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Cardiovascular Research Advance Access originally published online on November 17, 2008
Cardiovascular Research 2009 81(2):278-285; doi:10.1093/cvr/cvn311
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Leucocyte cathepsin K affects atherosclerotic lesion composition and bone mineral density in low-density lipoprotein receptor deficient mice

Jian Guo1, Ilze Bot1,*, Ramon de Nooijer1,2, Sandra J. Hoffman3, George B. Stroup3, Erik A.L. Biessen1, G. Martin Benson4, Pieter H.E. Groot4, Miranda Van Eck1 and Theo J.C. Van Berkel1

1 Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research (LACDR), Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands
2 Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands
3 Department of Bone and Cartilage Biology, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA 19406, USA
4 Atherosclerosis Department, GlaxoSmithKline Pharmaceuticals, Stevenage, UK

* Corresponding author. Tel: +31 71 5276213; fax: +31 71 5276032.E-mail address: i.bot{at}lacdr.leidenuniv.nl

Received 14 August 2008; revised 7 November 2008; accepted 11 November 2008

Time for primary review: 17 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: Cathepsin K (CatK), an established drug target for osteoporosis, has been reported to be upregulated in atherosclerotic lesions. Due to its proteolytic activity, CatK may influence the atherosclerotic lesion composition and stability. In this study, we investigated the potential role of leucocyte CatK in atherosclerotic plaque remodelling.

Methods and results: To assess the biological role of leucocyte CatK, we used the technique of bone marrow transplantation to selectively disrupt CatK in the haematopoietic system. Total bone marrow progenitor cells from CatK+/+, CatK+/–, and CatK–/– mice were transplanted into X-ray irradiated low-density lipoprotein receptor knockout (LDLr–/–) mice. The selective silencing of leucocyte CatK resulted in phenotypic changes in bone formation with an increased total bone mineral density in the CatK–/– chimeras and an effect of gene dosage. The absence of leucocyte CatK resulted in dramatically decreased collagen and increased macrophage content of the atherosclerotic lesions while lesion size was not affected. The atherosclerotic lesions also demonstrated less elastic lamina fragmentation and a significant increase in the apoptotic and necrotic area in plaques of mice transplanted with CatK–/– bone marrow.

Conclusion: Leucocyte CatK is an important determinant of atherosclerotic plaque composition, vulnerability, and bone remodelling, rendering CatK an attractive target for pharmaceutical modulation in atherosclerosis and osteoporosis.

KEYWORDS Bone marrow transplantation; Cathepsin K; Atherosclerosis; Bone remodelling; Atherosclerotic plaque composition


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Remodelling of the extracellular matrix of blood vessels as well as bone resorption vs. formation is a life-long continuously changing and dynamic process. Imbalance of this process could lead to the clinical manifestation of e.g. osteopetrosis/osteoporosis or phenotypic changes in atherosclerotic lesion development. Several research groups have cloned the cysteine protease cathepsin K (CatK), an enzyme with known elastolytic activity, from mouse, rabbit, and human cDNA libraries.1–4 Previous studies on the role of CatK have focused primarily on its function in bone remodelling. CatK, which is highly expressed in osteoclasts,5 has been shown to degrade bone collagen as well as other bone matrix proteins.6,7 Mutations in the CatK gene have been identified as the underlying cause of the relatively rare human osteopetrotic disease, pycnodysostosis.8,9 Targeted deletion of the CatK gene in mice results in many of the phenotypic features of pycnodysostosis, including increased bone mineral density (BMD) and bone deformity.10,11 Furthermore, administration of CatK antagonists in mice has been successful in inhibiting bone resorption both in vitro and in vivo.12 Interestingly, CatK is also highly expressed in human atheroma,13 raising the possibility of its functional role outside the bone, for instance, in remodelling of extracellular matrix of atherosclerotic lesions. In the plaque, CatK was observed in macrophages,14 consistent with their proteolytic activity, and in endothelial cells, where CatK expression was demonstrated to be shear stress induced.15 In addition, Lutgens et al.16 observed increased expression of CatK in smooth muscle cells (SMCs) of atherosclerotic lesions. From these findings, we hypothesize that endothelial CatK may be mainly involved in initial plaque formation, while macrophage CatK may be more important for plaque composition and stability.

Indeed, CatK promotes plaque progression and reduces plaque stability as demonstrated by reduced plaque size and enhanced collagen deposition in ApoE–/–/CatK–/– mice when compared with ApoE deficient mice.16 Whole body CatK deficiency also induced lipid uptake by macrophages, thereby aggravating foam cell formation.16,17 Recently, these data were confirmed by Samokhin et al.,18 demonstrating that CatK deficiency inhibited plaque progression and increased fibrous cap thickness in the brachiocephalic artery after cholate-containing high-fat diet feeding.

To specify the contribution of macrophage CatK to atherosclerotic lesion initiation, progression, and stability, we performed a bone marrow transplantation (BMT) study. Hereto, low-density lipoprotein receptor knockout (LDLr–/–) mice were lethally irradiated and reconstituted with bone marrow cells from CatK wild-type (CatK+/+), heterozygous (CatK+/–), and homozygous knockout (CatK–/–) mice. Also, the effect of CatK heterozygous and homozygous knockout bone marrow cell replacement on bone remodelling was monitored. We demonstrate that leucocyte CatK not only influences the BMD, but also importantly affects the composition of the atherosclerotic plaque.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Animals
All animal work was approved by the regulatory authority of Leiden University and performed in compliance with the Dutch government guidelines. LDLr–/– mice were obtained from the local animal breeding facility (Gorlaeus Laboratories, Leiden, The Netherlands). The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). CatK+/+, CatK+/–, and CatK–/– used as donor mice for BMT were generated as described previously.10 Mice were housed in sterilized filter-top cages and fed a chow diet (Special Diet Services, Witham, Essex, UK). Four weeks after BMT, the diet was switched to a western-type diet (0.25% w/w cholesterol and 15% w/w cocoa butter) for another 12 weeks in order to induce atherosclerosis. Drinking water was supplied with antibiotics (83 mg/L ciprofloxacin and 67 mg/L polymyxin B sulfate) and 6.5 g/L sugar.

To induce bone marrow aplasia, female LDLr–/– mice were exposed to a single dose of 9 Gy (0.19 Gy/min, 200 kV, 4 mA) total body irradiation, using an Andrex Smart 225 Röntgen source (YXLON International, Copenhagen, Denmark) with a 6 mm aluminium filter, 1 day before transplantation. The next day, the mice received 0.5 x 107 bone marrow progenitor cells isolated from CatK+/+, CatK+/–, and CatK–/– mice intravenously (n = 11–13 per group).

Throughout the study, serum lipid levels were measured. After an overnight fasting period, ~100 µL blood was drawn from each individual mouse by tail bleeding. The concentrations of serum total cholesterol and triglyceride levels were determined using enzymatic colorimetric assays. Precipath (standardized serum; Roche, Germany) was used as an internal standard.

The haematopoietic chimerism of the LDLr–/– mice was determined by genomic DNA genotyping from peripheral blood leucocytes, at 16 weeks after BMT. CatK forward: 5'-ATGGTCTCTCTAAACCTTTGG-3' and CatK reverse: 5'-ACCTGGTTCTTGACTGGAGTAACG-3' primers were used during PCR using the following programmes: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1.5 min, for 30 cycles; 72°C for 10 min. Semi-quantitative PCR was set up by mixing the genomic DNA from both wild-type and knockout animals, ranging from 0% up to 100% of knockout vs. wild-type. Final PCR products were separated on 1% agarose gel.

2.2 Peripheral quantitative computed tomographic analysis of tibias
To investigate the role of leucocyte CatK in bone remodelling, right proximal tibias of the transplanted mice were evaluated by peripheral quantitative computer tomography (pQCT) using the Stratec/Norland Research M (Norland Medical Systems, Inc., Fort Atkinson, WI, USA). Quality control of the instrument was carried out each day prior to and after sample analysis by scanning a cone phantom of known density. A standard scan was performed following sample analysis. Scans were made ex vivo on the right proximal tibia. A 3D 0.5 mm slice was taken at a point 15% of the length between the tibia–fibula junctions distal to the most proximal tibia–fibula junction. Settings for the mask were as follows: object length, 100 mm; voxel size, 0.07 mm; diameter, 10 mm; speed, 2 mm/s; number of blocks, 1; scout view (SV) speed, 20 mm/s; and SV distance between lines, 0.5 mm. BMD, bone mineral content, and bone area were determined for the total, trabecular, subcortical and cortical, and cortical regions. Analysis was as follows: Calcbd was set at contour mode 2, peel mode 2, inner threshold 400 mg/cm3 and Cortbd was separation mode 2 and threshold 400 mg/cm3.

2.3 Histology
To analyse the atherosclerotic lesions, LDLr–/– mice were sacrificed at 16 weeks after BMT (4 weeks on regular chow diet followed by 12 weeks on western-type diet). The arterial tree was perfusion-fixed (Zinc Formal Fixx, Shandon, UK) and atherosclerosis was analysed as described.19 Mean lesion area (in µm2) was calculated from 10 oil red O-stained sections from each individual mouse, starting at the appearance of the tricuspid valves. The macrophage infiltration in the atherosclerotic lesions was determined by immunohistochemistry using a rabbit-anti-mouse CD68 antibody (kindly provided by S. Gordon, Sir William Dunn School of Pathology, University of Oxford, UK; 1:500 dilution). The collagen content of the lesions was visualized by a Masson’s Trichrome staining according to the manufacturer’s instructions (Sigma Diagnostics). The CD68- and collagen-positive content of lesions were expressed as the level of staining-positive area as the percentage of total lesion area (five sections from each mouse). The mean necrotic core area was carefully examined as previously described by Virmani et al.20 and displayed as the necrotic area as the percentage of total lesion area (five sections from each mouse). For the detection of DNA fragmentation, terminal deoxynucleotide transferase-mediated dUTP nick end labelling (TUNEL) staining for apoptosis in atherosclerotic lesions was performed using the In Situ Cell Death Detection Kit (Roche) as previously described by Gavrieli et al.21 TUNEL-positive nuclei were visualized by Nova Red (Vector) and sections were counterstained with 0.3% methylgreen. Sections treated with DNase (2 U/section) served as a positive control. Cell death was expressed as the percentage of TUNEL-positive nuclei of the total nuclei number in the neointima. All quantifications were performed in a blinded fashion by computer-aided morphometric analysis using a Leica image analysis system.

To analyse the effect of CatK deficiency on the preservation of the elastic lamina, oil red O-stained sections of the aortic root were examined under a fluorescence microscope for the discontinuities of elastic lamina, which exhibits auto-fluorescence under a 465–495 nm excitation filter and a 515–555 nm emission filter. The number of breakdowns of elastic lamina of each mouse was added up together within each group and calculated as the frequency of fragmentation (=breakdowns/number of mice).

2.4 Statistical analysis
Values are displayed as mean ± SEM. Differences were statistically analysed for significance using an ANOVA test. A level of P < 0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Generation of LDLr–/– mice with specific deficiency in leucocyte cathepsin K
To assess the biological role of leucocyte CatK both in atherosclerosis and bone remodelling, we used the technique of BMT to selectively disrupt CatK in the haematopoietic system. Bone marrow progenitor cells from CatK+/+, CatK+/–, and CatK–/– mice were transplanted into irradiated LDLr–/– mice. Reconstitution of the haematopoietic system in recipients of CatK+/+, CatK+/–, and CatK–/– marrow was demonstrated by PCR of genomic DNA from peripheral blood leucocytes at 16 weeks after BMT (Figure 1A). PCR amplification of the wild-type allele produces a 518 bp product in recipients transplanted with CatK+/+ and CatK+/– marrow, and amplification of the mutant allele produces a 1.7 kb product in recipients transplanted with CatK+/– and CatK–/– marrow. Semi-quantitative PCR analysis demonstrated that more than 98% of the peripheral blood leucocytes were of CatK–/– donor origin (Figure 1B).


Figure 1
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  		Figure 1 Generation of LDLr–/– mice with specific deficiency in leucocyte cathepsin K (CatK). Genomic DNA from peripheral blood leucocytes was extracted and used as template for PCR amplification (A) lane 1, DNA marker; lane 2–4, CatK+/+ transplanted mice, which displayed 518 bp wild-type bands; lane 5–7, CatK+/– chimeras, which displayed both 518 bp wild-type bands and 1.7 kb knockout bands; lane 8–10, CatK–/– transplanted mice, which displayed prominent 1.7 kb knockout bands with only faint 518 bp wild-type bands. (B) Semi-quantitative PCR by mixing the genomic DNA from both wild-type and knockout animals, ranging from 0% up to 100% of knockout vs. wild-type, demonstrates that more than 98% of the peripheral blood leucocytes of mice transplanted with CatK–/– bone marrow are indeed of CatK–/– origin.

 
In order to induce atherosclerotic lesion formation, the transplanted LDLr–/– mice were fed a western-type diet. As a result, the total serum cholesterol levels in both the control and experimental groups increased during diet feeding; however, no differences could be detected in both serum total cholesterol and triglyceride levels between the CatK+/+, CatK+/–, and CatK–/– chimeras (Figure 2).


Figure 2
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  		Figure 2 Effects of leucocyte cathepsin K (CatK) deficiency on serum total cholesterol and triglycerides levels. The absence of leucocyte CatK does not affect serum total cholesterol (A) and triglyceride (B) levels, both on a chow diet and on western-type diet (WTD). Values represent the mean ± SEM.

 
3.2 Effect of leucocyte cathepsin K deficiency on bone remodelling
Peripheral quantitative computed tomography analysis of the proximal tibias revealed a CatK gene dosage effect on BMD. Significantly increased total BMD in CatK–/– chimeras to 793 ± 11.5 mg/cm3 was observed, compared with 735.9 ± 16.6 and 757.5 ± 12.6 mg/cm3 in CatK+/+ and CatK+/– transplanted mice, respectively (P < 0.05, Figure 3A). The increase in total BMD was a direct result of a larger amount of cortical bone, likely due to impaired osteoclastic bone resorption in the absence of CatK (Figure 3B). There was very little trabecular bone mass observed in any of the groups. Furthermore, a tendency to a decreased length of the tibia was observed due to the absence of CatK (Figure 3C).


Figure 3
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  		Figure 3 Effect of leucocyte cathepsin K (CatK) deficiency on bone morphology. Disruption of leucocyte CatK results in a gene dosage effect on total bone mineral density (A), cortical bone mineral density (B), and length of tibias (C). Values represent the mean ± SEM. *P < 0.05 compared with CatK+/+.

 
3.3 Effect of leucocyte cathepsin K deficiency on atherosclerotic lesion development
To determine the effects of leucocyte CatK deficiency on the formation of atherosclerotic lesions, the aortic root of the LDLr–/– mice, transplanted with either CatK+/+, CatK+/–, or CatK–/– bone marrow, was perfused and fixed at 16 weeks after BMT. Representative photomicrographs of the aortic roots of CatK+/+, CatK+/–, and CatK–/– transplanted mice are shown in Figure 4. The mean atherosclerotic lesion area of mice transplanted with CatK+/+, CatK+/–, or CatK–/– bone marrow was 5.9 ± 2.8 x 105, 5.9 ± 1.7 x 105, and 6.2 ± 1.1 x 105 µm2, respectively (Figure 4A, P = NS). The necrotic core area as the percentage of total lesion area was 5.6 ± 1.4% and 7.1 ± 4.3% in the CatK+/+ and CatK+/– chimeras, respectively, whereas a significant increase up to 11.9 ± 3.8% in the CatK–/– transplanted mice was observed (Figure 4B, P < 0.01, arrows: typical necrotic core areas). Quantitative morphological analysis of the atherosclerotic lesions revealed that 31.3 ± 9.8% and 35.3 ± 10.2% of the lesions consisted of infiltrated macrophages in CatK+/+ and CatK+/– transplanted animals, respectively, while lesional macrophage content in the CatK–/– group was increased to 53.1 ± 8.1% (Figure 5A, P < 0.05). Collagen content, however, decreased dramatically down to 8.6 ± 3.3% (P < 0.001) in the CatK–/– chimeras compared with 49.6 ± 11.5% and 42.8 ± 12.6% in lesions of mice transplanted with CatK+/+ or CatK+/– bone marrow (Figure 5B). The mice deficient in leucocyte CatK also showed a significantly lower frequency of elastic lamina fragmentation as indicated by arrows in Figure 6; 9 breakdowns from 13 CatK–/– transplanted mice, compared with 23 breakdowns from 13 mice and 16 breakdowns from 11 mice for CatK+/+ and CatK+/– chimeras, respectively (Figure 6A). TUNEL staining also demonstrated that the amount of cell death via apoptosis in the atherosclerotic lesions significantly increased up to 3.0 ± 0.8% (P < 0.01) in leucocyte CatK–/– mice, when compared with 1.8 ± 0.6% and 1.7 ± 0.6% in CatK+/+ and CatK+/– chimeras, respectively (Figure 6B).


Figure 4
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  		Figure 4 Leucocyte cathepsin K (CatK) deficiency did not affect plaque size, but enhanced necrotic area. (A) Plaque size did not differ between the CatK+/+, CatK+/–, and CatK–/– chimeras, while necrotic core size (B) was significantly enhanced in the lesions of mice transplanted with CatK–/– deficient bone marrow. Right panel: representative photos for lesion development in CatK+/+, CatK+/–, and CatK–/– chimeras. Typical necrotic areas are indicated by arrows. Values represent the mean ± SEM, **P < 0.01 compared with CatK+/+.

 


Figure 5
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  		Figure 5 Lesional macrophage and collagen content. (A) Macrophage content (CD68 staining) was significantly increased in the CatK–/– chimeras compared with CatK+/+ controls, while (B) collagen content as measured by a Masson’s Trichrome staining was decreased in these plaques. Lower panels: representative pictures of CD68 and Masson’s Trichrome staining. Values represent the mean ± SEM, *P < 0.05, ***P < 0.001 compared with CatK+/+.

 


Figure 6
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  		Figure 6 Elastic lamina fragmentation and apoptosis. (A) Leucocyte cathepsin K (CatK) deficiency reduced elastic lamina fragmentation, as measured by lamina auto-fluorescence under a 465–495 nm excitation filter and a 515–555 nm emission filter. Fragmentations are designated by arrows in the lower panels. (B) TUNEL staining for apoptosis in atherosclerotic lesions, as shown by representative pictures in the lower panels, was demonstrated to be enhanced in the CatK–/– chimeric mice. Values represent the mean ± SEM, **P < 0.01 compared with CatK+/+.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
Cathepsin K attracted extensive attention with respect to its role in bone remodelling, as systemic disruption of CatK resulted in impaired osteoclastic bone resorption.10,11 Interestingly, Sukhova et al.13 described that CatK is abundantly expressed in macrophages and vascular smooth muscle cells (VSMCs) in human atheroma, while CatK was also seen to be expressed in endothelial cells of atherosclerotic plaques.15,16

In ApoE–/–/CatK–/– mice at 26 weeks of age, plaque area was reduced for 41 ± 8% owing to a decrease in the number of advanced lesions as well as a decrease in individual plaque area.16 This suggests an important role for CatK in atherosclerosis progression; however, CatK disruption also accelerated macrophage foam cell formation.16,17 Thus, it remained unclear how CatK inhibited atherosclerosis progression and if indeed macrophage CatK was responsible for this decrease. In order to investigate the contribution of specifically leucocyte CatK in atherogenesis, we utilized a BMT protocol. This approach possesses the advantage to specifically elucidate the contribution of CatK from haematopoietic origin vs. non-haematopoietic origin to both bone remodelling and atherogenesis.

The absence of leucocyte CatK resulted in significantly increased BMD, which is consistent with previous findings.10,11 These data demonstrate that indeed CatK expression was dose-dependently reduced in the CatK+/– and the CatK–/– transplanted animals compared with CatK+/+ chimeras, although we did not specifically determine CatK activity in the bones. The absence of leucocyte CatK in bone marrow progenitor cells did not affect serum total cholesterol and triglyceride levels. In contrast to total body CatK deficiency, the deficiency of leucocyte CatK did not affect atherosclerotic lesion size, with plaque sizes of 5.9 ± 2.8 x 105, 5.9 ± 1.7 x 105, and 6.2 ± 1.1 x 105 µm2 for CatK+/+, CatK+/–, or CatK–/– bone marrow, respectively. These data indicate that the earlier observed decreased lesion size in total body CatK deficiency was caused by either endothelial cell or SMC CatK. In this respect, it is of interest that CatK in endothelial cells is induced by shear stress15 and its secretion of CatK may influence monocyte infiltration into the arterial wall and consequently plaque progression. The markedly similar lesion size in this study allows specific analysis of leucocyte CatK deficiency on lesion composition.

The absence of leucocyte CatK resulted in a less stable plaque phenotype, as judged from a dramatic decrease in collagen from 49.6 ± 11.5% to 8.6 ± 3.3%, which is accompanied by an increase in lesional macrophage staining and necrotic core area. Alteration of macrophage function by CatK deficiency was previously described in macrophages from ApoE–/–/CatK–/– mice, demonstrating increased lipid uptake and macrophage size.16 Microarray analysis of atherosclerotic aortic arches from ApoE–/– mice vs. ApoE–/–/CatK–/– mice demonstrated that CatK deficiency acts profibrotic by upregulation of genes involved in actin cytoskeleton formation, but is also lipogenic by enhancement of lipid uptake and foam cell formation by pointing towards an upregulation of a number of macrophage genes including CD36.17 While macrophage migration remained unaffected by CatK deficiency, incubation of CatK–/– macrophages with oxidized LDL indeed led to a 53.7% increase in cholesterol ester accumulation when compared with CatK+/+ macrophages, probably due to the upregulation of CD36 as induced by the absence of CatK.17 Our plaque phenotype demonstrating increased macrophage staining can be fully explained by this mechanistic insight, although we did not analyse lipid uptake of macrophages isolated from the CatK+/+, CatK+/–, and CatK–/– chimeras in vitro. However, we demonstrate full repopulation of our recipient mice with the specific donor genotype, implicating that bone marrow-derived macrophages from CatK–/– transplanted mice possess the same phenotype as macrophages isolated from total body CatK deficient mice with respect to lipid uptake and cellular homeostasis.

Importantly, leucocyte numbers were not altered in ApoE–/–/CatK–/– compared with ApoE–/–/CatK+/+ mice, as no difference was found in the number of CD3 positive T-cells and Gr-1 positive macrophages in lymph nodes, blood, and spleen, indicating the absence of systemic effects of CatK deficiency on circulating leucocytes.16 Also, the activation status of these T-cells is not altered as can be concluded from the CD4/CD8 ratio and the number of CD25+ T-cells. As mentioned earlier, we demonstrate full repopulation of our recipient mice with each of the donor genotypes, implying that also in our study the circulating leucocyte numbers remained unaffected by CatK deficiency.

Further characterization of the lesions revealed less elastic lamina fragmentation, again demonstrating the elastinolytic and matrix-degrading activity of CatK similarly as established by the increased BMD in CatK deficient chimeras. These data once more illustrate the consequences of the dose dependent reduction in CatK activity in CatK+/+, CatK+/–, and CatK–/– chimeras, respectively. Also, we found a significant increase in necrotic area in the CatK–/– chimeras, compared with CatK+/+ and CatK+/– transplanted mice. The markedly decreased collagen content is in contrast to the observed slight increase in lesion collagen in total body CatK deficiency.16,18 Apparently, SMC- or endothelial cell-derived CatK mainly contributes to collagen degradation or other collagenase-like enzymes are upregulated in response to macrophage CatK deficiency. VSMCs, the major source of collagen in arteries, which migrate from the tunica media into the intima during late-stage atherosclerosis, require proteolytic degradation of elastin.22 Due to the disruption of CatK, the most potent mammalian elastase yet described,6 in the freshly infiltrated monocytes during initial atherogenesis, this proteolytic degradation of elastin might be partially absent. This in turn may hinder the entrance of SMCs crossing the internal elastic membrane into the intima. The well-preserved elastic lamina structures in the CatK–/– chimeras due to the absence of leucocyte CatK thus might have resulted in the impaired migration of medial SMCs into the atherosclerotic lesions and thus decreased production of collagen. The greatly reduced amount of collagen was compensated by an increase in macrophage content, which may have also contributed to the reduced collagen content by enhanced release of other proteolytic enzymes such as matrix metalloproteinases, which results in accelerated degradation of collagen.23 The observed reduction in elastic lamina breaks do correspond with plaque morphology of lesions in whole body CatK–/– mice,16,18 establishing that indeed leucocyte CatK is mainly responsible for the degradation of elastin in the plaque and medial lamina.

We envision the following sequence of events in atherogenesis: (i) monocytes infiltrate into the arterial wall driven by an initial inflammatory event and differentiate into macrophages; (ii) these macrophages take up oxLDL and become foam cells, meanwhile producing inflammatory cytokines and chemokines that attract more monocytes; (iii) more and more monocytes infiltrate at the later stage, accumulate and die via apoptosis/necrosis due to the excessive production and uptake of oxLDL. Apoptotic macrophages are subsequently engulfed and cleared by newly infiltrated macrophages and other defence systems. However, macrophages death via necrosis results in the disruption of cell membrane/organelles and release of pro-inflammatory molecules, which attracts even more leucocytes into the atherosclerotic lesion area. In the absence of leucocyte CatK, the normal balance during atherogenesis between monocytes infiltration/foam cell accumulation vs. cell death appears to be disrupted, favouring towards augmented cell death via both apoptosis and necrosis. Lutgens et al.16 demonstrated that macrophages of CatK–/– mice display enhanced cholesterol accumulation, which enhances foam cell formation. This in turn may result in enhanced cell death as excessive free cholesterol accumulation in macrophages induces cell death,24,25 thereby explaining the enhanced apoptosis and necrotic core size in the CatK–/– chimeras.

Our study presents a well-defined role of leucocyte CatK in the remodelling of both atherosclerotic lesion phenotype and bone resorption vs. formation. Pharmaceutical intervention of leucocyte CatK thus accounts for an attractive strategy to treat osteoporosis. However, disruption of leucocyte CatK unexpectedly resulted in more unstable atherosclerotic lesions, indicating an important contribution of leucocyte CatK in plaque stability.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This work was supported by GSK Pharmaceuticals (J.G.), the Netherlands Heart Foundation [grant numbers 2001T041 (M.V.E.), M93.001 (R. de N.)] and by the Netherlands Organization for Scientific Research [grant number 916.86.046 (I.B.)].


   Acknowledgements
 
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 Programme for Research Priority 1 (Life Sciences, Genomics, and Biotechnology for Health; contract LSHM-CT-2003-503254).

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

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  6. Bossard MJ, Tomaszek TA, Thompson SK, Amegadzie BY, Hanning CR, Jones C, et al. Proteolytic activity of human osteoclast cathepsin K. Expression, purification, activation, and substrate identification. J Biol Chem (1996) 271:12517–12524.[Abstract/Free Full Text]
  7. Garnero P, Borel O, Byrjalsen I, Ferreras M, Drake FH, McQueney MS, et al. The collagenolytic activity of cathepsin K is unique among mammalian proteinases. J Biol Chem (1998) 273:32347–32352.[Abstract/Free Full Text]
  8. Gelb BD, Shi GP, Chapman HA, Desnick RJ. Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency. Science (1996) 273:1236–1238.[Abstract]
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Cathepsin K: boon or bale for atherosclerotic plaque stability?
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O. Hofnagel and H. Robenek
Cathepsin K: boon or bale for atherosclerotic plaque stability?
Cardiovasc Res, February 1, 2009; 81(2): 242 - 243.
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