Published online before print December 18, 2008, doi: 10.1161/ATVBAHA.108.181578
© 2009 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
Leukocyte Cathepsin S Is a Potent Regulator of Both Cell and Matrix Turnover in Advanced Atherosclerosis
From the Division of Biopharmaceutics (R.d.N., I.B., J.H.v.d.T., A.O.K., R.D., P.J.v.S., S.H.v.H., M.M.W., T.J.C.v.B., E.A.L.B.), Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, The Netherlands; the Department of Cardiology (R.d.N., A.O.K., J.W.J., E.E.v.d.W.), Leiden University Medical Centre, The Netherlands; the Department of Pathology (J.H.v.d.T.), Academic Medical Center, Amsterdam, The Netherlands; the Division of Bio-organic Synthesis (M.A.L., H.S.O.), Gorlaeus Laboratories, Leiden University, The Netherlands; Wihuri Research Institute (P.T.K.), Helsinki, Finland; the Department of Medicine (G.P.S.), Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass; and the Department of Pathology (E.A.L.B.), University of Maastricht, The Netherlands.
Correspondence to I. Bot, Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands. E-mail i.bot{at}lacdr.leidenuniv.nl
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Abstract |
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Objective— A dysbalance of proteases and their inhibitors is instrumental in remodeling of atherosclerotic plaques. One of the proteases implicated in matrix degradation is cathepsin-S (CatS). To address its role in advanced lesion composition, we generated chimeric LDLr–/– mice deficient in leukocyte CatS by transplantation with CatS–/–xLDLr–/– or with LDLr–/– bone marrow and administered a high-fat diet.
Methods and Results— No difference in aortic root lesion size could be detected between CatS+/+ and CatS–/– chimeras. However, leukocyte CatS deficiency markedly changed plaque morphology and led to a dramatic reduction in necrotic core area by 77% and an abundance of large foam cells. Plaques of CatS–/– chimeras contained 17% more macrophages, 62% less SMCs, and 33% less intimal collagen. The latter two could be explained by a reduced number of elastic lamina fractures. Moreover, macrophage apoptosis was reduced by 60% with CatS deficiency. In vitro, CatS was found to be involved in cholesterol metabolism and in macrophage apoptosis in a collagen and fibronectin matrix.
Conclusion— Leukocyte CatS deficiency results in considerably altered plaque morphology, with smaller necrotic cores, reduced apoptosis, and decreased SMC content and collagen deposition and may thus be critical in plaque stability.
To address the role of leukocyte cathepsin-S in advanced lesion composition, we generated chimeric LDLr–/– mice deficient in leukocyte CatS by transplantation with CatS–/–xLDLr–/– or with LDLr–/– bone marrow. CatS deficiency resulted in considerably altered plaque morphology, with smaller necrotic cores, reduced apoptosis, and decreased SMC content and collagen deposition.
Key Words: atherosclerosis • matrix • cathepsins • leukocytes • apoptosis
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Introduction |
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Proteolysis is an important process in the pathogenesis of atherosclerosis. Leukocyte transmigration through the endothelium, smooth muscle cell (SMC) migration through the elastic lamina, and intimal neoangiogenesis all rely on degradation of the extracellular matrix (ECM).1–3 Proteolytic enzymes like matrix metalloproteinases (MMPs) and cathepsins have been linked to ECM remodeling leading to arterial enlargement, aneurysm formation, and plaque disruption.4–7 Moreover, by releasing matrix-bound cytokines, chemokines, and growth factors, proteases actively participate in cell turnover and inflammation.2,8
Cathepsins are enzymes with strong elastolytic and collagenolytic properties and form a distinct subgroup of atherosclerosis-related proteases because their physiological actions not only affect ECM degradation, but also directly modulate inflammation, immunogenic responses, and cellular behavior.9–12 In fact, Cathepsin-B (CatB) has been shown to activate IL-1 converting enzyme (caspase-1)13 and Cathepsin-S (CatS) processes the invariant chain (Ii), a chaperone for MHC-II and -I, therewith affecting antigen presentation and NKT-cell maturation.14–16 Furthermore, CatS inhibits HDL3 induced cholesterol efflux from macrophages and several cathepsins (eg, D, F, S, and K) are able to modify apoB100 in LDL, thereby inducing foam cell formation.17–19 CatS expression is stimulated by the proinflammatory cytokines IL-1β, IFN-
, and TNF-
,20 all of which have been linked to atherosclerosis.21,22
Because this enzyme can be expressed by all atheroma-associated cells and both its expression and activity are stimulated by a range of proinflammatory cytokines that are highly expressed in atherosclerotic plaques, Sukhova et al recently proposed the involvement of CatS in atherogenesis.20,23–25 Also, CatS expression by macrophages at the shoulder regions was found to be increased, suggesting that this enzyme is involved in plaque rupture.20 CatS deficiency attenuates plaque growth in LDLr knockout (LDLr–/–) mice26 and impairs intimal neovascularization,27 a process that is thought to be of major importance in atherosclerotic plaque progression and stability.28
As CatS can exert a wide spectrum of physiological actions depending on its source, abundance, and microenvironment, it is important to dissect the cell-specific functions of this enzyme to fully understand its role in atherogenesis. With the present study we aimed to define the leukocyte-specific function of CatS. Chimeric LDLr–/– mice deficient in leukocyte CatS were generated by bone marrow transplantation (BMT) of LDLr–/– x CatS–/– mice to irradiated LDLr–/– mice. We found that leukocyte CatS deficiency leads to a markedly altered plaque morphology and composition, which could in part be attributed to fewer elastic lamina ruptures and a higher resistance of macrophages to apoptosis and necrosis.
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Materials and Methods |
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A detailed description of the Methods is given in the supplemental materials (available online at http://atvb.ahajournals.org).
Animals and Study Protocol
All animal work was approved by the regulatory authority of Leiden University and performed in compliance with the Dutch government guidelines. Female LDLr–/– mice (n=22) were obtained from our in-house breeding stock and irradiated with an X-ray dose of 9 Gy as previously described.29 Twenty-four hours after irradiation, mice were injected intravenously with 1x107 CatS-deficient bone marrow–derived cells obtained from CatS–/–xLDLr–/– mice that were generated as described previously.29 Bone marrow from CatS+/+xLDLr–/– littermates was used as control. Mice were placed on a high-fat diet containing 0.25% cholesterol (Special Diet Services) for 12 weeks starting 6 weeks after BMT. After a total of 12 weeks of diet feeding, in situ perfusion-fixation was performed, after which the aortic root lesions were analyzed. Bone marrow cells were obtained for genotyping to verify that recipient cells had been replaced by donor bone marrow by flushing both femurs and tibias with PBS. Double knockout genotypes were confirmed by PCR of genomic DNA as described.10
Statistics
Differences in plaque size were statistically analyzed for significance using the Mann–Whitney U test. Other plaque parameters and constituents as well as differences in 
Ct were compared using the 2-tailed Student t test. The incidence of elastic lamina rupture was compared using the Yate corrected 2-sided Fisher exact test. Values are displayed as mean±SEM. A level of P<0.05 was considered significant.
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Results |
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Bone Marrow Transplantation
Bone marrow transplantation was performed with freshly isolated bone marrow cells from CatS–/–xLDLr–/– mice or their CatS+/+xLDLr–/– littermates. Western-type diet feeding resulted in a steady elevation of plasma cholesterol levels (850±150 mg/dL to 1980±190 mg/dL) and bodyweight over a period of 12 weeks during which both parameters did not differ between groups.
To verify whether the transplantation was successful, CatS genotyping was performed on genomic DNA from bone marrow–derived cells after euthanization 18 weeks posttranplantation. This showed that CatS–/– BMT resulted in an almost complete depletion of autologue bone marrow (data not shown). Immunostaining revealed that the overall intimal CatS content had been reduced by approximately 50% (P=0.004) in CatS–/– transplanted animals, whereas CatS protein levels were preserved in SMC rich areas, such as the media and the fibrous cap (Figure 1A through 1C).
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No Effect on Lesion Size, but a Remarkable Change in Plaque Morphology
Advanced lesions developed within 12 weeks of high-fat diet and did not display any differences in size between both groups (Figure 2A). This study, however, aimed to analyze the effect of leukocyte CatS deficiency on the stability, morphology, and composition of advanced plaques, and this revealed a marked change in plaque phenotype. Mice with CatS deficient leukocytes tended to developed less progressed lesions compared to controls. Only 36% of the plaques in the CatS–/– BMT group showed a cap-core morphology compared to 82% in the control lesions (P=0.08, Table). Instead, the majority of these lesions were phenotypically similar to large fatty streaks, containing a high amount of macrophage derived foam cells, with few SMCs and little collagen and lacking a necrotic core with an overlying fibrous cap (Figure 2G).
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Leukocyte CatS Deficiency Resulted in Decreased SMC and Collagen Content
The observed changes in plaque morphology were confirmed by quantification of several plaque constituents. Only limited amount of fibronectin could be detected in lesions by immunohistochemistry, but overall expression levels did not differ between groups (data not shown). Intimal macrophage content however was slightly increased in lesions of the CatS–/– chimeras (P=0.02), whereas SMC content was decreased by a marked 62% (P=0.007, Figure 2B and 2C). In keeping, intimal collagen was reduced as well (–33%, P=0.02), albeit that the effect was smaller than that on VSMCs (Figure 2D). In fact, the collagen:SMC-ratio increased from 2.0±0.3 in controls to 6.2±0.8 in CatS–/– lesion (P=0.03), indicating that, in addition to reduced collagen synthesis as a consequence of decreased SMC numbers, collagen degradation might be impaired as well in the absence of leukocyte CatS. Alternatively, VSMCs present in plaques of CatS–/– chimeras may produce more collagen, resulting in a disproportional reduction in collagen content. To test this hypothesis, collagen synthesis was determined by VSMCs in vitro in the presence or absence of CatS inhibitors. Therefore, the selective CatS inhibitor CLIK6030 was synthesized and validated for its inhibitory capacity (supplemental Figure IA). CatS activity in RAW264.7 cells or peritoneal macrophages from C57Bl/6 or LDLr–/– mice was completely repressed by the specific CatS inhibitor CLIK60 at 10–6 mol/L. CatS inhibition by the general cathepsin inhibitor E64 as well as CLIK60 resulted in enhanced collagen synthesis, whereas Brefeldin A and serum deprivation decreased collagen synthesis as expected (supplemental Figure IB). This suggests that indeed plaque SMCs of CatS–/– chimeras may elaborate more collagen than SMCs in littermate plaques.
Leukocyte CatS Deficiency Resulted in Decreased Necrotic Core Development
Of the lesions in the CatS–/– chimeras, 64% did not develop necrotic cores, whereas plaques that did have smaller areas of necrosis. Necrotic core area was decreased by 77% from 22% in controls to only 5% in plaques of CatS–/– chimeras (P=0.001, Figure 2E). This effect was also observed when excluding lesions that had not developed necrotic cores at all (data not shown). Furthermore, leukocyte CatS deficiency led to lesions that contained a higher amount of large macrophage foam cells (Type III), whereas control plaques enclosed mainly small and medium sized macrophages and macrophage derived foam cells (Type I & II lesions, Table).31 This is reflected by a 38% increase of average foam cell size (P=0.0003, Figure 2F).
CatS Deficiency Impaired Elastolysis and Attenuated Apoptosis
SMC content depends on a variety of factors like proliferation, migration, and ECM degradation. On stimulation SMCs will transmigrate to the intima through the elastic lamina. Degradation of the elastic lamina is vital for migration of SMCs and is mediated by the action of various proteases, such as MMP-8, CatK, and -S and to a lesser extent MMP-2 and -9. Indeed, leukocyte CatS deficiency led to a marked decrease by 68% of elastic lamina ruptures per mouse (P=0.02), which suggests that leukocyte-derived CatS is a dominant elastolytic enzyme in elastic lamina disruption (Figure 3A through 3C).
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As intimal macrophage content and phenotype changed on CatS deficiency and various studies have tentatively proposed a role for cathepsins in cell death,7,12 lesions were TUNEL stained to assess apoptosis. Most commonly, TUNEL-positive staining was found in foam cell rich or necrotic areas, principally indicating macrophage cell death. In accordance with the decreased necrotic core area, apoptotic rate was reduced by approximately 60% in lesions that contained CatS deficient leukocytes (P=0.001; Figure 3D through 3F).
Macrophage apoptosis was further assessed in vitro. Apoptotic cell death was induced by 25 µg/mL Cu2+-oxidized LDL or 10 µmol/L cisplatin. Inhibition of CatS activity did not affect RNA expression of the apoptosis related genes Bcl-2, Bax, P53, or Flip (supplemental Figure IIA). XIAP expression tended to be increased by 70%, but this was not significant (P=0.16). Spontaneous or oxLDL/cisplatin induced apoptotic rate measured by flow cytometry analysis of AnnexinV+ cells was not affected by CLIK60 or E64 treatment, pointing to a more indirect role for CatS in apoptosis (supplemental Figure IIB).
Matrix Degradation Products and Apoptosis
To test the hypothesis that elastin degradation products, derived from CatS elastolytic activity, induced apoptosis, oxLDL-treated peritoneal macrophages were incubated with 10 µg/mL soluble elastin with or without 100 mmol/L lactose, an inhibitor of the laminin/elastin receptor. Elastin degradation products neither aggravated nor attenuated apoptotic or necrotic cell death of peritoneal macrophages nor did they affect macrophage proliferation (supplemental Figure IIC and IID).
Alternatively, CatS could effect a disruption of cell-matrix interaction of macrophages in the surrounding ECM. To test this, peritoneal macrophages were cultured on gelatin, collagen type I, or fibronectin coated dishes and stimulated with IFN-, a powerful CatS inducer,20 and the effect of CLIK60 on apoptosis in this context was assessed by AnnexinV/PI staining. Cells became progressively less viable when cultured on a fibronectin or collagen matrix compared to those that were cultured on gelatin. The amount of apoptotic cells, however, remained at the level of the gelatin cultured cells, when cells were treated with CLIK60 (Figure 4), suggesting that CatS contributes to the increased apoptosis of fibronectin or collagen attached macrophages.
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Furthermore, macrophages that were exposed to fibronectin displayed higher protein expression of focal adhesion kinase (FAK) compared to control cells (P=0.01), which was abolished when the macrophages were incubated with CatS-induced degradation products of fibronectin (supplemental Figure III). These data were confirmed by RT-PCR analysis, demonstrating that fibronectin degradation by CatS tended to reduce FAK mRNA expression compared to the expression in fibronectin exposed macrophages (relative FAK expression 0.039±0.008 compared to 0.064±0.002, respectively; P=0.09).
CatS Inhibition Increased Macrophage Cholesterol Accumulation and Efflux
As foam cells from lesions of hematopoietic CatS–/– chimeras were larger in size than those of littermate controls, the amount of cholesterol accumulation in macrophages was determined in vitro in the presence of CLIK60. Indeed, free cholesterol accumulation was more than 2-fold increased when CatS was inhibited by CLIK60 (P<0.01; Figure 5A). Because intracellular accumulation of free cholesterol has been reported to be an important inducer of apoptosis,32 also macrophage apoptosis was determined under these conditions. As shown previously, CatS inhibition per se did not affect macrophage apoptosis (Figure 5B).
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Furthermore, the effect of CatS inhibition on the cholesterol efflux capacity of peritoneal macrophages was studied. CLIK60 dose-dependently increased cholesterol efflux, enhancing HDL-induced cholesterol efflux almost 2-fold (P=0.001) at a concentration of 10–6 mol/L (Figure 5C). CatS inhibition did not affect the expression of genes involved in macrophage cholesterol metabolism, such as HMGCoA reductase, SR-A1, SR-B1, ABCA1, and ABCG1 (data not shown). Thus, although CatS appears to be implicated in the regulation of intracellular cholesterol metabolism, a direct involvement of CatS in cholesterol-induced macrophage apoptosis could not be established. Apparently CatS may act proapoptotic in a cholesterol-independent indirect manner.
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Discussion |
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Since the presence of CatS in human atherosclerotic plaques was first described,20 it has become increasingly clear that it is a key actor in atherogenesis and other related vasculopathies. Given its pleiotropic actions in inflammation, cell and matrix turnover, and cholesterol trafficking, it is important to carefully map the cell specific effects of this enzyme in atherosclerosis. In this study, we specifically establish the role of leukocyte CatS in atherosclerotic plaque composition. Cellular and matrix composition of advanced plaques was dramatically altered in mice that were transplanted with CatS-deficient bone marrow, showing a 62% decrease of SMC content, which could, at least in part, be explained by a marked decrease in elastic lamina ruptures, pivotal for SMC migration into the intima. The disproportional reduction of intimal collagen by only 33% suggests that CatS may also be directly involved in intimal collagen production and breakdown. Previous studies with CatS–/– mice already showed impaired lamina degradation and reduced intimal SMC and collagen content.26 Rodgers et al have recently demonstrated that CatS contributes significantly to plaque progression as demonstrated by a reduced plaque size and a reduced number of fibrous cap ruptures in apoE/CatS double knockout mice. Collagen levels and the number of elastic lamina ruptures remained unaffected by CatS deficiency.33 The present study establishes that leukocyte-, and not SMC-, derived CatS is instrumental in the degradation of the inner elastic lamina, which renders leukocyte CatS a potential target for plaque stabilization. Elastin degradation products have previously been reported to be able to promote SMC proliferation via the elastin/laminin receptor.34 Conceivably, in our study, the absence of the elastolytic CatS might have contributed to impaired intimal SMC accumulation. However, no effect of degraded elastin could be detected on macrophage proliferation, nor was there any effect of elastin degradation products on apoptosis, either spontaneous or induced with oxLDL or cisplatin.
Earlier studies demonstrated that systemic CatS deficiency impairs monocyte transmigration through an endothelial barrier.26 By contrast, monocyte infiltration was not repressed and lesional macrophage staining area was even increased in the absence of leukocyte CatS. Thus, whereas earlier studies showed impaired monocyte transmigration through the endothelium in CatS–/– mice,26 the present observations suggest that endothelial cell–, and not monocyte-, derived CatS is vital for leukocyte transmigration. The relative increase of intimal macrophages and the observed higher abundance of large foam cells could be explained by the vast reduction in necrotic core formation and reduced susceptibility of macrophages to apoptosis in the absence of CatS.
Several studies show that lysosomal enzymes, including Cathepsin B, D, and L, can directly induce apoptosis once released from the lysosomal compartment.12,35–37 We now show that CatS inhibition by CLIK60 did not directly affect apoptosis or necrosis in macrophages that were stimulated with oxLDL or cisplatin. Moreover, the expression levels of several apoptosis-related genes remained unaffected by CLIK60. Additionally, ECM constituents and degradation products have been reported to be important regulators of cell death.7,37,38 As mentioned earlier, soluble elastin degradation products did not affect apoptosis or proliferation of macrophages in vitro. Interestingly, macrophages cultured on fibronectin or collagen type I showed an increased apoptotic rate compared to macrophages that were cultured on gelatin. Inhibition of CatS activity prevented this induction of apoptosis and kept the rate of cell death at the baseline level of gelatin cultured macrophages. In vivo, fibronectin levels may be affected with reduced plaque CatS content, however only limited amount of fibronectin was detectable in atherosclerotic lesions and despite reduced lesional CatS content overall plaque fibronectin did not differ between groups. However, the in vitro data suggest that CatS may induce apoptosis by mediating pericellular fibronectin and collagen type I breakdown and may thus be an important regulator of macrophage apoptosis in vivo. These effects may be, at least partially, attributable to loss of FAK expression induced after CatS mediated degradation of fibronectin, resulting in detachment of macrophages from the extracellular matrix.
Finally, free cholesterol is a potent inducer of apoptosis in macrophages by triggering cytochrome C release and activating FasL.32,39,40 In line with earlier observations regarding the inhibitory action of CatS on cholesterol efflux,17 CatS inhibition was indeed found to potentiate both HDL and apoAI-induced cholesterol efflux from peritoneal macrophages. Intriguingly, CatS inhibition also led to enhanced free cholesterol accumulation, which did not affect macrophage apoptosis, pointing toward a more indirect role for CatS in the regulation of apoptosis.
In conclusion, our data suggest that leukocytic CatS contributes to plaque growth and stability. Medial SMC migration into the intima and subsequent proliferation and collagen deposition heavily relies on the elastolytic properties of macrophage derived CatS. Also, the critical contribution of macrophage CatS to necrotic core expansion in advanced plaques underpins its importance for plaque stability and eventually acute ischemic events.
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Acknowledgments |
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Sources of Funding
This work was supported by the Netherlands Heart Foundation (Grants no. M93.001 [R.d.N.], 2001D032 [J.W.J.], and 2003T201 [E.A.L.B.]) and the Netherlands Organization for Scientific Research (VENI award [J.H.v.d.T.], grants 916.86.046 [I.B.] and 016.026.019 [E.A.L.B.]). The Leiden University division of Biopharmaceutics belongs to the European Vascular Genomics Network (http://www.evgn.org), a Network of Excellence supported by the European Community’s Sixth Framework Program for Research Priority 1 (Life Sciences, Genomics, and Biotechnology for Health; contract LSHM-CT-2003-503254).
Disclosures
None.
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Footnotes |
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Received November 24, 2008; accepted December 4, 2008.
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