks, several recipients were switched to HFD. After 16 weeks, white blood cell chimerism, myeloid and lymphoid lineages, and plaque size were analyzed. n = 8–11.

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  Figure 5. Ex-4 prohibits myeloid cell production induced by LDL through ABCA1-independent pathways. Wild-type HSPCs were sorted out by FACS and equal amounts of HSPCs were seeded in U-bottom 96-well plates. (A) After 5 days of culture in 0 or 100 µM LDL with or without 50 nM Ex-4, cells were harvested to calculate the expansion. n = 8. (B and C) The cells were stained with anti-mouse CD11b and anti-mouse Gr-1 for FACS analysis. n = 4–6. (D–F) Equal amounts of BMCs were cultured with LDL in the presence or absence of Ex-4 for 24 h. Cells were harvested to extract RNA for qPCR analysis of ABCA1, ABCG1, and SR-BI expression. n = 8 or 9. (G) BMCs were stimulated with LDL with or without Ex-4 for 5 min. Cells were stained with HSPC surface markers and pERK antibody for FACS acquisition. Percentage of pERK+ HSPCs in the HSPC population was analyzed by FACS. (H) Representative FACS analysis of pERK+ HSPCs. Left: HSPCs defined as cKit+Sca-1+ cells when gated on lineage−/low cells. Right: pEKR+ cells when gated on HSPCs.

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  Figure 6. Metabolic profiles of HSPCs with Ex-4 treatment under HFD. LDLr−/− mice were placed on HFD for 12 weeks. After 6 weeks, mice received saline or Ex-4 treatment using minipump till the end of the experiment. HSPCs were FACS-sorted for metabolomics measurement. (A) Lipidome summary. (B) Plot of differential metabolites between HSPCs of chow diet and HFD groups. Blue dots represent the metabolites with decreased levels in HSPCs of HFD-fed mice receiving Ex-4, compared with that of HFD-fed mice. (C–H) Intracellular levels of PEP, α-ketoglutarate, FFA18:2, SM d18:1/24:0, UDP, and oxaloacetate in HSPCs. n = 3–8.

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  Figure 7. Protein expression and activity of enzymes related to PEP levels in the liver and BMCs of LDLr−/− mice on HFD with or without Ex-4. Mice on chow diet were served as controls. (A–F) Proteins were extracted from the liver and BMCs of mice. Protein expression (A–C) and enzyme activity (D–F) of pyruvate carboxylase, PCK1, and PCK2 in the liver and BMCs were quantified by ELISA. n = 5–8. (G) A proposed model of glucolysis and gluconeogenesis in HSPCs of HFD mice with or without Ex-4 treatment. Black line and red line represent parts of the glycolysis and gluconeogenesis pathways in cells. PEP is the key metabolite linking glycolysis and gluconeogenesis. In glucolysis, pyruvate is generated from PEP by PYK. By pyruvate dehydrogenase, pyruvate can be converted to acetyl CoA to enter TCA cycle. Oxaloacetate is one of the metabolites in TCA cycle that could be metabolized to generate PEP by PCK1 and PCK2, which reside in the cytosol and the mitochondria, respectively. By targeted metabolomics, intracellular levels of PEP and oxaloacetate in HSPCs were found increased in HFD mice but decreased in HFD mice with Ex-4 treatment. Ex-4 treatment reduced PCK activity in HSPCs from HFD mice.…