Search for author "Zhaohua Cai"

AMP-activated protein kinase (AMPK) α1 ablation increases atherosclerotic calcification of aortic roots in vivo. ApoE^−/−, ApoE^−/−/AMPKα1^−/−, and ApoE^−/−/AMPKα2^−/− mice were fed with Western diet for 24 wk. A, Representative images of alizarin red staining of aortic roots in indicated groups. B, ApoE^−/−/AMPKα1^−/− but not ApoE^−/−/AMPKα2^−/− mice had increased alizarin red staining in atherosclerotic lesion areas of aortic roots compared with ApoE^−/− mice. C, ApoE^−/−/AMPKα1^−/− mice had increased aortic calcium content compared with ApoE^−/− mice. No difference of aortic calcium content between ApoE^−/−/AMPKα2^−/− and ApoE^−/− groups (scale bar, 500 μm. ApoE^−/−: n=8; ApoE^−/−/AMPKα1^−/−: n=8; ApoE^−/−/AMPKα2^−/−: n=7; *P<0.05; n.s. P>0.05).

Metformin administration inhibits atherosclerotic calcification and Runx2 (Runt-related transcription factor) expression via AMP-activated protein kinase (AMPK) α1 in vivo. ApoE^−/− and ApoE^−/−/AMPKα1^−/− mice with or without metformin (100 mg/kg per d) were treated with Western diet for 24 wk. A, Representative alizarin red staining of aortic roots of indicated groups. B, ApoE^−/−/AMPKα1^−/− mice had increased alizarin red staining in atherosclerotic lesion areas of aortic roots compared with ApoE^−/− mice. Metformin treatment significantly reduced atherosclerotic calcification in ApoE^−/− mice, but had no such effect in ApoE^−/−/AMPKα1^−/− mice. C, ApoE^−/−/AMPKα1^−/− mice had increased aortic calcium content compared with ApoE^−/− mice. Metformin treatment significantly reduced aortic calcium content in ApoE^−/− mice, but had no such effect in ApoE^−/−/AMPKα1^−/− mice. D, Representative immunofluorescence staining of Runx2 in aortic roots of indicated groups. E, ApoE^−/−/AMPKα1^−/− had increased Runx2 staining in atherosclerotic lesions compared with ApoE^−/− mice. Metformin administration significantly inhibited Runx2 staining in ApoE^−/−, but the effect was abolished in ApoE^−/−/AMPKα1^−/− mice. F, Western blot results show ApoE^−/−/AMPKα1^−/− had increased aortic Runx2 expression compared with ApoE^−/− mice. Metformin administration significantly inhibited Runx2 expression in ApoE^−/−, but the effect was abolished in ApoE^−/−/AMPKα1^−/− mice (scale bar: 500 μmol/L for alizarin red staining images and 200 μmol/L for immunofluorescence staining of Runx2. ApoE^−/−: n=8; ApoE^−/−+metformin: n=7; ApoE^−/−/AMPKα1^−/−: n=7; ApoE^−/−/AMPKα1^−/− plus metformin: n=6; *P<0.05; n.s. P>0.05).

AMP-activated protein kinase (AMPK) α1 deficiency promotes osteoblastic differentiation of vascular smooth muscle cell (VSMC) via Runx2 (Runt-related transcription factor). A, Alizarin red staining of calcium nodule formation of murine wild-type (WT), AMPKα1^−/−, and AMPKα2^−/− VSMC treated with osteogenic media for 14 d. AMPKα1 but not AMPKα2 deficiency significantly induced calcium nodule formation compared with WT VSMC. B, AMPKα1-deficient murine VSMC had significantly increased Runx2 expression compared with WT VSMC. C, ox-LDL (100 μg/mL) significantly induced Runx2 expression, which could be inhibited by metformin (2 mmol/L). AMPKα1 silencing markedly increased Runx2 expression compared with scrambled siRNA-transfected (Scr siRNA) cells. Oxidized low-density lipoprotein (ox-LDL) further induced Runx2 expression, but could not be suppressed by metformin supplementation. D, Representative alizarin red staining of murine WT and AMPK^α1−/− VSMC with indicated treatment in osteogenic media for 14 d. AMPKα1 deficiency significantly induced calcium nodule formation compared with WT VSMC. Silencing of Runx2 by lentivirus Runx2 shRNA markedly suppressed the effect (n=3 for each experiment). Lenti-Runx2 shRNA indicates lentivirus Runx2 shRNA; and Lenti-Scr shRNA, lentivirus scramble shRNA. *P<0.05; n.s. P>0.05.

AMP-activated protein kinase (AMPK) α1 phosphorylates protein inhibitor of activated STAT-1 (PIAS1) at serine 510 and promotes Runx2 (Runt-related transcription factor) small ubiquitin-related modifier (SUMO)ylation in vascular smooth muscle cell (VSMC). A, Treatment of metformin (2 mmol/L) for 6 h significantly induced Runx2-SUMO1 expression (≈100-kD band). The effect could be blocked by addition of compound C (10 μmol/L). B, AMPKα1 and PIAS1 in whole-cell lysates were pulled down by the appropriate primary antibody and subjected to Western blot analysis to detect the binding of AMPKα1 and PIAS1. C, Serine phosphorylation of PIAS1 was determined by pull-down PIAS1 antibody and immunoblotting with phosphor-serine antibody. Six hours of metformin treatment induced PIAS1 phosphorylation as indicated by pulling down by PIAS1 and immunoblotting with phosphor-serine antibody (≈80-kD band). The effect could be abolished by compound C. Neither metformin nor compound C altered PIAS1 expression. D, Myc-tagged WT and site-directed mutants of PIAS1 were transfected into human aortic VSMC then treated with metformin for 6 h. Serine phosphorylation of PIAS1 was determined by pull-down with anti-Myc and Western blot analysis with antiphospho-serine antibody (n=3 for each experiment. *P<0.05).