Lysosomal Cysteine Proteases in Atherosclerosis
http://www.100md.com
《动脉硬化血栓血管生物学》
From the Department of Molecular and Cell Biology (J.L., J.-S.S., W.-H.X.), School of Life Science, University of Science and Technology of China, Hefei, Anhui, China; and Donald W. Reynolds Cardiovascular Clinical Research Center (G.K.S., P.L., G.-P.S.), Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass.
ABSTRACT
Atherosclerosis is an inflammatory disease characterized by extensive remodeling of the extracellular matrix architecture of the arterial wall. Although matrix metalloproteinases and serine proteases participate in these pathologic events, recent data from atherosclerotic patients and animals suggest the participation of lysosomal cysteine proteases in atherogenesis. Atherosclerotic lesions in humans overexpress the elastolytic and collagenolytic cathepsins S, K, and L but show relatively reduced expression of cystatin C, their endogenous inhibitor, suggesting a shift in the balance between cysteine proteases and their inhibitor that favors remodeling of the vascular wall. Extracts of human atheromatous tissue show greater elastolytic activity in vitro than do those from healthy donors. The cysteinyl protease inhibitor E64d limits this increased elastolysis, indicating involvement of cysteine proteases in elastin degradation during atherogenesis. Furthermore, inflammatory cytokines augment expression and secretion of active cysteine proteases from cultured monocyte-derived macrophages, vascular smooth muscle cells, and endothelial cells and increase degradation of extracellular elastin and collagen. Cathepsin S–deficient cells or those treated with E64d show significantly impaired elastolytic or collagenolytic activity. Additionally, recent in vivo studies of atherosclerosis-prone, LDL receptor–null mice lacking cathepsin S show participation of this enzyme in the initial infiltration of leukocytes, medial elastic lamina degradation, endothelial cell invasion, and neovascularization, illustrating an important role for cysteine proteases in arterial remodeling and atherogenesis.
Atherosclerosis is an inflammatory disease characterized by extensive remodeling of the extracellular matrix architecture of the arterial wall. Although matrix metalloproteinases and serine proteases participate in these pathologic events, recent data from atherosclerotic patients and animals suggest the participation of lysosomal cysteine proteases in atherogenesis.
Key Words: atherosclerosis ? cysteine proteases ? cathepsins ? cystatin ? cytokines
Introduction
The extracellular matrix (ECM) of the vascular wall, largely elastin and collagen, subserves many functions essential for vessel homeostasis. These macromolecules serve as an adhesive substrate for vascular endothelial cells (ECs) and smooth muscle cells (SMCs), furnish survival signals to resident cells, bind and retain lipoproteins, and provide a reservoir for growth factors.1 Elastin and collagen also contribute to the strength, resiliency, and structural integrity of the vascular wall.1,2 Normal tissues exhibit strict regulation of the expression and turnover of ECM. However, ECM damage or remodeling in conditions such as rheumatoid arthritis,3 malignant tumors,4 aortic aneurysm,5,6 and atherosclerosis7,8 contributes to the formation, progression, and clinical expressions of these disorders.
Atherosclerosis is an inflammatory disease whose complex developments encompass 3 main stages: initiation, progression, and complication.8 Throughout these stages, several pathologic events involve proteolysis, including initial translocations of mononuclear leukocytes from the vascular lumen through the basement membrane into the subendothelium; progressive migration of SMCs through the elastic laminas from the media into the intima; and finally, disruption of the arterial wall—outward in aneurysmal disease and luminally in athero-occlusive disease.
Many studies have documented augmented elastase, collagenase, and gelatinase activities within atherosclerotic lesions. Among the responsible proteolytic enzymes, matrix metalloproteinases (MMPs) and serine proteases have garnered the most attention.9–15 Increased expression of MMP-1, -2, -3, -7, -8, -9, and -13 and of metalloelastase-12 (MMP-12) occurs in macrophages bordering the lipid core adjacent to the fibrous cap and in macrophages and SMCs in the shoulder regions of well-developed atherosclerotic plaques, sites prone to rupture.7,12,13,16 Studies in animals have permitted assessment of the contribution of these proteases to arterial remodeling. Administration of small-molecule MMP inhibitors delayed SMC migration and intima formation in injured rat arteries.17 Overexpression of tissue inhibitors of MMPs (TIMPs) reduced atheroma progression18 or intimal thickening in veins.19 However, introduction of an MMP-1 transgene driven by a macrophage promoter into atherosclerosis-prone, apolipoprotein E (Apo E)–deficient mice surprisingly reduced atherosclerotic lesions20 for reasons that remain uncertain. Human atherosclerotic lesions overexpress both urokinase- and tissue-type plasminogen activators.21,22 Deficiency of urokinase-type plasminogen activator protected against medial elastic destruction and atherosclerotic aneurysm formation in Apo E–null mice, probably via impaired plasmin-mediated elastolytic MMP activation.23 In contrast, transgenic expression of rabbit urokinase-type plasminogen activator enlarged neointimal lesions and caused degradation of elastic laminas in cholesterol-fed rabbits.24 However, plasminogen deficiency accentuated atherogenesis in atherosclerosis-prone, Apo E–null mice.25 Recent findings have described expression of neutrophil elastase, a serine proteinase, in the macrophage-rich shoulders of human atherosclerotic lesions and its regulated secretion from monocytes by the inflammatory mediator CD40 ligand.26 In addition, serine proteases can activate MMPs secreted from SMCs and degrade fibrin within advanced plaques.27,28 Thus, different families of proteolytic enzymes may participate in atherogenesis.
Cathepsins of the cysteine protease family localize in lysosomes and endosomes and function there to degrade unwanted intracellular or endocytosed proteins. In the early 1990s, we recognized few of these proteases and knew little of their physiologic or pathologic functions. More recently, modern molecular biology permitted the characterization of a series of novel cathepsins, many widely expressed in humans and animals. However, the recognition of inducible cathepsins such as S, K, C, V, and W led to the unraveling of their roles in inflammatory and/or autoimmune diseases such as cancer, rheumatoid arthritis, osteoporosis, hyperkeratosis/periodontitis, and atherosclerosis.29 Most strikingly, we now recognize that these lysosomal cysteine proteases can function outside lysosomes or endosomes, although these intracellular organelles still contain the bulk of these enzymes. Detection of active cysteine proteases in culture media of SMCs, ECs, and macrophages significantly broadened our understanding of their potential roles in arterial pathobiology. This review will discuss some of the recent findings in this field and highlight the significance of lysosomal cysteine proteases in arterial ECM remodeling and atherogenesis.
Regulated Expression of Cysteine Proteases and Their Endogenous Inhibitor, Cystatin C
Overexpression of Lysosomal Cysteine Proteases and Deficiency of Cystatin C in Atherosclerosis
Atherosclerotic lesions contain much more cathepsin S and K mRNAs and proteins than do normal arteries. Either protease can degrade elastin and collagen.29,30 Cathepsin K, one of the most potent mammalian collagenases, participates in joint and bone collagen metabolism. Deficiency of this protease impairs bone growth in both humans and animals.31,32 Immunohistochemical studies demonstrated expression of cathepsins S and K mainly in macrophages in the shoulder regions of atheromata, in SMCs of the fibrous cap, and at sites of internal elastic laminae fragmentation.33 These findings suggest involvement of these proteases in degradation of elastic laminae in the vascular wall, which may facilitate SMC migration, and destabilization of atherosclerotic plaque by degrading collagen of the fibrous caps.34 Also, the ECs lining the lumen of the vessel (not shown) as well as those in the plaque microvessels (Figure 1) express cathepsin S, indicating a role for this protease in neovascularization, a process implicated in plaque growth and complication.35 In contrast, healthy human aortae contain no immunoreactive cathepsin S or K, although Northern blot analyses of healthy aortas have shown low levels of cathepsin K mRNA.33 Western blot analysis and elastase assay of tissue extracts from human atherosclerotic lesions demonstrated increased levels of active forms of cathepsins S and K with significantly elevated elastolytic activity, sensitive to the cysteine protease inhibitor E64d or the cathepsin S selective inhibitor morpholinurea leucine-homophenylalanine-vinylsulfone-phenyl (LHVS),33 suggesting an important contribution of cysteine proteases in arterial elastic tissue remodeling.
Figure 1. Colocalization of cathepsin S and cystatin C with microvascular endothelium. Frozen sections of human carotid lesions were double immunostained with rabbit anti-human cathepsin S (1:300)33 or cystatin C (1:1500, Vortex) polyclonal antibodies followed by biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, Calif). After application of the avidin/biotin blocking kit, anti-CD31 monoclonal antibody (1:30, Dako) for ECs was added. Subsequently, biotinylated horse anti-mouse secondary antibody was applied, followed by streptavidin-fluorescein isothiocyanate (FITC, Amersham). Nuclei were counterstained with 0.5 μg/mL bis-benzimide H33258 (Calbiochem) in PBS. Both cathepsin S (red, left; x400) and cystatin C (red, right; x400) colocalized with endothelial marker CD31 (green). Orange color on the top panels indicates microvascular ECs expressing both antigens. Lumen of the microvessel is indicated.
Atherosclerotic lesions in Apo E–deficient mice showed a similar increase in expression of lysosomal cathepsins S, L, and B.36 As described in human atherosclerotic lesions,33 cathepsin S localized mainly in intimal SMCs and macrophages and medial SMCs. Nonatherosclerotic arteries showed no expression of cathepsin S.36 This expression pattern affirms an association of these proteases with atherogenesis in humans. Consistent with this hypothesis, human atherosclerotic lesions have relatively low levels of cystatin C, an endogenous inhibitor of these cathepsins, whereas normal arteries express abundant cystatin C in medial SMCs and ECs.37 This expression profile suggests an imbalance between cysteine proteases and their inhibitors that favors arterial ECM breakdown. Similar loss of this counterbalance was observed in atherosclerotic lesions from Apo E–deficient mice. In mouse aortic atherosclerotic lesions, immunostaining for cystatin C was also much less intense than that in normal mouse aortas (Sukhova and Shi, unpublished data). Such inverse regulation of vascular proteases and protease inhibitors appears much less prominent in the case of MMPs and their tissue inhibitors (TIMPs). In human atherosclerotic lesions, TIMP expression changes little or increases compared with that in control vessels.38–40 Murine atherosclerotic lesion development showed similar expression patterns.36 Increased MMP-9 expression in Apo E–deficient, murine atherosclerotic lesions accompanies increased TIMP-1 levels, whereas expression of cysteine proteases and their inhibitor cystatin C remains inversely regulated.
Expression and Secretion of Lysosomal Cysteine Proteases and Cystatin C In Vitro
Macrophages, SMCs, and ECs account for most of the cysteine protease expression in human atherosclerotic arteries. To study the regulation of cysteine protease expression in these cells, we isolated human monocytes, vascular SMCs, and vascular ECs and then examined cysteine protease expression under different conditions. Although human blood monocytes express negligible levels of cathepsin K, maturation of monocytes into macrophages during incubation in 40% fetal calf serum markedly augments cathepsin K expression and elastolytic activity.41 These human monocyte-derived macrophages also express and secrete cathepsins S, L, and B.42,43 Similar to medial SMCs in the normal vessel wall, cultured SMCs do not express cathepsin S or K under basal conditions. However, incubation of these cells with inflammatory cytokines such as interleukin-1? (10 ng/mL), tumor necrosis factor- (10 ng/mL), and interferon (IFN)- (400 U/mL) for 24 hours significantly induces the expression and secretion of cathepsins S and K at both the mRNA and protein levels.33 Furthermore, increased cathepsin expression occurs in tandem with enhanced elastase activities, largely sensitive to E64, establishing a predominant contribution to the elastolytic activity of cysteine proteases in inflamed SMCs. More important, culture media from IFN-–stimulated SMCs contained active cathepsin S detected by both active-site labeling of de novo synthesized enzymes and by degradation of water-insoluble elastin.33 These findings indicate release of cathepsin S from SMCs and interaction with ECM proteins, a process that likely occurs in atherosclerotic lesion development during SMC migration and further neointima formation.
In contrast, cystatin C, the endogenous inhibitor of the cysteine proteases, shows distinct regulation by different cytokines.37 For example, IFN- or interleukin-1? increases cathepsin S expression 10- to 20-fold in SMCs but does not affect cystatin C expression. Tumor necrosis factor- induces cathepsin S expression in SMCs but reduces cystatin C expression in vascular ECs.37 We obtained similar results with the angiogenic stimulus basic fibroblast growth factor, which augments cathepsin S expression by >10-fold in ECs but also reduces cystatin C expression.35 Grainger et al44 detected significantly lower blood levels of transforming growth factor-?1 in patients with atherosclerosis, but transforming growth factor-?1 increases SMC cystatin C secretion by a posttranscriptional mechanism.37 The Table lists the effects of various cytokines and angiogenic factors on cathepsin S and cystatin C expression and secretion in these cells. These observations indicate that some inflammatory factors differentially regulate the expression and secretion of lysosomal cysteine proteases and their endogenous inhibitors in arterial cells.
Effect of Cytokines and Angiogenic Factors on Cathepsin S and Cystatin C Expression and Secretion
Proteolytic Activities of Extracellular Lysosomal Cysteine Proteases
Lysosomal cysteine proteases are synthesized and targeted to the acidic compartments, lysosomes and endosomes, via either the mannose-6-phosphate receptor–dependent pathway45,46 or the mannose-6-phosphate receptor–independent pathway,47 where they are activated to degrade their substrates. These compartments provide cysteine protease cathepsins with the optimal pH for their activity.29 Although cathepsin cDNA sequences do not appear to encode a conventional secretory signal peptide,48 several groups including our own have demonstrated the existence and activity (collagenolytic and/or elastolytic) of these proteases in media conditioned by SMCs, ECs, and monocyte-derived macrophages.33,37,42,43 Therefore, several questions remain. These include how these proteases are released and how they retain their activity once they have left the cells, because most of these proteases have a very narrow pH optimum (pH 4 to 6). For instance, cathepsins K and L lose their activity at neutral pH.49 Although cathepsin S does exceptionally retain some activity at neutral pH,50 it is questionable whether this partial activity explains all of the cysteine protease–dependent ECM degradation observed in vitro. Hence, in the recent proposal of a "focal contact" hypothesis, Punturieri et al43 used human monocyte-derived macrophages to demonstrate the formation of a localized acidic environment in a zone of contact that excludes the surrounding extracellular milieux. Such focal contact permits lysosomal or endosomal cysteine proteases to degrade extracellular elastin efficiently. After incubating cultures for 24 hours, they detected an acidic interface between the cell surface and elastin filaments by use of a fluorescent pH indicator. Punturieri and colleagues further demonstrated by use of bafilomycin, an inhibitor of acidification of both intracellular and extracellular compartments that inhibited monocyte-derived macrophage elastase activity, that these extracellular acidic milieux resulted from increased expression of vacuole-type H+-ATPase. These findings provide a reasonable explanation for ECM degradation by cysteine proteases released from macrophages.
Monocyte adhesion, migration, and differentiation into macrophages play essential roles in the pathogenesis of the atherosclerotic lesion. These cells may also use extracellular cysteine proteases to assist their migration during atherogenesis. We recently demonstrated in vitro that cathepsin S–deficient monocytes cannot migrate through artificial membranes containing SMCs, collagen mixtures, and an endothelial monolayer.51 This finding suggests a critical involvement of cysteine proteases in blood-borne cell transmigration from peripheral blood into the vessel wall. Therefore, it is reasonable to hypothesize that these monocytes/macrophages may degrade ECM via the pathways of vacuole-type H+-ATPase expression, extracellular acidic milieu formation, and extracellular collagen degradation. Validation of this hypothesis will require further investigation.
It is therefore conceivable that SMCs and ECs use the same focal contact mechanism to dissolve ECM as do macrophages.33,35 Medial SMCs contain immunoreactive cathepsins S, K, and L in atherosclerotic human and animal vessel walls, notably near sites of internal elastic lamina fragmentation33,51 (see below). These SMCs likely also form an extracellular acidic microenvironment that allows extracellular cathepsin S and possibly other elastolytic cathepsins (eg, K and L) to break down the elastic barriers. Several lines of evidence support this hypothesis. Stimulation of cultured human or murine SMCs with IFN- induced expression of elastolytic cathepsins and increased extracellular degradation of water-insoluble elastin. The cathepsin inhibitor E64d blocks much of this activity.33,51 Furthermore, SMCs lacking cathepsin S showed a significantly reduced ability to degrade extracellular elastic fibers. Although we do not have direct evidence of formation of a cell-substrate acidic interface, we observed attachment of SMCs to exogenous water-insoluble elastin fibers in culture (Figure 2). Nevertheless, it remains to be confirmed whether SMCs act as monocyte-derived macrophages do and form acidic focal contact compartments that facilitate degradation of extracellular elastin in culture and within human and animal vascular walls.
Figure 2. Focal contact of SMCs and elastin fibers. Human saphenous vein SMCs were cultured in monolayers. Water-insoluble elastin fibers were seeded on top of the cell monolayer (left). By 24 hours of culture, SMCs had formed direct focal contacts with elastin fibers (right).
Lysosomal Cysteine Proteases and Atherogenesis
Increased expression of cysteine proteases cathepsins S, K, B, H, and L and decreased expression of their endogenous inhibitor cystatin C in human atherosclerotic lesions suggested the involvement of cysteine proteases in atherogenesis. Recent findings from in vitro and in vivo experiments indicate that these cysteine proteases may participate in the main stages of atherogenesis.
Initiation
Atherogenesis initially involves leukocyte recruitment from the circulation by adhesion to the endothelium, followed by penetration through the endothelial layer and arterial basement membrane.8 Current understanding implicates specific adhesion molecules expressed on the surface of vascular ECs, eg, vascular cell adhesion molecule-1 (VCAM-1), and chemoattractant molecules, such as macrophage chemoattractant protein-1 (MCP-1), in this process.8 Deficiency or impaired function of these molecules significantly reduces atherogenesis in animals.52–54 We currently do not know whether cysteine proteases play any role in regulating MCP-1 or VCAM-1 expression or leukocyte adhesion. Data from studies of cathepsin S–knockout mice illustrated a significant reduction of these molecules in sera of mice initiated for atherosclerosis with a high-cholesterol diet.51 Therefore, cathepsin S may act like MMPs and release adhesion molecules from the surface of ECs.55 Alternatively, cathepsin S may indirectly influence the production of adhesion molecules by affecting the -T lymphocyte population,56 another possible source of VCAM production.57 On the other hand, the reduced levels of MCP-1 and VCAM-1 observed in cathepsin S–deficient mice may result from lower serum lipid levels58 or decreased lesional monocyte content59 and may not directly depend on cathepsin S activity.
After adhesion and transmigration through the endothelial layer and basement membrane, monocytes become macrophages, proliferate, and become lipid-laden foam cells. The arterial subendothelial basement membrane contains type IV collagen, laminin, and fibronectin.60,61 Migration of blood-borne leukocytes requires degradation of these ECM components, as does microvessel ingrowth from vasa vasorum of the adventitia and tunica media.62,63 Macrophages derived from human monocytes express and secrete substantial amounts of active cathepsins K, L, and S, which can degrade these subendothelial basement membrane components.64–67 The mononuclear cells may well use these proteolytic enzymes to aid their transmigration. Indeed, the subendothelial basement membrane in early atherosclerotic lesions (fatty streaks) contains large amounts of immunoreactive cathepsins S and K.33 To test for a role for cysteine proteases in monocyte transmigration, we recently used an in vitro preparation comprising layers of SMCs and a mixture of collagen types I and IV overlaid by a monolayer of ECs.51 Human monocytes seeded on top of this structure migrated through the collagen matrix into the SMC layer, where they matured into macrophages. Interestingly, treatment of human monocytes with a low concentration (8 nmol/L) of LHVS (a condition that selectively inhibits cathepsin S) or a high concentration (1 μmol/L) of LHVS (that also inhibits other cysteine proteases) significantly reduced monocyte translocation (Shi et al, unpublished data). Cathepsin S–null monocytes yielded similar results.51 More than 90% of cathepsin S–null monocytes remained atop the endothelial monolayer, whereas the majority of wild-type monocytes migrated into the SMC layer. These observations suggest involvement of cathepsin S and possibly other cysteine proteases in leukocyte transmigration.
Progression
During the progression of atherosclerosis, lipid-laden SMCs and macrophages accumulate along with T lymphocytes. These cells release inflammatory cytokines, and SMCs in particular elaborate ECM constituents. In addition to the transmigration of leukocytes from the lumen, SMC migration from the media also occurs. Medial SMC migration and neointimal formation are part of the main pathologic events during progression of atherosclerosis. This SMC migration requires traversal of the internal elastic laminas. Although the elastic laminae may contain fenestrae, dissolution of these elastic barriers may aid the migration of SMCs to the intima. In vitro experiments demonstrated that elastin peptides in a modified Boyden chamber halted aortic SMC migration.68 Furthermore, in SMCs, intact elastin regulates actin stress fiber organization, proliferation, and migration via a heterotrimeric G protein–coupled pathway.69 Thus, understanding atherogenesis requires identification of the proteases that mediate elastolysis. The ability of human monocyte-derived macrophages to release elastolytic cysteine proteases led us to hypothesize that SMCs may also use cysteine proteases for their migration through arterial elastin. We validated this hypothesis by showing reduced elastolytic activity in experiments in vitro with cysteine protease inhibitors with human SMCs and aortic SMCs isolated from cathepsin S–deficient mice.33,51 In vivo experiments further supported this hypothesis: studies of atherosclerosis-prone LDL receptor–deficient mice that consumed an atherogenic diet for 8, 12, and 26 weeks showed that SMCs at sites of elastin fragmentation expressed cathepsin S similar to those in human lesions. Mice lacking both the LDL receptor and cathepsin S on the same diet showed substantially less fragmentation of the arterial internal elastic lamina, reduced intimal SMC accumulation and collagen production, and significantly smaller atherosclerotic lesions. Indirect evidence comes from recent observations of Apo E–deficient mice. In these atherosclerosis-prone mice, deficiency of the endogenous cysteine protease inhibitor cystatin C increased aortic elastolysis mediated by SMC cysteine protease and consequently enhanced medial degradation of elastic laminae (Shi and Sukhova, manuscript in preparation).
Oxidized LDL (oxLDL) may favor formation of foam cells and necrotic or apoptotic lipid cores, hallmarks of both human and murine atherogenesis. Several experiments have implicated cysteine proteases in these pathologic processes. The lysosomal cysteine proteases cathepsins B and L may process caspases and induce apoptosis70–72 and localize in the cytoplasm and nuclei of apoptotic (caspase-3–positive) macrophages in human atheromata.73 Inhibition of cathepsins B and L with E64 or Z-Phe-Arg-FMK protects mononuclear cells from oxysterol-induced cell death. Thus, cytotoxic oxLDL and associated oxysterols in atheromata may cause lysosomal destabilization and release of cathepsins, fostering the apoptotic death of phagocytic cells and contributing to further atherosclerotic lesion evolution, including lipid core formation.73,74 This proapoptotic mechanism may also apply to other cells in atheromata, such as SMCs and ECs. In cathepsin S–deficient, LDL receptor–null atherosclerotic mice, we detected not only significantly less accumulation of lipid in lesions and lower serum levels of LDL or total cholesterol51 but also lower titers of autoantibody to both malondialdehyde-oxLDL and copper-oxLDL epitopes (Shi et al, unpublished data). Because some proteases have been linked to major histocompatibility class II–associated antigen processing and presentation, cathepsin S, and possibly other cathepsins, may participate in the adaptive humoral and cellular immune responses that operate during atherosclerosis.75–77
Neovascularization provides a portal for leukocyte trafficking6 and entry of plasma constituents, including lipoproteins, and occurs during progression of atherosclerotic lesions.78 This process requires lysis of the ECM to pave the way for neovessel formation.79 Although the microvessels of various human malignant tumors express cysteine proteases, direct evidence for involvement of these enzymes in neovascularization emerged only recently from studies of cathepsin S–deficient mice.35 Healing skin wounds in cathepsin S–null mice contained far fewer microvessels. Defining the contribution of impaired neovascularization due to loss of cathepsin S activity to reduced atherosclerosis in cathepsin S–null mice is difficult. However, in human atherosclerotic lesions, areas of microvascularization contain both cathepsin S and its inhibitor cystatin C (Figure 1), in contrast to other regions of plaque that contain less cystatin C.37 Therefore, cathepsin S and possibly other cysteine proteases may also regulate the growth of lesion microvessels during atherogenesis.
Complications
Thrombotic complications of atherosclerosis often involve plaque rupture. Lesions that have ruptured typically have a thin fibrous cap, prominent lipid deposition, and macrophage accumulation. Such thrombotic complications actually cause most of the acute manifestations of atherosclerosis, such as myocardial infarction or stroke.7,8 Indeed, thrombus formation usually results from physical disruption of atherosclerotic plaque and appears related to the level of collagen in the lesion’s fibrous cap. Therefore, the collagen content of the fibrous cap critically influences plaque stability and may depend in turn on the expression of interstitial collagen-degrading proteases.
Vulnerable human atherosclerotic plaques overexpress several MMP collagenases capable of degrading interstitial collagen types I and III.79 For example, macrophages in plaques contain abundant MMP-1, -8, and -13 and colocalize with sites of collagen degradation in situ. Macrophages appear to furnish the bulk of cysteine proteases in atheromata. Indeed, Chen et al80 used near-infrared imaging to detect cathepsin B activity in vivo in atheromatous lesions of Apo E–deficient mice. Increased expression of cathepsin B in atheromatous plaques colocalized with the macrophage marker Mac-3. We made similar observations in sections of human atherosclerotic lesions with cathepsins S, K, and L, all of which can degrade collagen.29,30 Cathepsins S and K were originally isolated from human macrophages, and inflammatory cytokines increase cathepsin S secretion from macrophages (the Table).81 In human atherosclerotic plaques, macrophages and fibrous cap SMCs express all 3 collagenolytic cathepsins33 (Shi, unpublished data). However, the possible involvement of cysteine proteases in plaque rupture requires further examination. Fibrous cap thickness is directly associated with plaque vulnerability. Cathepsin S/LDL receptor double-deficient mice have decreased SMC and collagen contents in the lesions and a reduction in the size of the fibrous cap compared with those in control LDL receptor–null mice. Such a reduction in fibrous cap size could be the result of a decrease in the number of SMCs in lesions, which may increase plaque vulnerability.34 Cystatin C/Apo E double-deficient mice consistently had increased lesional SMC and collagen contents and better developed fibrous caps (Sukhova et al, unpublished data), characteristics of stable plaques in humans. Thus, cysteine proteases could play dual roles. Medial SMCs release cathepsins to allow them to pass through the elastica and accumulate in the neointima, where they produce collagens and reinforce the fibrous caps.34 On the other hand, SMCs in the fibrous cap can also produce cathepsins,33 which are collagenolytic.29,30 These cathepsins may cause weakening of the fibrous cap and lead to plaque rupture, although this conjecture requires experimental validation.
To date, no direct experiment has tested for a role of cysteine proteases in thrombosis during atherogenic complications. Data from our laboratory and collaborators indicate that cathepsin S regulates thrombotic responses to arterial injury.82 After photochemical carotid artery injury, the time to the development of occlusive thrombosis decreased in cathepsin S–null mice. The accelerated thrombotic response to arterial injury and the shortening of plasma clotting times accompanied an increased activity of coagulation factors VIII, IX, and X and plasma von Willebrand factor, as measured by 1-stage clotting-based assays in cathepsin S–deficient mice. These results suggest that cathepsin S has antithrombotic properties. However, the mechanism by which cathepsin S affects thrombosis and whether its antithrombotic properties impact atherogenesis remain undetermined.
Conclusions
Numerous observations now support key functions of cysteine protease cathepsins in atherogenesis (Figure 3). Cathepsins may pave the way for entry of blood-borne monocytes and lymphocytes into the arterial vessel wall by degrading the subendothelial basement membrane and for the migration of SMCs through elastic laminae to enter the intima. Several cell types, including medial SMCs, neointimal SMCs, macrophages, and neointimal microvascular ECs, express cathepsins in atheromatous lesions. Exposure to inflammatory cytokines or growth factors released by transmigrated monocytes, T lymphocytes, or ECM degradation products may enhance the expression or release of cathepsins in these cells (Figure 3 and the Table), a hypothesis supported by observations from studies of cultured cells. Thus, the inflammatory process at the core of atherogenesis is linked tightly with proteolysis due to cathepsins. Furthermore, cathepsin function in thrombosis82 or even immunity76,83 also may contribute to the pathogenesis of atherosclerosis, although we lack direct evidence for these functions. Several recent reports related primarily to the remodeling of the ECM do establish an important role for cysteine proteases in atherogenesis in genetically altered mice. We therefore propose that multiple proteases, including MMPs and serine proteases, work in concert during the initiation, progression, and complication of atherosclerotic plaques.
Figure 3. Potential roles of cysteine protease cathepsins in atherogenesis. The diagram depicts a cross section of an atherosclerotic lesion. Immunohistologic analysis demonstrated localization of cysteine protease cathepsins in the subendothelium (regulating blood-borne leukocyte transmigration), in SMCs in the fibrous cap (influencing plaque stability), at sites of internal elastic lamina fragmentation (controlling SMC migration), in macrophages and foam cells at the lipid core (degrading matrix proteins and affecting cell apoptosis), and in ECs of the vessel wall and microvessels (involving neovascularization). Inflammatory cytokines released from infiltrated lymphocytes, growth factors from degradation of matrix protein (eg, collagens), or even oxLDL may regulate cathepsin expression during atherogenesis. Both macrophages and SMCs take up modified LDL and become foam cells, which become the major components of the lipid core.
Acknowledgments
This work was funded in part by grants to G.-P.S. from the National Heart, Lung, and Blood Institute (HL 60942, HL67283) and the American Heart Association (0355130Y), to G.K.S. from the National Heart, Lung, and Blood Institute (HL 67249), and to P.L. from the National Heart, Lung, and Blood Institute (HL-56985), and by a grant from the Donald W. Reynolds Foundation. We also thank Karen Williams for her editorial expertise.
References
Libby P, Lee RT. Matrix matters. Circulation. 2000; 102: 1874–1876.
Parks WC. Who are the proteolytic culprits in vascular disease. J Clin Invest. 1999; 104: 1167–1168.
Weber AJ, De Bandt M. Angiogenesis: general mechanisms and implications for rheumatoid arthritis. Joint Bone Spine. 2000; 67: 366–383.
Ohtani H. Stromal reaction in cancer tissue: pathophysiologic significance of the expression of matrix-degrading enzymes in relation to matrix turnover and immune/inflammatory reactions. Pathol Int. 1998; 48: 1–9.
Halloran BG. Pathogenesis of aneurysms. Semin Vasc Surg. 1995; 8: 85–92.
MacSweenety STR. Pathogenesis of abdominal aortic aneurysm. Br J Surg. 1994; 81: 935–941.
Libby P. Molecular bases of the acute coronary syndromes. Circulation. 1995; 91: 2844–2850.
Libby P. Changing concepts of atherogenesis. J Intern Med. 2000; 247: 349–358.
Galis ZS, Muszynski M, Sukhova GK, Simon-Morrissey E, Unemori EN, Lark MW, Amento E, Libby P. Cytokine-stimulated human vascular smooth muscle cells synthesize a complement of enzymes required for extracellular matrix digestion. Circ Res. 1994; 75: 181–189.
Newby AC, Southgate KM, Davies M. Extracellular matrix degrading metalloproteinases in the pathogenesis of atherosclerosis. Basic Res Cardiol Suppl. 1994; 89: 59–70.
Henney AM, Wakeley PR, Davies MJ, Foster K, Hembry R, Murphy G, Humphries S. Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization. Proc Natl Acad Sci U S A. 1991; 88: 8154–8158.
Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994; 94: 2493–2530.
Halpert I, Sires UI, Roby JD, Potter-Perigo S, Wight TN, Shapiro SD, Welgus HG, Wickline SA, Parks WC. Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme. Proc Natl Acad Sci U S A. 1996; 93: 9748–9753.
Woessner JF Jr. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 1991; 5: 2145–2154.
Suzuki K, Enghild JJ, Morodomi T, Salvesen G, Nagase H. Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin). Biochemistry. 1990; 29: 10261–10270.
Weisman HF, Bulkley BH. Pathophysiology of atherosclerotic heart disease. Cardiol Clin. 1984; 2: 555–569.
Bendeck MP, Irvin C, Reidy MA. Inhibition of matrix metalloproteinase activity inhibits smooth muscle cell migration but not neointimal thickening after arterial injury. Circ Res. 1996; 78: 38–43.
Ye S, Watts GF, Mandalia S, Humphries SE, Henney AM. Preliminary report: genetic variation in the human stromelysin promoter is associated with progression of coronary atherosclerosis. Br Heart J. 1995; 73: 209–215.
George SJ, Baker AH, Angelini GD, Newby AC. Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins. Gene Ther. 1998; 5: 1552–1560.
Lemaitre V, O’Byrne TK, Borczuk AC, Okada Y, Tall AR, D’Armiento J. ApoE knockout mice expressing human matrix metalloproteinase-1 in macrophages have less advanced atherosclerosis. J Clin Invest. 2001; 107: 1227–1234.
Steins MB, Padro T, Li CX, Mesters RM, Ostermann H, Hammel D, Scheld HH, Berdel WE, Kienast J. Overexpression of tissue-type plasminogen activator in atherosclerotic human coronary arteries. Atherosclerosis. 1999; 145: 173–180.
Kienast J, Padro T, Steins M, Li CX, Schmid KW, Hammel D, Scheld HH, van de Loo JC. Relation of urokinase-type plasminogen activator expression to presence and severity of atherosclerotic lesions in human coronary arteries. Thromb Haemost. 1998; 79: 579–586.
Carmeliet P, Moons L, Lijnen R, Baes M, Lemaitre V, Tipping P, Drew A, Eeckhout Y, Shapiro S, Lupu F, Collen D. Urokinase-generated plasmin activates matrix metalloproteinases during aneurysm formation. Nat Genet. 1997; 17: 439–444.
Falkenberg M, Tom C, DeYoung MB, Wen S, Linnemann R, Dichek DA. Increased expression of urokinase during atherosclerotic lesion development causes arterial constriction and lumen loss, and accelerates lesion growth. Proc Natl Acad Sci U S A. 2002; 99: 10665–10670.
Xiao Q, Danton MJ, Witte DP, Kowala MC, Valentine MT, Bugge TH, Degen JL. Plasminogen deficiency accelerates vessel wall disease in mice predisposed to atherosclerosis. Proc Natl Acad Sci U S A. 1997; 94: 10335–10340.
Dollery CM, Owen CA, Sukhova GK, Krettek A, Shapiro SD, Libby P. Neutrophil elastase in human atherosclerotic plaques: production by macrophages. Circulation. 2003; 107: 2829–2836.
Okumura Y, Sato H, Seiki M, Kido H. Proteolytic activation of the precursor of membrane type 1 matrix metalloproteinase by human plasmin: a possible cell surface activator. FEBS Lett. 1997; 402: 181–184.
Baramova EN, Bajou K, Remacle A, L’Hoir C, Krell HW, Weidle UH, Noel A, Foidart JM. Involvement of PA/plasmin system in the processing of pro-MMP-2 activation. FEBS Lett. 1997; 405: 157–162.
Chapman HA, Riese RJ, Shi GP. Emerging roles for cysteine proteases in human biology. Annu Rev Physiol. 1997; 59: 63–88.
Li Z, Yasuda Y, Li W, Bogyo M, Katz N, Gordon RE, Fields GB, Bromme D. Regulation of collagenase activities of human cathepsins by glycosaminoglycans. J Biol Chem. 2004; 279: 5470–5479.
Gelb BD, Shi GP, Chapman HA, Desnick RJ. Pyknodysostosis, a lysosomal disease caused by cathepsin K deficiency. Science. 1996; 273: 1236–1238.
Saftig P, Hunziker E, Wehmeyer O, Jones S, Boyde A, Rommerskirch W, Moritz JD, Schu P, von Figura K. Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice. Proc Natl Acad Sci U S A. 1998; 95: 13453–13458.
Sukhova GK, Shi GP, Simon DI, Chapman HA, Libby P. Expression of the elastolytic cathepsins S and K in human atheroma and regulation of their production in smooth muscle cells. J Clin Invest. 1998; 102: 576–583.
Shah PK, Falk E, Badimon JJ, Fernandez-Ortiz A, Mailhac A, Villareal-Levy G, Fallon JT, Regnstrom J, Fuster V. Human monocyte-derived macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques: potential role of matrix-degrading metalloproteinases and implications for plaque rupture. Circulation. 1995; 92: 1565–1569.
Shi GP, Sukhova GK, Kuzuya M, Ye Q, Du J, Zhang Y, Pan JH, Lu ML, Cheng XW, Iguchi A, Perrey S, Lee AM, Chapman HA, Libby P. Deficiency of the cysteine protease cathepsin S impairs microvessel growth. Circ Res. 2003; 92: 493–500.
Jormsjo S, Wuttge DM, Sirsjo A, Whatling C, Hamsten A, Stemme S, Eriksson P. Differential expression of cysteine and aspartic proteases during progression of atherosclerosis in apolipoprotein E-deficient mice. Am J Pathol. 2002; 161: 939–945.
Shi GP, Sukhova GK, Grubb A, Ducharme A, Rhode LH, Lee RT, Ridker PM, Libby P, Chapman HA. Cystatin C deficiency in human atherosclerosis and aortic aneurysms. J Clin Invest. 1999; 104: 1191–1197.
Fabunmi RP, Sukhova GK, Sugiyama S, Libby P. Expression of tissue inhibitor of metalloproteinases-3 in human atheroma and regulation in lesion-associated cells: a potential protective mechanism in plaque stability. Circ Res. 1998; 83: 270–278.
Fabunmi RP, Baker AH, Murray EJ, Booth RF, Newby AC. Divergent regulation by growth factors and cytokines of 95 kDa and 72 kDa gelatinases and tissue inhibitors or metalloproteinases-1, -2, and -3 in rabbit aortic smooth muscle cells. Biochem J. 1996; 315: 335–342.
Ishibashi S, Goldstein JL, Brown MS, Herz J, Burns DK. Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice. J Clin Invest. 1994; 93: 1885–1893.
Shi GP, Chapman HA, Bhairi SM, DeLeeuw C, Reddy VY, Weiss SJ. Molecular cloning of human cathepsin O, a novel endoproteinase and homologue of rabbit OC2. FEBS Lett. 1995; 357: 129–134.
Reddy VY, Zhang QY, Weiss SJ. Pericellular mobilization of the tissue-destructive cysteine proteases, cathepsin B, L, and S, by human monocyte-derived macrophages. Proc Natl Acad Sci U S A. 1995; 92: 3849–3853.
Punturieri A, Filippov S, Allen E, Caras I, Murray R, Reddy V, Weiss SJ. Regulation of elastinolytic cysteine proteinase activity in normal and cathepsin K-deficient human macrophages. J Exp Med. 2000; 192: 789–799.
Grainger DJ, Kemp PR, Metcalfe JC, Liu AC, Lawn RM, Williams NR, Grace AA, Schofield PM, Chauhan A. The serum concentration of active transforming growth factor-? is severely depressed in advanced atherosclerosis. Nat Med. 1995; 1: 74–79.
Nishimura Y, Kawabata T, Yano S, Kato K. Inhibition of intracellular sorting and processing of lysosomal cathepsins H and L at reduced temperature in primary cultures of rat hepatocytes. Arch Biochem Biophys. 1990; 283: 458–463.
Kuliawat R, Klumperman J, Ludwig T, Arvan P. Differential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic beta-cells. J Cell Biol. 1997; 137: 595–608.
Ahn K, Yeyeodu S, Collette J, Madden V, Arthur J, Li L, Erickson AH. An alternate targeting pathway for procathepsin L in mouse fibroblasts. Traffic. 2002; 3: 147–159.
Jackson RC, Blobel G. Post-translational cleavage of presecretory proteins with an extract of rough microsomes from dog pancreas containing signal peptidase activity. Proc Natl Acad Sci U S A. 1977; 74: 5598–5602.
Bromme D, Okamoto K, Wang BB, Biroc S. Human cathepsin O2, a matrix protein-degrading cysteine protease expressed in osteoclasts: functional expression of human cathepsin O2 in Spodoptera frugiperda and characterization of the enzyme. J Biol Chem. 1996; 271: 2126–2132.
Shi GP, Munger JS, Meara JP, Rich DH, Chapman HA. Molecular cloning and expression of human alveolar macrophage cathepsin S, an elastinolytic cysteine protease. J Biol Chem. 1992; 267: 7258–7262.
Sukhova GK, Zhang Y, Pan JH, Wada Y, Yamamoto T, Naito M, Kodama T, Tsimikas S, Witztum JL, Lu ML, Sakara Y, Chin MT, Libby P, Shi GP. Deficiency of cathepsin S reduces atherosclerosis in LDL receptor-deficient mice. J Clin Invest. 2003; 111: 897–906.
Gu L, Okada Y, Clinton SK, Gerard C, Sukhova GK, Libby P, Rollins BJ. Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol Cell. 1998; 2: 275–281.
Inoue S, Egashira K, Ni W, Kitamoto S, Usui M, Otani K, Ishibashi M, Hiasa K, Nishida K, Takeshita A. Anti-monocyte chemoattractant protein-1 gene therapy limits progression and destabilization of established atherosclerosis in apolipoprotein E-knockout mice. Circulation. 2002; 106: 2700–2706.
Dansky HM, Barlow CB, Lominska C, Sikes JL, Kao C, Weinsaft J, Cybulsky MI, Smith JD. Adhesion of monocytes to arterial endothelium and initiation of atherosclerosis are critically dependent on vascular cell adhesion molecule-1 gene dosage. Arterioscler Thromb Vasc Biol. 2001; 21: 1662–1667.
Hummel V, Kallmann BA, Wagner S, Fuller T, Bayas A, Tonn JC, Benveniste EN, Toyka KV, Rieckmann P. Production of MMPs in human cerebral endothelial cells and their role in shedding adhesion molecules. J Neuropathol Exp Neurol. 2001; 60: 320–327.
Riese RJ, Shi GP, Villadangos J, Stetson D, Driessen C, Lennon-Dumenil AM, Chu CL, Naumov Y, Behar SM, Ploegh H, Locksley R, Chapman HA. Regulation of CD1 function and NK1.1(+) T cell selection and maturation by cathepsin S. Immunity. 2001; 15: 909–919.
Leca G, Mansur SE, Bensussan A. Expression of VCAM-1 (CD106) by a subset of TCR -bearing lymphocyte clones: involvement of a metalloprotease in the specific hydrolytic release of the soluble isoform. J Immunol. 1995; 154: 1069–1077.
Kowalski J, Okopien B, Madej A, Makowiecka K, Zielinski M, Kalina Z, Herman ZS. Levels of sICAM-1, sVCAM-1 and MCP-1 in patients with hyperlipoproteinemia IIa and -IIb. Int J Clin Pharmacol Ther. 2001; 39: 48–52.
Wang G, Woo CW, Sung FL, Siow YL, O K. Increased monocyte adhesion to aortic endothelium in rats with hyperhomocysteinemia: role of chemokine and adhesion molecules. Arterioscler Thromb Vasc Biol. 2002; 22: 1777–1783.
Kittelberger R, Davis PF, Stehbens WE. Distribution of type IV collagen, laminin, nidogen and fibronectin in the haemodynamically stressed vascular wall. Histol Histopathol. 1990; 5: 161–167.
Navaratnam V. Organisation and reorganisation of blood vessels in embryonic development. Eye. 1991; 5: 147–150.
Del Maestro RF, Vaithilingam IS, McDonald W. Degradation of collagen type IV by C6 astrocytoma cells. J Neurooncol. 1995; 24: 75–81.
Libby P, Sch?nbeck U. Drilling for oxygen: angiogenesis involves proteolysis of the extracellular matrix. Circ Res. 2001; 89: 195–197.
Bromme D, Bonneau PR, Lachance P, Wiederanders B, Kirschke H, Peters C, Thomas DY, Storer AC, Vernet T. Functional expression of human cathepsin S in Saccharomyces cerevisiae: purification and characterization of the recombinant enzyme. J Biol Chem. 1993; 268: 4832–4838.
Dehrmann FM, Coetzer TH, Pike RN, Dennison C. Mature cathepsin L is substantially active in the ionic milieu of the extracellular medium. Arch Biochem Biophys. 1995; 324: 93–98.
Garnero P, Borel O, Byrjalsen I, Ferreras M, Drake FH, McQueney MS, Foged NT, Delmas PD, Delaisse JM. The collagenolytic activity of cathepsin K is unique among mammalian proteinases. J Biol Chem. 1998; 273: 32347–32352.
Ishidoh K, Kominami E. Procathepsin L degrades extracellular matrix proteins in the presence of glycosaminoglycans in vitro. Biochem Biophys Res Commun. 1995; 217: 624–631.
Ooyama T, Fukuda K, Oda H, Nakamura H, Hikita Y. Substratum-bound elastin peptide inhibits aortic smooth muscle cell migration in vitro. Arteriosclerosis. 1987; 7: 593–598.
Karnik SK, Brooke BS, Bayes-Genis A, Sorensen L, Wythe JD, Schwartz RS, Keating MT, Li DY. A critical role for elastin signaling in vascular morphogenesis and disease. Development. 2003; 130: 411–423.
Vancompernolle K, Van Herreweghe F, Pynaert G, Van de Craen M, De Vos K, Totty N, Sterling A, Fiers W, Vandenabeele P, Grooten J. Atractyloside-induced release of cathepsin B, a protease with caspase-processing activity. FEBS Lett. 1998; 438: 150–158.
Ishisaka R, Utsumi T, Kanno T, Arita K, Katunuma N, Akiyama J, Utsumi K. Participation of a cathepsin L-type protease in the activation of caspase-3. Cell Struct Funct. 1999; 24: 465–470.
Guicciardi ME, Deussing J, Miyoshi H, Bronk SF, Svingen PA, Peters C, Kaufmann SH, Gores GJ. Cathepsin B contributes to TNF--mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome C. J Clin Invest. 2000; 106: 1127–1137.
Li W, Dalen H, Eaton JW, Yuan XM. Apoptotic death of inflammatory cells in human atheroma. Arterioscler Thromb Vasc Biol. 2001; 21: 1124–1130.
Li W, Yuan XM, Olsson AG, Brunk UT. Uptake of oxidized LDL by macrophages results in partial lysosomal enzyme inactivation and relocation. Arterioscler Thromb Vasc Biol. 1998; 18: 177–184.
Shi GP, Villadangos JA, Dranoff G, Small C, Gu L, Haley KJ, Riese R, Ploegh HL, Chapman HA. Cathepsin S required for normal MHC class II peptide loading and germinal center development. Immunity. 1999; 10: 197–206.
Nakagawa T, Roth W, Wong P, Nelson A, Farr A, Deussing J, Villadangos JA, Ploegh H, Peters C, Rudensky AY. Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus. Science. 1998; 280: 450–453.
Hansson G, Libby P, Schoenbeck U, Yan Z-Q. Innate and adaptive immunity in the pathogenesis of atherosclerosis. Circ Res. 2002; 91: 281–291.
Zhang Y, Cliff WJ, Schoefl GI, Higgins G. Immunohistochemical study of intimal microvessels in coronary atherosclerosis. Am J Pathol. 1993; 143: 164–172.
Sukhova GK, Schonbeck U, Rabkin E, Schoen FJ, Poole AR, Billinghurst RC, Libby P. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques. Circulation. 1999; 99: 2503–2509.
Chen J, Tung CH, Mahmood U, Ntziachristos V, Gyurko R, Fishman MC, Huang PL, Weissleder R. In vivo imaging of proteolytic activity in atherosclerosis. Circulation. 2002; 105: 2766–2771.
Liuzzo JP, Petanceska SS, Moscatelli D, Devi LA. Inflammatory mediators regulate cathepsin S in macrophages and microglia: a role in attenuating heparan sulfate interactions. Mol Med. 1999; 5: 320–333.
Day SM, Reeve JL, Mundada L, Pan JH, Shi GP, Fay W. Cathepsin S–deficient mice display a prothrombotic phenotype. Circulation. 2002; 106: 195.
Villadangos JA, Bryant RA, Deussing J, Driessen C, Lennon-Dumenil AM, Riese RJ, Roth W, Saftig P, Shi GP, Chapman HA, Peters C, Ploegh HL. Proteases involved in MHC class II antigen presentation. Immunol Rev. 1999; 172: 109–120.(Jian Liu; Galina K. Sukho)
ABSTRACT
Atherosclerosis is an inflammatory disease characterized by extensive remodeling of the extracellular matrix architecture of the arterial wall. Although matrix metalloproteinases and serine proteases participate in these pathologic events, recent data from atherosclerotic patients and animals suggest the participation of lysosomal cysteine proteases in atherogenesis. Atherosclerotic lesions in humans overexpress the elastolytic and collagenolytic cathepsins S, K, and L but show relatively reduced expression of cystatin C, their endogenous inhibitor, suggesting a shift in the balance between cysteine proteases and their inhibitor that favors remodeling of the vascular wall. Extracts of human atheromatous tissue show greater elastolytic activity in vitro than do those from healthy donors. The cysteinyl protease inhibitor E64d limits this increased elastolysis, indicating involvement of cysteine proteases in elastin degradation during atherogenesis. Furthermore, inflammatory cytokines augment expression and secretion of active cysteine proteases from cultured monocyte-derived macrophages, vascular smooth muscle cells, and endothelial cells and increase degradation of extracellular elastin and collagen. Cathepsin S–deficient cells or those treated with E64d show significantly impaired elastolytic or collagenolytic activity. Additionally, recent in vivo studies of atherosclerosis-prone, LDL receptor–null mice lacking cathepsin S show participation of this enzyme in the initial infiltration of leukocytes, medial elastic lamina degradation, endothelial cell invasion, and neovascularization, illustrating an important role for cysteine proteases in arterial remodeling and atherogenesis.
Atherosclerosis is an inflammatory disease characterized by extensive remodeling of the extracellular matrix architecture of the arterial wall. Although matrix metalloproteinases and serine proteases participate in these pathologic events, recent data from atherosclerotic patients and animals suggest the participation of lysosomal cysteine proteases in atherogenesis.
Key Words: atherosclerosis ? cysteine proteases ? cathepsins ? cystatin ? cytokines
Introduction
The extracellular matrix (ECM) of the vascular wall, largely elastin and collagen, subserves many functions essential for vessel homeostasis. These macromolecules serve as an adhesive substrate for vascular endothelial cells (ECs) and smooth muscle cells (SMCs), furnish survival signals to resident cells, bind and retain lipoproteins, and provide a reservoir for growth factors.1 Elastin and collagen also contribute to the strength, resiliency, and structural integrity of the vascular wall.1,2 Normal tissues exhibit strict regulation of the expression and turnover of ECM. However, ECM damage or remodeling in conditions such as rheumatoid arthritis,3 malignant tumors,4 aortic aneurysm,5,6 and atherosclerosis7,8 contributes to the formation, progression, and clinical expressions of these disorders.
Atherosclerosis is an inflammatory disease whose complex developments encompass 3 main stages: initiation, progression, and complication.8 Throughout these stages, several pathologic events involve proteolysis, including initial translocations of mononuclear leukocytes from the vascular lumen through the basement membrane into the subendothelium; progressive migration of SMCs through the elastic laminas from the media into the intima; and finally, disruption of the arterial wall—outward in aneurysmal disease and luminally in athero-occlusive disease.
Many studies have documented augmented elastase, collagenase, and gelatinase activities within atherosclerotic lesions. Among the responsible proteolytic enzymes, matrix metalloproteinases (MMPs) and serine proteases have garnered the most attention.9–15 Increased expression of MMP-1, -2, -3, -7, -8, -9, and -13 and of metalloelastase-12 (MMP-12) occurs in macrophages bordering the lipid core adjacent to the fibrous cap and in macrophages and SMCs in the shoulder regions of well-developed atherosclerotic plaques, sites prone to rupture.7,12,13,16 Studies in animals have permitted assessment of the contribution of these proteases to arterial remodeling. Administration of small-molecule MMP inhibitors delayed SMC migration and intima formation in injured rat arteries.17 Overexpression of tissue inhibitors of MMPs (TIMPs) reduced atheroma progression18 or intimal thickening in veins.19 However, introduction of an MMP-1 transgene driven by a macrophage promoter into atherosclerosis-prone, apolipoprotein E (Apo E)–deficient mice surprisingly reduced atherosclerotic lesions20 for reasons that remain uncertain. Human atherosclerotic lesions overexpress both urokinase- and tissue-type plasminogen activators.21,22 Deficiency of urokinase-type plasminogen activator protected against medial elastic destruction and atherosclerotic aneurysm formation in Apo E–null mice, probably via impaired plasmin-mediated elastolytic MMP activation.23 In contrast, transgenic expression of rabbit urokinase-type plasminogen activator enlarged neointimal lesions and caused degradation of elastic laminas in cholesterol-fed rabbits.24 However, plasminogen deficiency accentuated atherogenesis in atherosclerosis-prone, Apo E–null mice.25 Recent findings have described expression of neutrophil elastase, a serine proteinase, in the macrophage-rich shoulders of human atherosclerotic lesions and its regulated secretion from monocytes by the inflammatory mediator CD40 ligand.26 In addition, serine proteases can activate MMPs secreted from SMCs and degrade fibrin within advanced plaques.27,28 Thus, different families of proteolytic enzymes may participate in atherogenesis.
Cathepsins of the cysteine protease family localize in lysosomes and endosomes and function there to degrade unwanted intracellular or endocytosed proteins. In the early 1990s, we recognized few of these proteases and knew little of their physiologic or pathologic functions. More recently, modern molecular biology permitted the characterization of a series of novel cathepsins, many widely expressed in humans and animals. However, the recognition of inducible cathepsins such as S, K, C, V, and W led to the unraveling of their roles in inflammatory and/or autoimmune diseases such as cancer, rheumatoid arthritis, osteoporosis, hyperkeratosis/periodontitis, and atherosclerosis.29 Most strikingly, we now recognize that these lysosomal cysteine proteases can function outside lysosomes or endosomes, although these intracellular organelles still contain the bulk of these enzymes. Detection of active cysteine proteases in culture media of SMCs, ECs, and macrophages significantly broadened our understanding of their potential roles in arterial pathobiology. This review will discuss some of the recent findings in this field and highlight the significance of lysosomal cysteine proteases in arterial ECM remodeling and atherogenesis.
Regulated Expression of Cysteine Proteases and Their Endogenous Inhibitor, Cystatin C
Overexpression of Lysosomal Cysteine Proteases and Deficiency of Cystatin C in Atherosclerosis
Atherosclerotic lesions contain much more cathepsin S and K mRNAs and proteins than do normal arteries. Either protease can degrade elastin and collagen.29,30 Cathepsin K, one of the most potent mammalian collagenases, participates in joint and bone collagen metabolism. Deficiency of this protease impairs bone growth in both humans and animals.31,32 Immunohistochemical studies demonstrated expression of cathepsins S and K mainly in macrophages in the shoulder regions of atheromata, in SMCs of the fibrous cap, and at sites of internal elastic laminae fragmentation.33 These findings suggest involvement of these proteases in degradation of elastic laminae in the vascular wall, which may facilitate SMC migration, and destabilization of atherosclerotic plaque by degrading collagen of the fibrous caps.34 Also, the ECs lining the lumen of the vessel (not shown) as well as those in the plaque microvessels (Figure 1) express cathepsin S, indicating a role for this protease in neovascularization, a process implicated in plaque growth and complication.35 In contrast, healthy human aortae contain no immunoreactive cathepsin S or K, although Northern blot analyses of healthy aortas have shown low levels of cathepsin K mRNA.33 Western blot analysis and elastase assay of tissue extracts from human atherosclerotic lesions demonstrated increased levels of active forms of cathepsins S and K with significantly elevated elastolytic activity, sensitive to the cysteine protease inhibitor E64d or the cathepsin S selective inhibitor morpholinurea leucine-homophenylalanine-vinylsulfone-phenyl (LHVS),33 suggesting an important contribution of cysteine proteases in arterial elastic tissue remodeling.
Figure 1. Colocalization of cathepsin S and cystatin C with microvascular endothelium. Frozen sections of human carotid lesions were double immunostained with rabbit anti-human cathepsin S (1:300)33 or cystatin C (1:1500, Vortex) polyclonal antibodies followed by biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, Calif). After application of the avidin/biotin blocking kit, anti-CD31 monoclonal antibody (1:30, Dako) for ECs was added. Subsequently, biotinylated horse anti-mouse secondary antibody was applied, followed by streptavidin-fluorescein isothiocyanate (FITC, Amersham). Nuclei were counterstained with 0.5 μg/mL bis-benzimide H33258 (Calbiochem) in PBS. Both cathepsin S (red, left; x400) and cystatin C (red, right; x400) colocalized with endothelial marker CD31 (green). Orange color on the top panels indicates microvascular ECs expressing both antigens. Lumen of the microvessel is indicated.
Atherosclerotic lesions in Apo E–deficient mice showed a similar increase in expression of lysosomal cathepsins S, L, and B.36 As described in human atherosclerotic lesions,33 cathepsin S localized mainly in intimal SMCs and macrophages and medial SMCs. Nonatherosclerotic arteries showed no expression of cathepsin S.36 This expression pattern affirms an association of these proteases with atherogenesis in humans. Consistent with this hypothesis, human atherosclerotic lesions have relatively low levels of cystatin C, an endogenous inhibitor of these cathepsins, whereas normal arteries express abundant cystatin C in medial SMCs and ECs.37 This expression profile suggests an imbalance between cysteine proteases and their inhibitors that favors arterial ECM breakdown. Similar loss of this counterbalance was observed in atherosclerotic lesions from Apo E–deficient mice. In mouse aortic atherosclerotic lesions, immunostaining for cystatin C was also much less intense than that in normal mouse aortas (Sukhova and Shi, unpublished data). Such inverse regulation of vascular proteases and protease inhibitors appears much less prominent in the case of MMPs and their tissue inhibitors (TIMPs). In human atherosclerotic lesions, TIMP expression changes little or increases compared with that in control vessels.38–40 Murine atherosclerotic lesion development showed similar expression patterns.36 Increased MMP-9 expression in Apo E–deficient, murine atherosclerotic lesions accompanies increased TIMP-1 levels, whereas expression of cysteine proteases and their inhibitor cystatin C remains inversely regulated.
Expression and Secretion of Lysosomal Cysteine Proteases and Cystatin C In Vitro
Macrophages, SMCs, and ECs account for most of the cysteine protease expression in human atherosclerotic arteries. To study the regulation of cysteine protease expression in these cells, we isolated human monocytes, vascular SMCs, and vascular ECs and then examined cysteine protease expression under different conditions. Although human blood monocytes express negligible levels of cathepsin K, maturation of monocytes into macrophages during incubation in 40% fetal calf serum markedly augments cathepsin K expression and elastolytic activity.41 These human monocyte-derived macrophages also express and secrete cathepsins S, L, and B.42,43 Similar to medial SMCs in the normal vessel wall, cultured SMCs do not express cathepsin S or K under basal conditions. However, incubation of these cells with inflammatory cytokines such as interleukin-1? (10 ng/mL), tumor necrosis factor- (10 ng/mL), and interferon (IFN)- (400 U/mL) for 24 hours significantly induces the expression and secretion of cathepsins S and K at both the mRNA and protein levels.33 Furthermore, increased cathepsin expression occurs in tandem with enhanced elastase activities, largely sensitive to E64, establishing a predominant contribution to the elastolytic activity of cysteine proteases in inflamed SMCs. More important, culture media from IFN-–stimulated SMCs contained active cathepsin S detected by both active-site labeling of de novo synthesized enzymes and by degradation of water-insoluble elastin.33 These findings indicate release of cathepsin S from SMCs and interaction with ECM proteins, a process that likely occurs in atherosclerotic lesion development during SMC migration and further neointima formation.
In contrast, cystatin C, the endogenous inhibitor of the cysteine proteases, shows distinct regulation by different cytokines.37 For example, IFN- or interleukin-1? increases cathepsin S expression 10- to 20-fold in SMCs but does not affect cystatin C expression. Tumor necrosis factor- induces cathepsin S expression in SMCs but reduces cystatin C expression in vascular ECs.37 We obtained similar results with the angiogenic stimulus basic fibroblast growth factor, which augments cathepsin S expression by >10-fold in ECs but also reduces cystatin C expression.35 Grainger et al44 detected significantly lower blood levels of transforming growth factor-?1 in patients with atherosclerosis, but transforming growth factor-?1 increases SMC cystatin C secretion by a posttranscriptional mechanism.37 The Table lists the effects of various cytokines and angiogenic factors on cathepsin S and cystatin C expression and secretion in these cells. These observations indicate that some inflammatory factors differentially regulate the expression and secretion of lysosomal cysteine proteases and their endogenous inhibitors in arterial cells.
Effect of Cytokines and Angiogenic Factors on Cathepsin S and Cystatin C Expression and Secretion
Proteolytic Activities of Extracellular Lysosomal Cysteine Proteases
Lysosomal cysteine proteases are synthesized and targeted to the acidic compartments, lysosomes and endosomes, via either the mannose-6-phosphate receptor–dependent pathway45,46 or the mannose-6-phosphate receptor–independent pathway,47 where they are activated to degrade their substrates. These compartments provide cysteine protease cathepsins with the optimal pH for their activity.29 Although cathepsin cDNA sequences do not appear to encode a conventional secretory signal peptide,48 several groups including our own have demonstrated the existence and activity (collagenolytic and/or elastolytic) of these proteases in media conditioned by SMCs, ECs, and monocyte-derived macrophages.33,37,42,43 Therefore, several questions remain. These include how these proteases are released and how they retain their activity once they have left the cells, because most of these proteases have a very narrow pH optimum (pH 4 to 6). For instance, cathepsins K and L lose their activity at neutral pH.49 Although cathepsin S does exceptionally retain some activity at neutral pH,50 it is questionable whether this partial activity explains all of the cysteine protease–dependent ECM degradation observed in vitro. Hence, in the recent proposal of a "focal contact" hypothesis, Punturieri et al43 used human monocyte-derived macrophages to demonstrate the formation of a localized acidic environment in a zone of contact that excludes the surrounding extracellular milieux. Such focal contact permits lysosomal or endosomal cysteine proteases to degrade extracellular elastin efficiently. After incubating cultures for 24 hours, they detected an acidic interface between the cell surface and elastin filaments by use of a fluorescent pH indicator. Punturieri and colleagues further demonstrated by use of bafilomycin, an inhibitor of acidification of both intracellular and extracellular compartments that inhibited monocyte-derived macrophage elastase activity, that these extracellular acidic milieux resulted from increased expression of vacuole-type H+-ATPase. These findings provide a reasonable explanation for ECM degradation by cysteine proteases released from macrophages.
Monocyte adhesion, migration, and differentiation into macrophages play essential roles in the pathogenesis of the atherosclerotic lesion. These cells may also use extracellular cysteine proteases to assist their migration during atherogenesis. We recently demonstrated in vitro that cathepsin S–deficient monocytes cannot migrate through artificial membranes containing SMCs, collagen mixtures, and an endothelial monolayer.51 This finding suggests a critical involvement of cysteine proteases in blood-borne cell transmigration from peripheral blood into the vessel wall. Therefore, it is reasonable to hypothesize that these monocytes/macrophages may degrade ECM via the pathways of vacuole-type H+-ATPase expression, extracellular acidic milieu formation, and extracellular collagen degradation. Validation of this hypothesis will require further investigation.
It is therefore conceivable that SMCs and ECs use the same focal contact mechanism to dissolve ECM as do macrophages.33,35 Medial SMCs contain immunoreactive cathepsins S, K, and L in atherosclerotic human and animal vessel walls, notably near sites of internal elastic lamina fragmentation33,51 (see below). These SMCs likely also form an extracellular acidic microenvironment that allows extracellular cathepsin S and possibly other elastolytic cathepsins (eg, K and L) to break down the elastic barriers. Several lines of evidence support this hypothesis. Stimulation of cultured human or murine SMCs with IFN- induced expression of elastolytic cathepsins and increased extracellular degradation of water-insoluble elastin. The cathepsin inhibitor E64d blocks much of this activity.33,51 Furthermore, SMCs lacking cathepsin S showed a significantly reduced ability to degrade extracellular elastic fibers. Although we do not have direct evidence of formation of a cell-substrate acidic interface, we observed attachment of SMCs to exogenous water-insoluble elastin fibers in culture (Figure 2). Nevertheless, it remains to be confirmed whether SMCs act as monocyte-derived macrophages do and form acidic focal contact compartments that facilitate degradation of extracellular elastin in culture and within human and animal vascular walls.
Figure 2. Focal contact of SMCs and elastin fibers. Human saphenous vein SMCs were cultured in monolayers. Water-insoluble elastin fibers were seeded on top of the cell monolayer (left). By 24 hours of culture, SMCs had formed direct focal contacts with elastin fibers (right).
Lysosomal Cysteine Proteases and Atherogenesis
Increased expression of cysteine proteases cathepsins S, K, B, H, and L and decreased expression of their endogenous inhibitor cystatin C in human atherosclerotic lesions suggested the involvement of cysteine proteases in atherogenesis. Recent findings from in vitro and in vivo experiments indicate that these cysteine proteases may participate in the main stages of atherogenesis.
Initiation
Atherogenesis initially involves leukocyte recruitment from the circulation by adhesion to the endothelium, followed by penetration through the endothelial layer and arterial basement membrane.8 Current understanding implicates specific adhesion molecules expressed on the surface of vascular ECs, eg, vascular cell adhesion molecule-1 (VCAM-1), and chemoattractant molecules, such as macrophage chemoattractant protein-1 (MCP-1), in this process.8 Deficiency or impaired function of these molecules significantly reduces atherogenesis in animals.52–54 We currently do not know whether cysteine proteases play any role in regulating MCP-1 or VCAM-1 expression or leukocyte adhesion. Data from studies of cathepsin S–knockout mice illustrated a significant reduction of these molecules in sera of mice initiated for atherosclerosis with a high-cholesterol diet.51 Therefore, cathepsin S may act like MMPs and release adhesion molecules from the surface of ECs.55 Alternatively, cathepsin S may indirectly influence the production of adhesion molecules by affecting the -T lymphocyte population,56 another possible source of VCAM production.57 On the other hand, the reduced levels of MCP-1 and VCAM-1 observed in cathepsin S–deficient mice may result from lower serum lipid levels58 or decreased lesional monocyte content59 and may not directly depend on cathepsin S activity.
After adhesion and transmigration through the endothelial layer and basement membrane, monocytes become macrophages, proliferate, and become lipid-laden foam cells. The arterial subendothelial basement membrane contains type IV collagen, laminin, and fibronectin.60,61 Migration of blood-borne leukocytes requires degradation of these ECM components, as does microvessel ingrowth from vasa vasorum of the adventitia and tunica media.62,63 Macrophages derived from human monocytes express and secrete substantial amounts of active cathepsins K, L, and S, which can degrade these subendothelial basement membrane components.64–67 The mononuclear cells may well use these proteolytic enzymes to aid their transmigration. Indeed, the subendothelial basement membrane in early atherosclerotic lesions (fatty streaks) contains large amounts of immunoreactive cathepsins S and K.33 To test for a role for cysteine proteases in monocyte transmigration, we recently used an in vitro preparation comprising layers of SMCs and a mixture of collagen types I and IV overlaid by a monolayer of ECs.51 Human monocytes seeded on top of this structure migrated through the collagen matrix into the SMC layer, where they matured into macrophages. Interestingly, treatment of human monocytes with a low concentration (8 nmol/L) of LHVS (a condition that selectively inhibits cathepsin S) or a high concentration (1 μmol/L) of LHVS (that also inhibits other cysteine proteases) significantly reduced monocyte translocation (Shi et al, unpublished data). Cathepsin S–null monocytes yielded similar results.51 More than 90% of cathepsin S–null monocytes remained atop the endothelial monolayer, whereas the majority of wild-type monocytes migrated into the SMC layer. These observations suggest involvement of cathepsin S and possibly other cysteine proteases in leukocyte transmigration.
Progression
During the progression of atherosclerosis, lipid-laden SMCs and macrophages accumulate along with T lymphocytes. These cells release inflammatory cytokines, and SMCs in particular elaborate ECM constituents. In addition to the transmigration of leukocytes from the lumen, SMC migration from the media also occurs. Medial SMC migration and neointimal formation are part of the main pathologic events during progression of atherosclerosis. This SMC migration requires traversal of the internal elastic laminas. Although the elastic laminae may contain fenestrae, dissolution of these elastic barriers may aid the migration of SMCs to the intima. In vitro experiments demonstrated that elastin peptides in a modified Boyden chamber halted aortic SMC migration.68 Furthermore, in SMCs, intact elastin regulates actin stress fiber organization, proliferation, and migration via a heterotrimeric G protein–coupled pathway.69 Thus, understanding atherogenesis requires identification of the proteases that mediate elastolysis. The ability of human monocyte-derived macrophages to release elastolytic cysteine proteases led us to hypothesize that SMCs may also use cysteine proteases for their migration through arterial elastin. We validated this hypothesis by showing reduced elastolytic activity in experiments in vitro with cysteine protease inhibitors with human SMCs and aortic SMCs isolated from cathepsin S–deficient mice.33,51 In vivo experiments further supported this hypothesis: studies of atherosclerosis-prone LDL receptor–deficient mice that consumed an atherogenic diet for 8, 12, and 26 weeks showed that SMCs at sites of elastin fragmentation expressed cathepsin S similar to those in human lesions. Mice lacking both the LDL receptor and cathepsin S on the same diet showed substantially less fragmentation of the arterial internal elastic lamina, reduced intimal SMC accumulation and collagen production, and significantly smaller atherosclerotic lesions. Indirect evidence comes from recent observations of Apo E–deficient mice. In these atherosclerosis-prone mice, deficiency of the endogenous cysteine protease inhibitor cystatin C increased aortic elastolysis mediated by SMC cysteine protease and consequently enhanced medial degradation of elastic laminae (Shi and Sukhova, manuscript in preparation).
Oxidized LDL (oxLDL) may favor formation of foam cells and necrotic or apoptotic lipid cores, hallmarks of both human and murine atherogenesis. Several experiments have implicated cysteine proteases in these pathologic processes. The lysosomal cysteine proteases cathepsins B and L may process caspases and induce apoptosis70–72 and localize in the cytoplasm and nuclei of apoptotic (caspase-3–positive) macrophages in human atheromata.73 Inhibition of cathepsins B and L with E64 or Z-Phe-Arg-FMK protects mononuclear cells from oxysterol-induced cell death. Thus, cytotoxic oxLDL and associated oxysterols in atheromata may cause lysosomal destabilization and release of cathepsins, fostering the apoptotic death of phagocytic cells and contributing to further atherosclerotic lesion evolution, including lipid core formation.73,74 This proapoptotic mechanism may also apply to other cells in atheromata, such as SMCs and ECs. In cathepsin S–deficient, LDL receptor–null atherosclerotic mice, we detected not only significantly less accumulation of lipid in lesions and lower serum levels of LDL or total cholesterol51 but also lower titers of autoantibody to both malondialdehyde-oxLDL and copper-oxLDL epitopes (Shi et al, unpublished data). Because some proteases have been linked to major histocompatibility class II–associated antigen processing and presentation, cathepsin S, and possibly other cathepsins, may participate in the adaptive humoral and cellular immune responses that operate during atherosclerosis.75–77
Neovascularization provides a portal for leukocyte trafficking6 and entry of plasma constituents, including lipoproteins, and occurs during progression of atherosclerotic lesions.78 This process requires lysis of the ECM to pave the way for neovessel formation.79 Although the microvessels of various human malignant tumors express cysteine proteases, direct evidence for involvement of these enzymes in neovascularization emerged only recently from studies of cathepsin S–deficient mice.35 Healing skin wounds in cathepsin S–null mice contained far fewer microvessels. Defining the contribution of impaired neovascularization due to loss of cathepsin S activity to reduced atherosclerosis in cathepsin S–null mice is difficult. However, in human atherosclerotic lesions, areas of microvascularization contain both cathepsin S and its inhibitor cystatin C (Figure 1), in contrast to other regions of plaque that contain less cystatin C.37 Therefore, cathepsin S and possibly other cysteine proteases may also regulate the growth of lesion microvessels during atherogenesis.
Complications
Thrombotic complications of atherosclerosis often involve plaque rupture. Lesions that have ruptured typically have a thin fibrous cap, prominent lipid deposition, and macrophage accumulation. Such thrombotic complications actually cause most of the acute manifestations of atherosclerosis, such as myocardial infarction or stroke.7,8 Indeed, thrombus formation usually results from physical disruption of atherosclerotic plaque and appears related to the level of collagen in the lesion’s fibrous cap. Therefore, the collagen content of the fibrous cap critically influences plaque stability and may depend in turn on the expression of interstitial collagen-degrading proteases.
Vulnerable human atherosclerotic plaques overexpress several MMP collagenases capable of degrading interstitial collagen types I and III.79 For example, macrophages in plaques contain abundant MMP-1, -8, and -13 and colocalize with sites of collagen degradation in situ. Macrophages appear to furnish the bulk of cysteine proteases in atheromata. Indeed, Chen et al80 used near-infrared imaging to detect cathepsin B activity in vivo in atheromatous lesions of Apo E–deficient mice. Increased expression of cathepsin B in atheromatous plaques colocalized with the macrophage marker Mac-3. We made similar observations in sections of human atherosclerotic lesions with cathepsins S, K, and L, all of which can degrade collagen.29,30 Cathepsins S and K were originally isolated from human macrophages, and inflammatory cytokines increase cathepsin S secretion from macrophages (the Table).81 In human atherosclerotic plaques, macrophages and fibrous cap SMCs express all 3 collagenolytic cathepsins33 (Shi, unpublished data). However, the possible involvement of cysteine proteases in plaque rupture requires further examination. Fibrous cap thickness is directly associated with plaque vulnerability. Cathepsin S/LDL receptor double-deficient mice have decreased SMC and collagen contents in the lesions and a reduction in the size of the fibrous cap compared with those in control LDL receptor–null mice. Such a reduction in fibrous cap size could be the result of a decrease in the number of SMCs in lesions, which may increase plaque vulnerability.34 Cystatin C/Apo E double-deficient mice consistently had increased lesional SMC and collagen contents and better developed fibrous caps (Sukhova et al, unpublished data), characteristics of stable plaques in humans. Thus, cysteine proteases could play dual roles. Medial SMCs release cathepsins to allow them to pass through the elastica and accumulate in the neointima, where they produce collagens and reinforce the fibrous caps.34 On the other hand, SMCs in the fibrous cap can also produce cathepsins,33 which are collagenolytic.29,30 These cathepsins may cause weakening of the fibrous cap and lead to plaque rupture, although this conjecture requires experimental validation.
To date, no direct experiment has tested for a role of cysteine proteases in thrombosis during atherogenic complications. Data from our laboratory and collaborators indicate that cathepsin S regulates thrombotic responses to arterial injury.82 After photochemical carotid artery injury, the time to the development of occlusive thrombosis decreased in cathepsin S–null mice. The accelerated thrombotic response to arterial injury and the shortening of plasma clotting times accompanied an increased activity of coagulation factors VIII, IX, and X and plasma von Willebrand factor, as measured by 1-stage clotting-based assays in cathepsin S–deficient mice. These results suggest that cathepsin S has antithrombotic properties. However, the mechanism by which cathepsin S affects thrombosis and whether its antithrombotic properties impact atherogenesis remain undetermined.
Conclusions
Numerous observations now support key functions of cysteine protease cathepsins in atherogenesis (Figure 3). Cathepsins may pave the way for entry of blood-borne monocytes and lymphocytes into the arterial vessel wall by degrading the subendothelial basement membrane and for the migration of SMCs through elastic laminae to enter the intima. Several cell types, including medial SMCs, neointimal SMCs, macrophages, and neointimal microvascular ECs, express cathepsins in atheromatous lesions. Exposure to inflammatory cytokines or growth factors released by transmigrated monocytes, T lymphocytes, or ECM degradation products may enhance the expression or release of cathepsins in these cells (Figure 3 and the Table), a hypothesis supported by observations from studies of cultured cells. Thus, the inflammatory process at the core of atherogenesis is linked tightly with proteolysis due to cathepsins. Furthermore, cathepsin function in thrombosis82 or even immunity76,83 also may contribute to the pathogenesis of atherosclerosis, although we lack direct evidence for these functions. Several recent reports related primarily to the remodeling of the ECM do establish an important role for cysteine proteases in atherogenesis in genetically altered mice. We therefore propose that multiple proteases, including MMPs and serine proteases, work in concert during the initiation, progression, and complication of atherosclerotic plaques.
Figure 3. Potential roles of cysteine protease cathepsins in atherogenesis. The diagram depicts a cross section of an atherosclerotic lesion. Immunohistologic analysis demonstrated localization of cysteine protease cathepsins in the subendothelium (regulating blood-borne leukocyte transmigration), in SMCs in the fibrous cap (influencing plaque stability), at sites of internal elastic lamina fragmentation (controlling SMC migration), in macrophages and foam cells at the lipid core (degrading matrix proteins and affecting cell apoptosis), and in ECs of the vessel wall and microvessels (involving neovascularization). Inflammatory cytokines released from infiltrated lymphocytes, growth factors from degradation of matrix protein (eg, collagens), or even oxLDL may regulate cathepsin expression during atherogenesis. Both macrophages and SMCs take up modified LDL and become foam cells, which become the major components of the lipid core.
Acknowledgments
This work was funded in part by grants to G.-P.S. from the National Heart, Lung, and Blood Institute (HL 60942, HL67283) and the American Heart Association (0355130Y), to G.K.S. from the National Heart, Lung, and Blood Institute (HL 67249), and to P.L. from the National Heart, Lung, and Blood Institute (HL-56985), and by a grant from the Donald W. Reynolds Foundation. We also thank Karen Williams for her editorial expertise.
References
Libby P, Lee RT. Matrix matters. Circulation. 2000; 102: 1874–1876.
Parks WC. Who are the proteolytic culprits in vascular disease. J Clin Invest. 1999; 104: 1167–1168.
Weber AJ, De Bandt M. Angiogenesis: general mechanisms and implications for rheumatoid arthritis. Joint Bone Spine. 2000; 67: 366–383.
Ohtani H. Stromal reaction in cancer tissue: pathophysiologic significance of the expression of matrix-degrading enzymes in relation to matrix turnover and immune/inflammatory reactions. Pathol Int. 1998; 48: 1–9.
Halloran BG. Pathogenesis of aneurysms. Semin Vasc Surg. 1995; 8: 85–92.
MacSweenety STR. Pathogenesis of abdominal aortic aneurysm. Br J Surg. 1994; 81: 935–941.
Libby P. Molecular bases of the acute coronary syndromes. Circulation. 1995; 91: 2844–2850.
Libby P. Changing concepts of atherogenesis. J Intern Med. 2000; 247: 349–358.
Galis ZS, Muszynski M, Sukhova GK, Simon-Morrissey E, Unemori EN, Lark MW, Amento E, Libby P. Cytokine-stimulated human vascular smooth muscle cells synthesize a complement of enzymes required for extracellular matrix digestion. Circ Res. 1994; 75: 181–189.
Newby AC, Southgate KM, Davies M. Extracellular matrix degrading metalloproteinases in the pathogenesis of atherosclerosis. Basic Res Cardiol Suppl. 1994; 89: 59–70.
Henney AM, Wakeley PR, Davies MJ, Foster K, Hembry R, Murphy G, Humphries S. Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization. Proc Natl Acad Sci U S A. 1991; 88: 8154–8158.
Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994; 94: 2493–2530.
Halpert I, Sires UI, Roby JD, Potter-Perigo S, Wight TN, Shapiro SD, Welgus HG, Wickline SA, Parks WC. Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme. Proc Natl Acad Sci U S A. 1996; 93: 9748–9753.
Woessner JF Jr. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 1991; 5: 2145–2154.
Suzuki K, Enghild JJ, Morodomi T, Salvesen G, Nagase H. Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin). Biochemistry. 1990; 29: 10261–10270.
Weisman HF, Bulkley BH. Pathophysiology of atherosclerotic heart disease. Cardiol Clin. 1984; 2: 555–569.
Bendeck MP, Irvin C, Reidy MA. Inhibition of matrix metalloproteinase activity inhibits smooth muscle cell migration but not neointimal thickening after arterial injury. Circ Res. 1996; 78: 38–43.
Ye S, Watts GF, Mandalia S, Humphries SE, Henney AM. Preliminary report: genetic variation in the human stromelysin promoter is associated with progression of coronary atherosclerosis. Br Heart J. 1995; 73: 209–215.
George SJ, Baker AH, Angelini GD, Newby AC. Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins. Gene Ther. 1998; 5: 1552–1560.
Lemaitre V, O’Byrne TK, Borczuk AC, Okada Y, Tall AR, D’Armiento J. ApoE knockout mice expressing human matrix metalloproteinase-1 in macrophages have less advanced atherosclerosis. J Clin Invest. 2001; 107: 1227–1234.
Steins MB, Padro T, Li CX, Mesters RM, Ostermann H, Hammel D, Scheld HH, Berdel WE, Kienast J. Overexpression of tissue-type plasminogen activator in atherosclerotic human coronary arteries. Atherosclerosis. 1999; 145: 173–180.
Kienast J, Padro T, Steins M, Li CX, Schmid KW, Hammel D, Scheld HH, van de Loo JC. Relation of urokinase-type plasminogen activator expression to presence and severity of atherosclerotic lesions in human coronary arteries. Thromb Haemost. 1998; 79: 579–586.
Carmeliet P, Moons L, Lijnen R, Baes M, Lemaitre V, Tipping P, Drew A, Eeckhout Y, Shapiro S, Lupu F, Collen D. Urokinase-generated plasmin activates matrix metalloproteinases during aneurysm formation. Nat Genet. 1997; 17: 439–444.
Falkenberg M, Tom C, DeYoung MB, Wen S, Linnemann R, Dichek DA. Increased expression of urokinase during atherosclerotic lesion development causes arterial constriction and lumen loss, and accelerates lesion growth. Proc Natl Acad Sci U S A. 2002; 99: 10665–10670.
Xiao Q, Danton MJ, Witte DP, Kowala MC, Valentine MT, Bugge TH, Degen JL. Plasminogen deficiency accelerates vessel wall disease in mice predisposed to atherosclerosis. Proc Natl Acad Sci U S A. 1997; 94: 10335–10340.
Dollery CM, Owen CA, Sukhova GK, Krettek A, Shapiro SD, Libby P. Neutrophil elastase in human atherosclerotic plaques: production by macrophages. Circulation. 2003; 107: 2829–2836.
Okumura Y, Sato H, Seiki M, Kido H. Proteolytic activation of the precursor of membrane type 1 matrix metalloproteinase by human plasmin: a possible cell surface activator. FEBS Lett. 1997; 402: 181–184.
Baramova EN, Bajou K, Remacle A, L’Hoir C, Krell HW, Weidle UH, Noel A, Foidart JM. Involvement of PA/plasmin system in the processing of pro-MMP-2 activation. FEBS Lett. 1997; 405: 157–162.
Chapman HA, Riese RJ, Shi GP. Emerging roles for cysteine proteases in human biology. Annu Rev Physiol. 1997; 59: 63–88.
Li Z, Yasuda Y, Li W, Bogyo M, Katz N, Gordon RE, Fields GB, Bromme D. Regulation of collagenase activities of human cathepsins by glycosaminoglycans. J Biol Chem. 2004; 279: 5470–5479.
Gelb BD, Shi GP, Chapman HA, Desnick RJ. Pyknodysostosis, a lysosomal disease caused by cathepsin K deficiency. Science. 1996; 273: 1236–1238.
Saftig P, Hunziker E, Wehmeyer O, Jones S, Boyde A, Rommerskirch W, Moritz JD, Schu P, von Figura K. Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice. Proc Natl Acad Sci U S A. 1998; 95: 13453–13458.
Sukhova GK, Shi GP, Simon DI, Chapman HA, Libby P. Expression of the elastolytic cathepsins S and K in human atheroma and regulation of their production in smooth muscle cells. J Clin Invest. 1998; 102: 576–583.
Shah PK, Falk E, Badimon JJ, Fernandez-Ortiz A, Mailhac A, Villareal-Levy G, Fallon JT, Regnstrom J, Fuster V. Human monocyte-derived macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques: potential role of matrix-degrading metalloproteinases and implications for plaque rupture. Circulation. 1995; 92: 1565–1569.
Shi GP, Sukhova GK, Kuzuya M, Ye Q, Du J, Zhang Y, Pan JH, Lu ML, Cheng XW, Iguchi A, Perrey S, Lee AM, Chapman HA, Libby P. Deficiency of the cysteine protease cathepsin S impairs microvessel growth. Circ Res. 2003; 92: 493–500.
Jormsjo S, Wuttge DM, Sirsjo A, Whatling C, Hamsten A, Stemme S, Eriksson P. Differential expression of cysteine and aspartic proteases during progression of atherosclerosis in apolipoprotein E-deficient mice. Am J Pathol. 2002; 161: 939–945.
Shi GP, Sukhova GK, Grubb A, Ducharme A, Rhode LH, Lee RT, Ridker PM, Libby P, Chapman HA. Cystatin C deficiency in human atherosclerosis and aortic aneurysms. J Clin Invest. 1999; 104: 1191–1197.
Fabunmi RP, Sukhova GK, Sugiyama S, Libby P. Expression of tissue inhibitor of metalloproteinases-3 in human atheroma and regulation in lesion-associated cells: a potential protective mechanism in plaque stability. Circ Res. 1998; 83: 270–278.
Fabunmi RP, Baker AH, Murray EJ, Booth RF, Newby AC. Divergent regulation by growth factors and cytokines of 95 kDa and 72 kDa gelatinases and tissue inhibitors or metalloproteinases-1, -2, and -3 in rabbit aortic smooth muscle cells. Biochem J. 1996; 315: 335–342.
Ishibashi S, Goldstein JL, Brown MS, Herz J, Burns DK. Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice. J Clin Invest. 1994; 93: 1885–1893.
Shi GP, Chapman HA, Bhairi SM, DeLeeuw C, Reddy VY, Weiss SJ. Molecular cloning of human cathepsin O, a novel endoproteinase and homologue of rabbit OC2. FEBS Lett. 1995; 357: 129–134.
Reddy VY, Zhang QY, Weiss SJ. Pericellular mobilization of the tissue-destructive cysteine proteases, cathepsin B, L, and S, by human monocyte-derived macrophages. Proc Natl Acad Sci U S A. 1995; 92: 3849–3853.
Punturieri A, Filippov S, Allen E, Caras I, Murray R, Reddy V, Weiss SJ. Regulation of elastinolytic cysteine proteinase activity in normal and cathepsin K-deficient human macrophages. J Exp Med. 2000; 192: 789–799.
Grainger DJ, Kemp PR, Metcalfe JC, Liu AC, Lawn RM, Williams NR, Grace AA, Schofield PM, Chauhan A. The serum concentration of active transforming growth factor-? is severely depressed in advanced atherosclerosis. Nat Med. 1995; 1: 74–79.
Nishimura Y, Kawabata T, Yano S, Kato K. Inhibition of intracellular sorting and processing of lysosomal cathepsins H and L at reduced temperature in primary cultures of rat hepatocytes. Arch Biochem Biophys. 1990; 283: 458–463.
Kuliawat R, Klumperman J, Ludwig T, Arvan P. Differential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic beta-cells. J Cell Biol. 1997; 137: 595–608.
Ahn K, Yeyeodu S, Collette J, Madden V, Arthur J, Li L, Erickson AH. An alternate targeting pathway for procathepsin L in mouse fibroblasts. Traffic. 2002; 3: 147–159.
Jackson RC, Blobel G. Post-translational cleavage of presecretory proteins with an extract of rough microsomes from dog pancreas containing signal peptidase activity. Proc Natl Acad Sci U S A. 1977; 74: 5598–5602.
Bromme D, Okamoto K, Wang BB, Biroc S. Human cathepsin O2, a matrix protein-degrading cysteine protease expressed in osteoclasts: functional expression of human cathepsin O2 in Spodoptera frugiperda and characterization of the enzyme. J Biol Chem. 1996; 271: 2126–2132.
Shi GP, Munger JS, Meara JP, Rich DH, Chapman HA. Molecular cloning and expression of human alveolar macrophage cathepsin S, an elastinolytic cysteine protease. J Biol Chem. 1992; 267: 7258–7262.
Sukhova GK, Zhang Y, Pan JH, Wada Y, Yamamoto T, Naito M, Kodama T, Tsimikas S, Witztum JL, Lu ML, Sakara Y, Chin MT, Libby P, Shi GP. Deficiency of cathepsin S reduces atherosclerosis in LDL receptor-deficient mice. J Clin Invest. 2003; 111: 897–906.
Gu L, Okada Y, Clinton SK, Gerard C, Sukhova GK, Libby P, Rollins BJ. Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol Cell. 1998; 2: 275–281.
Inoue S, Egashira K, Ni W, Kitamoto S, Usui M, Otani K, Ishibashi M, Hiasa K, Nishida K, Takeshita A. Anti-monocyte chemoattractant protein-1 gene therapy limits progression and destabilization of established atherosclerosis in apolipoprotein E-knockout mice. Circulation. 2002; 106: 2700–2706.
Dansky HM, Barlow CB, Lominska C, Sikes JL, Kao C, Weinsaft J, Cybulsky MI, Smith JD. Adhesion of monocytes to arterial endothelium and initiation of atherosclerosis are critically dependent on vascular cell adhesion molecule-1 gene dosage. Arterioscler Thromb Vasc Biol. 2001; 21: 1662–1667.
Hummel V, Kallmann BA, Wagner S, Fuller T, Bayas A, Tonn JC, Benveniste EN, Toyka KV, Rieckmann P. Production of MMPs in human cerebral endothelial cells and their role in shedding adhesion molecules. J Neuropathol Exp Neurol. 2001; 60: 320–327.
Riese RJ, Shi GP, Villadangos J, Stetson D, Driessen C, Lennon-Dumenil AM, Chu CL, Naumov Y, Behar SM, Ploegh H, Locksley R, Chapman HA. Regulation of CD1 function and NK1.1(+) T cell selection and maturation by cathepsin S. Immunity. 2001; 15: 909–919.
Leca G, Mansur SE, Bensussan A. Expression of VCAM-1 (CD106) by a subset of TCR -bearing lymphocyte clones: involvement of a metalloprotease in the specific hydrolytic release of the soluble isoform. J Immunol. 1995; 154: 1069–1077.
Kowalski J, Okopien B, Madej A, Makowiecka K, Zielinski M, Kalina Z, Herman ZS. Levels of sICAM-1, sVCAM-1 and MCP-1 in patients with hyperlipoproteinemia IIa and -IIb. Int J Clin Pharmacol Ther. 2001; 39: 48–52.
Wang G, Woo CW, Sung FL, Siow YL, O K. Increased monocyte adhesion to aortic endothelium in rats with hyperhomocysteinemia: role of chemokine and adhesion molecules. Arterioscler Thromb Vasc Biol. 2002; 22: 1777–1783.
Kittelberger R, Davis PF, Stehbens WE. Distribution of type IV collagen, laminin, nidogen and fibronectin in the haemodynamically stressed vascular wall. Histol Histopathol. 1990; 5: 161–167.
Navaratnam V. Organisation and reorganisation of blood vessels in embryonic development. Eye. 1991; 5: 147–150.
Del Maestro RF, Vaithilingam IS, McDonald W. Degradation of collagen type IV by C6 astrocytoma cells. J Neurooncol. 1995; 24: 75–81.
Libby P, Sch?nbeck U. Drilling for oxygen: angiogenesis involves proteolysis of the extracellular matrix. Circ Res. 2001; 89: 195–197.
Bromme D, Bonneau PR, Lachance P, Wiederanders B, Kirschke H, Peters C, Thomas DY, Storer AC, Vernet T. Functional expression of human cathepsin S in Saccharomyces cerevisiae: purification and characterization of the recombinant enzyme. J Biol Chem. 1993; 268: 4832–4838.
Dehrmann FM, Coetzer TH, Pike RN, Dennison C. Mature cathepsin L is substantially active in the ionic milieu of the extracellular medium. Arch Biochem Biophys. 1995; 324: 93–98.
Garnero P, Borel O, Byrjalsen I, Ferreras M, Drake FH, McQueney MS, Foged NT, Delmas PD, Delaisse JM. The collagenolytic activity of cathepsin K is unique among mammalian proteinases. J Biol Chem. 1998; 273: 32347–32352.
Ishidoh K, Kominami E. Procathepsin L degrades extracellular matrix proteins in the presence of glycosaminoglycans in vitro. Biochem Biophys Res Commun. 1995; 217: 624–631.
Ooyama T, Fukuda K, Oda H, Nakamura H, Hikita Y. Substratum-bound elastin peptide inhibits aortic smooth muscle cell migration in vitro. Arteriosclerosis. 1987; 7: 593–598.
Karnik SK, Brooke BS, Bayes-Genis A, Sorensen L, Wythe JD, Schwartz RS, Keating MT, Li DY. A critical role for elastin signaling in vascular morphogenesis and disease. Development. 2003; 130: 411–423.
Vancompernolle K, Van Herreweghe F, Pynaert G, Van de Craen M, De Vos K, Totty N, Sterling A, Fiers W, Vandenabeele P, Grooten J. Atractyloside-induced release of cathepsin B, a protease with caspase-processing activity. FEBS Lett. 1998; 438: 150–158.
Ishisaka R, Utsumi T, Kanno T, Arita K, Katunuma N, Akiyama J, Utsumi K. Participation of a cathepsin L-type protease in the activation of caspase-3. Cell Struct Funct. 1999; 24: 465–470.
Guicciardi ME, Deussing J, Miyoshi H, Bronk SF, Svingen PA, Peters C, Kaufmann SH, Gores GJ. Cathepsin B contributes to TNF--mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome C. J Clin Invest. 2000; 106: 1127–1137.
Li W, Dalen H, Eaton JW, Yuan XM. Apoptotic death of inflammatory cells in human atheroma. Arterioscler Thromb Vasc Biol. 2001; 21: 1124–1130.
Li W, Yuan XM, Olsson AG, Brunk UT. Uptake of oxidized LDL by macrophages results in partial lysosomal enzyme inactivation and relocation. Arterioscler Thromb Vasc Biol. 1998; 18: 177–184.
Shi GP, Villadangos JA, Dranoff G, Small C, Gu L, Haley KJ, Riese R, Ploegh HL, Chapman HA. Cathepsin S required for normal MHC class II peptide loading and germinal center development. Immunity. 1999; 10: 197–206.
Nakagawa T, Roth W, Wong P, Nelson A, Farr A, Deussing J, Villadangos JA, Ploegh H, Peters C, Rudensky AY. Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus. Science. 1998; 280: 450–453.
Hansson G, Libby P, Schoenbeck U, Yan Z-Q. Innate and adaptive immunity in the pathogenesis of atherosclerosis. Circ Res. 2002; 91: 281–291.
Zhang Y, Cliff WJ, Schoefl GI, Higgins G. Immunohistochemical study of intimal microvessels in coronary atherosclerosis. Am J Pathol. 1993; 143: 164–172.
Sukhova GK, Schonbeck U, Rabkin E, Schoen FJ, Poole AR, Billinghurst RC, Libby P. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques. Circulation. 1999; 99: 2503–2509.
Chen J, Tung CH, Mahmood U, Ntziachristos V, Gyurko R, Fishman MC, Huang PL, Weissleder R. In vivo imaging of proteolytic activity in atherosclerosis. Circulation. 2002; 105: 2766–2771.
Liuzzo JP, Petanceska SS, Moscatelli D, Devi LA. Inflammatory mediators regulate cathepsin S in macrophages and microglia: a role in attenuating heparan sulfate interactions. Mol Med. 1999; 5: 320–333.
Day SM, Reeve JL, Mundada L, Pan JH, Shi GP, Fay W. Cathepsin S–deficient mice display a prothrombotic phenotype. Circulation. 2002; 106: 195.
Villadangos JA, Bryant RA, Deussing J, Driessen C, Lennon-Dumenil AM, Riese RJ, Roth W, Saftig P, Shi GP, Chapman HA, Peters C, Ploegh HL. Proteases involved in MHC class II antigen presentation. Immunol Rev. 1999; 172: 109–120.(Jian Liu; Galina K. Sukho)