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

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.

Determination of aspartic protease gene dosage in the Onchocerca volvulus genome

Aspartic proteases are a relatively small group of enzymes which express in various nematodes including Onchocerca volvulus. An estimation of the gene copy number corresponding to the OV7A clone, which contains a cDNA insert encoding approximately two-thirds of the entire coding sequence of aspartic protease of O. volvulus, was made by slot blot analysis in a closely related species O. gibsoni genome. Nylon membrane was loaded with serial dilutions of genomic DNA alongside the OV7A plasmid DNA before hybridizing the membrane to that [32]P-labeled cDNA insert. To prepare the initial probe, OV7A cDNA insert was amplified using gene-specific primers. By comparing the signal intensity of slot blot hybridization of known amounts of genomic DNA and plasmid DNA containing the cDNA insert under similar conditions, the abundance of sequence homologues to the [32]P-labeled cDNA insert in the genome was calculated. For confirmation, southern blot analysis was performed by digesting genomic DNA with a panel of different restriction enzymes. Hybridizing patterns of the same probe revealed a single band except when predicted internal restriction sites were affected. It was confirmed that Onchocerca contains a single copy of the gene corresponding to this cDNA insert per haploid genome