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Effects of chymase inhibitor on angiotensin II-induced abdominal aortic aneurysm development in apolipoprotein E-deficient mice
Atherosclerosis 204 (2009) 359–364
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Effects of chymase inhibitor on angiotensin II-induced abdominal aortic aneurysm development in apolipoprotein E-deficient mice Nao Inoue a,b , Michiko Muramatsu a , Denan Jin a , Shinji Takai a,∗ , Tetsuya Hayashi c , Hiroshi Katayama b , Yasushi Kitaura c , Hiroshi Tamai b , Mizuo Miyazaki a a b c
Department of Pharmacology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki City, Osaka 569-8686, Japan Department of Pediatrics, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki City, Osaka 569-8686, Japan Third Department of Internal Medicine Osaka Medical College, 2-7 Daigaku-machi, Takatsuki City, Osaka 569-8686, Japan
a r t i c l e
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Article history: Received 27 May 2008 Received in revised form 16 September 2008 Accepted 18 September 2008 Available online 8 October 2008 Keywords: Aneurysm Apolipoprotein E Chymase Inhibitor MMP-9 Mouse
a b s t r a c t Objective: Chymase may play an important role in abdominal aortic aneurysm (AAA) development through matrix metalloproteinase (MMP)-9 activation. The purpose of this study was to determine whether chymase is involved in angiotensin (Ang) II-induced AAA development in apolipoprotein E (apoE)-deficient mice. Methods and results: In this study, Ang II (1000 ng/kg/min; vehicle group) or saline (saline group) was administered to 16-week-old, male, apoE-deficient mice for 4 weeks. To examine the effects of chymase inhibition on AAA development, oral NK3201 (30 mg/kg/day) was given for the same period as the Ang II infusion. AAAs developed at the suprarenal region of the abdominal aorta in the Ang II-treated vehicle group, but they were not observed in the saline group. On the other hand, the severity and luminal area of the AAAs in the Ang II-treated vehicle group were significantly suppressed by NK3201 treatment. MMP-9 activity was significantly lower in the Ang II-treated + NK3201-treated group than in the Ang IItreated vehicle group. Furthermore, there were significantly fewer monocyte/macrophage cells in the Ang II-treated + NK3201-treated group than in the Ang II-treated vehicle group. Conclusions: Chymase is involved in Ang II-induced AAA development in apoE-deficient mice. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Abdominal aortic aneurysm (AAA) is a vascular disorder that occurs among the elderly. Chronic inflammation is a common finding, and the pathological characteristics of AAA include medial destruction, loss of medial smooth muscle cells, and chronic inflammatory cell infiltration [1]. Although most patients with small AAAs are asymptomatic, AAAs expand at a rate of about 0.3–0.5 cm/year. If the diameter reaches 4.5 cm, the rates of rupture and mortality are greatly increased [1]. In addition, elective repair for small AAAs does not improve the mortality rate [2]. Nevertheless, other than surgical repair, no effective treatments are available to prevent AAA rupture, partially due to a lack of knowledge of the mechanisms of AAA development. Matrix metalloproteinase (MMP)-9 is the member of the MMP family that has the highest affinity for elastin as a substrate; it is thus considered to play an important role in the pathology of AAAs.
This hypothesis has been confirmed in several studies of human and animal experimental AAAs [3–5]. Chymase has been shown to activate promatrix metalloproteinase-9 (proMMP-9) to MMP-9, both in vitro and in vivo [6,7]. Chymase is a chymotrypsin-like serine protease located in the secretory granules of mast cells. Sun et al. [8] reported that mast cells play an important role in elastase-induced AAA development by releasing proinflammatory cytokines. These reports suggest that chymase might be involved in AAA development through MMP-9 activation. To confirm this hypothesis, we investigated whether the chymase inhibitor NK3201 suppresses angiotensin (Ang) II-induced AAA development in apolipoprotein E (apoE)-deficient mice. This is the first report to demonstrate that a chymase inhibitor could suppress Ang II-induced AAA development in apoE-deficient mice.
2. Materials and methods 2.1. Drug
∗ Corresponding author. Tel.: +81 72 684 7292; fax: +81 72 684 6518. E-mail address: [email protected] (S. Takai). 0021-9150/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2008.09.032
The drug 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[{3,4-dioxo-1-phenyl-7-(2-pyridyloxy)}-2-hep-
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tyl] acetamide (NK3201; Nippon Kayaku, Tokyo, Japan) was synthesized as a specific chymase inhibitor [9].
The interobserver correlation between individual measurement values obtained from two observers was significant (data not shown).
2.2. Animals 2.7. Enzyme activity Male, 14-week-old, apoE-deficient mice with a C57BL/6 background were obtained from Jackson Laboratories (Bar Harbor, ME, USA). All mice had ad libitum access to a normal diet and water. Ang II (Peptide Institute Inc., Osaka, Japan) (1000 ng/kg/min) (n = 32) or saline (n = 16, the saline group) was administered subcutaneously by Alzet osmotic minipump (model 2004; Durect, Cupertino, CA, USA) to 16-week-old mice for 4 weeks. To investigate the effect of a chymase inhibitor, either 30 mg/kg/day of NK3201 (n = 16) or vehicle (n = 16) was administered orally to the Ang II-treated group from the beginning of the Ang II infusion to the end of the study. NK3201 was mixed in feed for administration. At the end of the study, each group was randomly divided into two groups based on body weight. Half of the mice were used as histological assessments of AAA and the others were used as measurements of enzyme activities. All procedures involving animals were conducted in accordance with the guidelines of Osaka Medical College. 2.3. Determination of blood pressure Systolic blood pressure (SBP) was determined in conscious mice using a computerized tail-cuff method (BP-98A; Softron, Tokyo, Japan). SBP was recorded 1 week before and after starting the Ang II infusion. 2.4. Plasma total cholesterol level Blood was collected from each mouse. The total cholesterol concentration was measured with a clinical analyzer (H7070 Hitachi, Tokyo, Japan). 2.5. Preparation of aortic tissue The entire aorta from the ascending aorta to the ileal bifurcation was exposed. The aortic aneurysm region was removed and fixed for histological assessment with Carnoy’s fixative in 10% methanol overnight or immediately frozen at −80 ◦ C for enzymatic assessment. 2.6. Histological assessments Fixed aortic tissues were embedded in paraffin, and then cut from each block at a thickness of 3 m. To compare the extent of aortic dilatation, the luminal area at the suprarenal abdominal aortic level was the average of three different sections stained with Elastica von Gieson. To visualize monocyte/macrophage accumulation in the AAA, immunohistochemical staining was performed. In brief, sections were incubated with 3% H2 O2 in methanol to inhibit endogenous peroxidase, then incubated with proteinblocking solution (Dako, Tokyo, Japan) to block non-specific antigens. Sections were incubated with rat anti-mouse monocyte/macrophage monoclonal antibody (MOMA-2) (1:25; Serotec, Oxford, UK) overnight, and then with secondary biotinylated goat anti-rat IgG antibody (1:50; Invitrogen, Carlsbad, CA, USA). Sections were visualized using horseradish peroxidase and 3-amino-9ethylcarbozole as the substrate-chromogen (Dako). The nuclei were counterstained using hematoxylin. In each specimen, the ratio of the MOMA-2-positive area to the total area was measured using a computerized morphometry system, Fuji-BSA 2000 image analyzing system (Fuji Co., Tokyo, Japan). The observers were blinded, and the order in which the images were analyzed was also randomized.
Chymase activity was measured by incubating the tissue extracts with 5 mM Suc-Ala-Ala-Pro-Phe-4-methylcoumaryl-7amide (Peptide Institute Inc., Osaka, Japan) as the substrate [10]. One unit of chymase activity was defined as the amount of enzyme required to cleave 1 mol of 7-amino-4-methylcoumarin/min. The protein concentration was assayed using BCA Protein Assay Reagents (Pierce, Rockford, Illinois, USA), with bovine serum albumin as the standard. 2.8. Gelatin zymography MMP-2 and MMP-9 activities were assessed using gelatin zymography as described previously [10]. In brief, equal volumes of tissue extract (80 g of protein) were purified using gelatin sepharose 4B (GE Healthcare, Buckinghamshire, UK), followed by resolution under electrophoresis on 10% SDS-polyacrylamide gels containing 1 mg/mL of gelatin. Thereafter, the gels were renatured in 50 mM Tris–HCl containing 100 mM NaCl and 2.5% Triton X-100. They were then incubated with 50 mM Tris–HCl containing 10 mM CaCl2 . The gels were stained with Coomassie Brilliant Blue, and gelatinolytic activity was quantified using NIH-Image version 1.61 software. 2.9. Effect of chymase inhibitor on proMMP-9 activation in extracts of AAA To investigate whether chymase could activate proMMP-9 in AAA tissues from Ang II-treated vehicle mice, tissue extracts were incubated for 24 h at 4 ◦ C and 37 ◦ C before gelatin zymography. The effect of chymase inhibition was also examined after incubation with 1 M of NK3201 in 0.01% dimethyl sulfoxide (DMSO) for 24 h at 37 ◦ C. 2.10. Statistical analysis Data are expressed as mean ± standard error of the mean. Significant differences among the mean values of multiple groups were evaluated using 1-way analysis of variance followed by Fisher’s test. Values of p < 0.05 were considered statistically significant. 3. Results 3.1. Blood pressure One week before the Ang II infusion, at the age of 15 weeks, SBP was not significantly different among the three groups. After 1 week of the Ang II infusion, SBP was significantly higher in the Ang IItreated vehicle and Ang II-treated + NK3201-treated groups than in the saline group (saline group, 111 ± 1.6 mmHg; Ang II-treated vehicle group mmHg, 171 ± 3.7 mmHg; Ang II-treated + NK3201-treated group, 164 ± 4.2 mmHg). No differences in SBP were seen between the Ang II-treated vehicle and the Ang II-treated + NK3201-treated groups. 3.2. Total cholesterol level The plasma total cholesterol concentrations were 574 ± 15.3 mg/dL in the saline group, 619 ± 28.3 mg/dL in the Ang II-treated vehicle group, and 564 ± 20.3 mg/dL in the Ang
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Fig. 1. Typical whole aortae from the saline group, the Ang II-treated vehicle group, and the Ang II-treated + NK3201-treated group.
II-treated + NK3201-treated group; there was no significant difference among the three groups. 3.3. Analysis of AAA Gross examination showed AAA development in the region of the abdominal aorta in the Ang II-treated groups, but not in the saline group (Fig. 1). Luminal area was significantly larger in the Ang II-treated vehicle group than in the saline group (Fig. 2). In contrast, luminal area was significantly smaller in the Ang II-treated + NK3201-
Fig. 3. Immunohistochemical staining using an anti-MOMA-2 antibody (A). Saline group (left side); Ang II-treated vehicle group (center); and Ang II-treated + NK3201treated group (right side). Original magnification: 20× (upper side). Original magnification: 200× (lower side). Ratio of MOMA-2-positive areas to total vascular areas in the saline group, the Ang II-treated vehicle group, and the Ang IItreated + NK3201-treated group (B). Values represent mean ± SEM (n = 8). **P < 0.01 vs. the saline group. †† P < 0.01 vs. the Ang II-treated vehicle group.
treated group than in the Ang II-treated vehicle group (Fig. 2). No significant differences existed between the saline and Ang IItreated + NK3201-treated groups (Fig. 2). 3.4. Immunohistochemistry A representative picture of the immunohistochemical results is shown in Fig. 4. Many MOMA-2-positive cells were present in the adventitia in the Ang II-treated vehicle group, but not in the saline group. There were significantly fewer monocyte/macrophage cells in the Ang II-treated + NK3201-treated group than in the Ang IItreated vehicle group (Fig. 3). 3.5. Chymase activity Chymase activities were 0.15 ± 0.02 mU/mg protein in the saline group, 023 ± 0.02 mU/mg protein in the Ang II-treated vehicle group, and 0.21 ± 0.02 mU/mg protein in the Ang IItreated + NK3201-treated group. Chymase activity significantly increased in the Ang II-treated vehicle group compared to the saline group, but no significant differences were observed between the chymase inhibitor-treated group and the saline group or the Ang II-treated vehicle group. 3.6. Gelatin zymography
Fig. 2. Typical aorta slices stained with Elastica van Gieson stain. Aortae from the saline group, the Ang II-treated vehicle group, and the Ang II-treated + NK3201treated group (A). Original magnification: 20×. Luminal areas in the saline group, the Ang II-treated vehicle group, and the Ang II-treated + NK3201-treated group (B). Values represent mean ± SEM (n = 8). **P < 0.01 vs. the saline group. † P < 0.05 vs. the Ang II-treated vehicle group.
ProMMP-9 levels in the Ang II-treated vehicle group were about 2.8-fold higher than in the saline group, and 1.5-fold higher than in the Ang II-treated + NK3201-treated group (Fig. 4). MMP-9 activity was also significantly higher in the Ang II-treated vehicle group
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Fig. 4. Typical gelatin zymograms for the saline group, the Ang II-treated vehicle group, and the Ang II-treated + NK3201-treated group (A). ProMMP-9, MMP-9, proMMP-2, and MMP-2 levels in the Ang II-treated vehicle group (hatched bar) and the Ang II-treated + NK3201-treated group (closed bar), with each level in the saline group (open bar) represented as 100% (B). Values represent mean ± SEM (n = 8). *P < 0.05 and **P < 0.01 vs. the saline group. † P < 0.05 and †† P < 0.01 vs. the Ang IItreated vehicle group.
than in the saline and Ang II-treated + NK3201-treated groups. Unlike proMMP-9, levels of MMP-9 activity were almost the same in the Ang II-treated + NK3201-treated group as in the saline group (Fig. 4). ProMMP-2 and MMP-2 activity were significantly higher in the Ang II-treated vehicle group than in the saline group, whereas their levels were almost the same in the Ang II-treated vehicle group and in the Ang II-treated + NK3201-treated group (Fig. 4). 3.7. Effect of chymase inhibitor on proMMP-9 activation in extracts of AAA Zymography showed that the MMP-9 band in extracts was more dense after a 24 h incubation at 37 ◦ C than at 4 ◦ C (Fig. 5). In contrast, a proMMP-9 band appeared after incubation for 24 h at 4 ◦ C, but almost completely disappeared after incubation at 37 ◦ C (Fig. 5). In the presence of a chymase inhibitor at 37 ◦ C, the density of both the MMP-9 and proMMP-9 bands was the same as that after the incubation at 4 ◦ C (Fig. 5). 4. Discussion This is the first study to demonstrate that the chymase inhibitor NK3201 suppresses Ang II-induced AAA development in apoEdeficient mice. Loss of elastic fibers in the media is thought to be an initiating event in AAA development, and it may be caused
Fig. 5. Typical zymography photographs of a sample from Ang II-treated vehicle group incubated for 24 h at 4 ◦ C and samples incubated with solvent (0.01% DMSO) or a chymase inhibitor (Chy-I) for 24 h at 37 ◦ C (A). Ratios (%) of proMMP-9 and MMP-9 in samples incubated with solvent and a chymase inhibitor (Chy-I) for 24 h at 37 ◦ C compared to the samples incubated for 24 h at 4 ◦ C (B). Values represent mean ± SEM (n = 8). **P < 0.01 vs. incubation at 4 ◦ C. †† P < 0.01 vs. incubation with solvent at 37 ◦ C.
by imbalances in elastic fiber synthesis and destruction. MMP9 is thought to play a critical role in AAA development due to elastinolytic actions on elements such as elastin and type IV collagen in the vascular wall [11]. Pyo et al. [4] and Longo et al. [5] reported suppression of experimental AAA in MMP-9-deficient mice. In addition, doxycycline, a broad-spectrum MMP inhibitor, attenuated Ang II-induced AAA formation in mice [12]. MMP-9 is secreted as zymogen and activated by MMP-3 and serine proteases including chymase [6,7,13,14]. Recently, we demonstrated that chymase cleaved the Phe91 -Glu92 bond of proMMP-9 in extract from human AAA [15]. By contrast, MMP-3 and serine proteases other than chymase cleave proMMP-9 at the same site, the bond of Lys87 -Phe88 [6]. In the present study, proMMP-9 activity in the Ang II-treated + NK3201-treated group was not decreased to the same level as in the saline group. The present study demonstrated that AAA in the Ang II-treated vehicle group was associated with MMP9 activity. In contrast, AAA was suppressed in association with decreased MMP-9 activity in the Ang II-treated + NK3201-treated group. With respect to proMMP-2 and MMP-2, it has been reported that MMP-2 also plays an important role in AAA development [7], however, proMMP-2 and MMP-2 activities were not attenuated by NK3201 in the present study. These results suggest that the effect of NK3201 on Ang II-induced AAA development was not through MMP-2 inhibition but through MMP-9 inhibition.
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The mast cell is involved in atherosclerosis and AAA development in humans and experimental models [8,16]. NK3201 is a specific chymase inhibitor that does not inhibit other serine proteases, such as tryptase, thrombin, elastase, plasmin or plasminogen activator. The IC50 values of human, dog, rat and hamster chymases are 2.5, 1.2, 19, and 28 nM, respectively [17]. In dogs, the concentration of NK3201 in plasma, heart and aorta are about 470, 195 and 78 nM, respectively, 8 h after oral administration of 1 mg/kg of NK3201 [17]. No marked toxicity was observed following administration of 1000 mg/kg in a previous experiment using rats [17], and no major side effects were thought to have occurred in the present study. In the present study, no significant differences were observed between the chymase inhibitor group, which was given 30 mg/kg/day of NK3201, and the Ang II-treated vehicle group. However, unlike the Ang II-treated vehicle group, the chymase inhibitor-treated group did not have significant increases in chymase activity compared to the saline group. We recently reported, in a mouse model of ulcerative colitis, that intraperitoneal injection of 10 mg/kg/day of NK3201 inhibited chymase activity and strongly inhibited MMP-9 activity at the site of colitis [10]. In the present experiment, although no significant inhibition was observed for the vehicle group, a significant reduction in MMP-9 activity was observed in the chymase inhibitor group. Therefore, we considered that the dose of NK3201 administered was sufficient to suppress AAA development. Daugherty et al. [18] reported that Ang II administration to hyperlipidemic, apoE-deficient mice induced AAA development at the level of the suprarenal artery. The mechanisms underlying Ang II-induced AAA in apoE-deficient mice have not been completely elucidated, but Ang II appears to affect the inflammatory response, inducing monocyte chemoattractant protein-1 expression in monocytes, activation of macrophages, enhancement of oxidative stress, increased uptake of oxidized low density lipoprotein by macrophages, and increased MMP-9 expression [19,20]. MMP-9 is known to enhance inflammatory responses by degrading basement membranes, which is necessary for macrophages to traverse the extracellular matrix and enter inflammatory sites [21]. Macrophage infiltration was reported to be decreased in MMP-9-knockout mice [22]. In addition, elastin- and lamininderived fragments generated by MMP-9 have been reported to affect inflammation to promote leukocyte chemotaxis and increase proMMP expression [23]. In this way, MMP-9 is involved in AAA development not only by directly degrading elastic fibers, but also by indirectly enhancing inflammatory responses. In the present study, MMP-9 activity in AAA tissues from Ang II-induced vehicle mice was significantly higher after incubation for 24 h-incubation at 37 ◦ C than at 4 ◦ C; this suggests that the extract included proMMP-9-activating enzymes. However, the incubated samples to which NK3201 was applied had significantly attenuated the MMP-9 activity. We thus confirmed the importance of chymasedependent MMP-9 activation in AAA tissues in vitro. Furthermore, in the presence of NK3201 at 37 ◦ C, the density of both the MMP-9 and proMMP-9 bands was the same as that after the incubation at 4 ◦ C. This finding suggests that chymase inhibition might attenuate the accumulation of proMMP-9 by inhibiting the conversion of proMMP-9 to MMP-9. NK3201 might stop this vicious cycle initially induced by Ang II by inhibiting MMP-9 activation. We also hypothesized that NK3201 inhibited AAA development by suppressing monocyte/macrophage accumulation. In the present study, Ang II infusion induced macrophage accumulation and AAA development, as reported previously [18]. Chymase functions as a potent chemoattractant and that it could induce chemotactic migration of monocytes [24]. Our study showing that monocyte/macrophage infiltration was decreased by inhibition of chymase was consistent with these reports. In the present study,
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not only MMP-9 level but also proMMP-9 level was decreased by treatment with NK3201. The suppression of monocyte/macrophage accumulation by NK3201 might be involved in the decrease of proMMP-9 level. In summary, the results of the present study demonstrate that the chymase inhibitor NK3201 suppresses Ang II-induced AAA development in apoE-deficient mice. NK3201 might offer a new strategy for preventing human AAA. Acknowledgment This study was partly supported by grants from the Science Research Promotion Fund (No.9 in 2007) and High-Tech Research Center of the Promotion and Mutual Aid Corporation for Private School of Japan. References [1] Thompson RW. Basic science of abdominal aortic aneurysms: emerging therapeutic strategies for an unresolved clinical problem. Curr Opin Cardiol 1996;11:504–18. [2] Ballotta E, Toniato A. Small abdominal aortic aneurysms. N Engl J Med 2002;347:1112–5. [3] Thompson RW, Holmes DR, Mertens RA, Liao S, Botney MD, Mecham RP, et al. Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms. An elastolytic metalloproteinase expressed by aneurysminfiltrating macrophages. J Clin Invest 1995;96:318–26. [4] Pyo R, Lee JK, Shipley JM, Curci JA, Mao D, Ziporin SJ, et al. Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. J Clin Invest 2000;105: 1641–9. [5] Longo GM, Xiong W, Greiner TC, Zhao Y, Fiotti N, Baxter BT. Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms. J Clin Invest 2002;110:625–32. [6] Fang KC, Raymond WW, Blount JL, Caughey GH. Dog mast cell alpha-chymase activates progelatinase B by cleaving the Phe88 -Gln89 and Phe91 -Glu92 bonds of the catalytic domain. J Biol Chem 1997;272:25628–35. [7] Tchougounova E, Lundequist A, Fajardo I, Winberg JO, Abrink M, Pejler G. A key role for mast cell chymase in the activation of pro-matrix metalloprotease-9 and pro-matrix metalloprotease-2. J Biol Chem 2005;280:9291–6. [8] Sun J, Sukhova GK, Yang M, Wolters PJ, MacFarlane LA, Libby P, et al. Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice. J Clin Invest 2007;117:3359–68. [9] Takai S, Jin D, Nishimoto M, Yuda A, Sakaguchi M, Kamoshita K, et al. Oral administration of a specific chymase inhibitor, NK3201, inhibits vascular proliferation in grafted vein. Life Sci 2001;69:1725–32. [10] Ishida K, Takai S, Murano M, Nishikawa T, Inoue T, Murano N, et al. Role of chymase-dependent matrix metalloproteinase-9 activation in mice with dextran sodium sulfate-induced colitis. J Pharmacol Exp Ther 2008;324:422–6. [11] Murphy G, Cockett MI, Ward RV, Docherty AJ. Matrix metalloproteinase degradation of elastin, type IV collagen and proteoglycan. A quantitative comparison of the activities of 95 kDa and 72 kDa gelatinases, stromelysins-1 and -2 and punctuated metalloproteinase (PUMP). Biochem J 1991;277:277–9. [12] Manning MW, Cassis LA, Daugherty A. Differential effects of doxycycline, a broad-spectrum matrix metalloproteinase inhibitor, on Ang II-induced atherosclerosis and abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol 2003;23:483–8. [13] Lijnen HR. Plasmin and matrix metalloproteinases in vascular remodelling. Thromb Haemost 2001;8:324–33. [14] Carmeliet P, Moons L, Lijnen R, Baes M, Lemaître V, Tipping P, et al. Urokinasegenerated plasmin activates matrix metalloproteinases during aneurysm formation. Nat Genet 1997;17:439–44. [15] Furubayashi K, Takai S, Jin D, Miyazaki M, Katsumata T, Inagaki S, et al. Chymase activates promatrix metalloproteinase-9 in human abdominal aortic aneurysm. Clin Chim Acta 2008;388:214–6. [16] Sun J, Sukhova GK, Wolters PJ, Yang M, Kitamoto S, Libby P, et al. Mast cells promote atherosclerosis by releasing proinflammatory cytokines. Nat Med 2007;13:719–24. [17] Takai S, Miyazaki M. Application of a chymase inhibitor, NK3201, for prevention of vascular proliferation. Cardiovasc Drug Rev 2003;21:185–98. [18] Daugherty A, Manning MW, Cassis LA. Ang II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J Clin Invest 2000;105:1605–12. ˜ [19] Hernández-Presa M, Bustos C, Ortego M, Tunon J, Renedo G, Ruiz-Ortega M, et al. Ang-converting enzyme inhibition prevents arterial nuclear factor- B activation, monocyte chemoattractant protein-1 expression, and macrophage infiltration in a rabbit model of early accelerated atherosclerosis. Circulation 1997;95:1532–41.
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[20] Yanagitani Y, Rakugi H, Okamura A, Moriguchi K, Takiuchi S, Ohishi M, et al. Ang II type 1 receptor-mediated peroxide production in human macrophages. Hypertension 1999;33:335–9. [21] Watanabe H, Nakanishi I, Yamashita K, Hayakawa T, Okada Y. Matrix metalloproteinase-9 (92 kDa gelatinase/type IV collagenase) from U937 monoblastoid cells: correlation with cellular invasion. J Cell Sci 1993;104:991–9. [22] Luttun A, Lutgens E, Manderveld A, Maris K, Collen D, Carmeliet P, et al. Loss of matrix metalloproteinase-9 or matrix metalloproteinase-12 protects
apolipoprotein E-deficient mice against atherosclerotic media destruction but differentially affects plaque growth. Circulation 2004;109:1408–14. [23] Corcoran ML, Kibbey MC, Kleinman HK, Wahl LM. Laminin SIKVAV peptide induction of monocyte/macrophage prostaglandin E2 and matrix metalloproteinases. J Biol Chem 1995;270:10365–8. [24] Tani K, Ogushi F, Kido H, Kawano T, Kunori Y, Kamimura T, et al. Chymase is a potent chemoattractant for human monocytes and neutrophils. J Leukoc Biol 2000;67:585–9.
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Effects of chymase inhibitor on angiotensin II-induced abdominal aortic aneurysm development in apolipoprotein E-deficient mice Nao Inoue a,b , Michiko Muramatsu a , Denan Jin a , Shinji Takai a,∗ , Tetsuya Hayashi c , Hiroshi Katayama b , Yasushi Kitaura c , Hiroshi Tamai b , Mizuo Miyazaki a a b c
Department of Pharmacology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki City, Osaka 569-8686, Japan Department of Pediatrics, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki City, Osaka 569-8686, Japan Third Department of Internal Medicine Osaka Medical College, 2-7 Daigaku-machi, Takatsuki City, Osaka 569-8686, Japan
a r t i c l e
i n f o
Article history: Received 27 May 2008 Received in revised form 16 September 2008 Accepted 18 September 2008 Available online 8 October 2008 Keywords: Aneurysm Apolipoprotein E Chymase Inhibitor MMP-9 Mouse
a b s t r a c t Objective: Chymase may play an important role in abdominal aortic aneurysm (AAA) development through matrix metalloproteinase (MMP)-9 activation. The purpose of this study was to determine whether chymase is involved in angiotensin (Ang) II-induced AAA development in apolipoprotein E (apoE)-deficient mice. Methods and results: In this study, Ang II (1000 ng/kg/min; vehicle group) or saline (saline group) was administered to 16-week-old, male, apoE-deficient mice for 4 weeks. To examine the effects of chymase inhibition on AAA development, oral NK3201 (30 mg/kg/day) was given for the same period as the Ang II infusion. AAAs developed at the suprarenal region of the abdominal aorta in the Ang II-treated vehicle group, but they were not observed in the saline group. On the other hand, the severity and luminal area of the AAAs in the Ang II-treated vehicle group were significantly suppressed by NK3201 treatment. MMP-9 activity was significantly lower in the Ang II-treated + NK3201-treated group than in the Ang IItreated vehicle group. Furthermore, there were significantly fewer monocyte/macrophage cells in the Ang II-treated + NK3201-treated group than in the Ang II-treated vehicle group. Conclusions: Chymase is involved in Ang II-induced AAA development in apoE-deficient mice. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Abdominal aortic aneurysm (AAA) is a vascular disorder that occurs among the elderly. Chronic inflammation is a common finding, and the pathological characteristics of AAA include medial destruction, loss of medial smooth muscle cells, and chronic inflammatory cell infiltration [1]. Although most patients with small AAAs are asymptomatic, AAAs expand at a rate of about 0.3–0.5 cm/year. If the diameter reaches 4.5 cm, the rates of rupture and mortality are greatly increased [1]. In addition, elective repair for small AAAs does not improve the mortality rate [2]. Nevertheless, other than surgical repair, no effective treatments are available to prevent AAA rupture, partially due to a lack of knowledge of the mechanisms of AAA development. Matrix metalloproteinase (MMP)-9 is the member of the MMP family that has the highest affinity for elastin as a substrate; it is thus considered to play an important role in the pathology of AAAs.
This hypothesis has been confirmed in several studies of human and animal experimental AAAs [3–5]. Chymase has been shown to activate promatrix metalloproteinase-9 (proMMP-9) to MMP-9, both in vitro and in vivo [6,7]. Chymase is a chymotrypsin-like serine protease located in the secretory granules of mast cells. Sun et al. [8] reported that mast cells play an important role in elastase-induced AAA development by releasing proinflammatory cytokines. These reports suggest that chymase might be involved in AAA development through MMP-9 activation. To confirm this hypothesis, we investigated whether the chymase inhibitor NK3201 suppresses angiotensin (Ang) II-induced AAA development in apolipoprotein E (apoE)-deficient mice. This is the first report to demonstrate that a chymase inhibitor could suppress Ang II-induced AAA development in apoE-deficient mice.
2. Materials and methods 2.1. Drug
∗ Corresponding author. Tel.: +81 72 684 7292; fax: +81 72 684 6518. E-mail address: [email protected] (S. Takai). 0021-9150/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2008.09.032
The drug 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[{3,4-dioxo-1-phenyl-7-(2-pyridyloxy)}-2-hep-
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tyl] acetamide (NK3201; Nippon Kayaku, Tokyo, Japan) was synthesized as a specific chymase inhibitor [9].
The interobserver correlation between individual measurement values obtained from two observers was significant (data not shown).
2.2. Animals 2.7. Enzyme activity Male, 14-week-old, apoE-deficient mice with a C57BL/6 background were obtained from Jackson Laboratories (Bar Harbor, ME, USA). All mice had ad libitum access to a normal diet and water. Ang II (Peptide Institute Inc., Osaka, Japan) (1000 ng/kg/min) (n = 32) or saline (n = 16, the saline group) was administered subcutaneously by Alzet osmotic minipump (model 2004; Durect, Cupertino, CA, USA) to 16-week-old mice for 4 weeks. To investigate the effect of a chymase inhibitor, either 30 mg/kg/day of NK3201 (n = 16) or vehicle (n = 16) was administered orally to the Ang II-treated group from the beginning of the Ang II infusion to the end of the study. NK3201 was mixed in feed for administration. At the end of the study, each group was randomly divided into two groups based on body weight. Half of the mice were used as histological assessments of AAA and the others were used as measurements of enzyme activities. All procedures involving animals were conducted in accordance with the guidelines of Osaka Medical College. 2.3. Determination of blood pressure Systolic blood pressure (SBP) was determined in conscious mice using a computerized tail-cuff method (BP-98A; Softron, Tokyo, Japan). SBP was recorded 1 week before and after starting the Ang II infusion. 2.4. Plasma total cholesterol level Blood was collected from each mouse. The total cholesterol concentration was measured with a clinical analyzer (H7070 Hitachi, Tokyo, Japan). 2.5. Preparation of aortic tissue The entire aorta from the ascending aorta to the ileal bifurcation was exposed. The aortic aneurysm region was removed and fixed for histological assessment with Carnoy’s fixative in 10% methanol overnight or immediately frozen at −80 ◦ C for enzymatic assessment. 2.6. Histological assessments Fixed aortic tissues were embedded in paraffin, and then cut from each block at a thickness of 3 m. To compare the extent of aortic dilatation, the luminal area at the suprarenal abdominal aortic level was the average of three different sections stained with Elastica von Gieson. To visualize monocyte/macrophage accumulation in the AAA, immunohistochemical staining was performed. In brief, sections were incubated with 3% H2 O2 in methanol to inhibit endogenous peroxidase, then incubated with proteinblocking solution (Dako, Tokyo, Japan) to block non-specific antigens. Sections were incubated with rat anti-mouse monocyte/macrophage monoclonal antibody (MOMA-2) (1:25; Serotec, Oxford, UK) overnight, and then with secondary biotinylated goat anti-rat IgG antibody (1:50; Invitrogen, Carlsbad, CA, USA). Sections were visualized using horseradish peroxidase and 3-amino-9ethylcarbozole as the substrate-chromogen (Dako). The nuclei were counterstained using hematoxylin. In each specimen, the ratio of the MOMA-2-positive area to the total area was measured using a computerized morphometry system, Fuji-BSA 2000 image analyzing system (Fuji Co., Tokyo, Japan). The observers were blinded, and the order in which the images were analyzed was also randomized.
Chymase activity was measured by incubating the tissue extracts with 5 mM Suc-Ala-Ala-Pro-Phe-4-methylcoumaryl-7amide (Peptide Institute Inc., Osaka, Japan) as the substrate [10]. One unit of chymase activity was defined as the amount of enzyme required to cleave 1 mol of 7-amino-4-methylcoumarin/min. The protein concentration was assayed using BCA Protein Assay Reagents (Pierce, Rockford, Illinois, USA), with bovine serum albumin as the standard. 2.8. Gelatin zymography MMP-2 and MMP-9 activities were assessed using gelatin zymography as described previously [10]. In brief, equal volumes of tissue extract (80 g of protein) were purified using gelatin sepharose 4B (GE Healthcare, Buckinghamshire, UK), followed by resolution under electrophoresis on 10% SDS-polyacrylamide gels containing 1 mg/mL of gelatin. Thereafter, the gels were renatured in 50 mM Tris–HCl containing 100 mM NaCl and 2.5% Triton X-100. They were then incubated with 50 mM Tris–HCl containing 10 mM CaCl2 . The gels were stained with Coomassie Brilliant Blue, and gelatinolytic activity was quantified using NIH-Image version 1.61 software. 2.9. Effect of chymase inhibitor on proMMP-9 activation in extracts of AAA To investigate whether chymase could activate proMMP-9 in AAA tissues from Ang II-treated vehicle mice, tissue extracts were incubated for 24 h at 4 ◦ C and 37 ◦ C before gelatin zymography. The effect of chymase inhibition was also examined after incubation with 1 M of NK3201 in 0.01% dimethyl sulfoxide (DMSO) for 24 h at 37 ◦ C. 2.10. Statistical analysis Data are expressed as mean ± standard error of the mean. Significant differences among the mean values of multiple groups were evaluated using 1-way analysis of variance followed by Fisher’s test. Values of p < 0.05 were considered statistically significant. 3. Results 3.1. Blood pressure One week before the Ang II infusion, at the age of 15 weeks, SBP was not significantly different among the three groups. After 1 week of the Ang II infusion, SBP was significantly higher in the Ang IItreated vehicle and Ang II-treated + NK3201-treated groups than in the saline group (saline group, 111 ± 1.6 mmHg; Ang II-treated vehicle group mmHg, 171 ± 3.7 mmHg; Ang II-treated + NK3201-treated group, 164 ± 4.2 mmHg). No differences in SBP were seen between the Ang II-treated vehicle and the Ang II-treated + NK3201-treated groups. 3.2. Total cholesterol level The plasma total cholesterol concentrations were 574 ± 15.3 mg/dL in the saline group, 619 ± 28.3 mg/dL in the Ang II-treated vehicle group, and 564 ± 20.3 mg/dL in the Ang
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Fig. 1. Typical whole aortae from the saline group, the Ang II-treated vehicle group, and the Ang II-treated + NK3201-treated group.
II-treated + NK3201-treated group; there was no significant difference among the three groups. 3.3. Analysis of AAA Gross examination showed AAA development in the region of the abdominal aorta in the Ang II-treated groups, but not in the saline group (Fig. 1). Luminal area was significantly larger in the Ang II-treated vehicle group than in the saline group (Fig. 2). In contrast, luminal area was significantly smaller in the Ang II-treated + NK3201-
Fig. 3. Immunohistochemical staining using an anti-MOMA-2 antibody (A). Saline group (left side); Ang II-treated vehicle group (center); and Ang II-treated + NK3201treated group (right side). Original magnification: 20× (upper side). Original magnification: 200× (lower side). Ratio of MOMA-2-positive areas to total vascular areas in the saline group, the Ang II-treated vehicle group, and the Ang IItreated + NK3201-treated group (B). Values represent mean ± SEM (n = 8). **P < 0.01 vs. the saline group. †† P < 0.01 vs. the Ang II-treated vehicle group.
treated group than in the Ang II-treated vehicle group (Fig. 2). No significant differences existed between the saline and Ang IItreated + NK3201-treated groups (Fig. 2). 3.4. Immunohistochemistry A representative picture of the immunohistochemical results is shown in Fig. 4. Many MOMA-2-positive cells were present in the adventitia in the Ang II-treated vehicle group, but not in the saline group. There were significantly fewer monocyte/macrophage cells in the Ang II-treated + NK3201-treated group than in the Ang IItreated vehicle group (Fig. 3). 3.5. Chymase activity Chymase activities were 0.15 ± 0.02 mU/mg protein in the saline group, 023 ± 0.02 mU/mg protein in the Ang II-treated vehicle group, and 0.21 ± 0.02 mU/mg protein in the Ang IItreated + NK3201-treated group. Chymase activity significantly increased in the Ang II-treated vehicle group compared to the saline group, but no significant differences were observed between the chymase inhibitor-treated group and the saline group or the Ang II-treated vehicle group. 3.6. Gelatin zymography
Fig. 2. Typical aorta slices stained with Elastica van Gieson stain. Aortae from the saline group, the Ang II-treated vehicle group, and the Ang II-treated + NK3201treated group (A). Original magnification: 20×. Luminal areas in the saline group, the Ang II-treated vehicle group, and the Ang II-treated + NK3201-treated group (B). Values represent mean ± SEM (n = 8). **P < 0.01 vs. the saline group. † P < 0.05 vs. the Ang II-treated vehicle group.
ProMMP-9 levels in the Ang II-treated vehicle group were about 2.8-fold higher than in the saline group, and 1.5-fold higher than in the Ang II-treated + NK3201-treated group (Fig. 4). MMP-9 activity was also significantly higher in the Ang II-treated vehicle group
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Fig. 4. Typical gelatin zymograms for the saline group, the Ang II-treated vehicle group, and the Ang II-treated + NK3201-treated group (A). ProMMP-9, MMP-9, proMMP-2, and MMP-2 levels in the Ang II-treated vehicle group (hatched bar) and the Ang II-treated + NK3201-treated group (closed bar), with each level in the saline group (open bar) represented as 100% (B). Values represent mean ± SEM (n = 8). *P < 0.05 and **P < 0.01 vs. the saline group. † P < 0.05 and †† P < 0.01 vs. the Ang IItreated vehicle group.
than in the saline and Ang II-treated + NK3201-treated groups. Unlike proMMP-9, levels of MMP-9 activity were almost the same in the Ang II-treated + NK3201-treated group as in the saline group (Fig. 4). ProMMP-2 and MMP-2 activity were significantly higher in the Ang II-treated vehicle group than in the saline group, whereas their levels were almost the same in the Ang II-treated vehicle group and in the Ang II-treated + NK3201-treated group (Fig. 4). 3.7. Effect of chymase inhibitor on proMMP-9 activation in extracts of AAA Zymography showed that the MMP-9 band in extracts was more dense after a 24 h incubation at 37 ◦ C than at 4 ◦ C (Fig. 5). In contrast, a proMMP-9 band appeared after incubation for 24 h at 4 ◦ C, but almost completely disappeared after incubation at 37 ◦ C (Fig. 5). In the presence of a chymase inhibitor at 37 ◦ C, the density of both the MMP-9 and proMMP-9 bands was the same as that after the incubation at 4 ◦ C (Fig. 5). 4. Discussion This is the first study to demonstrate that the chymase inhibitor NK3201 suppresses Ang II-induced AAA development in apoEdeficient mice. Loss of elastic fibers in the media is thought to be an initiating event in AAA development, and it may be caused
Fig. 5. Typical zymography photographs of a sample from Ang II-treated vehicle group incubated for 24 h at 4 ◦ C and samples incubated with solvent (0.01% DMSO) or a chymase inhibitor (Chy-I) for 24 h at 37 ◦ C (A). Ratios (%) of proMMP-9 and MMP-9 in samples incubated with solvent and a chymase inhibitor (Chy-I) for 24 h at 37 ◦ C compared to the samples incubated for 24 h at 4 ◦ C (B). Values represent mean ± SEM (n = 8). **P < 0.01 vs. incubation at 4 ◦ C. †† P < 0.01 vs. incubation with solvent at 37 ◦ C.
by imbalances in elastic fiber synthesis and destruction. MMP9 is thought to play a critical role in AAA development due to elastinolytic actions on elements such as elastin and type IV collagen in the vascular wall [11]. Pyo et al. [4] and Longo et al. [5] reported suppression of experimental AAA in MMP-9-deficient mice. In addition, doxycycline, a broad-spectrum MMP inhibitor, attenuated Ang II-induced AAA formation in mice [12]. MMP-9 is secreted as zymogen and activated by MMP-3 and serine proteases including chymase [6,7,13,14]. Recently, we demonstrated that chymase cleaved the Phe91 -Glu92 bond of proMMP-9 in extract from human AAA [15]. By contrast, MMP-3 and serine proteases other than chymase cleave proMMP-9 at the same site, the bond of Lys87 -Phe88 [6]. In the present study, proMMP-9 activity in the Ang II-treated + NK3201-treated group was not decreased to the same level as in the saline group. The present study demonstrated that AAA in the Ang II-treated vehicle group was associated with MMP9 activity. In contrast, AAA was suppressed in association with decreased MMP-9 activity in the Ang II-treated + NK3201-treated group. With respect to proMMP-2 and MMP-2, it has been reported that MMP-2 also plays an important role in AAA development [7], however, proMMP-2 and MMP-2 activities were not attenuated by NK3201 in the present study. These results suggest that the effect of NK3201 on Ang II-induced AAA development was not through MMP-2 inhibition but through MMP-9 inhibition.
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The mast cell is involved in atherosclerosis and AAA development in humans and experimental models [8,16]. NK3201 is a specific chymase inhibitor that does not inhibit other serine proteases, such as tryptase, thrombin, elastase, plasmin or plasminogen activator. The IC50 values of human, dog, rat and hamster chymases are 2.5, 1.2, 19, and 28 nM, respectively [17]. In dogs, the concentration of NK3201 in plasma, heart and aorta are about 470, 195 and 78 nM, respectively, 8 h after oral administration of 1 mg/kg of NK3201 [17]. No marked toxicity was observed following administration of 1000 mg/kg in a previous experiment using rats [17], and no major side effects were thought to have occurred in the present study. In the present study, no significant differences were observed between the chymase inhibitor group, which was given 30 mg/kg/day of NK3201, and the Ang II-treated vehicle group. However, unlike the Ang II-treated vehicle group, the chymase inhibitor-treated group did not have significant increases in chymase activity compared to the saline group. We recently reported, in a mouse model of ulcerative colitis, that intraperitoneal injection of 10 mg/kg/day of NK3201 inhibited chymase activity and strongly inhibited MMP-9 activity at the site of colitis [10]. In the present experiment, although no significant inhibition was observed for the vehicle group, a significant reduction in MMP-9 activity was observed in the chymase inhibitor group. Therefore, we considered that the dose of NK3201 administered was sufficient to suppress AAA development. Daugherty et al. [18] reported that Ang II administration to hyperlipidemic, apoE-deficient mice induced AAA development at the level of the suprarenal artery. The mechanisms underlying Ang II-induced AAA in apoE-deficient mice have not been completely elucidated, but Ang II appears to affect the inflammatory response, inducing monocyte chemoattractant protein-1 expression in monocytes, activation of macrophages, enhancement of oxidative stress, increased uptake of oxidized low density lipoprotein by macrophages, and increased MMP-9 expression [19,20]. MMP-9 is known to enhance inflammatory responses by degrading basement membranes, which is necessary for macrophages to traverse the extracellular matrix and enter inflammatory sites [21]. Macrophage infiltration was reported to be decreased in MMP-9-knockout mice [22]. In addition, elastin- and lamininderived fragments generated by MMP-9 have been reported to affect inflammation to promote leukocyte chemotaxis and increase proMMP expression [23]. In this way, MMP-9 is involved in AAA development not only by directly degrading elastic fibers, but also by indirectly enhancing inflammatory responses. In the present study, MMP-9 activity in AAA tissues from Ang II-induced vehicle mice was significantly higher after incubation for 24 h-incubation at 37 ◦ C than at 4 ◦ C; this suggests that the extract included proMMP-9-activating enzymes. However, the incubated samples to which NK3201 was applied had significantly attenuated the MMP-9 activity. We thus confirmed the importance of chymasedependent MMP-9 activation in AAA tissues in vitro. Furthermore, in the presence of NK3201 at 37 ◦ C, the density of both the MMP-9 and proMMP-9 bands was the same as that after the incubation at 4 ◦ C. This finding suggests that chymase inhibition might attenuate the accumulation of proMMP-9 by inhibiting the conversion of proMMP-9 to MMP-9. NK3201 might stop this vicious cycle initially induced by Ang II by inhibiting MMP-9 activation. We also hypothesized that NK3201 inhibited AAA development by suppressing monocyte/macrophage accumulation. In the present study, Ang II infusion induced macrophage accumulation and AAA development, as reported previously [18]. Chymase functions as a potent chemoattractant and that it could induce chemotactic migration of monocytes [24]. Our study showing that monocyte/macrophage infiltration was decreased by inhibition of chymase was consistent with these reports. In the present study,
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not only MMP-9 level but also proMMP-9 level was decreased by treatment with NK3201. The suppression of monocyte/macrophage accumulation by NK3201 might be involved in the decrease of proMMP-9 level. In summary, the results of the present study demonstrate that the chymase inhibitor NK3201 suppresses Ang II-induced AAA development in apoE-deficient mice. NK3201 might offer a new strategy for preventing human AAA. Acknowledgment This study was partly supported by grants from the Science Research Promotion Fund (No.9 in 2007) and High-Tech Research Center of the Promotion and Mutual Aid Corporation for Private School of Japan. References [1] Thompson RW. Basic science of abdominal aortic aneurysms: emerging therapeutic strategies for an unresolved clinical problem. Curr Opin Cardiol 1996;11:504–18. [2] Ballotta E, Toniato A. Small abdominal aortic aneurysms. N Engl J Med 2002;347:1112–5. [3] Thompson RW, Holmes DR, Mertens RA, Liao S, Botney MD, Mecham RP, et al. Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms. An elastolytic metalloproteinase expressed by aneurysminfiltrating macrophages. J Clin Invest 1995;96:318–26. [4] Pyo R, Lee JK, Shipley JM, Curci JA, Mao D, Ziporin SJ, et al. Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. J Clin Invest 2000;105: 1641–9. [5] Longo GM, Xiong W, Greiner TC, Zhao Y, Fiotti N, Baxter BT. Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms. J Clin Invest 2002;110:625–32. [6] Fang KC, Raymond WW, Blount JL, Caughey GH. Dog mast cell alpha-chymase activates progelatinase B by cleaving the Phe88 -Gln89 and Phe91 -Glu92 bonds of the catalytic domain. J Biol Chem 1997;272:25628–35. [7] Tchougounova E, Lundequist A, Fajardo I, Winberg JO, Abrink M, Pejler G. A key role for mast cell chymase in the activation of pro-matrix metalloprotease-9 and pro-matrix metalloprotease-2. J Biol Chem 2005;280:9291–6. [8] Sun J, Sukhova GK, Yang M, Wolters PJ, MacFarlane LA, Libby P, et al. Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice. J Clin Invest 2007;117:3359–68. [9] Takai S, Jin D, Nishimoto M, Yuda A, Sakaguchi M, Kamoshita K, et al. Oral administration of a specific chymase inhibitor, NK3201, inhibits vascular proliferation in grafted vein. Life Sci 2001;69:1725–32. [10] Ishida K, Takai S, Murano M, Nishikawa T, Inoue T, Murano N, et al. Role of chymase-dependent matrix metalloproteinase-9 activation in mice with dextran sodium sulfate-induced colitis. J Pharmacol Exp Ther 2008;324:422–6. [11] Murphy G, Cockett MI, Ward RV, Docherty AJ. Matrix metalloproteinase degradation of elastin, type IV collagen and proteoglycan. A quantitative comparison of the activities of 95 kDa and 72 kDa gelatinases, stromelysins-1 and -2 and punctuated metalloproteinase (PUMP). Biochem J 1991;277:277–9. [12] Manning MW, Cassis LA, Daugherty A. Differential effects of doxycycline, a broad-spectrum matrix metalloproteinase inhibitor, on Ang II-induced atherosclerosis and abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol 2003;23:483–8. [13] Lijnen HR. Plasmin and matrix metalloproteinases in vascular remodelling. Thromb Haemost 2001;8:324–33. [14] Carmeliet P, Moons L, Lijnen R, Baes M, Lemaître V, Tipping P, et al. Urokinasegenerated plasmin activates matrix metalloproteinases during aneurysm formation. Nat Genet 1997;17:439–44. [15] Furubayashi K, Takai S, Jin D, Miyazaki M, Katsumata T, Inagaki S, et al. Chymase activates promatrix metalloproteinase-9 in human abdominal aortic aneurysm. Clin Chim Acta 2008;388:214–6. [16] Sun J, Sukhova GK, Wolters PJ, Yang M, Kitamoto S, Libby P, et al. Mast cells promote atherosclerosis by releasing proinflammatory cytokines. Nat Med 2007;13:719–24. [17] Takai S, Miyazaki M. Application of a chymase inhibitor, NK3201, for prevention of vascular proliferation. Cardiovasc Drug Rev 2003;21:185–98. [18] Daugherty A, Manning MW, Cassis LA. Ang II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J Clin Invest 2000;105:1605–12. ˜ [19] Hernández-Presa M, Bustos C, Ortego M, Tunon J, Renedo G, Ruiz-Ortega M, et al. Ang-converting enzyme inhibition prevents arterial nuclear factor- B activation, monocyte chemoattractant protein-1 expression, and macrophage infiltration in a rabbit model of early accelerated atherosclerosis. Circulation 1997;95:1532–41.
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