A potential key mechanism in ascending aortic aneurysm development: Detection of a linear relationship between MMP-14/TIMP-2 ratio and active MMP-2

Abstract

Objectives: Elevated matrix metalloproteinase-2 (MMP-2) tissue levels have been associated with ascending thoracic aortic aneurysm (aTAA). As MMP-2 activation is controlled by interactions among matrix metalloproteinase-14 (MMP-14), a tissue inhibitor of metalloproteinases-2 (TIMP-2) and Pro-MMP-2 in cell culture, this activation process might also play a role in aTAA.

Methods: Via gelatin zymography we analyzed tissue levels of MMP-2 isoforms (Pro-MMP-2, active MMP-2, total MMP-2) and via enzyme-linked immunosorbent assay (ELISA,) MMP-14,TIMP-2 and total MMP-2 tissue levels in N = 42 patients with aTAA. As controls, MMP-14 and TIMP-2 aortic tissue levels in N = 9 patients undergoing coronary artery bypass surgery were measured via ELISA, and levels of MMP-2 isoforms in N = 11 patients via gelatin zymography.

Results: Active MMP-2 was significantly higher in aTAA than in controls. Patients with aTAA exhibited significantly lower Pro-MMP-2 and TIMP-2 levels. Total MMP-2 and MMP-14 did not differ significantly between groups. Regression analysis revealed a linear relationship between TIMP-2 and the MMP-14/TIMP-2 ratio, as well as active MMP-2 in aTAA. Aneurysmatic tissue can be accurately distinguished from control aortic tissue (AUC = 1) by analyzing the active MMP-2/Pro-MMP-2 ratio with a cutoff value of 0.11, whereas MMP-14 and TIMP-2 roles are negligible in ROC analysis.

Conclusion: A larger amount of MMP-2 is activated in aTAA than in control aortic tissue-a factor that seems to be a central process in aneurysm development. When active MMP-2 exceeds 10% compared to Pro-MMP-2, we conclude that it originates from aneurysmatic tissue, which we regard as a starting point for further studies of aTAA biomarkers. The tissue's MMP-14/TIMP-2 ratio may regulate the degree of Pro-MMP-2 activation as a determining factor, while the enzymatic activities of MMP-14 and TIMP-2 do not seem to play a key role in aneurysm development.

Fig 1

Representative zymograms of A. 8 patients (P1-8) with ascending aortic aneurysm and B. 6 control patients (C1-6) undergoing coronary bypass surgery (CABG). Lane 1: protein ladder. Lane 2: human full length MMP-2. Lane A. 3–10 and B. 3–8: tissue samples zymograms showing different gelatinolytic activities corresponding to Pro-MMP-2 and active MMP-2.

Fig 2. Results of zymography.

Comparison between aneurysm and control group (A) Pro-MMP-2, (B) active MMP-2, (C) total MMP-2 reveals significantly larger amounts of active MMP-2 and significant smaller amounts of Pro-MMP-2 in the aneurysm group, whereas total MMP-2 did not differ significantly between the two groups (MMP-2 isoforms given in AU).

Fig 3. Results of ELISA.

Comparison between aneurysm and control group (D) MMP-14 and (E) TIMP-2 shows significant lower amounts of TIMP-2 in the aneurysm group whereas MMP-14 did not differ significantly between the two groups (MMP-14 and TIMP-2 given in ng/mL).

Fig 4. ROC curve of active MMP-2/Pro-MMP-2 ratio.

The AUC (A) analyzing active MMP-2/Pro-MMP-2 ratio with a cutoff value of 0.11 illustrates perfect discrimination (AUC = 1) of aneurysmatic from control tissue.