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Mast cell chymase inhibition reduces atherosclerotic plaque progression and improves plaque stability in ApoE−/− mice

Ilze Bot, Martine Bot, Sandra H. van Heiningen, Peter J. van Santbrink, Inge M. Lankhuizen, Peter Hartman, Sabine Gruener, Hans Hilpert, Theo J.C. van Berkel, Jürgen Fingerle, Erik A.L. Biessen
DOI: http://dx.doi.org/10.1093/cvr/cvq260 244-252 First published online: 6 August 2010

Abstract

Aims Mast cells have been shown to accumulate in the adventitia of human atherosclerotic plaques and were recently demonstrated by us to contribute to plaque progression and instability. In this study, we investigated whether selective inhibition of mast cell chymases would affect the lesion development and stability.

Methods and results The protease inhibitor RO5066852 appeared to be a potent inhibitor of chymase activity in vitro and ex vivo. With this inhibitor, we provide three lines of evidence that chymase inhibition can prevent many pro-atherogenic activities. First, oral administration of RO5066852 reduced spontaneous atherosclerosis in the thoracic aorta of apoE−/− mice. Second, chymase inhibition prevented the accelerated plaque progression observed in apoE−/− mice that were exposed to repetitive episodes of systemic mast cell activation. Furthermore, RO5066852 enhanced lesional collagen content and reduced necrotic core size. Third, RO5066852 treatment almost completely normalized the increased frequency and size of intraplaque haemorrhages observed in apoE−/− mice after acute perivascular mast cell activation in advanced atherosclerosis.

Conclusion Our data indicate that chymase inhibition can inhibit pro-atherogenic and plaque destabilizing effects which are associated with perivascular mast cell activation. Our study thus identifies pharmacological chymase inhibition as a potential therapeutic modality for atherosclerotic plaque stabilization.

  • Atherosclerosis
  • Mast cells
  • Proteases
  • Chymase
  • Inhibitors

1. Introduction

The clinical outcomes of acute cardiovascular syndrome such as myocardial infarction and stroke are generally caused by rupture of an atherosclerotic plaque, but the actual cause of a plaque to rupture is not yet established.13 Interestingly, pathology studies have shown an increased presence of the mast cell, an important inflammatory effector cell, in the plaque and the perivascular tissue during plaque progression.46 Recently, we and others have conclusively demonstrated that (peri)vascular mast cells contribute to atherosclerotic plaque progression and destabilization in mice.79 Mast cells are laden with granules containing a wide variety of mediators, such as the vasoactive histamine, heparin, and proinflammatory cytokines.10,11 Mast cells also contain a range of proteases such tryptase and chymase.1113 Chymase is known as a leucocyte chemoattractant14 and has been demonstrated to induce apoptosis of vascular smooth muscle cells,15 endothelial cells,16 and macrophages,7 which all could translate in reduced plaque stability. Furthermore, chymase can, besides degrading extracellular matrix molecules itself, activate matrix metalloproteinases leading to degradation of collagen and elastin.1719 Chymase can also convert angiotensin I (AngI) into the proinflammatory, vasoactive angiotensin II (AngII),20 although this activity differs considerably between species.21 In atherosclerotic plaques of the aorta, mouse Mast Cell Protease (mMCP)-4 positive mast cells were shown to be also present in mice,8 although they are rather sparse; in the perivascular tissue, the number of mast cells was seen to be similar to that in human adventitia.7 Recently, we demonstrated that perivascular mast cell activation induces plaque destabilization as evidenced by an increased incidence of intraplaque haemorrhage, macrophage apoptosis, and vascular leakage.7 The major players in mast cell-mediated atherosclerotic plaque destabilization in vivo, however, are not fully elucidated yet. In this study, we aimed to determine the contribution of mast cell chymase to the previously observed adverse phenomena in atherosclerotic plaque stability by treatment of apoE deficient (apoE−/−) mice with a potent chymase inhibitor. Here, we are the first to demonstrate that inhibition of chymase reduces plaque progression, which was paralleled by enhanced plaque stability as demonstrated by increased collagen levels and reduced necrotic core size. Moreover, chymase inhibition during acute and focal mast cell activation markedly diminished frequency and cross-sectional area of intraplaque haemorrhages.

2. Methods

A detailed description of the Methods is given in the Supplementary material online.

2.1 IC50 determination

Human chymase (0.75 nM) was expressed in E. coli and hamster chymase II (2 nM) was expressed in HEK 293 cells and purified to homogeneity.1 Human chymotrypsin (0.5 nM) and cathepsin G (30 nM) were purchased from Sigma-Aldrich, Zwijndrecht, The Netherlands. For all enzymes, the substrate CAAPFW (Biosyntan) was used at 1 µM in 100 mM HEPES, pH 7.4, and 0.01% Triton X-100; fluorescence assays were all done in the presence of 80 mg/mL heparin. Validation of the inhibitory potential of the chymase inhibitor RO5066852 was done using a MR121-CAAPFW probe (Roche, Basel, Switzerland) based quenching assay. The C-terminal tryptophan of the uncleaved peptide quenches the fluorescence of the N-terminal MR121 fluorophore (MR121-CAAPFW mixed with unlabeled peptide 1:10), while fluorescence dequenching only occurs once the peptide has been cleaved. Samples were titrated with four-fold dilutions over the appropriate concentration range and fluorescence monitored for 20 measurements over a period of 18 min (λex=612 nm, λem=670 nM).

2.2 Cell culture

Peritoneal mast cells (PMCs) were seeded at 2 × 106 cells/mL and allowed to attach for 1 h. Non-adhered PMCs and MC/9 cells were seeded at 2.5 × 105 cells/mL and used for degranulation experiments as previously described7 in the presence and or absence of 1 µM of RO5066852. Chymase and tryptase activity in the releasate was determined as described previously.7

2.3 Chymase expression in mouse atherosclerosis

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). All animal work was approved by the regulatory authority of Leiden University and performed in compliance with Dutch and Swiss government guidelines. Male LDL receptor deficient (LDLr−/−) mice were fed a western-type diet (Special Diet Services, Sussex, UK) 2 weeks prior to surgery and throughout the experiment. To determine mouse chymase gene expression levels in mouse plaques (n = 20), atherosclerotic carotid artery lesions were induced by perivascular collar placement. Gene expression profiles of carotid artery plaques in LDLr−/− mice were determined at 0–8 weeks after perivascular collar placement. Total RNA was extracted and RNA was reverse transcribed by M-MuLV reverse transcriptase (RevertAid, MBI Fermentas, Leon-Roth) and used for the quantitative analysis of mouse chymase gene expression with an ABI PRISM 7700 Taqman apparatus (Applied Biosystems, Foster City, CA, USA).

2.4 RO5066852 plasma concentration

ApoE−/− mice (Jackson Laboratories) were fed a cholesterol-rich diet (01.25%, diet 2481, Kliba, Kaiseraugst, Switzerland) to which the chymase inhibitor RO5066852 was added as food mixture (3, 10, or 30 mg/kg/day, n = 4–8 per group). Two hundred microlitre plasma was extracted with 400 µL acetone. The precipitate was spun down and 500 µL of the supernatant dried under nitrogen. The residue was taken up in 167 µL of standard chymase assay buffer with heparin, four-fold dilution series were made, and 80 µL of each dilution were assayed for remaining chymase activity.

2.5 Plaque progression

ApoE−/− mice (8 weeks old, Jackson Laboratories) were fed a cholesterol-rich diet (01.25%, diet 2481, Kliba, Kaiseraugst, Switzerland) for 14 weeks. For the last 7 weeks, the chymase inhibitor RO5066852 was added as food mixture (10 mg/kg/day, n = 10 per group). Mice were perfusion-fixed with 4% paraformaldehyde via a ventricular cannula. Aortas were carefully excised and longitudinally opened for Sudan III staining and morphometric analysis (IPLab).

2.6 Systemic mast cell activation

ApoE−/− mice (10–12 weeks old), obtained from the Gorlaeus animal breeding facility in Leiden, were fed a western-type diet (SDS, Sussex, UK) for 6 weeks. Then, carotid artery plaque formation was induced by perivascular collar placement in male apoE−/− mice as described previously,22 after which the mice were fed either western-type diet as control diet or diet supplemented with chymase inhibitor RO5066852 (30 mg/kg/day) for another 6 weeks. One week after surgery, the mice were skin sensitized as previously described.23 One week later, the mice were challenged intravenously by injection of dinitrophenyl-albumin (DNP, 1 mg/animal, Sigma-Aldrich), which was repeated once weekly for another 3 weeks to induce episodes of systemic mast cell activation. After a total of 12 weeks of diet feeding, in situ perfusion–fixation was performed.

2.7 Focal mast cell activation

ApoE−/− mice were fed a western-type diet 2 weeks prior to perivascular collar placement. After 5 weeks of lesion development, mice were skin sensitized on the abdomen. Five days later, the mice were perivascularly challenged by applying control pluronic F-127 gel (25% w/v, Sigma-Aldrich) or pluronic F-127 gel containing DNP (50 µg/animal, Sigma-Aldrich) at the carotid artery segment carrying the advanced atherosclerotic plaque,7 while receiving a western-type diet supplemented with the chymase inhibitor RO5066852 as described above (n = 11 per group). Three days after the challenge, the animals were anaesthetized and in situ perfusion–fixation was performed.

2.8 Histology and morphometry

Plaques were stained with haematoxylin–eosin to determine plaque size. Mast cells and neutrophils were visualized by staining of 5 µm cryosections with naphthol AS-D chloroacetate esterase (Sigma-Aldrich). Apoptosis was visualized using a terminal deoxytransferase dUTP nick-end labelling (TUNEL) kit (Roche Diagnostics). Collagen was visualized using a Masson's Trichrome staining kit (Sigma-Aldrich). Morphometric analysis was performed using Leica Qwin image analysis software. Intraplaque haemorrhage was defined as masses of red blood cells free in the plaque matrix or filling the necrotic core.7,9 All histological and morphometric analyses were performed by blinded independent operators (S.H.v.H. and I.B.).

2.9 Microvascular leakage

Microvascular permeability was assessed essentially as described previously.7 In short, C57Bl/6 mice (n = 5–6 per group), fed with the chymase inhibitor RO5066852 or control diet for 2 weeks as described above, were injected intradermally at randomized sites with either PBS, 5 × 105 MC/9 mast cells suspended in PBS or 5 × 105 MC/9 cells activated with 50 µg/mL compound 48/80 in the absence of presence of 1 µM RO5066852. Immediately after intradermal injection of the cell suspensions, 100 µL 1.25% Evans Blue was injected intravenously and after 30 min, the surface area of Evans Blue stained skin was measured.

2.10 Statistical analysis

Data are expressed as mean ± SEM. A two-tailed Student's t-test was used to compare individual groups, while multiple groups were compared with a one-way ANOVA and a subsequent Student–Newman–Keuls multiple comparisons test. Frequency data analysis was performed by means of the Fisher's exact test. A level of P < 0.05 was considered significant.

3. Results

3.1 Chymase inhibition and mast cell activation in vitro

In vitro enzyme inhibition studies showed that the chymase inhibitor RO5066852 is a potent and selective inhibitor of human chymase and hamster chymase II displaying IC50 values in the low nanomolar level, while being relatively inert for related proteases such as human tryptase, cathepsin G, and chymotrypsin (Table 1). Inhibition of mouse mast cell chymases by RO5066852 was demonstrated by the analysis of chymase activity in lysates of freshly isolated murine PMCs in the presence of RO5066852. RO5066852 (1 µM) inhibited mast cell chymase in PMC lysates by 86 ± 1% (P < 0.01). To determine whether this chymase inhibitor was also able to inhibit mast cell activation, we analysed the releasate of activated MC/9 cells (a murine mast cell line) and of PMCs for β-hexosaminidase and chymase activity. Pre-incubation of compound 48/80 activated MC/9 mast cells or PMCs with RO5066852 (1 µM) was seen to attenuate mast cell activation, as illustrated by a decreased β-hexosaminidase activity in the releasate (Figure 1A). Tryptase activity in the releasates remained unaffected by the chymase inhibitor (data not shown). As expected, chymase activity in the releasate was almost ablated after incubation with the chymase inhibitor (Figure 1B). These data firmly establish that RO5066852 is a potent inhibitor of mast cell chymase activity and to some extent also of mast cell activation in vitro.

View this table:
Table 1

Structure and IC50 of the chymase inhibitor RO5066852 on purified enzymes

EnzymeExpression systemnM
Human chymaseE. coli11Embedded Image
Hamster chymase IIHek2933
Human tryptasePurified>100 × 103
Human chymotrypsinPurified1.5 × 103
Human cathepsin GPurified300
Figure 1

Pre-incubation of MC/9 mast cells or freshly isolated PMCs with 1 µM of RO5066852 resulted in a significant reduction in β-hexosaminidase activity (A) as well as in a marked decrease in chymase activity (B) in mast cell releasate after activation with 0.5 µg/mL compound 48/80 (**P < 0.01, ***P < 0.001, n.d., not detectable).

3.2 Chymase expression in mouse atherosclerosis

Mast cells accumulate in the perivascular tissue of human atherosclerotic plaques during disease progression6 and also in mice we observed an increased presence of perivascular mast cells during plaque progression (Figure 2A). Previously, we demonstrated these mast cells mostly to be connective tissue-type mast cells (98.4%) by Alcian blue/saphranin O staining7 and these mast cells are generally chymase and tryptase positive.10 To determine the expression of mouse chymases during atherosclerotic lesion progression, mRNA from plaques at different stages of plaque development was isolated and gene expression determined by qPCR. Mouse chymases mMCP-1 and -5 were found to be increasingly expressed during lesion progression (Figure 2B), while the expression of mMCP-4 was only highly expressed in advanced plaques. The expression of mMCP-9 remained unchanged. These data demonstrate that mouse chymases are expressed in mouse models of atherosclerosis due to the increased accumulation of mast cells and by differential chymase expression during plaque progression, rendering the mouse a qualified model to examine chymase inhibitors.

Figure 2

(A) Mast cells accumulate in the perivascular tissue of aortic atherosclerotic plaques during the progression of atherosclerosis. (B) Mouse chymases mMCP-1 (upper left panel) and mMCP-5 (lower left panel) mRNA are increasingly expressed during atherosclerotic lesion progression in apoE−/− mice (0–8 weeks of collar-induced carotid artery plaque development, n = 3 samples of 2–3 plaques pooled per time point). mMCP-4 is mainly expressed in highly advanced plaques (upper right panel), while mMCP-9 (lower right panel) is not regulated during the development of atherosclerosis (*P < 0.05 compared to 0 weeks).

3.3 RO5066852 treatment and plaque progression

Next we studied the pharmacokinetics of RO5066852. Hereto, the chymase inhibitor was orally administered to mice by supplementation of western-type diet with RO5066852. Figure 3A depicts RO5066852 plasma levels in mice treated with 3, 10, or 30 mg/kg/day of RO5066852, showing peak plasma levels of ∼1.2, 2.2, and 8.4 µM, respectively. RO5066852 plasma levels over the day were on average 0.7, 1.2, and 4.8 µM. These data indicate that oral treatment of mice, when given 10 or 30 mg/kg/day, leads to effective plasma concentrations of the chymase inhibitor.

Figure 3

(A) Oral administration of RO5066852 via western-type diet leads to plasma peak levels at 4–6 h after start of diet change (n = 4–8 per group). (B) Treatment of apoE−/− mice with RO5066852 resulted in an inhibition of spontaneous plaque surface area in the thoracic aorta (*P < 0.05, control: n = 10, RO5066852: n = 10) as demonstrated by en face Sudan III staining of lipid deposits representing atherosclerotic plaques (C).

To determine the effects of chymase inhibition on spontaneous atherosclerotic plaque progression in the aorta, apoE−/− mice received either control cholesterol-rich diet or diet supplemented with RO5066852 (10 mg/kg/day) as described above. In these mice, inhibition of chymase led to a significant 22% reduction in en face plaque surface area in the thoracic aortas compared with control mice (P < 0.05, Figure 3B) as demonstrated by Sudan III staining (Figure 3C), suggesting that chymase contributes to spontaneous plaque progression.

3.4 Systemic mast cell activation

In human atherosclerosis, the number of activated perivascular mast cells increases during plaque progression,6 while, as we have previously demonstrated, systemic mast cell activation aggravates atherosclerosis in apoE−/− mice.7 To determine the contribution of chymase in mast cell-induced plaque progression, in the next experiment we exposed RO5066852 (30 mg/kg/day) and mock-treated apoE−/− mice to multiple episodes of systemic mast cell activation by repeated intravenous challenges with the hapten DNP during plaque development. ApoE−/− mice were fed a western-type diet as control diet or diet supplemented with a high dose of the chymase inhibitor RO5066852 (30 mg/kg/day), which resulted in pharmacologically effective RO5066852 plasma levels of 3 µM on average (Figure 4A) throughout the experiment. In a separate experiment, we determined whether this RO5066852 plasma concentration was effective in inhibiting mouse chymase in vivo after 1 week of treatment (30 mg/kg/day). We peritoneally challenged apoE−/− mice with the mast cell activator compound 48/80. Chymase activity in the peritoneum was almost two-fold reduced by RO5066852 treatment (OD405: 0.85 ± 0.14 in controls vs. 0.48 ± 0.10 in RO5066852 treated mice), demonstrating that this chymase inhibitor indeed reduced mouse chymase activity in vivo.

Figure 4

(A) Oral administration of RO5066852 to apoE−/− mice that were systemically challenged during plaque progression to induce mast cell activation, resulted in sustained, therapeutically effective plasma concentrations. (B) Brachiocephalic artery plaque size was significantly reduced after RO5066852 treatment during systemic mast cell activation (*P < 0.05, controls: n = 10, RO5066852: n = 12). Right panel: H&E stained brachiocephalic artery plaques of control (upper) and RO5066852 treated (lower) mice (×100 magnification). (C) Representative H&E stained carotid artery plaques of control and RO5066852 treated mice (×100 magnification) demonstrate a significant reduction in relative necrotic core content (as indicated by arrows) of the lesions (upper panels, **P < 0.01, controls: n = 12, RO5066852: n = 13), while collagen content (in blue) tended to be enhanced (lower panels, ×100 magnification, controls: n = 12, RO5066852: n = 13). (D) Ex vivo RO5066852 (1 µM) treatment of skin segments of apoE−/− mice resulted a significant reduction in tryptase activity in the skin releasate indicative of and inhibition of mast cell activation (*P < 0.05).

Throughout the experiment, body weight, plasma total cholesterol, and triglyceride levels did not differ between the groups (data not shown), while also blood cell numbers and plasma TNFα levels were not affected by the chymase inhibitors (data not shown).

Chymase inhibition by RO5066852 treatment resulted in a significant 30% inhibition of plaque progression in the brachiocephalic artery (control: 200 ± 20 × 103 µm2 and RO5066852: 140 ± 13 × 103 µm2, Figure 4B, P < 0.05), which corresponded with a reduction in intima/lumen ratio (control: 0.70 ± 0.02 and RO5066852: 0.54 ± 0.04, P < 0.05). Media size was not significantly affected by RO5066852 treatment (control: 64 ± 3 × 103 µm2 and RO5066852: 59 ± 4 × 103 µm2, P = NS). Adventitial mast cell numbers and activation status did not differ between the groups (control: 16 ± 3 mast cells/mm2 adventitial tissue vs. 16 ± 2 in the RO5066852 group). In collar-induced carotid artery plaques, the relative necrotic core size was significantly reduced from 53 ± 4% in controls to 35 ± 3% in RO5066852 treated mice (P < 0.01, Figure 4C), which corresponded with a trend towards an increased relative plaque collagen content (0.8 ± 0.2% in control mice vs. 1.8 ± 0.6% in RO5066852 treated mice; P = 0.13, Figure 4C), reflective of increased plaque stability.

Because at 1 week after final challenge effects of the chymase inhibitor on adventitial mast cell activation in situ are largely undetectable, we isolated skin segments and ex vivo challenged them with DNP after which mast cell activation was measured by means of β-hexosaminidase and tryptase. Co-treatment of skin segments with RO5066852 resulted in a trend towards a decreased β-hexosaminidase activity in the supernatant (OD405: 1.16 ± 0.07 vs. 1.50 ± 0.20 in controls, P = 0.1), while significantly reducing tryptase activity in skin releasates (OD405: 0.29 ± 0.05 vs. 0.44 ± 0.04 in controls, P < 0.05, Figure 4D). These data suggest that also in vivo, the chymase inhibitor RO5066852 affects mast cell activation.

Overall, these data demonstrate that inhibition of mast cell chymases ameliorates both spontaneous as well as systemic mast cell activation-induced plaque progression and stability.

3.5 Focal mast cell activation

Mast cell density and activation are particularly high in perivascular tissue of advanced and ruptured human atherosclerotic lesions5 and previously, we already showed that in mice, focal mast cell activation in perivascular tissue of advanced plaques leads to plaque destabilization.7 Therefore, we assessed the effect of chymase inhibition after a single episode of focal adventitial mast cell activation on advanced atherosclerotic plaques in apoE−/− mice. Again, apoE−/− mice were fed RO5066852 supplemented or regular western-type diet as described above and at 3 days after focal perivascular mast cell activation, plaques were analysed.

Plaque size did not differ between groups at 3 days after challenge (control: 50 ± 10 × 103 µm2 and RO5066852: 40 ± 7 × 103 µm2, Figure 5A, P = NS), while media size also remained unaffected (control: 31 ± 2 × 103 µm2, RO5066852: 30 ± 2 × 103 µm2, Figure 5B, P = NS). Focal perivascular mast cell activation resulted in an enhanced incidence of intraplaque haemorrhage (IPH, defined as masses of red blood cells free in the plaque matrix or filling the necrotic core24) in control animals (5 out of 22 carotid artery plaques, 23%) as described previously.7 IPH frequency tended to be inhibited in RO5066852 treated mice (1 out of 22 plaques, 5%, P = 0.18, Figure 5C), while IPH size as measured by erythrocyte positive area (Figure 5D) was reduced by 98% in RO5066852 treated compared with control mice (21 ± 15 vs. 1232 ± 551 µm2 in controls, P < 0.05, Figure 5E). Perivascular mast cell numbers were slightly reduced in RO5066852 treated mice (2.7 ± 0.9 vs. 4.0 ± 1.4 mast cells/mm2 perivascular tissue in controls), while mast cell activation status was unaffected at 3 days after challenge (data not shown). Interestingly, perivascular neutrophil numbers were markedly reduced upon RO5066852 treatment (17 ± 4 vs. 48 ± 10 neutrophils/mm2 perivascular tissue in untreated mice, P < 0.05).

Figure 5

Chymase inhibition inhibits intraplaque haemorrhage after focal mast cell activation. Oral RO5066852 treatment of atherosclerotic apoE−/− mice that were perivascularly challenged to activate adventitial mast cells at the carotid artery lesion site, did not affect plaque (A) and media (B) size (controls: n = 11, RO5066852: n = 11). However, it reduced the incidence of intraplaque haemorrhage (C). (D) Representative picture of an intraplaque haemorrhaged carotid artery plaque after focal mast cell activation (×100 magnification). (E) Also, haemorrhage size as represented by the erythrocyte surface area (indicated by arrow) was significantly decreased in the chymase inhibitor treatment group (*P < 0.05, controls: n = 11, RO5066852: n = 11).

Previously, we have described that mast cell activation, besides increasing the incidence of IPH, also induces plaque macrophage apoptosis in a tryptase, chymase, and histamine-dependent manner.7 In this study, apoptotic cell numbers were slightly lower in the RO5066852 treatment group (controls: 1.0 ± 0.4% vs. RO5066852: 0.6 ± 0.2% in IPH negative plaques), while apoptosis in IPH positive plaques was 1.2 ± 0.4%, demonstrating that chymase inhibition may, at least in part, inhibit plaque cell apoptosis induced by acute mast cell activation.

As mast cells are major inducers of vascular leakage,7 we have determined whether chymase inhibition affected acute microvascular leakage. Hereto, C57Bl6 mice were treated with RO5066852 or control diet for 2 weeks, after which we induced vascular leakage in the skin of these mice. We indeed observed that in control mice intradermal injection of 5 × 105 compound 48/80 activated MC/9 cells promoted acute vascular leakage as judged by Evans Blue spot size (37 ± 4 vs. 0 ± 0 mm2 for the PBS control; P < 0.0001). RO5066852 treatment did not affect microvascular leakage elicited by activated MC/9 injection (RO5066852: 32 ± 4 mm2, P = NS compared with control mice). Similarly, pre-incubation of MC/9 cells with RO5066852 (1 µM) also did not affect vascular leakage in these mice (data not shown).

4. Discussion

Mast cell chymase is a potent chymotrypsin-like serine protease12 involved in extracellular matrix degradation, conversion of AngI into active AngII, apoptosis, and leucocyte recruitment, which all are key processes in the development and progression of atherosclerosis. Chymase is also associated with the incidence of aortic aneurysms.25,26 This combined with the recent finding that mast cell activation exacerbates plaque progression and destabilization suggests that chymase inhibition could be beneficial for plaque stability. In this study, we aimed to determine the role of mast cell-derived chymase in atherosclerotic plaque stability in mice by oral administration of a potent and specific chymase inhibitor, RO5066852. RO5066852 was shown to specifically inhibit human chymase at low nanomolar levels. Interestingly, RO5066852 was also demonstrated to inhibit mouse chymase, while furthermore inhibiting mouse mast cell activation to a certain extent, which is in line with previous findings.27 Concordant with the progressive presence of mast cells in human and mouse plaque tissue during disease progression,46 several chymase isoforms were detectable in mouse atherosclerotic plaque material at the mRNA level, indicating that the chymase inhibitor RO5066852 could be effective in vivo as well.

The chymase inhibitor RO5066852 was administered orally to mice as diet supplement (3, 10, and 30 mg/kg/day) to determine the effective dosage in vivo. Plasma samples were taken during 24 h of food intake, resulting in peak RO5066852 levels of 1.2, 2.2, and 8.4 µM within 4–6 h which decline due to varying food intake during the day as a consequence of the circadian rhythm of mice. Average RO5066852 plasma levels over the day were 0.7, 1.2, and 4.8 µM at 3, 10, and 30 mg/kg/day and our in vitro studies demonstrated that 1 µM of RO5066852 already effectively inhibited mouse chymases. Although we were unable to measure RO5066852 concentrations in the atherosclerotic plaque and perivascular tissue, we assume perivascular tissue is heavily vascularized and perfused especially during atherosclerosis, suggesting that local inhibitor concentrations will at least be very similar to plasma levels. This indicates that tissue concentration in the adventitia and lesion is similar to free drug in plasma, implying that oral administration of 10 or 30 mg/kg/day to mice will lead to potentially effective plasma concentrations of the chymase inhibitor.

In this study, we provide three lines of evidence that mast cell chymase inhibition has a beneficial effect on atherosclerosis. First, oral RO5066852 treatment of apoE−/− mice inhibited spontaneous atherosclerotic plaque progression in the thoracic aorta, which is in line with previous data from Uehara et al.,28 demonstrating that chymase inhibition reduced lipid deposition in hamster aortas. Second, RO5066852 treatment was shown to inhibit the increased atherogenic response in the brachiocephalic arteries of apoE−/− mice that were subjected to multiple episodes of systemic mast cell activation,7 which furthermore resulted in increased plaque collagen content and reduced necrotic core size. This suggests that chymase is one of the major mast cell constituents, responsible for mast cell-induced plaque progression.7 These findings are in line with previous data published by Saarinen et al.,18 which demonstrate that chymase can activate procollagenase, resulting in collagen degradation. Interestingly, we and others have previously shown that chymase is one of the mast cell mediators involved in the induction of macrophage7 and smooth muscle cell15 apoptosis, suggesting that chymase, at least in part, indeed accounts for the necrotic core size expansion after focal mast cell activation. Our data also coincide with the findings by Guo et al.,29 who suggests that chymase inhibition by tranilast results in reduced plaque progression combined with enhanced plaque stability. However, tranilast is commonly known as a general mast cell stabilizer and anti-inflammatory compound which is not highly specific for mast cell chymases.

Third, RO5066852 treatment was seen to prevent the plaque destabilizing effect of perivascular mast cell challenge in advanced carotid artery atherosclerosis. This was particularly intriguing as mast cell numbers have been demonstrated to be predominantly high in patients with unstable advanced atherosclerotic plaques and in ruptured plaques, while also activation of mast cells was seen to increase during plaque progression.46 To elucidate the contribution of chymase released from adventitial mast cells in advanced atherosclerosis to plaque destabilization, we treated apoE−/− mice with RO5066852 during the perivascular mast cell activation strategy that we previously have pursued to study mast cell function.7 As expected, focal mast cell activation resulted in an increased incidence and size of intraplaque haemorrhages, a widely appreciated feature of plaque destabilization.30 The chymase inhibitor largely reduced the incidence and size of the mast cell-induced haemorrhage. RO5066852 was ineffective in the prevention of mast cell-induced acute vascular leakage, which is completely in line with previous data pointing to a major role for mast cell-derived histamine in this process.7 These data imply that chymase inhibitors mainly act on plaque stability and in particular intraplaque haemorrhage via pathways such as a decrease of extracellular matrix degradation. Furthermore, chymase inhibition appeared to inhibit plaque apoptosis. This is in line with our earlier findings that chymase, in concert with tryptase and histamine, is responsible for the induction of macrophage apoptosis.7 As previously reported, mast cell chymases can also convert AngI into AngII,19 which suggests that the observed adverse effects on plaque stability in mice may be partly due to inhibition of AngII production. Our data demonstrate, in particular, effects of chymase inhibition on atherosclerotic plaque stability features, such as necrotic core size, collagen content, and the incidence of intraplaque haemorrhage. Additional treatment with either an ACE-inhibitor or an AngI receptor antagonist (such as AT1 antagonists) to dissect out AngI-dependent effects of our chymase inhibitor will already affect lesion formation to such an extent,31,32 that effects of chymase inhibition on established lesions will be difficult to assess. As AngI converting activity of mouse chymases is significantly lower than that of human chymases,21 we anticipate that the profound effects of chymase inhibition seen in our study are probably only for a minor part attributable to inhibition of AngI converting chymase activity.

Chymase has previously been reported to enhance neutrophil recruitment in vitro14 and interestingly, RO5066852 treatment resulted in reduced perivascular neutrophil levels. Plaque neutrophils have recently been implicated in enhanced plaque progression,33 among others by releasing proteases such as neutrophil elastase, cathepsins, and myeloperoxidase.34,35 As we have implicitly shown, mast cell activation is accompanied by CXCR2-dependent recruitment of leucocyte subsets, and mainly neutrophils, to perivascular tissue.7 Conceivably, by reducing mast cell activation or by direct chymase-induced neutrophil recruitment,14 chymase inhibitors could target neutrophils as well, which may partly underlie their plaque stabilizing activity.

In conclusion, we are the first to demonstrate that mast cell chymase contributes significantly to the adverse effects on plaque stability observed after mast cell activation in mice. Chymase inhibition results in reduced progression and improved stability of atherosclerotic plaques and almost completely prevents mast cell activation-associated intraplaque haemorrhages, identifying chymase inhibition as a new therapeutic approach in the prevention of acute coronary syndromes.

Funding

This work was supported by grant number (916.86.046) from the Netherlands Organization for Scientific Research (I.B.) and grant number (2003T201) from the Netherlands Heart Foundation (E.A.L.B.).

Acknowledgements

The authors would like to thank J. Beauchamp and D. Schlatter for technical assistance. The authors belong to the European Vascular Genomics Network (http://www.evgn.org), a Network of Excellence supported by the European Community's Sixth Framework Program for Research Priority 1 (Life Sciences, Genomics, and Biotechnology for Health; contract LSHM-CT-2003-503254).

Conflict of interest: employment by F. Hoffmann-La Roche: P.H., S.G., H.H., and J.F.

References

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