Circulating and Tissue Levels of Matrix Metalloproteinases in Experimental Abdominal Aortic Aneurysms Exacerbated by Chronic Tobacco Exposure

Amy Hackmann, MD, Michel Bergoeing, MD, Batool Arif, Terri Ennis, Robert W. Thompson, MD and John A. Curci, MD
Washington University School of Medicine, St. Louis, MO

Objectives: Clinically, there is a very strong correlation between cigarette smoking and abdominal aortic aneurysm (AAA) disease. Based on the similarities in matrix changes between AAA and pulmonary emphysema, it has been speculated that there may be a prominent role for elastolytic proteases, particularly matrix metalloproteases (MMP). Recently we described a mouse model of AAA which is exacerbated by chronic tobacco smoke. We hypothesized that increased production of MMP-9 and MMP-12 by infiltrating inflammatory cells may be responsible for the larger aneurysms in smoke exposed mice. In our model of AAA, we examined the expression of these enzymes in the aortas and peripheral blood of control mice and mice exposed to cigarette smoke.
Methods: Male 129 SvEv mice were pre-conditioned with cigarette smoke (N=16) or placed in control cages (N=8) for four weeks prior to inducing aortic aneurysm development with elastase perfusion. The mice were maintained in smoking or control environments for an additional 8 weeks. At the time of sacrifice, aortic diameter measurements were made in vivo and the change in pre-perfusion to harvest diameter calculated (ΔAD). The aorta was harvested and blood was collected using the cardiac puncture technique. The RNA was extracted from circulating leukocytes (Smokers: N=6, control: N=3) and aorta (Smokers: N=6, control: N=4), and processed for real-time PCR. Expression of MMP-9 and MMP-12 was normalized to GAPDH and expressed as arbitrary units (mean ± standard error). Representative aortic specimens were formalin fixed and stained using elastin-specific stain.
Results: All mice had developed AAA at sacrifice, although smokers exhibited significantly larger aneurysms than non-smoking controls (ΔAD = 171.88 ± 27.62% vs. 116.25 ± 19.96%, p<0.0001). There were no significant differences in MMP-9 expression (see Figure) between smokers and controls in blood (9.29x10^-2 ± 5.28x10^-3 vs. 2.51x10^-1 ± 8.67x10^-2, p=0.6) or aortic wall (7.43x10^-3 ± 2.40x10^-3 vs. 6.48x10^-3 ± 1.97x10^-3, p=0.8). There was also no significant difference in MMP-12 expression in circulating leukocytes (1.52x10^-2 ± 9.77x10^-3 vs. 6.65x10^-3 ± 1.44x10^-3, p=0.6) or in aortic wall in the smoke exposed animals (1.05 ± 0.366 vs. 0.393 ± 0.146, p=0.1). Although there was a trend toward increased MMP-12 in smokers in both tissues, the data was highly variable. There was no correlation between enzyme expression and ΔAD, and no gross difference in histology between the two groups was found.
Conclusions: In this model of AAA and smoking, increased expression of MMP-9 and MMP-12 does not appear central to the deleterious effects of smoke exposure on AAA growth. Other mechanisms may play a more important role in smoke-mediated aneurysm exacerbation than increased elastolytic enzyme expression.