Deficiency of cathepsin S attenuates angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein E-deficient mice

Abstract

Aims

Abdominal aortic aneurysm (AAA) is characterized by extensive aortic wall matrix degradation that contributes to the remodelling and eventual rupture of the arterial wall. Elastinolytic cathepsin S (Cat S) is highly expressed in human aneurysmal lesions, but whether it contributes to the pathogenesis of AAA remains unknown.

Methods and results

AAAs were induced in apolipoprotein E (ApoE) and Cat S compound mutant (Apoe^–/–Ctss^–/–) mice and in ApoE-deficient Cat S wild-type littermates (Apoe^–/–Ctss^+/+) by chronic angiotensin II infusion, and AAA lesions were analysed after 28 days. We found that Cat S expression increased significantly in mouse AAA lesions. The AAA incidence in Apoe^–/–Ctss^–/– mice was much lower than that in Apoe^–/–Ctss^+/+ mice (10 vs. 80%). Cat S deficiency significantly reduced external and luminal abdominal aortic diameters, medial elastin fragmentation, and adventitia collagen content. Cat S deficiency reduced aortic lesion expression and the activity of matrix metalloproteinase (MMP)-2, MMP-9, and Cat K, but not the activity of other major cathepsins, such as Cat B and Cat L. Absence of Cat S significantly reduced AAA lesion media smooth muscle cell (SMC) apoptosis, lesion adventitia microvessel content, and inflammatory cell accumulation and proliferation. In vitro studies proved that Cat S helps promote SMC apoptosis, angiogenesis, monocyte and T-cell transmigration, and T-cell proliferation—all of which are essential to AAA pathogenesis.

Conclusions

These data provide direct evidence that Cat S plays an important role in AAA formation and suggest that Cat S is a new therapeutic target for human AAA.

1. Introduction

Abdominal aortic aneurysms (AAAs) are permanent dilations of the arterial wall in the abdominal aorta that cause life-threatening bleeding after rupture. The pathophysiology of AAA formation is complicated. Several risk factors—such as sex, hypertension, age, atherosclerosis, and genetic predisposition—all associate with AAA development.^1,2 AAAs are characterized by destruction of extracellular matrix (ECM) in the media and adventitia, by loss of medial smooth muscle cells (SMCs) with thinning of the arterial wall, and by transmural infiltration of inflammatory cells. ECM remodelling is critical to AAA pathogenesis.^2–5 Arterial wall ECM consists of elastins, collagens, and proteoglycans, and is largely synthesized by SMCs.⁶ Proteolytic enzymes, such as matrix metalloproteinases (MMPs) and cysteine protease cathepsins, can degrade ECM and therefore contribute to aneurysm formation.^7–10

Cysteinyl cathepsins, including cathepsins (Cat) K, L, and S, are highly expressed in human atherosclerotic and aneurysmal lesions and localize to all major cell types, including SMCs, macrophages, and endothelial cells (ECs).^11,12 Emerging evidence indicates that cathepsins and their inhibitors are important in atherosclerosis and AAA formation.^8,10 Cat S deficiency in atherosclerosis-prone low-density lipoprotein receptor-deficient (Ldlr^–/–) mice significantly reduces the atherosclerotic plaque area and decreases elastin breaks and elastinolytic activities.¹³ Deficiency of cystatin C, an endogenous inhibitor of cysteinyl cathepsins, increases elastic lamina degradation and aortic dilatation in apolipoprotein E-null (Apoe^–/–) mice¹⁴ and promotes inflammation in angiotensin II (Ang II)-induced AAAs in atherosclerotic mice.¹⁵ In contrast, Cat K deficiency does not affect murine AAA formation induced by Ang II infusion in Apoe^–/– mice.¹⁶ Cat S, like Cat K, exerts potent elastinolytic activity.¹⁷ It not only affects ECM degradation, but also directly modulates inflammation, immunogenic responses, and SMC apoptosis during atherogenesis.^18–20 Whether this protease participates directly in AAA formation, however, remains unknown—although human AAA lesions express Cat S highly, and AAA patients have significantly higher plasma Cat S levels than do age-matched controls.^21,22 The present study was designed to test the hypothesis that Cat S participates in Ang II-induced AAA formation in mice.

2. Methods

2.1. Apoe^–/–Ctss^–/– mouse generation and experimental AAA

To generate Apoe^–/–Ctss^–/– mice, we crossbred Apoe^–/– mice (C57BL/6, N12, The Jackson Laboratory, Bar Harbor, ME, USA) with Ctss^–/– mice (C57BL/6, N>10) to generate Apoe^+/–Ctss^+/– mice, which were consequently used as breeding pairs.^13,19 This study used both Apoe^–/–Ctss^–/– mice and their littermates, Apoe^–/–Ctss^+/+ mice, as experimental controls (10-week-old males; 10 mice per group). Mice were anaesthetized by intraperitoneal injection (ip) with one dose of ketamine (200 mg/kg) and xylazine (10 mg/kg) in 50 µL of saline. Anaesthesia was monitored by pinching the toe. Aneurysm formation was then induced by subcutaneous insertion of a mini-osmotic pump (model 2004; ALZA Scientific Products, Inc., Mountain View, CA, USA) containing Ang II (1000 ng/kg/min) for 28 days, as described previously.²³ Post-operative analgesia (buprenophine, 0.05 mg/kg/12 h, ip) was administered for 48 h. Mice were sacrificed with carbon dioxide narcosis, followed by cardiac puncture blood collection and aortic tissue harvest. Aortas from Apoe^–/–Ctss^+/+ and Apoe^–/–Ctss^–/– mice infused with saline (n = 10) were used as negative controls. The Institutional Animal Care and Use Committee of Capital Medical University, Beijing, China, approved all studies. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

2.2. Systolic blood pressure and plasma lipid measurements

Mean blood pressure was measured weekly on conscious, restrained mice using the Visitech tail cuff system (Apex, NC, USA). To avoid procedure-induced anxiety, mice were initially accustomed to the instrument for 5 consecutive days before the actual recorded measurements. Moreover, the first 10 of 30 blood pressure values recorded at each session were disregarded, and the remaining 20 values were averaged and used for analysis. Plasma triglycerides and total cholesterol levels were determined using Infinity triglyceride or cholesterol lipid stable reagent with the appropriate standards (Thermo Scientific, Middletown, VA, USA).

2.3. Vascular pathology

Mice were sacrificed after 28 days of Ang II infusion. Aortic diameter expansion ≥100% of that before Ang II infusion was considered an AAA, and aneurysms in the abdominal aorta were quantified by the per cent incidence, as described previously.²³

2.4. Histological analysis

Mice were perfused with saline containing 0.4% heparin, and then perfused with a 4% paraformaldehyde solution before aorta harvesting. The aortas were dissected and divided into segments (200 µm intervals), starting at the middle of the aneurysm. The aortic piece was fixed overnight in 4% paraformaldehyde and then embedded in paraffin. Serial sections (5 μm) of the aortas were prepared for elastin staining, Sirius red staining, and immunohistochemical analysis for Cat S (1:200, Abcam, Cambridge, UK), smooth-muscle α-actin (α-SMA, 1:400, Abcam), Ki67 (1:200, Santa Cruz Biotech Inc., Santa Cruz, CA, USA), CD4 (1:200, Santa Cruz), Mac-2 (1:200, Santa Cruz), CD31 (1:200, Santa Cruz), chemokine monocyte chemotactic protein-1 (MCP-1, 1:200, Santa Cruz), TUNEL (Promega, Madison, WI, USA), Cat K (1:200, Abcam), MMP-2 (1:200, Santa Cruz), and vascular endothelial growth factor (VEGF, 1:200, Santa Cruz), as described previously.¹³ The relative Cat S, Cat K, MMP-2, collagen, VEGF, macrophage, and MCP-1 contents within the aortas were quantified by measuring the immunostaining signal-positive area. Lesion TUNEL-positive cells, media TUNEL-positive SMCs, CD4⁺ T cells, and Ki67⁺CD3⁺ cells were quantified as cell numbers per aortic section. Lesion CD31⁺ microvessels and Ki67⁺ cells were quantified as cell numbers per square millimetres. Lesion SMC loss was graded as previously described.²⁴ Images were viewed and captured with a Nikon Labophot 2 microscope equipped with a Sony CCD-Iris/RGB colour video camera attached to a computerized imaging system, and images were analysed by ImagePro Plus 3.0 software (ECLIPSE80i/90i; Nikon, Japan).

2.5. Analysis of elastin fragmentation, gelatin gel zymogram, and JPM labelling

Aortic wall elastin fragmentation was graded based on the degree of elastin filament break, as described previously.^13,24 Sirius red staining for collagen was analysed by polarization microscopy. Lesion MMP activity was assessed with a gelatin gel zymogram assay, as described previously.²⁵ Equal protein loading (7 μg lesion protein/lane) was confirmed by SDS–PAGE, followed by Coomassie staining. AAA lesion cathepsin activities were assessed using JPM labelling, as described elsewhere.¹³ Equal amounts of proteins (2 µg AAA lesion extract/sample) from Apoe^–/–Ctss^–/– and Apoe^–/–Ctss^+/+ mice were labelled with biotinylated JPM, followed by separation on a 12% SDS–PAGE.

2.6. Western blot analysis and ELISA

Aortic tissues or cells were extracted in a lysis buffer containing 20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 90 mM KCl, 2 mM EDTA, 1% Triton X-100, 1 mM Na₃VO₄, 10 mM NaF, and protease and phosphatase inhibitor cocktail (Thermo Scientific). Equal amounts of protein extracts (30 µg/lane) were loaded on a 12% SDS–PAGE gel, transferred to nitrocellulose membrane, and probed with goat anti-mouse Cat S polyclonal antibodies (1:1000).¹³ Immunoblot for housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:3000, Santa Cruz) was performed to assure equal protein loading. We determined MCP-1 (Pierce Chemical Co., Rockford, IL, USA) and interferon-γ (IFN-γ) (Bender MedSystems, Vienna, Austria) levels in the plasma and AAA tissue extracts from Apoe^–/–Ctss^–/– and Apoe^–/–Ctss^+/+ mice using ELISA kits, according to the manufacturer's instructions.

2.7. Cell proliferation and apoptosis assays

CD3⁺ T cells were isolated²⁶ and their proliferation was assessed with three methods: First, we used Cell Counting Kit-8 (Dojindo, Kumomoto, Japan), according to the manufacturer's instructions. Cell proliferation was expressed as the optical density of the responder cells (OD_{450 nm}).²⁷ Second, an equal volume of carboxyfluorescein succinimidyl ester (CFSE) (Dojindo) was added to the unstimulated T cells. After 3 days, T cells were harvested, and CD3⁺CFSE⁺ cells were quantified using flow cytometry.²⁸ Third, T cells were cultured on an anti-mouse CD3 antibody-coated 96-well plate. After 3 days, medium IL-2 level was determined by ELISA, according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA).²⁹

Mouse aortic SMCs were prepared and cultured in 50 μM of pyrrolidine dithiocarbamate (PDTC) (Sigma-Aldrich, St Louis, MO, USA) to induce SMC apoptosis, as previously described.²⁴ Apoptotic SMCs were detected using the in situ Cell Death Detection Kit (Promega), according to the manufacturer's instructions. We counted the number of TUNEL-positive cells.

2.8. Aortic ring assay

An aortic ring assay was performed to test the role of Cat S in angiogenesis.²⁴ In brief, a 96-well plate was coated with Matrigel (50 µL/well, BD Biosciences, Bedford, MA, USA). Two 1 mm long mouse aortic rings were laid on the top of the Matrigel, and covered with 100 μL of Matrigel per well. After solidification, 150 μL of RPMI [10% FBS and 10 ng/mL basic fibroblast growth factor (bFGF), PeproTech, Rocky Hill, NJ, USA] was added to each well. After 7–10 days of culture, the aortas were photographed, and the endothelial outgrowth was analysed using Image-Pro Plus software and presented as square millimetres.

2.9. Statistical analysis

All data were expressed as the mean ± SEM. Statistical analysis was performed using SPSS 18.0 software. Owing to the small sample size and often abnormally distributed data, we used the Mann–Whitney U test to examine the statistical significance between Apoe^–/–Ctss^–/– and Apoe^–/–Ctss^+/+ mice. A P-value <0.05 denoted a statistically significant difference.

3. Results

3.1. Mouse AAA lesions contain Cat S and its deficiency inhibits Ang II-induced AAA formation in mice

We used an established Ang II perfusion model to produce AAA in Apoe^–/– mice, to investigate the role of Cat S in the development of AAA.²³ Apoe^–/– mice were infused with Ang II for 28 days and then analysed for Cat S expression in AAA tissues by both western blot analysis and immunohistology. Consistent with previous studies in human aortic aneurysms,²¹ western blot analysis showed elevated Cat S expression in AAA tissue extracts, whereas mouse aortas from mice infused with saline contained negligible Cat S (Figure 1A). Immunohistochemistry further confirmed that the expression of Cat S was significantly higher in AAA lesions than in normal aortas (Figure 1B). Increased expression of Cat S in mouse AAA lesions suggests a role of this protease in AAA formation.

Figure 1.

Role of Cat S in Ang II-induced AAA in Apoe^–/– mice. (A) Apoe^–/– mice were infused with Ang II for 28 days and then analysed for Cat S expression in AAA lesions by immunoblot. Representative immunoblots are shown on the left, and the relative gel density ratios of Cat S/GAPDH are shown in the bar graph on the right (n = 10). (B) Immunohistochemistry showed Cat S-positive areas in Apoe^–/– mice perfused with or without Ang II (n = 10). Scale bar: 50 µm. AAA incidence in percentage (C, Fisher's exact test) and aortic external and luminal diameters (D) in both Apoe^–/–Ctss^–/– and Apoe^–/–Ctss^+/+ mice. n = 10 mice per group. Representative photographs in (B) and (D) are shown on the left. Data represent the mean ± SEM. P < 0.05 is considered statistically significant; Mann–Whitney U test was used in all panels except (C).

Morphologically, the aortas of Apoe^–/–Ctss^–/– mice infused with saline did not differ from those of control Apoe^–/–Ctss^+/+ mice (data not shown). There was no AAA formation in the control groups (saline infusion) in both Apoe^–/–Ctss^+/+ and Apoe^–/–Ctss^–/– mice (n = 10, respectively). As previously reported,^16,23 we found that treatment with Ang II for 28 days promoted AAA formation in Apoe^–/– mice (Figure 1A and B). We performed Ang II infusion in both Apoe^–/–Ctss^–/– and Apoe^–/–Ctss^+/+ mice to examine the role of Cat S in AAA formation. Systolic blood pressures did not differ between Apoe^–/–Ctss^–/– and Apoe^–/–Ctss^+/+ mice before (106.9 ± 7.6 vs. 110.6 ± 10.8 mmHg, P = 0.133) or after (140.3 ± 8.7 vs. 145.7 ± 9.6 mmHg, P = 0.246) 28 days of Ang II infusion. Total cholesterol levels also did not differ between Apoe^–/–Ctss^–/– and Apoe^–/–Ctss^+/+ mice before (646.3 ± 92.6 vs. 632.7 ± 89.2 mg/dL, P = 0.632) or after (678.2 ± 89.5 vs. 658.3 ± 93.4 mg/dL, P = 0.588) Ang II perfusion. The incidence of AAA formation was significantly lower in Apoe^–/–Ctss^–/– mice (10%) than in Apoe^–/–Ctss^+/+ mice (80%) (P = 0.006, Fisher's exact test, Figure 1C). Furthermore, maximal aortic diameters and luminal diameters were significantly smaller in Apoe^–/–Ctss^–/– mice than in Apoe^–/–Ctss^+/+ mice after Ang II infusion (Figure 1D). AAA lesion areas were also smaller in Apoe^–/–Ctss^–/– mice than in Apoe^–/–Ctss^+/+ mice (1.38 ± 0.27 vs. 0.07 ± 0.04 mm², P = 0.006), suggesting that Cat S deficiency confers protection from Ang II infusion-induced AAA formation.

3.2. Cat S deficiency reduces AAA lesion ECM degradation and SMC apoptosis

Aortic wall elastin fragmentation is one of the most important signatures of human and animal AAA. As expected, the elastic lamina was frequently disrupted and degraded in Ang II-treated Apoe^–/–Ctss^+/+ mice. In contrast, Cat S deficiency significantly prevented elastin fragmentation in the lamina (Figure 2A) and collagen degradation in the adventitia (Figure 2B). Preserved media elastin and adventitia collagen in Apoe^–/–Ctss^–/– mice yielded elastin-to-collagen ratios in these mice that were not significantly different from those in Apoe^–/–Ctss^+/+ mice (0.29 ± 0.36 vs. 0.28 ± 0.60, P = 0.866). Earlier studies demonstrated that, in aortic elastase perfusion-induced experimental AAA lesions, the absence of Cat L reduced the expression and activities of other cysteinyl cathepsins and MMPs.³⁰ Reduced elastinolysis and collagenolysis in AAA lesions from Apoe^–/–Ctss^–/– mice can also result from impaired activities of other cathepsins or MMPs. To test this hypothesis, we assessed both cysteinyl cathepsin and MMP activities using cathepsin active site labelling with biotinylated JPM and gelatin gel zymogram assays. JPM labelling did not show a clear impact of Cat S deficiency on other cysteinyl cathepsins, such as Cat B and Cat L (Figure 2C). In contrast, the absence of Cat S greatly suppressed both MMP-2 and MMP-9 activities (Figure 2D). Compared with Cat B and Cat L, both Cat S and Cat K activities are often difficult to detect by JPM labelling due to their relative low abundance. Both Cat S and Cat K are potent elastases, and examining whether Cat S deficiency affects Cat K expression is therefore important. Immunostaining revealed significantly lower Cat K levels in AAA lesions from Apoe^–/–Ctss^–/– mice than in those from Apoe^–/–Ctss^+/+ mice (Figure 2E). Immunostaining for MMP-2 (Figure 2F) affirmed the observations from the gelatin gel zymogram assay, shown in Figure 2D. Reduced collagen and elastin degradation in AAA lesions from Apoe^–/–Ctss^–/– mice therefore could be due in part to reduced levels of Cat K, MMP-2, MMP-9, and other untested proteases.

Figure 2.

AAA lesion ECM degradation and protease expression. (A) Medial elastin fragmentation grades between Apoe^–/–Ctss^+/+ and Apoe^–/–Ctss^–/– mice. Grading keys are shown on the left. (B). Sirius red staining showed adventitial collagen contents in AAA lesions. Representative photographs are shown on the right. (C) AAA lesion extract JPM labelling to detect active cathepsins. Coomassie staining was used for protein loading control. (D) Gelatin gel zymograph. Both active MMP-2 and MMP-9 and their molecular weights are indicated. (E). AAA lesion Cat K-positive areas. (F) AAA lesion MMP-2-positive areas. Representative graphs of (E) and (F) are shown on the left. Data represent the mean ± SEM (n = 10 per group). P < 0.05 is considered statistically significant; Mann–Whitney U test. Scale bar: 50 µm.

Medial SMC loss is one of the major events in AAA formation, and contributes to arterial wall thinning, expansion, and rupture. Immunostaining with anti-α-SMA antibodies revealed significantly reduced SMC loss in AAA lesions from Apoe^–/–Ctss^–/– mice, compared with those from Apoe^–/–Ctss^+/+ mice (Figure 3A). Consistent with these observations, TUNEL staining demonstrated significantly reduced apoptotic cell numbers in AAA lesions from Apoe^–/–Ctss^–/– mice, compared with those from Apoe^–/–Ctss^+/+ mice (Figure 3B). These observations suggest that Cat S participates in the apoptosis of arterial cells, such as SMCs. A role of Cat S in aortic SMC apoptosis was confirmed by AAA lesion SMC (α-actin) and TUNEL co-immunostaining and by the in vitro aortic SMC apoptosis assay. SMC and TUNEL co-immunostaining demonstrated much more media SMC apoptosis in AAA lesions from Apoe^–/–Ctss^+/+ mice than in those from Apoe^–/–Ctss^–/– mice (Figure 3C). In vitro, cultured aortic SMCs from Apoe^–/–Ctss^–/– mice were protected significantly from PDTC-induced apoptosis, compared with SMCs from Apoe^–/–Ctss^+/+ mice. There was no difference in SMC apoptosis between groups in the absence of PDTC (Supplementary material online, Figure S1). These observations suggest that Cat S deficiency preserves SMCs, thereby increasing ECM synthesis and stabilizing the arterial wall.

Figure 3.

Cat S deficiency reduces AAA lesion cell apoptosis and loss. (A) AAA lesion medial SMC loss between Apoe^–/–Ctss^+/+ and Apoe^–/–Ctss^–/– mice. SMC grading keys are shown on the left. (B) TUNEL staining to detect AAA lesion cell apoptosis (green fluorescence). Representative graphs are shown on the left. Data are presented as average apoptotic cell numbers on each AAA section. (C) TUNEL and α-actin double staining to detect apoptotic SMCs (arrows) in AAA lesions. Data represent the mean ± SEM. n = 10 per group. P < 0.05 is considered statistically significant; Mann–Whitney U test. Scale bar: 50 µm.

3.3. Absence of Cat S impairs angiogenesis in AAA lesions

Microvessels are abundant in human AAA lesions,³¹ and neovascularization is essential to AAA formation, providing paths for lesion inflammatory cell recruitment.³² Inhibition of neovascularization or angiogenesis prevents AAA growth.³³ To test whether Cat S deficiency influences AAA lesion angiogenesis, we immunostained AAA lesions with both anti-mouse CD31 and VEGF antibodies, and found that CD31⁺ microvessel numbers (Figure 4A) and VEGF-positive areas (Figure 4B) in the adventitia were significantly lower in Apoe^–/–Ctss^–/– mice than in Apoe^–/–Ctss^+/+ mice. In AAA lesions, VEGF is rich in areas with abundant inflammatory cell infiltrates (Figure 4B, left panels), indicating a role of Cat S in recruiting inflammatory cells. Although not tested in this study, high numbers of CD31⁺ microvessels and VEGF-positive areas in Apoe^–/–Ctss^+/+ mice suggested that other pro-angiogenic factors were also higher in AAA lesions from these mice than in those from Apoe^–/–Ctss^–/– mice. To confirm that Cat S participates in angiogenesis, we performed an in vitro aortic ring assay using aortic rings from Apoe^–/–Ctss^+/+ and Apoe^–/–Ctss^–/– mice without Ang II infusion, and demonstrated that microvessel-sprouting areas from aortic rings from Apoe^–/–Ctss^–/– mice were significantly decreased, compared with those from Apoe^–/–Ctss^+/+ mice in the presence of bFGF (Figure 4C). Therefore, both in vitro and in vivo observations suggest the importance of Cat S in angiogenesis.

Figure 4.

Role of Cat S in neovascularization. (A) Representative CD31 staining in AAA lesions from Apoe^–/–Ctss^+/+ and Apoe^–/–Ctss^–/– mice (left). The bar graph shows AAA lesion quantitative analysis of CD31⁺ microvessel numbers (right). Scale bar: 100 µm. (B) Representative VEGF staining in AAA lesions from Apoe^–/–Ctss^+/+ and Apoe^–/–Ctss^–/– mice (left). The bar graph shows AAA lesion quantitative analysis of VEGF-positive areas (right). Scale bar: 50 µm. (C) In vitro aortic ring assay showed microvessel sprouting in aortas from Apoe^–/–Ctss^+/+ and Apoe^–/–Ctss^–/– mice (left panels). Microvessel growth areas in square millimetres are shown to the right. Data represent the mean ± SEM (n = 10 per group). P < 0.05 is considered statistically significant; Mann–Whitney U test.

3.4. Cat S deficiency decreases inflammatory response in AAA lesions

Inflammatory cells are important components of AAA lesions, and they play essential roles in AAA initiation and progression in humans and animals.^4,34 Reduced angiogenesis in AAA lesions from Apoe^–/–Ctss^–/– mice suggests that inflammatory cell contents in these mice are also different from those in AAA lesions from Apoe^–/–Ctss^+/+ mice. Immunostaining with antibodies against Mac-2, CD4, and MCP-1 demonstrated that Cat S deficiency reduced AAA lesion Mac-2⁺-positive macrophage areas, CD4⁺ T-cell numbers, and chemokine MCP-1-positive areas (Figure 5A–C). Impaired accumulation of macrophages and T cells in AAA lesions from Apoe^–/–Ctss^–/– mice suggested a role of Cat S in inflammatory cell recruitment, which was tested in vitro in a cell transmigration assay. Peripheral blood mononuclear cells (PBMC; mainly monocytes) (Figure 5D) and T cells (Figure 5E) from Apoe^–/–Ctss^–/– mice migrated toward MCP-1 significantly more slowly through collagen-coated transwells than did those from Apoe^–/–Ctss^+/+ mice. Impaired migration of Cat S-deficient monocytes and T cells toward MCP-1 may explain, in part, fewer Mac-2⁺ macrophages and CD4⁺ T cells in AAA lesions from Apoe^–/–Ctss^–/– mice (Figure 5A and B). To assess Cat S-deficiency-associated suppression of inflammation quantitatively, we performed ELISA to determine both MCP-1 and inflammatory cytokine IFN-γ in AAA lesion extracts and plasma. Both MCP-1 and IFN-γ levels in AAA lesion extracts and plasma were significantly lower in Apoe^–/–Ctss^–/– mice than those in Apoe^–/–Ctss^+/+ mice (Supplementary material online, Figure S2).

Figure 5.

Role of Cat S in AAA lesion inflammatory cell accumulation. Immunohistochemistry for Mac-2 (A), CD4 (B), or MCP-1 (C) in AAA lesions from Apoe^–/–Ctss^–/– and Apoe^–/–Ctss^+/+ mice (left panels). The bar graphs on the right show the percentages of Mac-2-positive area, CD4⁺ T-cell numbers per aortic section, and MCP-1-positive areas. Data are presented as mean ± SEM (n = 10 per group). Peripheral blood mononuclear cell (PBMC) (D) and T-cell (E) transmigration assays through collagen-coated transwells. Cell genotypes are indicated. Data are mean ± SEM of three independent experiments. P < 0.05 is considered statistically significant; Mann–Whitney U test. Scale bar: 50 µm.

Reduced cell proliferation in the absence of Cat S can also reduce the number of inflammatory cells in AAA lesions from Apoe^–/–Ctss^–/– mice. Immunostaining with anti-mouse Ki67 polyclonal antibodies revealed significantly fewer Ki67⁺ cells in AAA lesions from Apoe^–/–Ctss^–/– mice than in those from Apoe^–/–Ctss^+/+ mice (Figure 6A). These Ki67⁺ cells are probably mostly infiltrated inflammatory cells. We performed co-immunostaining with anti-Ki67 and anti-CD3 antibodies to test this hypothesis, and found that the numbers of Ki67⁺CD3⁺ proliferating T cells were significantly lower in AAA lesions from Apoe^–/–Ctss^–/– mice than in those from Apoe^–/–Ctss^+/+ mice (Figure 6B). In vitro cell proliferation assays further confirmed this hypothesis. We examined the optical density of T cells with Cell Counting Kit-8 (Dojindo). Cat S deficiency suppressed cell proliferation significantly (Figure 6C). When splenic CD3⁺ cells from Apoe^–/–Ctss^–/– and Apoe^–/–Ctss^+/+ mice were incubated with CFSE for 3 days, CD3⁺CFSE⁺ T-cell numbers were significantly reduced in the absence of Cat S, as determined by FACS analysis (Figure 6D). Media IL-2 production yielded the same result. After cells were incubated in an anti-CD3 antibody-coated culture dish for 3 days, media IL-2 production was significantly reduced in cells from Apoe^–/–Ctss^–/– mice, as measured by IL-2 ELISA (Figure 6E). Cat S deficiency therefore affects not only inflammatory cell migration (Figure 5D and E), but also inflammatory cell proliferation in vitro.

Figure 6.

Role of Cat S in AAA lesion cell proliferation. (A) Ki67 staining to detect AAA lesion cell proliferation. Data are presented as Ki67-positive cells in each square millimetre. (B) Ki67 and CD3 double staining to detect proliferating CD3⁺ T cells (arrows). Representative graphs are shown on the left. n = 10 per group. Data are mean ± SEM. Scale bar: 50 µm. In vitro CD3⁺ T-cell proliferation was assessed with Cell Counting Kit-8 (C), CFSE proliferation assay by FACS (D), and IL-2 production (ng/mL) in cell culture supernatants by ELISA (E). Data are mean ± SEM of six independent experiments. P < 0.05 is considered statistically significant; Mann–Whitney U test. Mouse genotypes are indicated.

4. Discussion

AAA formation involves extensive medial matrix protein proteolysis, SMC loss, angiogenesis, and inflammatory cell accumulation, leading to progressive dilatation and eventual rupture.³⁵ This study found that cysteinyl Cat S contributed to all of these pathological events, thereby promoting Ang II-induced AAA formation in Apoe^–/– mice.

Arterial wall medial SMC apoptosis is an important hallmark of AAA, and it increases during aneurysmal lesion formation and associates with the accumulation of inflammatory cells and the expression of pro-apoptotic molecules, such as p53 and death receptors.^36,37 Accumulating evidence suggests that cysteinyl cathepsins are important in cell apoptosis. In response to pro-inflammatory mediators, lysosomes release cathepsins to the cytoplasm, triggering cell apoptosis.^38,39 Cat L promotes caspase-dependent apoptosis.⁴⁰ Inhibition of Cat B and Cat L protects against oxysterol-induced mononuclear cell death.³⁸ Selective inhibition or deficiency of Cat S decreases IFN-γ-induced epithelial cell apoptosis in pulmonary emphysema in mice.⁴¹ Consistent with these prior studies, our results showed that Cat S deficiency reduced aortic SMC apoptosis in vitro (Supplementary material online, Figure S1) and reduced SMC loss in AAA lesions (Figure 3A–C), supporting a direct role of Cat S in SMC apoptosis during AAA formation.

Neovascularization recently was identified as a marker of plaque vulnerability, and it associates with AAA lesion rupture.⁴² The effect of cathepsins on microvessel formation and growth has been studied extensively in malignant tumours,^43,44 but less so in cardiovascular disease. Although Cat S deficiency did not affect proliferation or adhesion of mouse ECs, Cat S-deficient ECs showed reduced elastinolytic and type IV collagenolytic activities.⁴⁵ Further, several studies have demonstrated that Cat S and Cat B contribute to neovascularization by generating pro-angiogenic factors, stimulating cell proliferation, and enhancing endothelial capillary-like tubular formation.^43,44 In contrast, Cat S inhibition (with its selective inhibitor) effectively blocks microtubule formation.⁴⁵ Cat S deficiency showed an 80% reduction in microvessel numbers in mouse skin wound healing.⁴⁵ VEGF-A was overexpressed in the aortic wall of human and experimental AAA.⁴⁶ Consistent with these observations, the current study showed that Cat S deficiency significantly reduced CD31⁺ microvessel numbers and VEGF expression in AAA lesions and sprouting areas in an aortic ring assay (Figure 4A–C), suggesting that Cat S facilitates microvessel formation in AAA lesions.

Inflammatory cell infiltration into the arterial wall of AAA lesions mediates tissue destruction and leads to aortic wall weakening and eventual rupture.^34,47 Macrophages, T cells, neutrophils, and mast cells are common inflammatory cell types that brought broad interest to the field. These cells accumulate at the site of inflammation and release cytokines, chemokines, and proteases, such as tumour necrosis factor-α, interleukin-1β, and interleukin-6, MCP-1, MMPs, and cathepsins, which recruit more inflammatory cells and induce SMC death.³⁵ Cat S participates in inflammatory cell recruitment in atherosclerosis-prone Ldlr^–/– mice. Cat S deficiency significantly reduced macrophages, T cells, IFN-γ expression, and lipid content in atherosclerotic lesions.¹³ The current study established dual roles of Cat S in macrophage and T-cell accumulation and proliferation in AAA lesions. Although we do not know whether Cat S contributed directly to AAA lesion chemokine MCP-1 expression, reduced MCP-1 expression in AAA lesions from Apoe^–/–Ctss^–/– mice (Figure 5C, Supplementary material online, Figure S2) may have resulted from fewer macrophages and T cells in these lesions (Figure 5A and B). In vitro assays demonstrated that macrophage and T-cell migration requires Cat S (Figure 5D and E). Although we did not test whether monocyte or macrophage proliferation requires Cat S, its absence directly affected CD3⁺ T-cell proliferation in lesions (Figure 6A and B) and in cultures (Figure 6C–E). Cat S activities in inflammatory cell recruitment and proliferation therefore may contribute to reduced macrophage and T-cell contents in Apoe^–/–Ctss^–/– mouse AAA lesions.

Protease activities contribute to AAA formation by degrading elastin, collagen, fibronectin, laminin, and many other ECM proteins, to promote angiogenesis and blood-borne leucocyte migration and consequent inflammation. MMPs also participate in arterial wall remodelling and are important in the initiation and progression of AAA.^48,49 In this study, we found reduced AAA lesion activities of MMP-2 and MMP-9 (Figure 2D and F). Resistance to AAA formation in Apoe^–/–Ctss^–/– mice may be partially due to reduced activities of these MMPs. Prior studies showed that Cat K deficiency reduced aortic elastase perfusion-induced AAA, but not chronic Ang II perfusion-induced AAA, in mice.^16,50 Although Cat S deficiency also reduced Cat K expression in AAA lesions (Figure 2E), reduced Cat K expression may not explain suppressed Ang II perfusion-induced AAA lesions in Apoe^–/–Ctss^–/– mice.

Taken together, the results of this study demonstrate an important role of Cat S in Ang II perfusion-induced experimental AAA in mice. Although whether genetic deficiency of Cat S is AAA-resistant in other experimental models remains unknown, many of the molecular mechanisms described in this study— including ECM protein degradation, SMC apoptosis, angiogenesis, and lesion inflammatory cell recruitment and proliferation—are shared with other large or small vascular diseases.^43,44 We recently demonstrated that plasma Cat S levels (total Cat S, pre-Cat S, and active Cat S) were significantly higher in 476 male AAA patients than in 200 age-matched male controls. Both univariate and multivariate linear regression analyses demonstrated positive and significant correlations between plasma total, active and pre-Cat S levels, and aortic diameters.²² Thus, Cat S might serve as a potential therapeutic target for human AAA and other large or small arterial diseases. A Phase I trial recently was completed of a Cat S selective and potent inhibitor from Eli Lilly and Company designed to treat patients with AAA, atherosclerosis, and autoimmune diseases. Our study supports a role of Cat S in human AAA, and that Cat S may be a target of this arterial disease.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Funding

This study is partially supported by grants from the National Natural Science Foundation of China (81070251, 81100222, 81270389) to Y.W.Q.;(81170283) to H.J.Z.; Program for Changjiang Scholars and Innovative Research Team in University (IRT1074) and International Science & Technology Cooperation Program of China (2010DFB30040) to J.D.; grants from the U.S. National Institutes of Health (HL60942, HL81090, HL88547) to G.P.S.; and by an Established Investigator Award from the American Heart Association (0840118N) to G.P.S.