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Naoki Fujimura, MD, PhD, Jiang Xiong, MD, PhD, Ellen B

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1 Metformin treatment status and abdominal aortic aneurysm disease progression 
Naoki Fujimura, MD, PhD, Jiang Xiong, MD, PhD, Ellen B. Kettler, BS, Haojun Xuan, MD, Keith J. Glover, MD, Matthew W. Mell, MD, MPH, Baohui Xu, MD, PhD, Ronald L. Dalman, MD  Journal of Vascular Surgery  Volume 64, Issue 1, Pages e8 (July 2016) DOI: /j.jvs Copyright © 2016 Society for Vascular Surgery Terms and Conditions

2 Fig 1 Abdominal aortic aneurysm (AAA) enlargement as a function of demographic and environmental risk factors. The median rate of AAA enlargement for diabetic patients not receiving oral hypoglycemic therapy was 1.5 mm/y. Nominal logistic regression was used to calculate odds ratios (ORs) for individual variables. All data are ORs with the 95% confidence interval. Likelihood ratio test, .05 < #P < .1, ∗P < .05, and ∗∗P < .01. ACE, Angiotensin-converting enzyme; ARBs, angiotensin II type 1 receptor blockers; BMI, body mass index. Journal of Vascular Surgery  , e8DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

3 Fig 2 Effect of metformin treatment on experimental abdominal aortic aneurysm (AAA). AAAs were created through intra-aortic porcine pancreatic elastase (PPE) infusion in 10- to 12-week old male C57BL/6J mice. These mice were treated with either metformin in vehicle (250 mg/kg/d; n = 10 mice) or vehicle alone (PBS; n = 7 mice) beginning 3 days before PPE infusion for a total of 17 days. Aneurysm formation and progression were monitored by ultrasound in vivo. A, Representative ultrasound images before and 14 days after PPE infusion in metformin- and vehicle-treated mice. B, Mean and standard error of aortic diameters before and indicated days after AAA creation in both treatment groups. Two-way analysis of variance, ∗P < .05 and ∗∗P < .01 compared with vehicle group. C, Influence of metformin treatment on AAA incidence. An AAA is defined as a 50% or greater increase over the baseline diameter. Kaplan-Meier test, ∗P < .05 compared with vehicle group. Journal of Vascular Surgery  , e8DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

4 Fig 3 Representative histology from metformin- and vehicle-treated, porcine pancreatic elastase (PPE)-infused mice. Aortic frozen sections were prepared 14 days after abdominal aortic aneurysm (AAA) creation. Elastin integrity was assessed by the Verhoeff-van Gieson (EVG) stain. Mural macrophages and angiogenesis were stained with monoclonal antibodies against CD68 and CD31, respectively, by immunohistochemical staining. Representative images are shown. n = 7 and 10 mice in vehicle and metformin groups, respectively. Original magnification ×200. Journal of Vascular Surgery  , e8DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

5 Fig 4 Effects of metformin treatment on medial elastin and smooth muscle cell (SMC) integrity and mural inflammation in experimental abdominal aortic aneurysm (AAA). Aortic frozen sections from metformin-treated (n = 10) and vehicle-treated (n = 7) mice 14 days after porcine pancreatic elastase (PPE) infusion were stained for elastin (Verhoeff-van Gieson [EVG]), SMC (SMC α-actin), leukocyte subsets (CD68 for macrophages, CD4 for CD4 T cells, CD8 for CD8 T cells, B220 for B cells), and CD31 for angiogenesis. Elastin degradation, SMC depletion, and mural macrophage infiltration were graded as score I (mild) to score IV (severe). Mural CD4 T cells, CD8 T cells, B cells, and angiogenesis were quantified as positive cells or blood vessels per aortic cross section (ACS). Nonparametric Mann-Whitney test, ∗P < .05 and ∗∗P < .01 compared with vehicle treatment group. Journal of Vascular Surgery  , e8DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

6 Supplementary Fig 1 (online only)
There was no statistically significant correlation between aneurysm enlargement rate and follow-up years. Linear regression, r = 0.05; P = .70; N = 58. Journal of Vascular Surgery  , e8DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

7 Supplementary Fig 2 (online only)
Influence of metformin use on aneurysm enlargement rate after stratification for the presence or absence of each aneurysm risk factor. The numbers in each of the parentheses indicate the cases of patients with and without metformin use, respectively. All data are mean plus standard error. .05 < #P < .1, ∗P < .05, and ∗∗P < .01 compared with no metformin use group. BMI, Body mass index. Journal of Vascular Surgery  , e8DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

8 Supplementary Fig 3 (online only)
Influence of metformin use on aneurysm enlargement rate after stratification for the presence or absence of each comorbidity. The numbers in each of the parentheses indicate the cases of patients with and without metformin use, respectively. All data are mean plus standard error. .05 < #P < .1, ∗P < .05, and ∗∗P < .01 compared with no metformin use group. Journal of Vascular Surgery  , e8DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

9 Supplementary Fig 4 (online only)
Influence of metformin use on aneurysm enlargement rate after stratification for the presence or absence of each medication. The numbers in each of the parentheses indicate the cases of patients with and without metformin use, respectively. All data are mean plus standard error. .05 < #P < .1 and ∗P < .05 compared with no metformin use group. ACE, Angiotensin-converting enzyme; ARBs, angiotensin II type 1 receptor blockers. Journal of Vascular Surgery  , e8DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

10 Supplementary Fig 5 (online only)
Influence of metformin use on aneurysm enlargement rate after stratification for three major aneurysm risk factors (age, gender, and smoking status or age, gender, and obesity). The numbers in each of the parentheses indicate the cases of patients with and without metformin use, respectively. All data are mean plus standard error. ∗P < .05 compared with no metformin use group. BMI, Body mass index. Journal of Vascular Surgery  , e8DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

11 Supplementary Fig 6 (online only)
Influence of statin use on aneurysm enlargement rate after stratification for major aneurysm risk factors, comorbidity, and medications. The numbers in each of the parentheses indicate the cases of patients with and without statin use, respectively. All data are mean plus standard error. .05 < #P < .1 and ∗P < .05 compared with no statin use group. ACE, Angiotensin-converting enzyme; ARB, angiotensin II type 1 receptor blocker; BMI, body mass index. Journal of Vascular Surgery  , e8DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

12 Supplementary Fig 7 (online only)
Influence of metformin treatment on body weight gain and nonfasting glucose level. Male C57BL/6J mice at 10 to 12 weeks of age were treated with metformin (250 mg/kg/d; n = 10 mice) or vehicle (phosphate-buffered saline; n = 7 mice) through oral gavage beginning 3 days before porcine pancreatic elastase (PPE) infusion for a total of 17 days. Body weight and nonfasting glucose level (9:00-10:00 am) were measured before metformin or vehicle treatment (baseline level) and days 3, 7, and 14 after PPE infusion. Changes in body weight and nonfasting glucose level were presented as the percentage of the baseline level. All data are mean plus standard error. Two-way analysis of variance followed by Bonferroni post-test, ∗∗P < .01 compared with vehicle treatment group. Journal of Vascular Surgery  , e8DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

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