Laminar shear stress inhibits cathepsin L activity in endothelial cells

Abstract

Objective-The cysteine proteases, cathepsins, have been implicated in vascular remodeling and atherosclerosis, processes known to be regulated by shear stress. It is not known, however, whether shear regulates cathepsins. We examined the hypothesis that shear stress regulates cathepsin activity in endothelial cells. Methods and Results-Mouse aortic endothelial cells (MAECs) exposed to atheroprotective, unidirectional laminar shear (LS) degraded significantly less BODIPY-labeled elastin and gelatin in comparison to static and proatherogenic oscillatory shear (OS). The cathepsin inhibitor E64 also reduced this activity. Gelatin zymography showed that cathepsin activity of MAECs was blunted by LS exposure and by a cathepsin L inhibitor but not by cathepsin B and S inhibitors, whereas a cathepsin K inhibitor had a minor effect. Cathepsin L siRNA knocked down cathepsin L expression, gelatinase, and elastase activity in OS and static MAECs. A partial reduction of cathepsin B protein raised the possibility that the siRNA effect on the matrix protease activity could have been attributable to cathepsin L or B. Cathepsin B activity study using the synthetic peptide showed it was not regulated by shear. Key Words: shear stress Ⅲ cathepsin Ⅲ elastase Ⅲ gelatinase Ⅲ atherosclerosis V ascular endothelial cells are constantly exposed to fluid shear stress, the frictional force generated by blood flow over the vascular endothelium. The importance of shear stress in vascular biology and pathophysiology has been highlighted by the focal development patterns of atherosclerosis in hemodynamically defined regions. For example, the regions of branched and curved arteries exposed to disturbed flow conditions including oscillatory and low mean shear stresses (OS) correspond to atheroprone areas. In contrast, straight arteries exposed to pulsatile high levels of laminar shear stress (LS) are relatively well protected from atherosclerotic plaque development. 1 Changes in blood flow have been shown to be a critical factor inducing arterial remodeling. 1-7 Increases in arterial wall shear stress prevent vascular remodeling leading to thickening of the vascular wall and inflammation, 2 whereas decreases in arterial wall shear stress promote arterial remodeling and inflammation. 2,3 Additionally, low wall shear stress leads to degradation of the internal elastic lamina (IEL). 5 Despite these findings, the underlying mechanisms by which shear regulates proteases degrading vessel wall matrix and IEL are not well described. Although there is a report demonstrating that shear regulates matrix metalloproteases (MMPs) in endothelial cells, 8 it is not clear whether other proteases are also regulated by shear. Conclusions-TheseCathepsins are the papain family of cysteine proteases which degrade elastin in addition to collagen. 9 Unlike MMPs, the role for cathepsins in blood vessel remodeling and cardiovascular disease has been understudied until recently. Cathepsins K, L, and S, potent elastinolytic proteases, have been identified in atheroscleroti...

Elevated cyclic stretch alters matrix remodeling in aortic valve cusps: implications for degenerative aortic valve disease

“…The associated increase in collagen content at 15% and 20% stretch led us to further speculate that cathepsin L is a key enzyme involved in normal collagen turnover in valve cusps, wherein its downregulation under 15% and 20% stretch resulted in increased collagen content. Previous studies in our laboratory showed that cathepsin L was downregulated by altered levels of cyclic pressure (25) and laminar shear stress (23,24), further lending strength to this speculation.…”

Toll-like receptor 4 deficiency causes pulmonary emphysema

“…Endothelial cells have been found to possess elastase-like activity (36)(37)(38)(39). In addition, Platt et al used fluorescently labeled gelatin and elastin as matrix substrates to demonstrate that live endothelial cells exhibit varying degrees of gelatinase and elastase activities depending upon the degree of shear stress (62). We found that TLR4-deficient endothelial cells and WT lung endothelial cells, in which TLR4 expression was silenced, exhibited exaggerated levels of elastolytic activity that was reversed in the presence of Nox3 siRNA.…”

Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow

“…Because it is well known that microarray results obtained by different laboratories significantly vary, 40 we first compared our mouse array result obtained using the mouse Illumina BeadChip array with that of HUVEC microarray using the human Illumina BeadChip array, as we recently reported (GSE20739). As shown in supplemental (12,24, and 48 hours) after ligation were collected. Differentially expressed genes were selected for quantitative PCR analyses based on 48-hour microarray results.…”

“…We selected 46 genes (10 up-regulated, 30 downregulated genes plus 6 additional genes of interests, although they were not significantly changed at 48 hours after ligation) and tested by quantitative PCR assay with either the TaqMan (28 genes, supplemental Table 3) or SYBR Green (18 genes, supplemental Table 4) method using total endothelial RNAs obtained from LCAs or RCAs collected at 3 different time points (12,24, and 48 hours after ligation). These RNAs used for quantitative PCR validation were entirely independent from those used in the microarray study.…”

“…[5][6][7][8] To define molecular mechanisms responsible for these changes, investigators have carried out DNA microarray studies using endothelial cells [9][10][11][12][13][14][15][16][17] and have subsequently identified numerous shear-sensitive genes, such as kruppel-like factor 2 and 4 (Klf2, Klf4), endothelial nitric oxide synthase (eNOS), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), bone morphogenic protein 4 (BMP-4), cathepsins, and angiopoietin-2 (Angpt2). 11,14,[18][19][20][21][22][23][24][25][26][27][28][29] Functional studies based on these shear-sensitive genes and their protein products have revealed the critical roles that they play in regulation of inflammation, thrombosis, vascular remodeling, angiogenesis, and arteriogenesis. 11,[19][20][21][22][26][27][28][29][30] Although these in vitro studies have provided critical insights regarding shear-sensitive mechanisms in cultured endothelial cells using modeled flow conditions, it cannot be assumed that identical mechanosensitive genes and pathways are involved in vivo regulating flow-dependent vascular responses and diseases.…”