Quantitative expression and localization of cysteine and aspartic proteases in human abdominal aortic aneurysms

Abstract

Cysteine and aspartic proteases possess high elastolytic activity and might contribute to the degradation of the abdominal aortic aneurysm (AAA) wall. The aim of this study was to analyze, in detail, the proteases (cathepsins B, D, K, L and S, and inhibitor cystatin C) found in human AAA and healthy aortic tissue samples. The vessel walls from AAA patients (n=36) and nonaneurysmal aortae (n=10) were retrieved using conventional surgical repair and autopsy methods. Serum samples from the same AAA patients and 10 healthy volunteers were also collected. Quantitative expression analyses were performed at the mRNA level using real-time reverse transcriptase-PCR (RT-PCR). Furthermore, analyses at the protein level included western blot and immunoprecipitation analyses. Cellular sources of cysteine/aspartic proteases and cystatin C were identified by immunohistochemistry (IHC). All cysteine/aspartic proteases and cystatin C were detected in the AAA and control samples. Using quantitative RT-PCR, a significant increase in expression was observed for cathepsins B (P=0.021) and L (P=0.018), compared with the controls. Cathepsin B and cystatin C were also detected in the serum of AAA patients. Using IHC, smooth muscle cells (SMCs) and macrophages were positive for all of the tested cathepsins, as well as cystatin C; in addition, the lymphocytes were mainly positive for cathepsin B, followed by cathepsins D and S. All cysteine/aspartic proteases analyzed in our study were detected in the AAA and healthy aorta. The highest expression was found in macrophages and SMCs. Consequently, cysteine/aspartic proteases might play a substantial role in AAA.

Figure 1

Expression of cathepsins B, D, K, L and S, cystatin C, MMP-2 and -9 and TIMP-1 at the mRNA level in the AAA tissue samples compared with the control healthy aorta samples, as analyzed by quantitative real-time PCR and SYBR green fluorescence dye; the expression levels were normalized to GAPDH. AAA, abdominal aortic aneurysm; Cath, cathepsin; CysC, cystatin C; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP, matrix metalloprotease; TIMP-1, tissue inhibitor of metalloproteinase 1.

Figure 2

Ratio of the mRNA expression of cathepsins B, D, K, L and S against cystatin C and of MMP-2 and -9 to TIMP-1 in the AAA tissue samples compared with the control healthy aorta samples, as analyzed by quantitative real time PCR and SYBR green fluorescence dye, as shown in Figure 1. AAA, abdominal aortic aneurysm; Cath, cathepsin; CysC, cystatin C; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP, matrix metalloprotease; TIMP-1, tissue inhibitor of metalloproteinase 1.

Figure 3

Expression of cathepsins B, D, K, L and S and cystatin C at the protein level in AAA tissue samples, as analyzed by western blot (a), and the summary of quantitative analysis of the band intensity of the single proteinases and their inhibitor cystatin C (b). Fresh tissues samples were used. The intensities of the bands following blotting and chemiluminescence detection were normalized to GAPDH. Expression was adjusted to that of cystatin C, which was set as 100%. AAA, abdominal aortic aneurysm; Cath, cathepsin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. **P<0.01, ***P<0.001.

Figure 4

Immunoprecipitation of cathepsin B and cystatin C from the blood serum of AAA patients and healthy volunteers, as analyzed by western blot (a) and the quantitative analysis of the band intensities for cathepsin B and cystatin C, as well as their ratio (b). AAA, abdominal aortic aneurysm; Cath, cathepsin; CysC, cystatin C. **P<0.01.

Figure 5

Selective immunohistochemical staining of cathepsins B, D, K, L and S and cystatin C in smooth muscle cells (a), inflammatory cells (lymphocytes) (b) and neovessels (c) within the human abdominal aortic aneurysm (AAA) tissues samples. Cathepsin and cystatin C staining is brown, and the cells are counterstained with hematoxylin and eosin, shown in blue.