Rosemeire M Kanashiro Takeuchi, Lauro M Takeuchi, Chen C Yuan, Raul A Dulce, Darrell Hardin, Marilia Zuttion, Wayne Balkan, Ren Z Cai, Andrew V Schally and Joshua M Hare
AMP-activated protein kinase (AMPK) α2 deletion enhances Western diet–induced features of atherosclerotic plaque instability in the brachiocephalic arteries (BA). A, Representative images from BA lesions of Apoe−/− and Apoe−/−AMPKα2−/− mice with hematoxylin-eosin (H&E) staining for intraplaque hemorrhage (black arrow) and buried fibrous cap (black arrowhead). B–D, Incidence for intraplaque hemorrhage (B), presence of buried fibrous cap (C), and presence of fibrous cap discontinuity (D) in the BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. E and F, Representative images and quantification of necrotic core area in the BA based on H&E staining (black arrow) of Apoe−/− and Apoe−/−AMPKα2−/− mice. G and H, Representative images and quantification for the area of fibrous cap staining (black arrow) in BA based on Sirius Red staining (red staining) of Apoe−/− and Apoe−/−AMPKα2−/− mice. I and J, Representative images and quantification of plaque collagen content in BA based on Masson trichrome staining (blue staining, black arrow) of Apoe−/− and Apoe−/−AMPKα2−/− mice. K and L, Representative images and quantification of plaque macrophage content in BA based on CD68 immunohistochemistry (IHC) staining (brown staining, black arrow) of Apoe−/− and Apoe−/−AMPKα2−/− mice. n=20 to 21 in each group. Values represent the mean±SEM. *P<0.05 vs Apoe−/− mice. Scale bar, 100 µm.
AMP-activated protein kinase (AMPK) α2 deletion induces vascular smooth muscle cell phenotypic switching in advanced atherosclerotic plaque in the brachiocephalic arteries (BA). A, Representative images of immunohistochemistry staining of smooth muscle actin (SMA)-α (dark pink) in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. Arrow represents representative staining of SMA-α. Scale bar, 100 µm. B, Quantification of plaque SMA-α coverage on the plaque cap in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. C, Quantification of total plaque SMA-α content in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. D, Representative images of immunofluorescence staining of vimentin (red) in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. DAPI=blue staining of nucleus. Scale bar, 100 µm. E, Quantification of plaque vimentin coverage on the plaque cap in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. F, Quantification of total plaque vimentin content in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. n=10 in each group. Values represent the mean±SEM. *, P<0.05 vs Apoe−/− mice.
AMP-activated protein kinase (AMPK) α2 deletion upregulates Kruppel-like factor 4 (KLF4) expression in advanced atherosclerotic plaque in the brachiocephalic arteries (BA). A, Immunofluorescence staining of KLF4 (red) in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. DAPI=blue staining of nucleus. Scale bar, 100 µm. B, Quantification of KLF4 expression in BA of Apoe−/− and Apoe−/−AMPKα2−/− mice. n=10 in each group. Values represent the mean±SEM. *P<0.05 vs Apoe−/− mice.
Pravastatin treatment alleviates Western diet–induced plaque instability in Apoe−/−AMPKα2sm+/+ mice, but not in Apoe−/−AMPKα2sm−/− mice.A, Representative images from hematoxylin-eosin staining of the brachiocephalic arteries (BA) in Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice treated with or without pravastatin. Scale bar, 100 µm. B, Quantification of plaque size and (C) necrotic core size in the BA of Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice treated with or without pravastatin. D and E, Representative images and quantification of plaque collagen content in the BA based on Masson trichrome staining of Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice treated with or without pravastatin. Scale bar, 100 µm. F, Quantification of fibrous cap area in the BA of Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice treated with or without pravastatin. n=10 in each group. Values represent the means±SEM. *P<0.05 vs Apoe−/−AMPKα2sm+/+ mice without pravastatin treatment. #P<0.05 vs Apoe−/−AMPKα2sm+/+ mice with pravastatin treatment. G, Western blot analysis of pAMPKα (Thr172) in human aortic smooth muscle cell (HASMC) treated with 0.01 to 50 µmol/L pravastatin for 24 h. H, Western blot analysis of pAMPKα (Thr172) in aorta from Apoe−/−AMPKα2sm+/+ mice fed with Western diet for 10 wk and treated with or without pravastatin for 4 wk.
Vascular smooth muscle cell (VSMC) AMP-activated protein kinase (AMPK) α2 knockdown eliminates the effect of pravastatin treatment on VSMC phenotypic switching signaling in vivo. A, Representative images of immunohistochemistry staining of smooth muscle actin (SMA)-α (dark pink) in the brachiocephalic arteries (BA) of Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice treated with or without pravastatin. Scale bar, 100 µm. B and C, Quantification of plaque SMA-α coverage on the plaque cap (B) and total plaque SMA-α content (C) in BA of Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice treated with or without pravastatin. D, Representative images of immunofluorescence (IF) staining of vimentin (red) in the BA of Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice treated with or without pravastatin. DAPI=blue staining of nucleus. Scale bar, 100 µm. E and F, Quantification of plaque vimentin coverage on the plaque cap (E) and total plaque vimentin content (F) in BA of Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice treated with or without pravastatin. G, Representative images of IF staining of Kruppel-like factor 4 (KLF4; red) in the BA of Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice treated with or without pravastatin. DAPI=blue staining of nucleus. Scale bar, 100 µm. H, Quantification of KLF4 expression in BA of Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice treated with or without pravastatin. n=10 in each group. Values represent the means±SEM. *P<0.05 vs Apoe−/−AMPKα2sm+/+ mice without pravastatin treatment. #P<0.05 vs Apoe−/−AMPKα2sm+/+ mice with pravastatin treatment.
AMP-activated protein kinase (AMPK) α2 deficiency induces vascular smooth muscle cell (VSMC) phenotypic switching in vitro. A, Western blot analysis of contractile (smooth muscle actin [SMA]-α, calponin, and SM-MHC [smooth muscle-mysion heavy chain]) and synthetic proteins (vimentin and osteopontin) in VSMC isolated from wild-type (WT) and AMPKα2−/− mice (n=5). Values represent the mean±SEM. *P<0.05 vs WT. B, Quantitative real-time polymerase chain reaction (PCR) of contractile (SMA-α, calponin, and SM-MHC) and synthetic markers (vimentin and osteopontin) in VSMC isolated from WT and AMPKα2−/− mice (n=5). Values represent the mean±SEM. *P<0.05 vs WT. C, Western blot analysis of contractile and synthetic proteins in human aortic smooth muscle cell (HASMC) treated with con siRNA and AMPKα2 siRNA (n=5). Values represent the means±SEM. *P<0.05 vs con siRNA. D, Quantitative real-time PCR of contractile and synthetic markers in HASMC treated with con siRNA and AMPKα2 siRNA (n=5). Values represent the mean±SEM. *P<0.05 vs con siRNA.
AMP-activated protein kinase (AMPK) α2 deficiency–induced vascular smooth muscle cell (VSMC) phenotype switching is in Kruppel-like factor 4 (KLF4)–dependent manner. A, Western blot analysis of KLF4 protein expression in wild-type (WT) and AMPKα2−/− mouse VSMC (n=5). *P<0.05 vs WT. B, Quantitative real-time polymerase chain reaction (PCR) analysis of KLF4 mRNA level in WT and AMPKα2−/− mouse VSMC (n=5). *P<0.05 vs WT. C, Western blot analysis of KLF4 protein expression in HASMC treated with con siRNA and AMPKα2 siRNA (n=5). *P<0.05 vs con siRNA. D, Quantitative real-time PCR analysis of KLF4 mRNA level in human aortic smooth muscle cells (HASMCs) treated with con siRNA and AMPKα2 siRNA (n=5). *P<0.05 vs con siRNA. E, Western blot analysis of protein expression of smooth muscle actin (SMA)-α, calponin, vimentin, and osteopontin in WT and AMPKα2−/− mouse VSMC treated with con siRNA and KLF4 siRNA for 48 h (n=5). *P<0.05 vs WT+con siRNA. #P<0.05 vs AMPKα2−/−+con siRNA. F, Quantitative real-time PCR analysis of mRNA level of SMA-α, calponin, vimentin, and osteopontin in WT and AMPKα2−/− mouse VSMC treated with con siRNA and KLF4 siRNA for 48 h (n=5). *P<0.05 vs WT+con siRNA. #P<0.05 vs AMPKα2−/−+con siRNA. G, Western blot analysis of protein expression of SMA-α, calponin, vimentin, and osteopontin in HASMC treated with con siRNA, AMPKα2 siRNA, and KLF4 siRNA for 48 h (n=5). *P<0.05 vs con siRNA. #P<0.05 vs AMPKα2 siRNA. H, Quantitative real-time PCR analysis of mRNA level of SMA-α, calponin, vimentin, and osteopontin in HASMC treated with con siRNA, AMPKα2 siRNA and KLF4 siRNA for 48 h (n=5). *P<0.05 vs con siRNA. #P<0.05 vs AMPKα2 siRNA.
AMP-activated protein kinase (AMPK) α2 deletion upregulates Kruppel-like factor 4 (KLF4) through nuclear factor-κB (NF-κB) signaling. A, Western blot analysis of KLF4 expression in wild-type (WT) and AMPKα2−/− mouse VSMC treated with NF-κB control and NF-κB inhibitor (n=5). *P<0.05 vs WT VSMC treated with NF-κB control. #P<0.05 vs AMPKα2−/− VSMC treated with NF-κB control. B, Quantitative real-time polymerase chain reaction (PCR) analysis of mRNA level of KLF4 in WT and AMPKα2−/− mouse VSMC treated with NF-κB control and NF-κB inhibitor (n=5). *P<0.05 vs WT VSMC treated with NF-κB control. #P<0.05 vs AMPKα2−/− VSMC treated with NF-κB control. C, Western blot analysis of KLF4 expression in human aortic smooth muscle cells (HASMCs) treated with NF-κBp65 siRNA and AMPKα2 siRNA (n=5). *, P<0.05 vs con siRNA. #P<0.05 vs AMPKα2 siRNA. D, Quantitative real-time PCR analysis of mRNA level of KLF4 in HASMC treated with NF-κBp65 siRNA and AMPKα2 siRNA (n=5). *P<0.05 vs con siRNA. #P<0.05 vs AMPKα2 siRNA. E, The KLF4 promoter was analyzed using the Transcription Factor Database software, suggesting one binding site within the promoter. 2600 bp and 1583 bp KLF4 promoter luciferase constructs are shown. F, HASMC were transfected with 2600- and 1583-bp KLF4 promoter luciferase constructs and treated with con siRNA or AMPKα2 siRNA, and luciferase activity was measured after 24 h. Results of the luciferase reporter assay are presented as fold changes±SEM of the Firefly/Renilla luciferase activities (n=5). *P<0.05 vs HASMC transfected with 2600-bp KLF4 promoter luciferase construct and con siRNA. #P<0.05 vs HASMC transfected with 2600-bp KLF4 promoter luciferase construct and AMPKα2 siRNA. G, HASMC were transfected with either WT or the mutant KLF4 promoter-reporter and treated with con siRNA or AMPKα2 siRNA for 24 h to detect the luciferase activity (n=5). *P<0.05 vs HASMC transfected with WT 2600-bp KLF4 promoter reporter and con siRNA. #P<0.05 vs HASMC transfected with WT 2600-bp KLF4 promoter reporter and AMPKα2 siRNA. H, Chromatin immunoprecipitation assay for NF-κBp65 binding with KLF4 promoter in WT and AMPKα2−/− mouse VSMC.
