Cigarette smoking increases aortic dilatation without affecting matrix metalloproteinase- 9 and -12 expression in a modified mouse model of aneurysm formation

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Cigarette smoking increases aortic dilatation without affecting matrix metalloproteinase- 9 and -12 expression in a modified mouse model of aneurysm formation Michel P. Bergoeing, MD, Batool Arif, BS, Amy E. Hackmann, MD, Terri L. Ennis, BS, Robert W. Thompson, MD, John A. Curci, MD Journal of Vascular Surgery Volume 45, Issue 6, Pages 1217-1227.e2 (June 2007) DOI: 10.1016/j.jvs.2007.01.058 Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 1 A-C, Effect of varying doses of elastase on aortic aneurysm (AAA) dilatation. C57BL/6 mice underwent transient aortic perfusion with a solution of high-dose (HDE), standard-dose (SDE) or low-dose active elastase (LDE). Heat-inactivated elastase (HIE) was used as a control. A, Immediate aortic dilatation, B, interval aortic dilatation, and C, overall aortic dilatation. Comparisons were calculated with one-way analysis of variance and the Tukey multiple comparisons test. D-F, Effect of varying incubation time (postperfusion to harvest) in LDE-perfused mice. C57BL/6 mice were subjected to transient aortic perfusion with a solution of LDE. Mice were then harvested at the standard 14-day time-point or 21 days after perfusion (LDE D21). D, Immediate aortic dilatation, E, interval aortic dilatation, and F, and overall aortic dilatation. Comparisons were calculated with unpaired t test assuming unequal variances. NS, Not significant. Journal of Vascular Surgery 2007 45, 1217-1227.e2DOI: (10.1016/j.jvs.2007.01.058) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 2 Effect of smoking on abdominal aortic aneurysm (AAA) dilatation. C57BL/6 mice underwent transient aortic perfusion with a solution of standard-dose (SDE) or low-dose active elastase (LDE). SDE mice (n = 14) and LDE mice (n = 20) were exposed to cigarette smoke for 2 weeks before surgery and then continued smoking until aortic harvest. A-C, Effect of smoking on SDE-perfused mice. A, Immediate aortic dilatation, B, interval aortic dilatation, and C, overall aortic dilatation. Comparisons were calculated with unpaired t test assuming unequal variances. D-F, Effect of smoking on LDE-perfused mice. D, Immediate aortic dilatation, E, interval aortic dilatation, and F, overall aortic dilatation. Statistical comparisons were calculated on the mean change in aortic size with the unpaired t-test assuming unequal variances. NS, Not significant. Journal of Vascular Surgery 2007 45, 1217-1227.e2DOI: (10.1016/j.jvs.2007.01.058) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 3 A and B, Effect of varying doses elastase on matrix metalloproteinase (MMP) expression. C57BL/6 mice underwent transient aortic perfusion with a solution of standard-dose (SDE) or low-dose active elastase (LDE). Heat-inactivated elastase (HIE) was used as control. At harvest aortic tissue was promptly processed for messenger RNA (mRNA) extraction. A, MMP-9 expression. B, MMP-12 expression. Results were normalized to log base 10 for analysis and differences were calculated with one-way analysis of variance and Tukey-Kramer multiple comparison post test. C and D, Effect of smoking on MMP expression. C57BL/6 mice were subjected to transient aortic perfusion with a solution of SDE or LDE. SDE mice (n = 14) and LDE mice (n = 20) were exposed to cigarette smoke for 2 weeks before surgery and then continued smoking until aortic harvest. At harvest aortic tissue was promptly processed for mRNA extraction. C, MMP-9 expression in SDE and LDE mice. D, MMP-12 expression in SDE and LDE mice. Results were normalized to log base 10 for analysis, and differences were calculated with unpaired t test assuming unequal variances. NS, Not significant. Journal of Vascular Surgery 2007 45, 1217-1227.e2DOI: (10.1016/j.jvs.2007.01.058) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 4 Murine aortic wall histology. A–F, Staining of aortic sections was performed with Verhoeff-van Gieson stain to emphasize the elastic fibers in the tissue. Aortas shown in panels A, B, and C were all perfused with low dose elastase and panels D and E were perfused with standard-dose elastase (SDE). Panel F was perfused with SDE that underwent heat inactivation. Panel G plots the mean ± standard error of the blinded grading of sections from low-dose elastase (LDE) perfused aortas. Both elastin and inflammation were graded on a scale from 0 to 4, with higher numbers representing greater amounts of elastin or inflammation, respectively. Samples in A and D were fixed immediately after perfusion and demonstrate intact elastin fibers and no appreciable cellular infiltrate. D, After 2 weeks, marked inflammatory infiltration and severe elastin damage is seen in SDE-perfused animals. B, Relatively modest elastin damage is seen 2 weeks after LDE perfusion, despite a significant inflammatory infiltrate. C and G, There is significantly more inflammation and elastin degradation in LDE-perfused animals that are also exposed to cigarette smoke. All photomicrographs are high-power views (original magnification ×100), and insets are at low-power view (original magnification ×20). *P < .001 compared with day 0; #P < .001 compared with control. Journal of Vascular Surgery 2007 45, 1217-1227.e2DOI: (10.1016/j.jvs.2007.01.058) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions