Supplementary information Activation of AMP-activated protein kinase

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1 Supplementary information Activation of AMP-activated protein kinase 2 by nicotine instigates formation of abdominal aortic aneurysms in mice in vivo Shuangxi Wang 1,2,5, Cheng Zhang 1,2,5, Miao Zhang 1, Bin Liang 1, Huaiping Zhu 1, Jiyeon Lee 1, Benoit Viollet 3, Lijun Xia 4, Yun Zhang 2 & Ming-Hui Zou 1,2 1 Division of Molecular Medicine, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA. 2 The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong University, Qilu Hospital, Jinan City, Shandong, China. 3 Institut Cochin, Université Paris Descartes, Centre national de la recherche scientifique (CNRS(UMR 8104), Paris, France. 4 Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA. 5 These authors contributed equally to this work. Correspondence should be addressed to M.-H.Z. (ming-hui-zou@ouhsc.edu). Supplementary Figure 1-12 and legends Supplementary Table

Supplementary Figure 1 AMPK-α2 deficiency prevents high dose nicotine-induced AAA formation in Apoe / mice.

(a) Representative photographs showing macroscopic features of aneurysms induced by nicotine. Vehicle or nicotine was administered to mice of the indicated genotypes.

Ration is the aortic weight (g) to the total body weight (g) of the mouse and given as a percentage. * P<0.05 compared to vehicle-infused Apoe / mice. # P<0.

2 Supplementary Figure 1 AMPK-α2 deficiency prevents high dose nicotine-induced AAA formation in Apoe / mice. Apoe /, Apoe / ; Prkaa1 / and Apoe / ; Prkaa2 / mice were infused with nicotine (5 mg/kg/day) or vehicle for 6 weeks by an osmotic pump. (a) Representative photographs showing macroscopic features of aneurysms induced by nicotine. Vehicle or nicotine was administered to mice of the indicated genotypes. Arrows indicate typical AAAs. (b) The incidence of nicotine-induced AAA and (c) maximal abdominal aortic diameter. BW means body weight. Ration is the aortic weight (g) to the total body weight (g) of the mouse and given as a percentage. * P<0.05 compared to vehicle-infused Apoe / mice. # P<0.05 compared to vehicle-infused Apoe / ; Prkaa1 / mice. $ P<0.05 compared to nicotine-infused Apoe / mice. N is 8-10 in each group. The P values were obtained by a 2 test in b and one-way analysis of variance plus a post hoc analysis using a Bonferroni test in c. The error bars in c are s.e.m. 2

Supplementary Figure 2 Ablation of AMPK- 2 prevents AngII-induced AAA formation in Apoe / mice. (a) Representative photographs showing the macroscopic features of aneurysms induced by AngII.

3 Supplementary Figure 2 Ablation of AMPK- 2 prevents AngII-induced AAA formation in Apoe / mice. (a) Representative photographs showing the macroscopic features of aneurysms induced by AngII. Saline or AngII (1.44 mg/kg/day) was administered to mice of the indicated genotypes for 4 weeks. Arrows indicate typical AAAs. (b) The incidence of AngII-induced AAA, (c) maximal abdominal aortic diameter and (d) total aortic weights in mice of the indicated genotypes after saline or AngII infusion. BW means body weight. Ration is the aortic weight (g) to the total body weight (g) of the mouse and given as a percentage. (e) Representative staining with HE, elastin and -actin in the suprarenal aortas of mice of the indicated genotypes after saline or AngII infusion.the magnification of the two insets is 40-fold in the middle row. (f) Grade of elastin degradation in the aortic wall of mice of the indicated genotypes after saline or AngII infusion. *P < 0.05 compared to vehicle-infused Apoe / or Apoe / ; Prkaa1 / mice, #P < 0.05 compared to AngII- infused Apoe / mice. N is in each group. The P values were obtained by a 2 test in b and one-way analysis of variance plus a post hoc analysis using a Bonferroni test in c, d, and f. The error bars in c, d and f are s.e.m. 3

4 Supplementary Figure 3 Real-time PCR to repeat all samples of RT-PCR. Total RNA was extracted from mice aortas or cultured cells and transcripted to cdna by a kit. Real-time PCR was used to amplify cdna. (a, b) MMP2 mrna levels in Apoe /, Apoe / ; Prkaa1 / and Apoe / ; Prkaa2 / mice aortas infused by (a) nicotine or (b) AngII. N is in each group. * P<0.05 compared to control Apoe / mice, # P<0.05 compared to control Apoe / ; Prkaa1 / mice, $ P<0.05 compared to nicotine or AngII-infused Apoe / mice. (c, d) MMP2 mrna levels in human VSMCs untreated (control) or treated with (c) nicotine or (d) AngII in presence of compound C. N is 3 in each group. * P<0.05 compared to control without compound C, # P<0.05 compared to nicotine or AngII alone. (e) MMP2 mrna levels in human VSMCs transfected with control sirna, AMPK-α1 sirna, or AMPK-α2 sirna and incubated with nicotine or metformin. N=3 in each group. * P<0.05 compared to control sirna, # P<0.05 compared to AMPK-α1 sirna, $ P<0.05 compared to control sirna plus nicotine or metformin. (f) MMP2 mrna levels in human VSMCs transfected with control sirna, AMPK-α1 sirna, or AMPK-α2 sirna and incubated with AngII or AICAR. N=3. * P<0.05 compared to control sirna alone, # P<0.05 compared to AMPK-α1 sirna, $ P<0.05 compared to control sirna plus AngII or AICAR. (g) MMP2 mrna levels in mouse VSMCs (WT, Prkaa1 /, and Prkaa2 / ) incubated with nicotine. N=3. * P<0.05 compared to WT, # P<0.05 compared to Prkaa1 /, $ P<0.05 compared to WT plus nicotine. (h) MMP2 mrna levels in mouse VSMCs (WT, Prkaa1 /, and Prkaa2 / ) incubated with AngII. N=3 in each group. * P<0.05 compared to WT, # P<0.05 compared to Prkaa1 /, $ P<0.05 compared to WT plus AngII. The P values were obtained by one-way analysis of variance plus a post hoc analysis using a Bonferroni test. All error bars are s.e.m. 4

5 Supplementary Figure 4 AngII infusion results in oxidative stress and enhances the expression of MMP2 and MMP9 through AMPK- 2 in vivo. (a) Representative immunostaining of MMP2, MMP9, MDA and 3-NT in the suprarenal aortas of saline- or AngII-infused mice. (b, c) MMP2 and MMP9 activity by zymography, MMP2 and GAPDH mrna by RT-PCR and protein expression by Western blot in b, and ROS production in c as assessed by measurement of DHE levels by HPLC in the aortas of saline- or AngII-infused mice of the indicated genotypes. Quantitative results are shown below in b. (Pro-MMP9)2 means dimer of pro-mmp9. Pro-MMP means full-length MMP. Lipocalin/Pro-MMP9 means Pro-MMP9 which binds with lipocalin. * P < 0.05 compared to saline-infused Apoe / mice, # P < 0.05 compared to saline-infused Apoe / ; Prkaa1 / mice, $ P < 0.05 compared to AngII-infused Apoe / mice. All reseults presents 5-10 mice. The P values were obtained by one-way analysis of variance plus a post hoc analysis using a Bonferroni test in b and c. The error bars in b,c are s.e.m. 5

6 Supplementary Figure 5 MMP2 is crucial for AngII- or nicotine-induced elastin degradation in Apoe / mice. After in vivo trnasfection of MMP2 sirna, Apoe / mice were infused with AngII (1.44 mg/kg/day) or nicotine (1 mg/kg/day) for 7 days. Mice aortas were subjected to measure (a) MMP2 activity by zymography, MMP2, pro-mmp2, collagen IV, pampk, AMPK and GAPDH protein expressions by western blot and quantitative results are shown below. (b) Elastin degradation by HE staining and collagen IV levels by immunohistochemistry and (c) elevation of elastin degradation by quantitative analysis. N is 5 in each group. * P < 0.05 compared to control sirna mice, # P < 0.05 compared to control sirna mice infused with AngII or nicotine. NS, no significant. The P values were obtained by one-way analysis of variance plus a post hoc analysis using a Bonferroni test. The error bars in a,c are s.e.m. 6

7 Supplementary Figure 6 Both nicotine and AngII activate AMPK in human VSMCs. (a, b) Human VSMCs were pre-incubated with AICAR (2 mm), ONOO - (50 μm), or nicotine ( μm) for 1 hour. Cells were used to determine (a) pampk and AMPK levels by Western blot and (b) AMPK activity by 32 P-ATP in vitro kinase phosphorylation. N is 3. * P < 0.05 compared to control group. (c) Cultured human VSMCs were incubated with nicotine (0.1 μm) for indicated times. AMPK phosphorylation and AMPK was assayed by Western blot by using Thr172-phosphorylated antibody. (d, e) Cultured human VSMCs were incubated with AngII (1 μm) for indicated times. Cell lysates were subjected to detect pampk and pacc by Western blot and AMPK activity by 32 P -ATP in vitro kinase phosphorylation. N is 3. * P < 0.05 compared to control (0 ) group. The P values were obtained by one-way analysis of variance plus a post hoc analysis using a Bonferroni test. The error bars are s.e.m. 7

8 Supplementary Figure 7 Inhibition of AMPK by compound C abolished both nicotine and AngII induced MMP2 upregulations in human VSMCs. (a) Cultured human VSMCs were pre-incubated with or without compound C (10 μm) for 30 and then treated with nicotine (0.1 μm) for 24 hours. The pro-mmp2 protein and mrna expression in total cell lysates was assayed by Western blot and RT-PCR, and activity in culture medium were detected by zymography. GAPDH protein and mrna, pampk and AMPK are also shown. * P<0.05 vs. control, # P<0.05 vs. Nicotine. (b) Cultured human VSMCs were pre-incubated with compound C (10 μm) or DMSO for 30 and then treated with AngII (1 μm) for 24 hours. The pro-mmp2 protein and mrna expressions, activity in culture medium were analyzed as in a. n = 3 independent experiments for all quantitative data. * P < 0.05 compared to control group, # P < 0.05 compared to Nicotine or AngII alone. The P values were obtained by one-way analysis of variance plus a post hoc analysis using a Bonferroni test. The error bars are s.e.m. 8

9 Supplementary Figure 8 AngII and nicotine promote co-localization of AMPK-α2 to AP-2α in human VSMCs. Cultured human VSMCs were incubated with AngII (1 μm) or nicotine (0.1 μm) for 2 h in the presence of compound C (10 μm). Cells were mounted in formalin and subjected to immunofluoresence staining by AP-2α- or AMPK-α2 primary antibidy to determine the subcellular locations of AP-2α and AMPK-α2. Red, AMPK-α2; Green, AP-2α; Yellow, AMPK-α2 plus AP-2α overlap; Blue, DAPI/nucleus; Merge, AMPK-α2/AP-2α/DAPI. The images (40-fold) are representative of three independent experiments. 9

10 Supplementary Figure 9 The domains and amino acid sequence of human AP-2α. (a) Full length of human AP-2α amino acid sequence. (b) The domains and structure of human AP-2α protein. (c) Alignments of human AP-2α serine residues relative to an optimal AMPK motif by positional scanning peptide library screening. 10

11 Supplementary Figure 10 Mutant of AP-2α S219A suppressed AngII/nicotine-induced MMP2 upregulation in VSMCs. Human VSMCs were transfected with WT and S219A constructs of AP-2α for 48 hours and then treated with AngII (1 μm) or nicotine (0.1 μm) for 24 hours. (a) MMP2 and GAPDH mrna by RT-PCR, pro-mmp2 and GAPDH protein by Western blot in total cell lysates, and MMP2 activity in culture medium by zymography, (b) AP-2α DNA affinity by EMSA, and (c) the binding of AP-2α to MMP2 gene promoter by ChIP were measured. All pictures are a representative picture from 3 independent experiments. 11

12 Supplementary Figure 11 Increased AMPK-α2 activity, AMPK Thr172 phosphorylation, and ACC Ser79 phosphorylation in human AAA. Abdominal aortic tissues from age-matched patients with AAA or non-aaa patients were homogenated. The phosphorylations of AMPK Thr172, ACC Ser79, AMPK-α2 activity were assayed. (a) Increased detection of pampk and pacc in aortic tissues from patients with AAA. The blot is a representive blot obtained from 6 individuals; (b) Increased AMPK-α2 activity in aortic tissues from patients with AAAs. N is 6. # P < 0.05 compared to non-aaa; (c) cigarette smoking increased the phosphorylations of both AMPK Thr172 and ACC Ser79 and AMPK-α2 activity (d) in the plasma. The blot is a representive blot obtained from six blots. # P < 0.05 compared to non-smoking. The P values were obtained by t-test between two groups. All error bars are s.e.m. 12

Supplementary Figure 12 A proposed schem of AngII/nicotine-induced AAA formation via AMPK-α2 and AP-2α in mice.

NADPH oxidase produces a lot of reactive oxygen species (O 2 -, ONOO -, H 2 O 2, etc.

In nucleus, AMPK-α2 binds to AP-2α and phosphorylates AP-2α at serine 219, resulting in the formation of AMPK-α2/AP-2α/MMP2 promoter complex.

13 Supplementary Figure 12 A proposed schem of AngII/nicotine-induced AAA formation via AMPK-α2 and AP-2α in mice. AngII/nicotine binds to its receptors in vascular smooth muscle cell to activate G-protein, which further activates NADPH oxidase via unknown mechanisms. NADPH oxidase produces a lot of reactive oxygen species (O 2 -, ONOO -, H 2 O 2, etc.) and secretions of cytokines (IFN-γ, TNF-α, CypA, etc) to phosphorylate AMPK-α at Thr172, which triggers the nuclear translocation of AMPK-α2 from cytoplasm. In nucleus, AMPK-α2 binds to AP-2α and phosphorylates AP-2α at serine 219, resulting in the formation of AMPK-α2/AP-2α/MMP2 promoter complex. Activated AP-2α triggers the gene transcription of MMP2, and subsequent protein synthesis, MMP2 secretion to outside of cells and cleaved to active MMP2 by MT1-MMP. In vascular walls, active MMP2 degrades extracellular matrix, which weakens the resistant of vascular wall to blood flow, causing the formation of aortic aneurysm. 13

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Supplementary Figure 1. ERK signaling is not activated at early hypertension. a, Western blot analysis for the level of phospho-erk (perk) and total

Supplementary Figure 1. ERK signaling is not activated at early hypertension. a, Western blot analysis for the level of phospho-erk (perk) and total ERK in the aortic tissue from the saline- or AngII-infused