Collagen degradation in the abdominal aneurysm: a conspiracy of matrix metalloproteinase and cysteine collagenases

Abstract

Growth and rupture of abdominal aortic aneurysms (AAAs) result from increased collagen turnover. Collagen turnover critically depends on specific collagenases that cleave the triple helical region of fibrillar collagen. As yet, the collagenases responsible for collagen degradation in AAAs have not been identified. Increased type I collagen degradation products confirmed collagen turnover in AAAs (median values: <1, 43, and 108 ng/mg protein in control, growing, and ruptured AAAs, respectively). mRNA and protein analysis identified neutrophil collagenase [matrix metalloproteinase (MMP)-8] and cysteine collagenases cathepsin K, L, and S as the principle collagenases in growing and ruptured AAAs. Except for modestly increased MMP-14 mRNA levels, collagenase expression was similar in growing and ruptured AAAs (anterior-lateral wall). Evaluation of posttranslational regulation of protease activity showed a threefold increase in MMP-8, a fivefold increase in cathepsins K and L, and a 30-fold increase in cathepsin S activation in growing and ruptured AAAs. The presence of the osteoclastic proton pump indicated optimal conditions for extracellular cysteine protease activity. Protease inhibitor mRNA expression was similar in AAAs and controls, but AAA protein levels of cystatin C, the principle cysteine protease inhibitor, were profoundly reduced (>80%). We found indications that this secondary deficiency relates to cystatin C degradation by (neutrophil-derived) proteases.

Figure 1

Collagen degradation (CTX assay for collagen type I degradation) in aortic wall samples of control aorta, growing AAAs, and ruptured aneurysms (ruptured AAAs). *P < 0.02; ^#P < 0.001.

Figure 2

A: Relative mRNA expression (GAPDH = 1) of MMP collagenases and the gelatinase MMP-9 in the infrarenal aorta of controls, growing AAAs, and ruptured aneurysms. *P < 0.05 versus controls. B: Relative mRNA expression (GAPDH = 1) of the cysteine collagenases in the infrarenal aorta of controls, growing AAAs, and ruptured AAAs. *P < 0.05 versus controls.

Figure 3

Immunohistochemical staining of MMP-8 (top) and the osteoclastic proton pump v-H⁺-ATPase (bottom) in normal control aorta, AAA, and ruptured AAA. MMP-8 is expressed in infiltrating neutrophils, whereas v-H⁺-ATPase is primarily expressed in monocytes/macrophages and to a lesser extent in smooth muscle cells.

Figure 4

Increased expression of the activated forms of the collagenases MMP-8, cathepsin K, L (24- and 28-kd bands1), and S in growing AAAs (light gray boxes) and ruptured aneurysms (dark gray boxes) compared with control aorta (white boxes). *P < 0.05 versus controls.

Figure 5

Relative protein expression of the principal inhibitor of MMP activity (TIMP-1) and of cystatin C, the principal inhibitor of cysteine protease activity in control infrarenal aorta (white boxes), growing AAAs (light gray boxes), and ruptured AAAs (dark gray boxes). *P < 0.05 versus controls; ⁺P < 0.05 AAA versus ruptured AAAs.

Figure 6

Cystatin C degradation by neutrophil proteases MMP-8, MMP-9, and neutrophil elastase in vitro. Cystatin C and respective proteases were incubated for 24 hours in a 100:1 mol/mol ratio.