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Expression of the Elastolytic Cathepsins S and K in Human Atheroma and Regulation of Their Production in Smooth Muscle Cells

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Expression of the Elastolytic Cathepsins S and K in Human Atheroma and Regulation of Their Production in Smooth Muscle Cells Expression of the elastolytic cathepsins S and K in human atheroma and regulation of their production in smooth muscle cells The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Sukhova, G K, G P Shi, D I Simon, H A Chapman, and P Libby. 1998. “Expression of the Elastolytic Cathepsins S and K in Human Atheroma and Regulation of Their Production in Smooth Muscle Cells.” J. Clin. Invest. 102 (3) (August 1): 576–583. doi:10.1172/jci181. http://dx.doi.org/10.1172/JCI181. Published Version doi:10.1172/JCI181 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:13506487 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Expression of the Elastolytic Cathepsins S and K in Human Atheroma and Regulation of their Production in Smooth Muscle Cells Galina K. Sukhova, Guo-Ping Shi, Daniel I. Simon, Harold A. Chapman, and Peter Libby Brigham and Women’s Hospital, Vascular Medicine and Atherosclerosis Unit and Cardiovascular and Respiratory Divisions, Department of Medicine, Boston, Massachusetts 02115 Abstract sclerotic lesions. Early in formation of the thickened intima, smooth muscle cells (SMC)1 that may migrate from the tunica Formation of the atherosclerotic intima must involve al- media into the developing intima must penetrate the internal tered metabolism of the elastin-rich arterial extracellular elastic lamina. The compensatory enlargement characteristic matrix. Proteases potentially involved in these processes re- of the evolving atherosclerotic lesion must likewise involve re- main unclear. This study examined the expression of the po- modeling of constituents of the arterial extracellular matrix, tent elastases cathepsins S and K in human atheroma. Nor- including elastin. Recent clinical and pathological evidence mal arteries contained little or no cathepsin K or S. In suggests that inflammatory cells, especially macrophages, ac- contrast, macrophages in atheroma contained abundant im- cumulate in regions of plaques most susceptible to dissolution munoreactive cathepsins K and S. Intimal smooth muscle and rupture (1–3). The mechanisms of degradation of the arte- cells (SMC), especially cells appearing to traverse the inter- rial extracellular matrix in these various contexts remain un- nal elastic laminae, also contained these enzymes. Extracts clear. Substantial evidence supports the involvement of matrix of atheromatous tissues had approximately twofold greater metalloproteinases (MMP) in this process (4–6). Some studies elastase-specific activity than extracts of uninvolved arter- have also implicated serine proteases in vascular remodeling ies, mostly due to cysteine proteases. Cultured human SMC (7, 8). Cysteine proteases have received much less consider- displayed no immunoreactive cathepsins K and S and ex- ation in this regard, even though macrophages and SMC with hibited little or no elastolytic activity when incubated with greatly expanded lysosomal compartments figure prominently insoluble elastin. SMC stimulated with the atheroma-associ- in the histopathology of atherosclerotic plaques. ated cytokines IL-1b or IFN-g secreted active cathepsin S Cysteine proteases have several properties that suggest and degraded substantial insoluble elastin (15–20 mg/106 that they might contribute to arterial remodeling during cells/24 h). A selective inhibitor of cathepsin S blocked . 80% atherogenesis, if they were expressed in these lesions. Several of this elastolytic activity. The presence of cathepsins K and cysteine endoproteases, i.e., cathepsins K, L, and S, have po- S at sites of vascular matrix remodeling and the ability of tent elastolytic activity (9–12). Cathepsin K is the most potent SMC and macrophages to use these enzymes to degrade mammalian elastase yet described (13, 14), and also possesses elastin supports a role for elastolytic cathepsins in vessel collagenolytic activity (15). Although all members of this en- wall remodeling and identifies novel therapeutic targets in zyme family have acidic pH optima, cathepsin S is stable at regulating plaque stability. (J. Clin. Invest. 1998. 102:576– neutral pH, where it retains nearly as much elastase activity as 583.) Key words: atherosclerosis • cysteine protease • leukocyte elastase (10, 11, 16, 17). Although these enzymes are smooth muscle cell • elastolytic activity thought to generally reside in and function optimally within acidic lysosomes, recent evidence indicates that these elastases Introduction may also function extracellularly at or near the cell surface. Monocyte-derived human macrophages become markedly Remodeling of the extracellular matrix of blood vessels, and in elastolytic during in vitro culture and predominantly use cys- particular, the degradation of elastin, contributes importantly teine proteases to degrade extracellular elastin (12). Acquisi- to numerous aspects of formation and complication of athero- tion of a degradative phenotype correlates with the ability of the macrophages to contact elastin, create an acidic pericellu- lar space, and secrete elastolytic cysteine proteases, especially cathepsins S and L, into the contact zone (12, 18). This degra- These data were presented in part in abstract form at the Annual dative mechanism is reminiscent of the bone macrophage, os- Meeting of the American Heart Association, 13–16 November 1995, teoclasts. Indeed, in osteoclasts, cathepsin K appears to be the Anaheim, CA, and at the Ninth International Vascular Biology major protease involved in extracellular matrix metabolism Meeting, 4–8 September 1996, Seattle, WA. necessary for normal bone growth and remodeling (13). Defi- Galina K. Sukhova and Guo-Ping Shi contributed equally to this ciency of this enzyme results in accumulation of bone matrix work. mostly around rather than in osteoclasts, consistent with the Address correspondence to Peter Libby, M.D., 221 Longwood primary site of action of cathepsin K in the pericellular space Avenue, LMRC 307, Boston, MA 02115. Phone: 617-732-6628; FAX: 617-732-6961; E-mail: [email protected] (19). Together, these studies establish that under certain con- Received for publication 20 March 1997 and accepted in revised ditions macrophages utilize the elastolytic cathepsins to re- form 1 June 1998. J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 1. Abbreviations used in this paper: E64, trans-epoxysuccinyl-L-leucyl- 0021-9738/98/08/0576/08 $2.00 amido-(4-guanidino)butane; LHVS, morpholinurea-leucine-homo- Volume 102, Number 3, August 1998, 576–583 phenylalanine-vinylsulfone-phenyl; MMP, matrix metalloproteinase; http://www.jci.org SMC, smooth muscle cells. 576 Sukhova et al. model extracellular matrix, although cathepsin K protein has developed using chemiluminescence (ECL system; New England Nu- only been observed in bone, one study generally demonstrated clear, Boston, MA). cathepsin K mRNA and protein expression in human breast Northern blot analyses. Four nonatherosclerotic aortas and five carcinoma (20). atherosclerotic carotid plaques were pulverized and total RNAs pre- Atherosclerotic plaques contain abundant macrophages. cipitated from a modified guanidium isothiocyanate lysate with iso- propanol according to the manufacturer’s instructions (Stratagene These cells accumulate at sites of weakening of the extracellu- Inc., La Jolla, CA). Total RNAs were dissolved in diethylpyrocarbon- lar matrix, which predispose to plaque rupture. Disruption of ate-treated distilled water and 20 mg of total RNA from each sample atherosclerotic plaques frequently causes thrombosis and pre- was separated on 1.2% agarose gels followed by electroblotting onto cipitates the acute manifestations of atherosclerosis, including membrane (Maximum Strength Nytran Plus; Schleicher and Schuell). unstable angina and acute myocardial infarction (2, 3). In view To analyze SMC RNA, 15,000,000 SMCs from each culture were lysed of the proteolytic potential of cathepsins K and S, and their in guanidinium isothiocyanate lysis buffer and the RNA was pre- possible presence in the very cell types found at sites of plaque pared as described previously (22, 23). RNA blots were then pre- liability, we investigated the expression of these proteases in hybridized and hybridized with a-[32P]dCTP (New England Nuclear) normal and atherosclerotic human arteries and their regula- labeled cathepsins S and K full-length cDNA fragments, and glyceral- tion and function in vitro in cultured vascular SMC. dehyde-3-phosphate dehydrogenase cDNA fragment (Clontech, Palo Alto, CA) respectively, in Express Hyb Solution (Clontech) for 1 h followed by sequential 20 min of washing in 23 SSC/2% SDS, 0.53 Methods SSC/0.5% SDS, and 0.13 SSC/0.1% SDS, respectively. Membranes were then exposed to X-omat film (Eastman Kodak, Rochester, NY), Human tissue. Atherosclerotic plaques from human coronary (n 5 9) and the autoradiograms were quantified by densitometric analysis us- and carotid (n 5 15) arteries and nonatherosclerotic arteries (n 5 12) ing National Institutes of Health Image software. were obtained from transplantation donors, at endarterectomy, and Elastase assays. Tissue from five nonatherosclerotic aortas and at autopsy. Immunohistochemical localization of cathepsin S or K did seven carotid plaques was snap-frozen in liquid nitrogen
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