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Cardiovascular Research 2004 62(3):610-620; doi:10.1016/j.cardiores.2004.02.002
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Cathepsin S expression is up-regulated following balloon angioplasty in the hypercholesterolemic rabbit

Cynthia L Burns-Kurtis*,a, Alan R Olzinskia, Saul Needlea, Josephine H Foxb, Elizabeth A Capperb, Fiona M Kellyc, Michael S McQueneyd and Anne M Romanica

aDepartment of Vascular Inflammatory Diseases, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406, USA
bDepartment of Respiratory and Inflammatory Diseases, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406, USA
cDepartment of Quantitative Expression, GlaxoSmithKline Pharmaceuticals, Stevenage, Hertfordshire, SG1 2NY, UK
dAssay Development and Compound Profiling, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406, USA

* Corresponding author. Tel.: +1-610-270-4170; fax: +1-610-270-6206. Email address: cynthia_l_kurtis{at}gsk.com

Received 22 August 2003; revised 19 January 2004; accepted 3 February 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Neointimal development following balloon angioplasty involves many factors including smooth muscle cell (SMC) migration and proliferation and extracellular matrix (ECM) remodeling. Further, in hypercholesterolemic (HC) conditions, there is an influx of macrophage foam cells (FCs) into the restenotic lesion, which also involves degradation of the basement membrane and surrounding ECM. The ECM remodeling that occurs during restenosis has been shown to be mediated by various proteases. Here we have investigated the role of cathepsin S (CatS), a cysteine protease, in this process. Methods and results: We have demonstrated by Taqman quantitative PCR, Western blot, and immunohistochemistry that CatS is up-regulated in restenotic lesions of HC rabbits following balloon injury of the iliofemoral artery. CatS mRNA expression was elevated 28-fold in balloon-injured vessels relative to uninjured contralateral vessels in HC rabbits 8 weeks post-angioplasty (p<0.05). CatS protein expression was detected within 1 day post-injury, persisted throughout the entire time course evaluated (60 days post-injury), and was co-localized with SMCs, macrophages, and FCs. In contrast, cystatin C (CysC), the endogenous inhibitor of cathepsins, was only minimally up-regulated following injury. CysC mRNA expression was elevated 3.5-fold in balloon-injured vessels relative to uninjured contralateral vessels in HC rabbits 8 weeks post-angioplasty (p<0.005), and up-regulation of protein expression was not detected until days 28 and 60 post-injury. Additional biochemical studies using recombinant rabbit CatS revealed that rabbit CatS digests laminin, fibronectin, and type I collagen. Further, CatS expression was evaluated in SMCs that were induced to migrate through a matrix-coated Boyden chamber upon platelet-derived growth factor (PDGF) stimulation. The addition of a selective CatS inhibitor reduced SMC migration dose-dependently with an 80% reduction in migration at 30 nM (p<0.005). Additionally, we have shown that CatS protein expression by human macrophages was increased upon stimulation with oxidized low density lipoprotein (ox-LDL), implying augmentation of CatS production during foam cell formation. Conclusion: Taken together, our results indicate an enhanced expression of CatS during neointima formation and it is associated with invading SMCs, macrophages, and FCs, highlighting the importance of CatS in the pathogenesis of restenosis.

KEYWORDS Cathepsin; Restenosis; Atherosclerosis; Smooth muscle; Macrophage; Remodeling; Extracellular matrix


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Percutaneous transluminal coronary angioplasty (PTCA) is an increasingly used procedure for treating patients with ischemic coronary artery disease; however, approximately 20–30% of patients suffer from vascular restenosis within 6 months after the procedure [3]. Two of the principal cellular events that contribute to lesion formation following arterial injury due to angioplasty are smooth muscle cell (SMC) proliferation and migration [3,16]. One essential factor required for SMC migration is degradation of the basement membrane and surrounding extracellular matrix (ECM) [8,18]. Further, as many patients who undergo PTCA are hypercholesterolemic and have some degree of atherosclerosis, there is often an influx of macrophage foam cells (FCs) into the restenotic lesion [4,9,23,26]. Here too, degradation of the basement membrane and ECM is involved.

The ECM is a multifunctional complex of proteins (e.g., elastin, types I and III collagen) and proteoglycans assembled in a highly organized manner that contributes to the structural integrity of cells and tissues. The basement membrane, which serves as the foundation for cell attachment, plays an important role in the organization and support of SMCs located within the vessel wall. The basement membrane is comprised of ECM molecules such as type IV collagen, laminin, and entactin [27]. Various factors are involved in maintaining the integrity of the ECM and the tissues it supports. However, in certain pathological circumstances, the ECM is modulated such that the structure of the tissue becomes damaged, destroyed, or remodeled. There are various classes of enzymes that are capable of degrading components of the ECM including the matrix metalloproteinases (MMPs), plasmins, and cathepsins.

Cathepsins are a family of cysteine proteases found in lysosomes and in the extracellular milieu [2,14]. These enzymes are secreted as inactive pro forms that are processed to yield mature, active enzymes. Cathepsin S (CatS), a member of this family, is capable of degrading many components of the basement membrane [19]. Its substrates include fibrillar collagen, elastin, laminin, fibronectin, and heparan sulfate proteoglycans [6,13,17,21,25]. The degree of proteolysis depends, in part, upon the balance of inhibitor and protease. The most abundant extracellular endogenous inhibitor of the cathepsins is cystatin C (CysC). The high concentrations discovered in biological fluids suggests that CysC is an important extracellular cathepsin inhibitor [5]. CatS has been demonstrated to play a role in atherosclerosis, angiogenesis, inflammation, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), among others [1,2,10,15,17,19,21,22]. In restenosis, CatS is hypothesized to play a role in ECM remodeling, breakdown of the internal elastic lamina, and the migration of SMCs and FCs contributing to neointimal thickening. The resulting lesion often results in re-occlusion of the blood vessel.

The present series of experiments were performed to define the time course and cellular source(s) of CatS and CysC expression following balloon angioplasty in the iliofemoral artery of New Zealand white rabbits fed a high fat/cholesterol diet (hypercholesterolemic [HC]). CatS and CysC expression were also evaluated in oxidized low density lipoprotein (ox-LDL)-stimulated macrophages. Additional biochemical analyses were conducted to examine the ability of rabbit CatS to degrade laminin, fibronectin, and type I collagen. Finally, CatS-mediated cell migration was evaluated in cultured human coronary artery SMCs in a modified Boyden chamber.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Rabbit balloon angioplasty
Male New Zealand white rabbits (3 kg, 3–4 months old) were fed a HC diet (2.5% peanut oil and 0.5% cholesterol; TD 98263, Harlan Teklad) 1 week before the procedure and were maintained on this diet until the end of the study. For balloon injury, an incision was created in the thigh and the iliofemoral artery was isolated. The distal end of the vessel was ligated and a moistened 3.0 French Fogarty balloon catheter (Baxter) was inserted 10 cm into the artery. The balloon was inflated with 0.09–0.11 ml of saline while pulling with a twisting motion through the artery to denude the endothelial cell layer. This procedure was repeated three times and the artery was ligated after the catheter was withdrawn. Rabbits were euthanized at specific time points (days 1, 3, 7, 14, 28, or 60 post-angioplasty; day 0 represents no injury) with Fatal Plus (1 ml/3 kg, Vortech Pharmaceuticals). For histochemical studies only, the rabbits were perfused via the left ventricle with saline followed by 10% neutral buffered formalin (Sigma). For Taqman quantitative PCR studies, rabbits were maintained on the HC diet for 2 weeks prior to angioplasty and were then euthanized 8 weeks post-balloon angioplasty. The contralateral iliofemoral artery of each animal served as the non-injured control vessel. In addition, for the PCR studies, a parallel series of rabbits were maintained on a normal diet (5326, Purina Mills Institute) for the same length of time and served as non-HC/non-injured controls. All experiments were conducted in accordance with the Guide for Care and Use of Laboratory Animals (US National Institutes of Health, Publication 85-23, revised 1996).

2.2. Nucleic acid extraction and Taqman quantitative PCR analysis
To analyze CatS mRNA expression patterns in control and balloon-injured arteries, RNA extracts of the tissues were prepared. The injured left as well as the uninjured right iliofemoral arteries were excised from each animal (n=4) 8 weeks post-injury. Comparable tissue samples were collected from non-injured animals on a normal diet (n=3). Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. Contaminating genomic DNA was removed through incubation (37 °C, 10 min) with 2 U Dnase/10 µg RNA (Ambion).

RNA (2 µg) was reverse transcribed using muMLV reverse transcriptase according to the manufacturer's instructions (Applied Biosystems). Primers and internal oligonucleotide probes are described in Table 1 and were synthesized by Sigma-Genosys. Each PCR reaction included cDNA from 20 ng of RNA, 900 nM primers, 100 nM probe, and Taqman Universal PCR Master Mix (Applied Biosystems). Amplification and detection were performed using the ABI PRISM 7900HT Sequence Detection System (Perkin Elmer) with the following profile: 1 cycle of 50 °C (2 min), 1 cycle of 95 °C (10 min), 40 cycles of 95 °C (15 s), and 60°C (1 min). A linear regression line calculated from the standard curves of serially diluted rabbit genomic DNA allowed relative transcript levels in RNA-derived cDNA samples to be determined.


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  		Table 1 Primers and probes used for Taqman quantitative PCR

 
2.3. Preparation of tissue extracts
To analyze CatS and CysC protein expression patterns in control and balloon-injured arteries, protein extracts were prepared. The iliofemoral artery was excised from each animal at various times following balloon injury (n=3 per time point). Comparable tissue samples were collected from non-injured animals on a HC diet (n=3 per time point). Tissues were minced and incubated in extraction buffer (PBS containing 0.5% Triton X-100 (Sigma), 0.5 U/ml aprotinin (Sigma), and 0.01% sodium azide) while rotating (4 °C, 18 h). After centrifugation (14,000 rpm, 15 min, 4 °C), supernatants were collected and protein concentrations were determined with the DC Protein Assay (BioRad).

2.4. Western blot analysis for CatS and CysC
Samples (60 µg) were resolved by electrophoresis through a 12% or 16% Tris–glycine gel (Novex) under reducing conditions and then transferred to a nitrocellulose membrane for CatS and CysC analysis, respectively. As standards, 25 ng of recombinant mouse mature CatS and 2 ng of purified human CysC (Calbiochem) were used. After blocking overnight at 4 °C with 5% nonfat powdered milk in a 0.1 M Tris–HCl, 1.5 M NaCl, 0.5% Triton X-100, pH 8.0 (TBST buffer), the blot was incubated with a goat anti-CatS antibody (antibody M19; 1:700; Santa Cruz) or a goat anti-CysC antibody (1:200; Strategic Biosolutions), washed with TBST, and then incubated with an anti-goat IgG secondary antibody conjugated to horseradish peroxidase (HRP) (1:3000; Santa Cruz). The blots were developed using the enhanced chemiluminescence method (Amersham) according to the manufacturer's instructions.

2.5. Oil Red O (ORO) staining and morphometric analysis
For ORO evaluation of the arteries (n=4 per time point; days 0–28), a section 1 cm distal to the bifurcation of the abdominal aorta and 1 cm proximal to the incision site was excised and fixed in 10% neutral buffered formalin (Sigma) for 24 h. The vessels were placed in 15% sucrose/PBS, frozen, and sectioned (5 µm). For staining, sections were incubated (10 min) in 0.3% ORO (Sigma) in 60% isopropanol, rinsed in tap water, counter-stained in Gill's Hematoxylin (Vector Laboratories), and aquamounted (Polysciences).

ORO stained sections of injured arteries were used for quantification of lipid accumulation. Morphometric analysis was performed using ImagePro Plus image analysis software (Media Cybernetics). Measurements from four non-overlapping fields from each of four vessels were obtained using 20 x magnification on an Olympus BX60 microscope. Areas of ORO-positive staining and area of interest (AOI) were quantified and an average ORO area/AOI was calculated.

2.6. Immunohistochemical analysis
For histological evaluation of the arteries (n=3–5 per time point), tissues were collected and fixed as described above and then paraffin embedded and sectioned (6 µm). Immunostaining was performed using the Vectastain Elite ABC kit (Vector Laboratories) according to the manufacturer's instructions. Immunostaining for CatS was performed using a goat-anti CatS antibody (antibody M19; 1:300; Santa Cruz). As a negative control, serial sections were prepared in which the primary antibody was pre-incubated (18 h, 4 °C) with 10-fold excess CatS antigenic peptide (Santa Cruz) prior to application. Immunostaining for SMCs and macrophages was performed using a mouse anti-SMC {alpha}-actin antibody (clone 1A4; 1:7000; Sigma) and a mouse anti-macrophage antibody (clone Ram11; 1:50; Dako), respectively. As negative controls, serial sections were used in which the primary antibodies were omitted. All sections were then incubated with appropriate biotinylated secondary antibodies (7.5 µg/ml; Vector), followed by incubation with the Vectastain Elite ABC reagent solution. Immunoglobulin complexes were visualized upon incubation with 3,3'-diaminobenzidine (0.5 mg/ml; Vector). Sections were counterstained with Gill's Hematoxylin and examined by light microscopy using an Olympus BX60 microscope.

2.7. Proteolysis of ECM molecules by rabbit CatS
To evaluate the ability of CatS to degrade matrix proteins, recombinant rabbit CatS (2 µM) was incubated with 0.4 mg/ml laminin (from Engelbreth-Holm-Swarm murine sarcoma; Sigma) for 0–6 h; 0.4 mg/ml fibronectin (from human fibroblasts; Calbiochem) for 0–60 min; or 0.7 mg/ml type I collagen (from NZW rabbit skin; reconstituted in 5 M acetic acid, pH 3; Sigma) for 0–5 min. All reactions occurred at 37 °C in 150 mM HEPES, 150 mM NaCl, 20 mM L-cysteine, 5 mM EDTA, 5 mM DTT, pH 7.0 and stopped by the addition of 0.2 mM E64 (Sigma), a broad spectrum cysteine protease inhibitor. Mock digestions without CatS were also performed as negative controls. All samples were reduced and resolved by electrophoresis through a 4–20% Tris–glycine gel (Novex) and stained with Coomassie Brilliant Blue (Sigma).

2.8. Proteolysis of Matrigel by CatS
To examine the ability of CatS to degrade components of a basement membrane in vitro, CatS was incubated with Matrigel, a basement membrane preparation consisting of laminin, collagen IV, entactin, and heparan sulfate proteoglycan (Becton Dickinson), and the reaction products were visualized by SDS-PAGE. For assay, 1 µM recombinant human CatS was incubated with 75 µg Matrigel in the presence or absence of a CatS small molecule compound inhibitor, SB 432383 (Benzofuran-2-carboxylic acid {(S)-2-cyclohexyl-1-[3-oxo-1-(pyridine-2-sulfonyl-)-azepan-4-ylcarbamoyl]-ethyl}-amide) [7] at concentrations ranging from 0 to 3 µM. All reactions proceeded for 4 h at 37 °C in 0.1 M Tris–HCl (pH 7.4), 0.1 M NaCl, 10 mM CaCl2, and 0.05% Brij. Reaction products were reduced and resolved by electrophoresis through a 4–20% Tris–glycine gel (Novex) and stained with Coomassie Brilliant Blue (Sigma).

2.9. Vascular SMC migration assay
Platelet-derived growth factor-BB (PDGF; Biosource International)-stimulated migration of human coronary artery SMCs (Clonetics) was measured by labeling the cells fluorescently with 5 µM Calcein-AM (Molecular Probes) and loading them (10,000 cells/well) into the top of a 96-well modified Boyden chamber (Neuroprobe). The migration chamber was assembled with a Matrigel (0.5 mg/ml; growth factor-depleted)-coated polycarbonate membrane (8-µm pore-size) sandwiched between the upper chamber containing the cells and the lower containing the chemoattractant, PDGF-BB (20 ng/ml). The chamber was then incubated at 37 °C for 3.75 h. Following migration, the membrane was removed and the upper surface was wiped to remove nonmigrated cells. The membrane was placed into a fluorescent plate reader (Labsystems Fluoroskan) and SMCs that migrated through the pores and onto the undersurface of the membrane were measured. To measure the effect of CatS inhibition on SMC migration, the CatS small molecule compound inhibitor, SB 432383, was added to both the upper and lower wells (1–30 nM) prior to incubation.

2.10. Isolation of human monocytes and ox-LDL stimulation of differentiated macrophages
Human monocytes (n=3 donors) were isolated from monocyte-enriched leukopacks (Biological Specialties) using a Monocyte Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions. Monocytes were grown for 2 weeks in RPMI-1640 medium containing 2 mM L-glutamine, 5% human AB serum, 100 U/ml penicillin/100 µg/ml streptomycin, and 1.0 ng/ml granulocyte–monocyte colony stimulating factor to promote macrophage differentiation.

Following differentiation, cells were rinsed and incubated with 25 µg/ml ox-LDL (Intracell) in RPMI-1640 medium containing 2 mM L-glutamine and 100 U/ml penicillin/100 µg/ml streptomycin for 1, 16, 24, or 48 h. For each time point, culture medium was collected and cell extracts were prepared for subsequent Western blot analysis. Briefly, cells were lysed in TBS containing 1% Triton X-100 and a protease cocktail (Sigma; leupeptin (400 µM), aprotinin (20 µg/ml), soybean trypsin inhibitor (20 µg/ml) and PMSF (1 mM)). The extracts were incubated on ice (10 min) and then centrifuged (14,000 rpm, 4 °C, 10 min). Supernatants were collected and protein concentrations were determined using the DC Protein Assay (BioRad). Western blot analysis of the cell extracts (7.5 µg) and culture medium (5 µl for CysC analysis; 60 µl for CatS analysis) was conducted as described above using the following antibodies: goat anti-CatS (1:700; antibody C19; Santa Cruz) followed by an HRP conjugated anti-goat IgG secondary antibody (1:3000; Santa Cruz) or rabbit anti-CysC (1:1000; Cortex Biochem) followed by an HRP conjugated anti-rabbit IgG secondary antibody (1:5000; BioRad).

2.11. Statistical analysis
CatS and CysC mRNA expression is reported as the ratio of CatS or CysC (copies/50 ng total RNA) relative to β-Actin (copies/50 ng total RNA) mRNA. For statistical analysis of mRNA expression in the uninjured arteries of rabbits on a HC diet versus a normal diet, a two-sample unpaired t-test was performed. For statistical analysis of mRNA expression in the uninjured versus injured iliofemoral arteries of rabbits on a HC diet, a two-sample paired t-test was performed.

For the quantitated ORO staining analysis, data are expressed as mean±S.E.M. To compare differences in FC content of the iliofemoral arteries at days 1, 3, 7, 14 and 28 post-balloon injury versus day 0, one-way ANOVA was performed followed by a-posteriori comparisons of means (Dunnett's Test). For the SMC migration assay, data are expressed as mean±S.E.M. To compare differences in the level of SMC migration in SB 432383-treated groups relative to the group stimulated with PDGF only, one-way ANOVA was performed followed by a-posteriori comparisons of means (Dunnett's Test). A probability level of p<0.05 was considered statistically significant. All statistical analyses were conducted using SAS (SAS Institute).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. CatS and CysC mRNA expression are up-regulated 8 weeks post-balloon angioplasty of the iliofemoral artery of New Zealand rabbits fed an HC diet
CatS (A) and CysC (B) mRNA expression was assessed 8 weeks post-balloon angioplasty of the iliofemoral artery of injured and sham-operated animals on a HC diet and in sham-operated animals on a normal diet by Taqman quantitative PCR. Fig. 1A shows that CatS mRNA expression was similar in the uninjured arteries of rabbits fed a HC diet relative to a normal diet. However, CatS mRNA expression was significantly elevated (28-fold; p<0.05) in balloon-injured vessels relative to sham-operated (uninjured) contralateral vessels in HC rabbits. Fig. 1B shows that CysC mRNA expression was similar in the uninjured arteries of rabbits fed a HC diet relative to a normal diet. However, CysC mRNA expression was significantly elevated (3.5-fold; p<0.005) in balloon-injured vessels relative to sham-operated contralateral vessels in HC rabbits.


Figure 1
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  		Fig. 1 Iliofemoral arteries were analyzed for CatS and CysC mRNA expression 8 weeks post-balloon angioplasty or sham-injury by Taqman quantitative PCR. (A) CatS mRNA was significantly up-regulated in injured vessels of rabbits on a HC diet (0.77±0.1; p<0.05) relative to sham-operated vessels of rabbits on either an HC diet (0.03±0.01) or a normal diet (0.008±0.001). (B) CysC mRNA was significantly up-regulated in injured vessels of rabbits on a HC diet (1.95±0.4; p<0.005) relative to sham-operated vessels of rabbits on either an HC diet (0.82±0.1) or a normal diet (0.83±0.1). Values represent mean relative mRNA expression±S.E.M. NS, not significant.

 
3.2. CatS and CysC protein expression are up-regulated following balloon angioplasty of the iliofemoral artery of New Zealand rabbits fed an HC diet
The time course of CatS and CysC protein expression following balloon angioplasty of the iliofemoral artery was assessed in tissue extracts prepared from injured and sham-operated vessels by Western blot. Fig. 2 shows that CatS protein expression was detected in the balloon-injured artery within 7 days post-angioplasty and increased throughout the 60-day time course. Notably, a single 28-kDa band representing the mature/active form of CatS was observed. CysC protein expression was up-regulated at days 28 and 60 post-injury. CatS and CysC were also evaluated in the sham-operated arteries of rabbits fed a HC diet. CatS was not detected in these samples and constitutive CysC protein expression was observed (data not shown).


Figure 2
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  		Fig. 2 Iliofemoral arteries from balloon-injured HC rabbits were analyzed for CatS and CysC expression over a time-course of 0 to 60 days by Western blot. A 28-kDa CatS band, consistent with the size of the mature/active enzyme, was up-regulated 7 days post-injury and increased throughout the 60-day time course. CysC was up-regulated at days 28 and 60 post-injury. Data are representative examples (n=3 per time point).

 
3.3. CatS is immunolocalized with SMCs, macrophages, and macrophage FCs in restenotic lesions
The HC rabbit model of balloon injury in the iliofemoral artery is characterized by progressive arterial luminal narrowing due to a continuously enlarging intimal cellular infiltrate comprised of SMCs, macrophages, and FCs. Macrophages are an early component (beginning by day 3) of the cellular response to injury. At later time points (7 days and thereafter), both SMCs and macrophages contribute to the developing cellular infiltrate [20]. In addition, we show that FCs constitute a substantial part of the expanding neointima. As shown in Fig. 3B, stained FCs were negligible on days 0, 1, and 3. However, FC formation was evident at day 7 (Fig. 3A) and robust at days 14, and 28 as measured by ORO positive area/AOI (Fig. 3A and B).


Figure 3
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  		Fig. 3 Iliofemoral arteries from balloon-injured HC rabbits were analyzed for FC formation over a time-course of 0 to 28 days by ORO Staining. (A) Representative sections (n=4 per time point) from days 0, 7, 14, and 28 post-injury. Magnification x 20. FC formation was not evident until day 7 and was robust on days 14 and 28. (B) Quantitated data for FC formation. Data are expressed as ORO area/AOI and represent the mean±S.E.M. of four fields measured for each of four vessels. ***p<0.005 versus day 0.

 
To investigate the cellular and temporal expression of CatS in the HC rabbit model of balloon injury, immunohistochemical analysis was conducted. Fig. 4 shows that CatS was not detected at day 0, but was up-regulated within the media 24 h post-angioplasty and remained elevated throughout the 60-day time course. Serial sections evaluated for SMC {alpha}-actin and Ram11 indicated that at days 1 and 3, CatS expression was co-localized with medial SMCs and infiltrating macrophages (data not shown). From days 7 to 60, expression was detected throughout the restenotic lesion (Fig. 4). Notably, at day 7 expression was localized with SMCs, which were migrating and proliferating outward toward the lumen, as well as with macrophages infiltrating into the medial layer (Fig. 5A). As demonstrated in Fig. 3A, low levels of FCs were also present within the neointima by day 7. As such, CatS expression co-localized with macrophages, as indicated by Ram11 staining, may in fact originate from both macrophages and FCs. At day 14, CatS was detected within the media, now structurally disrupted by infiltrating macrophages and FCs, as well as within the expanding neointimal lesion (Figs. 4 and 5B)Go. Ram11 staining indicated that macrophages/FCs were localized along the internal elastic lamina of the developing intima. Also, SMC {alpha}-actin staining indicated that SMCs predominated in the portion of the intimal lesion adjacent to the lumen of the vessel with a smaller proportion at the base of the neointimal lesion. CatS expression was localized with both SMCs and macrophages/FCs at this time point (Fig. 5B). At days 28 and 60, CatS expression was still detected throughout the entire restenotic lesion (Fig. 4). However, at these later time points, SMCs and macrophages/FCs were now equally distributed throughout the lesion and CatS expression was localized with all cell types (Fig. 5C and day 60 data not shown).


Figure 4
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  		Fig. 4 Iliofemoral arteries from balloon-injured HC rabbits were analyzed for CatS protein by immunohistochemistry. CatS expression was not detected at day 0 but was modestly detected at day 1 and strongly detected throughout the remainder of the time course. Expression was localized in the medial smooth muscle cell layer and in the expanding neointima. Representative sections illustrating CatS immunoreactivity (n=3–5 per time point). D60 Neg represents a serial section incubated with antigenic peptide blocked CatS antibody demonstrating specificity of staining. Magnification x 20.

 

Figure 5
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  		Fig. 5 Serial sections of iliofemoral arteries from balloon-injured HC rabbits were analyzed for CatS, SMC {alpha}-actin, and Ram11 protein by immunohistochemistry. (A) Day 7 post-injury. (B) Day 14 post-injury. (C) Day 28 post-injury. CatS expression was detected throughout the restenotic lesion at these time points and was associated with both SMCs and macrophage/FCs. Representative sections (n=3–5 per time point). Magnification x 20.

 
In contrast to the CatS expression detected in the arteries following balloon injury, sham-operated vessels were devoid of expression (data not shown). Further, serial sections that were incubated with antigenic peptide-blocked CatS antibody were negative (Fig. 4). Importantly, immunohistochemical results demonstrated CatS expression throughout the entire 60-day time course, whereas Western blot analysis did not demonstrate CatS expression until 7 days post-injury. It is speculated that the sensitivity of detection was greater by immunohistochemistry compared to Western blot.

3.4. Rabbit CatS degrades laminin, fibronectin, and type I collagen
To demonstrate the ability of rabbit CatS to degrade ECM proteins, soluble preparations of laminin, fibronectin, and type I collagen were incubated with recombinant rabbit CatS. SDS-PAGE analysis demonstrated that rabbit CatS effectively degraded these purified proteins (Fig. 6). Mock digestions of laminin, fibronectin, and type I collagen, which were devoid of CatS, demonstrated no degradation over a 24-h time course (data not shown).


Figure 6
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  		Fig. 6 Laminin (A), fibronectin (B), or type I collagen (C) was incubated with recombinant rabbit CatS and the products collected at various time points were analyzed by SDS-PAGE. Rabbit CatS effectively degraded each of these matrix molecules.

 
3.5. CatS degrades Matrigel
To demonstrate the ability of CatS to degrade components of a basement membrane in vitro, Matrigel was incubated with recombinant human CatS. The results demonstrated that CatS effectively degraded the Matrigel (Fig. 7A). For further analysis, a CatS tool compound was utilized in the degradation assay. The compound, SB 432383, a potent (Ki=0.05 nM) inhibitor of CatS and selective against cathepsins K and L (unpublished observations), inhibited CatS-mediated degradation in a concentration-dependent manner (Fig. 7A).


Figure 7
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  		Fig. 7 CatS-mediated human coronary artery SMC migration through an in vitro basement membrane matrix was evaluated. (A) Recombinant human CatS was incubated with Matrigel in the presence or absence of the CatS inhibitor, SB 432383, and the reaction products were analyzed by SDS-PAGE. CatS effectively degraded Matrigel and SB 432383 inhibited this degradation in a concentration-dependent manner. (B) Fluorescently labeled SMCs, stimulated with PDGF, were applied to the upper compartment of a modified Boyden culture chamber in the presence or absence of SB 432383 at concentrations of 1–30 nM. Following an incubation period, cells that had migrated through the filter were counted. SB 432383 inhibited PDGF-induced vascular SMC migration in a concentration-dependent manner. *p<0.05, **p<0.005, ***p<0.001 versus cells stimulated with PDGF only.

 
3.6. CatS facilitates vascular SMC migration through an in vitro basement membrane matrix
Having demonstrated that CatS is able to effectively degrade Matrigel, it was then evaluated for its ability to facilitate SMC migration through a Matrigel-coated Boyden chamber. As shown in Fig. 7B, SMC migration through Matrigel was stimulated by PDGF. Notably, the ratio of PDGF-treated to untreated cell migration was approximately 10-fold providing an adequate window to demonstrate inhibitory activity. The data demonstrated that SB 432383 inhibited PDGF-induced vascular SMC migration in a concentration-dependent manner with approximately 80% inhibition (p<0.005) in migration at the highest dose tested (30 nM).

3.7. CatS protein expression increases during macrophage differentiation to foam cells
CatS protein expression by human macrophages in response to ox-LDL was examined by Western blot. Fig. 8A shows that intracellular levels of CatS increased over time in response to ox-LDL treatment. Notably, CysC protein expression remained unchanged in response to ox-LDL over this time course. Further, while it was observed that macrophages deprived of serum exhibited an increase in basal CatS levels, exposure to ox-LDL increased the ratio of mature CatS to pro-CatS secreted into the FC conditioned media over time (Fig. 8B). Also, in response to ox-LDL, the expression of CysC was not affected significantly compared to control.


Figure 8
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  		Fig. 8 Cell extracts and culture medium collected from human macrophages following a time course of ox-LDL stimulation were analyzed for CatS and CysC protein expression by Western analysis. (A) Immunoblot for CatS and CysC in the cell extracts. Intracellular CatS (active form) was up-regulated by ox-LDL at 16 h post-treatment and remained up-regulated throughout the 48-h time course. CysC expression remained unchanged following ox-LDL treatment. (B) Immunoblot for CatS and CysC in the cell culture medium. Exposure of ox-LDL increased the ratio of extracellular active CatS to pro-CatS in a time-dependent manner. Data are representative examples (n=3 donors).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The migration and proliferation of SMCs are pivotal cellular events that contribute to neointimal lesion formation during restenosis. Our data suggest that CatS-mediated modulation of the underlying basement membrane contributes to this process. In pro-atherosclerotic conditions, FCs are also involved in lesion growth/development. We have shown that CatS mRNA and protein expression are significantly up-regulated following balloon angioplasty in the HC rabbit and that SMCs and macrophage/FCs are the principal cellular sources of these enzymes. The time course of CatS protein expression correlates with SMC and macrophage FC migration during neointimal thickening. In particular, CatS is highly expressed within 1–3 days following injury, at a time when SMC migration begins in this animal model. In contrast, CysC was only minimally up-regulated and only at later time points post-injury suggesting that there is a net proteolytic increase during the initial stages of neointimal development.

As the ECM of the vessel wall undergoes significant remodeling during neointimal lesion development, cathepsins and MMPs have been implicated in the process. These enzymes degrade various components of the ECM. CatS has been shown to degrade elastin [17,21,25]. Notably, the elastic lamina is disrupted, allowing smooth muscle cells to migrate from the media into the newly forming intima [11,12,16]. We have demonstrated for the first time that recombinant rabbit CatS can degrade ECM components of the basement membrane. The role of CatS in basement membrane degradation and SMC migration was evaluated further in a modified Boyden chamber cell migration assay. Our results confirmed, also for the first time, that CatS plays a significant role in SMC migration through a basement membrane-like barrier. Specifically, our studies demonstrated that selective inhibition of CatS with a small-molecule compound inhibited SMC migration in a concentration-dependent manner. These results are similar to previous reports that endothelial cells from CatS deficient mice show reduced invasion across a Matrigel membrane [19]. Our results suggest that SMCs utilize CatS to degrade the ECM of their underlying basement membrane to allow them to migrate from the media and into the developing neointima.

The expression of CatS by macrophages/FCs was also demonstrated. It is hypothesized that these cells utilize CatS to aid in their invasion into the developing restenotic lesion where they possess the ability to evoke a number of inflammatory events. For example, stimulated macrophages and FCs secrete cytokines such as TNF-{alpha}, IFN-{gamma}, IL-1, IL-6, PDGF, TGF-β, bFGF, among others, that stimulate SMC migration and proliferation and leukocyte influx [24], thus compounding neointimal formation. Our data show that, as macrophages differentiated into FCs upon exposure to ox-LDL, mature CatS expression increased while CysC expression was not affected significantly. Thus, the results support the hypothesis that macrophages utilize CatS to migrate into the lesion and, upon exposure to a proatherosclerotic environment, they become FCs. As FCs, they express greater levels of active CatS which contributes to ECM remodeling of the developing intimal lesion. Further, these infiltrating macrophages are capable of creating a locally acidified extracellular environment through increased expression of vacuolar-type H+-ATPase components [19]. Since pro-CatS is autocatalytically processed to its active form at acidic pH, this environment may thus provide an additional level of regulation of CatS activity during lesion development.

In summary, we have shown that CatS expression is up-regulated in vascular SMCs and macrophage/FCs following balloon angioplasty in the HC rabbit iliofemoral artery. These results indicate that CatS is involved in SMC and macrophage/FC migration and tissue remodeling that occurs with restenosis. Additional in vivo studies are planned to evaluate the effect of a small molecule CatS inhibitor at reducing neointimal lesion growth following angioplasty and to confirm the role of CatS in restenosis.


   Acknowledgements
 
The authors are appreciative of Christine Webb and Leli Sarov-Blat for their technical assistance.


   Notes
 
Time for primary review 26 days


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

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