Comparative analysis of arterial compliance in mice genetically null for cathepsins K, L, or S.

Cysteine cathepsins are potent proteases implicated in cardiovascular disease for degrading extracellular matrix (ECM) whose structure and integrity determine the mechanical behavior of arteries. Cathepsin knockout mouse models fed atherogenic diets have been used to study their roles in cardiovascular disease, but the impacts of cathepsin knockout on non-atherosclerotic arterial mechanics are scarce. We examine arterial mechanics in several cathepsin knockout mouse lines (CatK, CatLApoE and CatSApoE) and controls (C57/Bl6, apolipoprotein E).

Human cathepsins K, L, and S: Related proteases, but unique fibrinolytic activity.

Background: Fibrin formation and dissolution are attributed to cascades of protease activation concluding with thrombin activation, and plasmin proteolysis for fibrin breakdown. Cysteine cathepsins are powerful proteases secreted by endothelial cells and others during cardiovascular disease and diabetes. Their fibrinolytic activity and putative role in hemostasis has not been well described.

Computational predictions of cysteine cathepsin-mediated fibrinogen proteolysis.

Fibrin clot formation is a proteolytic cascade of events with thrombin and plasmin identified as the main proteases cleaving fibrinogen precursor, and the fibrin polymer, respectively. Other proteases may be involved directly in fibrin(ogen) cleavage, clot formation, and resolution, or in the degradation of fibrin-based scaffolds emerging as useful tools for tissue engineered constructs. Here, cysteine cathepsins are investigated for their putative ability to hydrolyze fibrinogen, since they are potent proteases, first identified in lysosomal protein degradation and known to participate in extracellular proteolysis.

Multiplex Cathepsin Zymography to Detect Amounts of Active Cathepsins K, L, S, and V.

Cysteine cathepsins are powerful proteases that can degrade other proteins, among which are the extracellular matrix proteins collagen and elastin. Multiplex cathepsin zymography is an assay that can quantify the amount of active cathepsins in a cell or tissue preparation. This method works for measuring the amounts of active cathepsins K, L, S, and V in a cell or tissue preparation without requiring the use of antibodies for specific identification which tremendously reduces cost.

Systematic optimization of multiplex zymography protocol to detect active cathepsins K, L, S, and V in healthy and diseased tissue: compromise among limits of detection, reduced time, and resources.

Cysteine cathepsins are a family of proteases identified in cancer, atherosclerosis, osteoporosis, arthritis, and a number of other diseases. As this number continues to rise, so does the need for low cost, broad use quantitative assays to detect their activity and can be translated to the clinic in the hospital or in low resource settings. Multiplex cathepsin zymography is one such assay that detects subnanomolar levels of active cathepsins K, L, S, and V in cell or tissue preparations observed as clear bands of proteolytic activity after gelatin substrate SDS-PAGE with conditions optimal for cathepsin renaturing and activity.

Patient specific proteolytic activity of monocyte-derived macrophages and osteoclasts predicted with temporal kinase activation states during differentiation.

Patient-to-patient variability in disease progression continues to complicate clinical decisions of treatment regimens for cardiovascular diseases, metastatic cancers and osteoporosis. Here, we investigated if monocytes, circulating white blood cells that enter tissues and contribute to disease progression by differentiating into macrophages or osteoclasts, could be useful in understanding this variability. Monocyte-derived macrophages and osteoclasts produce cysteine cathepsins, powerful extracellular matrix proteases which have been mechanistically linked to accelerated atherosclerotic, osteoporotic, and tumor progression.

Tumor necrosis factor alpha stimulates cathepsin K and V activity via juxtacrine monocyte-endothelial cell signaling and JNK activation.

Inflammation and damage promote monocyte adhesion to endothelium and cardiovascular disease (CVD). Elevated inflammation and increased monocyte-endothelial cell interactions represent the initial stages of vascular remodeling associated with a multitude of CVDs. Cathepsins are proteases produced by both cell types that degrade elastin and collagen in arterial walls, and are upregulated in CVD.

Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues.

Cathepsins K, L, S, and V are cysteine proteases that have been implicated in tissue-destructive diseases such as atherosclerosis, tumor metastasis, and osteoporosis. Among these four cathepsins are the most powerful human collagenases and elastases, and they share 60% sequence homology. Proper quantification of mature, active cathepsins has been confounded by inhibitor and reporter substrate cross-reactivity, but is necessary to develop properly dosed therapeutic applications.

Multiplex zymography captures stage-specific activity profiles of cathepsins K, L, and S in human breast, lung, and cervical cancer.

Background: Cathepsins K, L, and S are cysteine proteases upregulated in cancer and proteolyze extracellular matrix to facilitate metastasis, but difficulty distinguishing specific cathepsin activity in complex tissue extracts confounds scientific studies and employing them for use in clinical diagnoses. Here, we have developed multiplex cathepsin zymography to profile cathepsins K, L, and S activity in 10 μg human breast, lung, and cervical tumors by exploiting unique electrophoretic mobility and renaturation properties.

Methods: Frozen breast, lung, and cervix cancer tissue lysates and normal organ tissue lysates from the same human patients were obtained (28 breast tissues, 23 lung tissues, and 23 cervix tissues), minced and homogenized prior to loading for cathepsin gelatin zymography to determine enzymatic activity.