AMPKα2 deficiency prevents nicotine-induced AAA formation in ApoE^−/− mice. ApoE^−/−, ApoE^−/−/AMPKα1^−/− and ApoE^−/−/AMPKα2^−/− mice were infused with nicotine (1 mg/kg/day) for 6 weeks using an osmotic pump. N=10–15 in each group. (a) Representative photographs showing macroscopic features of aneurysms induced by nicotine. The arrow indicates a typical AAA. (b) The incidence of nicotine-induced AAA, (c) Maximal abdominal aortic diameter, and (d) Total aortic weights in ApoE^−/−, ApoE^−/−/AMPKα1^−/− and ApoE^−/−/AMPKα2^−/− mice after nicotine infusion. (e) Representative staining with HE, elastin, and α-actin in suprarenal aortas of ApoE^−/−, ApoE^−/−/AMPKα1^−/− and ApoE^−/−/AMPKα2^−/− mice. (f) Grade of elastin degradation in aorta wall. *P<0.05 vs. vehicle-infused ApoE^−/− mice. ^#P<0.05 vs. vehicle-infused ApoE^−/−/AMPKα1^−/− mice. ^$P<0.05 vs. nicotine-infused ApoE^−/− mice.

Nicotine infusion results in oxidative stress and enhances MMP2/9 expressions via AMPKα2 in vivo. ApoE^−/, ApoE^−/−/AMPKα1^−/− and ApoE^−/−/AMPKα2^−/− mice were infused with nicotine (1 mg/kg/day) for 6 weeks. N is 10–15 in each group. (a) Representative stainings of MMP2, MMP9, MDA, and 3-NT in suprarenal aortas in nicotine-infused mice. (b, c and d) MMPs activity, protein expressions, and mRNA levels assayed by Zymagraphy, western blot and RT-PCR in mice aortas. (e) ROS productions detected by DHE/HPLC in mice aortas. P<0.05 vs. control ApoE^−/− mice. *P<0.05 vs. vehicle-infused ApoE^−/− mice. ^#P<0.05 vs. vehicle-infused ApoE^−/−/AMPKα1^−/− mice. ^$P<0.05 vs. nicotine-infused ApoE^−/− mice.

Bone marrow (BM) reconstitution shows a key role for vascular-specific AMPKα2 deficiency in AAA formation. After irradiation, ApoE^−/− and ApoE^−/−/AMPKα2^−/− mice were immediately injected with BM-derived cells from ApoE^−/− and ApoE^−/−/AMPKα2^−/− mice. (a–c) 6 weeks later, mice were infused with nicotine (1 mg/kg/day) for 6 weeks. (a) The incidence of AAA, (b) Maximal abdominal aortic diameters, and (c) Total aortic weights in nicotine-infused mice. N=10–15 in each group. *P<0.05 vs. nicotine-infused ApoE^−/− plus ApoE^−/−AMPKα2^+/+ BM-derived cells. NS indicates no significant difference. (d–f) 1 week later, mice were infused with AngII (1.44 mg/kg/day) for 4 weeks by using an osmotic pump. (d) The incidence of AAA, (e) Maximal abdominal aortic diameters, and (f) Total aortic weights in AngII-infused mice. N=10–15 in each group. *P<0.05 vs. AngII-infused ApoE^−/− plus ApoE^−/−AMPKα2^+/+ BM-derived cells. NS indicates no significant difference.

Inhibition of AMPKα2, but not AMPKα1, abolished nicotine/AngII-induced pro-MMP2 mRNA, protein expression, and MMP2 activity in VSMCs. (a) Cultured human VSMCs were transfected with control siRNA, AMPKα1 siRNA, or AMPKα2 siRNA for 48 h and then incubated with nicotine (0.1 μM) or metformin (2 mM) for 24 h. pro-MMP2 protein and MMP2 mRNA level in cell lysates, and MMP2 activity in culture medium were detected by Western blot, RT-PCR, and zymography, respectively. N=3. *P<0.05 vs. control siRNA. ^#P<0.05 vs. AMPKα1 siRNA. ^$P<0.05 vs. control siRNA plus nicotine or metformin. (b) Cultured mouse VSMCs (WT, AMPKα1^−/−, and AMPKα2^−/−) from the descending aorta were incubated with nicotine (0.1 μM) for 24 h. pro-MMP2 protein and mRNA levels in cell lysates and MMP2 activity in culture medium were assayed. N=3. *P<0.05 vs. WT. ^#P<0.05 vs. AMPKα1^−/−. ^$P<0.05 vs. WT plus nicotine. (c) Cultured human VSMCs were transfected with control siRNA, AMPKα1 siRNA, or AMPKα2 siRNA for 48 h and then incubated with AngII (1 μM) or AICAR (2 mM) for 24 h. pro-MMP2 protein and MMP2 mRNA level in cell lysates, and MMP2 activity in culture medium were detected by Western blot, RT-PCR, and zymography, respectively. N=3. *P<0.05 vs. control siRNA. ^#P<0.05 vs. AMPKα1 siRNA. ^$P<0.05 vs. control siRNA plus AngII or AICAR. (d) Cultured mouse VSMCs (WT, AMPKα1^−/−, and AMPKα2^−/−) from the descending aorta were incubated with AngII(1 μM) for 24 h. pro-MMP2 protein and mRNA levels in cell lysates and MMP2 activity in culture medium were assayed. N=3. *P<0.05 vs. WT. ^#P<0.05 vs. AMPKα1^−/−. ^$P<0.05 vs. WT plus AngII.

AP-2α mediates nicotine/AngII-induced AMPKα2-dependent MMP2 protein expression in VSMCs. (a) Cultured human VSMCs were transfected with control siRNA, AMPKα2 siRNA, or AP-2α siRNA for 48 h and then incubated with nicotine (0.1 μM) for 24 h. MMP2 activity in culture medium and pro-MMP2 protein expression in cell lysates were detected by Zymography and western blot, respectively. Representative results from three independent experiments are shown. *P<0.05 vs. Vehicle, ^#P<0.05 vs. Nicotine alone. (b) Cultured human VSMCs were transfected with control siRNA and AP-2α siRNA for 48 h and then incubated with AngII (1 μM) for 24 h. pro-MMP2 and AP-2α protein expression in cell lysates, mRNA level in cells, and MMP2 activity in culture medium were detected by western blot, RT-PCR, and zymography, respectively. N=3. *P<0.05 vs. control siRNA, ^#P<0.05 vs. control siRNA plus AngII. (c) Cultured human VSMCs were transfected with control siRNA or AMPKα2 siRNA for 48 h and then incubated with AngII (1 μM) or metformin (2 mM) for 2 h. Nuclear extract was subjected to EMSA to measure AP-2α DNA affinity. Negative control (−) consists of biotin-labeled DNA probe without extract. Positive control (+) consists of biotin-labeled DNA probe with nuclear extract provided by the kit. N=3. *P<0.05 vs. control siRNA, ^#P<0.05 vs. control siRNA plus metformin or AngII. (d–h) Cultured human VSMCs were pre-incubated with or without compound C (10 μM) for 30 min and then treated with nicotine (0.1 μM) or AngII (1 μM) for 2 h. AP-2α activity was detected by EMSA (d, e) or luciferase reporter gene (f) and binding of AP-2α to the MMP2 gene promoter were detected by ChIP (g, h), respectively. (d, e) N=3, *P<0.05 vs. control or vehicle, ^#P<0.05 vs. AngII or Nicotine. (f) N is 3. *P<0.05 vs. control, ^#P<0.05 vs. AngII alone, ^$P<0.05 vs. Nicotine alone. For ChIP experiments, the complex of chromatin/protein was pulled down by AP-2α primary antibody and the MMP2 promoter was amplified by PCR. Positive control, 10% of the total chromatin in the absence of immunoprecipitation (lane 1–4, input). Negative control, chromatin immunoprecipitated with IgG and amplified with MMP2 promoter primers (IgG, lane 5–8). The expected PCR product is 200 bp. Representative results from three independent experiments are shown. (i) Quantifications of ChIP. N is 3. *P<0.05 vs. control, ^#P<0.05 vs. AngII alone, ^$P<0.05 vs. Nicotine alone.

Nicotine/AngII promotes the binding of AMPKα2 to AP-2α and AMPKα2-dependent AP-2α serine 219 phosphorylation. (a, b) Cultured human VSMCs were incubated with nicotine (0.1 μM) or AngII (1 μM) for 2 h in the presence of compound C (10 μM). (a) Cell fractions were subjected to western blot analysis to determine the subcellular localization of AMPKα1/2 and AP-2α. LDH, cytoplasmic marker; H2AX, nuclear marker. N=3. (b) AMPKα1, AMPKα2, or AP-2α proteins in total cell lysates were pulled down by the appropriate primary antibody and subjected to western blot analysis to detect the binding of AP-2α to AMPKα1/α2. The blot is representative of three independent blots. (c) GST-AMPKα2 and GST-AP-2α protein were co-incubated in reaction buffer for 30′. Mixture was subjected to run native gel or pulled down by primary antibody followed by western blot. 3 independent experiments were performed. (d) Serine phosphorylation of AP-2α was assayed by IP with primary AP-2α antibody followed by western blotting with anti-phospho-serine antibody. The experiment was repeated three times. (e) Cultured HEK293 cells were transfected with wild type AMPKα2 (WT-AMPKα2) or dominant negative (DN-AMPKα2) for 24 h. WT-AMPKα2 and DN-AMPKα2 protein were purified by IP with primary AMPKα2 antibody. AP-2α protein and IP-derived AMPKα2 protein were co-incubated in kinase buffer in the presence of P³²-labeled ATP and phosphorylation of AP-2α was visualized by radioautography. N=3. (f) Peptides (SAMS, AP-2α 176-185/214-233/228-237/247-226/315-324) were co-incubated with AMPKα2 protein in reaction buffer in the presence of P³²-ATP. Phosphorylation of AP-2α and SAMS peptides was visualized by radioautography. N=3. (g) Peptides (SAMS, SAMS S6A, AP-2α 214–223, AP-2α 214–233 S219A) were co-incubated with AMPKα2 protein in reaction buffer in the presence of P³²-ATP. Phosphorylation of AP-2α and SAMS peptides was visualized by radioautography. N=3. (h) HA-tagged WT and site-directed mutants of full-length AP-2α were transfected into HEK293 cells for 24 h and then treated with AICAR (2 mM) for 2 h. Serine phosphorylation of AP-2α was determined by pull-down with anti-HA and western blot analysis with anti-phosphor-serine antibody. N=3.