Increased Expression of Elastolytic Cathepsins S, K, and V and Their Inhibitor Cystatin C in Stenotic Aortic Valves

Abstract

Objective-To investigate the possible role of elastolytic cathepsins S, K, and V and their endogenous inhibitor cystatin C in adverse extracellular matrix remodeling of stenotic aortic valves. Methods and Results-Stenotic aortic valves were collected at valve replacement surgery and control valves at cardiac transplantations. The expression of cathepsins S, K, and V and cystatin C was studied by conventional and real-time polymerase chain reaction and by immunohistochemistry. Total cathepsin activity in the aortic valves was quantified by a fluorometric microassay. When compared with control valves, stenotic valves showed increased mRNA expression of cathepsins S, K, and V (PϽ0.05 for each) and a higher total cathepsin activity (PϽ0.001). In stenotic valves, cystatin C mRNA was increased (PϽ0.05), and cystatin C protein was found particularly in areas with infiltrates of inflammatory cells. Both cathepsin S and cystatin C were present in bony areas of the valves, whereas cathepsin V localized to endothelial cells in areas rich of neovascularization. Incubation of thin sections of aortic valves with cathepsins S, K, and V resulted in severe disruption of elastin fibers, and this cathepsin effect could be blocked by adding cystatin C to the incubation system. Key Words: aortic stenosis Ⅲ cathepsin Ⅲ cystatin C Ⅲ elastin Ⅲ heart valves A ortic stenosis is an active inflammatory process resembling atherosclerosis. The pathophysiological changes in the stenotic aortic valves include inflammatory cell infiltration, neoangiogenesis, accumulation of lipids, and active mediators of calcification with extracellular matrix (ECM) degradation. 1-7 Furthermore, aortic valvular myofibroblasts undergo phenotypic changes toward an osteoblast-like phenotype, and active bone formation and remodeling occur in the stenotic valves. 8,9 These processes lead to loss of normal valvular geometry, disruption of the elastic fibers, and accumulation of fibrocalcific masses. This adverse remodeling of the valves is associated with the loss of their mechanical and elastic properties, leading to thickening and stiffening of the valve leaflets and to progression of aortic stenosis. Conclusions-StenoticThe pathophysiological mechanisms involved in the abnormal accumulation and degradation of ECM in the valves are incompletely understood. In atherosclerotic lesions, elastolytic cysteine proteases, including cathepsins S and K, are overexpressed at sites of arterial elastin damage, whereas their endogenous inhibitor, cystatin C, is severely reduced. 10,11 Moreover, a marked deficiency of cystatin C expression with accentuated cathepsin S and K expression in aortic aneurysms and a negative correlation of cystatin C serum levels with the diameter of abdominal aorta has been reported. 11 These observations support the role of an imbalance between cathepsins and cystatin C in diseases characterized by pathological elastin degradation. In addition, cathepsin K has a crucial role in bone matrix degradation necessary for normal bone growth and remodeling...

Cardiovascular Effects of Exercise Training

“…93,94 Using low-density lipoprotein receptor knockout mice, we recently demonstrated that regular exercise training as primary prevention prevents the development of aortic valve sclerosis. 95 It has been proposed that apoptosis of endothelial cells and myofibroblasts in the valve, 96 elastin degradation and collagen synthesis by MMPs and/or cathepsin, 97 increased oxidative stress, 98 and enhanced osteogenesis 99 contribute to aortic valve degeneration. In consequence, procalcific gene expression such as bone morphogenetic protein 2 (BMP2), CBFA1/Runx2 (runt-related transcription factor 2), and osteocalcin was amplified.…”

Role for Cysteine Protease Cathepsins in Heart Disease

“…Although cathepsins are abundantly present in human and animal cardiac wall 22 and valve tissues, 25,26,79 the exact role each specific cathepsin plays in heart disease development and the mechanism and significance behind their function are largely unknown.…”

“…This hypothesis has been confirmed in human stenotic aortic valves, which contain much greater amounts of cathepsin S, K, and V mRNAs and proteins than controls; moreover, levels of enzymatically active cathepsins are significantly higher than in controls. 25 Recent ex vivo work demonstrates that cyclic stretch increases cathepsins S, K, and L in porcine valves, accelerating the destruction of aortic valvular ECM and the progression of aortic stenosis. 27 Stenotic aortic valves are characterized by atherosclerosis-like lesions containing activated inflammatory cells, calcified nodules, and bone tissue.…”

“…21 Most strikingly, we have now discovered that these cathepsins can be secreted into and function within the extracellular spaces. The observations of cathepsin expression and activity in failing cardiac tissues [22][23][24] and valve tissues [25][26][27] from humans and animals and in cultured media of cardiomyocytes, 22,24 cardiac fibroblasts, 22 vascular smooth muscle cells, 28 endothelial cells, 12 and macrophages 6,10 have significantly broadened our understanding of their potential roles in cardiovascular pathogenesis. Furthermore, recent studies have shown that pharmacological cathepsin inhibition exhibits cardiovascular protective actions in animal models.…”

Cholate-Containing High-Fat Diet Induces the Formation of Multinucleated Giant Cells in Atherosclerotic Plaques of Apolipoprotein E−/− Mice

“…Cathepsin V represents the most potent mammalian elastase, and its expression has been demonstrated in aortic extracts. 35,40 In vitro incubation of cryostat sections of human aortic valves with recombinant human cathepsins K, S, and V at a pH of 6.5 revealed that cathepsin S reduced the valvular elastin content from 45% to 25%; cathepsins K and V reduced the elastin content to 33% and 34%, respectively. These data represent the potential contributions of these cathepsins to the extracellular elastin degradation that is biased toward cathepsin S because a pH of 6.5 is optimal for cathepsin S. 37 However, MGCs and macrophages are likely to also degrade a substantial part of elastin fibers lysosomally, where the activities of cathepsins K and V are favored because of their lower optimal pH.…”