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Increased local angiotensin II formation in aneurysmal aorta
Life Sciences 71 (2002) 2195 – 2205 www.elsevier.com/locate/lifescie
Increased local angiotensin II formation in aneurysmal aorta Masayoshi Nishimoto a,b, Shinji Takai a, Hitoshi Fukumoto b, Koutaro Tsunemi a,c, Atsushi Yuda a,c, Yoshihide Sawada c, Mayumi Yamada a, Denan Jin a, Masato Sakaguchi a, Yasuhisa Nishimoto b, Shinjiro Sasaki c, Mizuo Miyazaki a,* a
Department of Pharmacology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki City, Osaka 569-8686, Japan b Osaka Mishima Critical Care Medical Center, Takatsuki City, Osaka 569-1124, Japan c Department of Thoracic and Cardiovascular Surgery, Osaka Medical College, Takatsuki City, Osaka 569-8686, Japan Received 22 August 2001; accepted 24 May 2002
Abstract We investigated the levels and locations of angiotensin II-forming enzymes, angiotensin converting enzyme (ACE) and chymase, in aneurysmal and normal aortas. Aneurysmal aortic specimens (n = 14) were obtained at the time of operative aneurysm repair from 14 patients ranging in age from 57 to 84 y. Normal aortic specimens (n = 16) were obtained from 16 patients (48 to 72 y) who underwent coronary artery bypass surgery. The ACE and chymase activities were determined using each specimen. Sections of each specimen were immunostained with antibodies for ACE and chymase. The ACE activities in the aneurysmal and normal aortas were 0.82 F 0.10 and 0.14 F 0.05 mU/mg protein, respectively, and this difference was significant. The chymase activities in the aneurysmal and normal aortas were 17.9 F 2.40 and 1.02 F 0.18 mU/mg protein, respectively, and this difference was also significant. In the aneurysmal aorta, ACE-positive cells were detected with macrophages in the intima and media and chymase-positive cells were detected with mast cells in the media and adventitia, whereas positive ACE and chymase cells in the normal aorta were located only in the endothelium and adventitia, respectively. Angiotensin IIforming enzymes, chymase and ACE, were significantly increased in the aneurysmal aorta, and increased angiotensin II may be associated with the development of aneurysmal formations. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Aneurysm; Angiotensin II; Angiotensin-converting enzyme; Chymase
*
Corresponding author. Tel.: +81-726-84-6418; fax: +81-726-84-6518. E-mail address: [email protected] (M. Miyazaki). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 9 9 8 - 7
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Introduction Aneurysmal aorta, which represents a chronic degenerative condition associated with atherosclerosis [1,2], is characterized by segmental weakening and dilatation of the aortic wall, and carries a lifethreatening risk of rupture. Although the immediate risk associated with small symptomatic aneurysmal aorta is quite low, the natural history of these lesions involves gradual expansion over a period of years with a substantial number of aneurysmal aorta eventually rupturing [3–5]. The pathophysiology of aneurysmal aorta includes aortic atheroscrelosis, chronic inflammation within the outer aortic wall, and an imbalance between the production and degradation of structural extracellular matrix proteins [6]. Angiotensin II is known to play crucial roles in the remodelling of vascular tissues via activation of growth factors and cytokines, in addition to the regulation of blood pressure [7,8]. Recent papers demonstrated that angiotensin II also activates macrophages, and such activated macrophages could substantially oxidize low-density lipoprotein (LDL) and possess additional atherogenic properties such as an increase of oxidized LDL receptors [9–11]. Such activated macrophages are thought to induce the development of atherosclerosis [12–14]. In fact, angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor antagonists decrease significantly the development of atherosclerosis in monkeys fed a high cholesterol diet, suggesting a role for angiotensin II in the development of atherosclerosis [15–20]. However, the relationship between local angiotensin II formation and aneurysmal aorta has remained unclear. Angiotensin II is thought to be generated from angiotensin I by ACE, however human vessels also contain an alternative angiotensin II-forming enzyme, chymase [21,22]. To clarify the relationship between aneurysmal aorta and local angiotensin II formation, we investigated the levels and locations of the angiotensin II-forming enzymes, ACE and chymase, in human aneurysmal aorta in the present study.
Methods Tissue procurement Aneurysmal aortic specimens (n = 14) (12 men and 2 women) were obtained at the time of operative aneurysm repair from patients ranging in age from 57 to 84 y. All aneurysmal tissues were obtained 3 to 5 cm below the renal arteries. As a control, normal aortic specimens (n = 16) were obtained from 16 patients (13 men and 3 women, 48 to 72 y) who underwent coronary artery bypass surgery. No patient received ACE inhibitors or angiotensin II antagonists before surgery. All subjects gave written consent after a full explanation of the purpose of this study. The protocol of this study complied with the principles of the Helsinki Declaration. Preparation of vascular tissue The aneurysmal and normal aortic specimens were minced and homogenized in 10 volumes (w/v) of 20 mM Na-phosphate buffer, pH 7.4. The homogenate was centrifuged at 20,000 g for 30 min. The supernatant was discarded, and the pellet was re-suspended and homogenized in 5 volumes (w/v) of 10
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mM Na-phosphate buffer, pH 7.4, containing 2 M KCl and 0.1% Nonidet P-40. The homogenate was stored overnight at 4 jC, and centrifuged at 20,000 g for 30 min. The supernatant was used for measurement of the ACE and chymase activities. Measurement of enzyme activities ACE activity was measured using a synthetic substrate, hippuryl-His–Leu, specifically designed for ACE (Peptide Institute Inc., Osaka, Japan). Tissue extract was incubated for 30 min at 37 jC with 5 mM hippuryl-His–Leu in 10 mM phosphate buffer, pH 8.3, containing 600 mM NaCl [23]. The reaction was terminated by addition of 3% metaphosphoric acid, and then the mixture was centrifuged at 20,000 g for 5 min at 4 jC. The supernatant was applied to a reversed-phase column (4 mm i.d. 250 mm, IRICA Instrument, Kyoto, Japan), which had been equilibrated with 10 mM KH2PO4 and CH3OH (1:1, pH 3.0), and eluted with the same solution at a rate of 0.3 ml/min. Hippuric acid was detected by ultraviolet absorbance at 228 nm. One unit of ACE activity was defined as the amount of enzyme that cleaved 1 Amol hippuric acid/min. Chymase activity was measured by incubating tissue extracts for 30 min at 37 jC with 4 mM angiotensin I in 150 mM borax–borate buffer, pH 8.5, containing 8 mM dipyridyl, 770 AM diisopropyl fluorophosphate and 5 mM ethylenediaminetetraacetic acid, as described previously [23]. The reaction was terminated by addition of 15% trichloroacetic acid, and then the mixture was centrifuged at 20,000 g for 5 min at 4 jC. For fluorometric quantitation of His–Leu formed from an angiotensin I substrate, 10% o-phthaldialdehyde (dissolved in methanol) was added to the supernatant in the alkaline state, and was incubated for 10 min at room temperature. The reaction was terminated by addition of 6 M HCl and the fluorescence was measured at 340 nm excitation and 455 nm emission. One unit of chymase activity was defined as the amount of enzyme that cleaved 1 Amol His–Leu/min. A blank was also included, with an addition of 500 AM chymostatin. Protein concentration was assayed with BCA Protein Assay Reagents (Pierce, Rockford, IL), using bovine serum albumin as a standard. Histological analysis The segments of aneurysmal or normal aortic specimens were fixed in 10% methanol-Carnoy’s fixative overnight and embedded in paraffin. Sections of 3 Am thickness were cut from each block. The sections were stained with hematoxylin–eosin and van Gieson’s elastin stain, respectively. Other sections were used for immunohistochemical staining. These sections were fixed in cold acetone ( 20 jC) and preincubated with 0.3% hydogen peroxide in Dulbecco’s phosphate-buffered saline (PBS) to block endogenous peroxidase activity. The sections were incubated with primary antibodies diluted in PBS containing 10% horse serum at room temperature for 60 min. After the sections were washed in PBS containing 2% horse serum, species-appropriate biotinylated secondary antibodies were applied, followed by reaction with an avidin–biotin–peroxidase kit (Dako LSAB kit, DAKO Corporation, Carpinteria, CA) with 3-amino-9-ethylcarbazole color development. As a negative control, the primary antibody was omitted and replaced by an equal concentration of nonimmune mouse monoclonal IgG. The primary antibodies used in the present study were the following: mouse antihuman chymase monoclonal antibody (1:1000 dilution, Chemicon International Inc., Temecula, CA);
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Table 1 Demographic characteristics of patients used as aneurysmal and normal aorta Age (y) Male:Female Hypertention Diabetes Hyperlipidemia Smoking
Aneurysmal aorta patients (n = 9)
Normal aorta patients (n = 10)
66 F 4 12:2 9 2 (2) 7 (7) 6 (6)
68 F 3 13:3 9 3 (2) 6 (5) 5 (4) (Hypertention)
mouse anti-human ACE monoclonal antibody, 9B9 (1:1000 dilution, Chemicon International Inc.). Mouse anti-human macrophage monoclonal antibody, HAM-56 (1:100 dilution, DAKO Corporation). Mast cells were detected by staining with 0.5% toluidine blue acidified with 100 mM citrate (pH 4.8). Statistical analysis All data indicated in the text are expressed as means F standard error (S.E.). Statistical significance was determined with Student’s t-test. Differences were considered significant for P < 0.05.
Results Table 1 shows the demographic characteristics of the 9 aneurysmal and 10 control patients enrolled in the study. Hyperlipidemia and hypertension were more frequently observed in the aneurysmal patients than in the control patients, while the number of other diseases was almost the same. The ACE activities in the aneurysmal and normal aortas were 0.82 F 0.10 and 0.14 F 0.05 mU/mg protein, respectively, and this difference was significant (Fig. 1). The chymase activity in the aneurysmal
Fig. 1. The ACE and chymase activities in the aneurysmal and normal aorta. * * P < 0.01 vs. normal aorta.
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aorta was 17.9 F 2.40 mU/mg protein, while that in the normal aorta was 1.02 F 0.18 mU/mg protein, and this difference was also significant (Fig. 1). The ACE and chymase activities in the aneurysmal aorta were about 5.9 and 17.5 times, respectively, greater than those in the normal aorta.
Fig. 2. Typical photographs of the aneurysmal (upper panels) and normal aorta (lower panels) stained with hematoxylin – eosin (left panels) and van Gieson’s elastin (right panels). I: intima, M: Media. Magnification 36.
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Fig. 2 shows sections of the aneurysmal and normal aortas stained with hematoxylin–eosin and van Gieson’s elastin. The wall of an aneurysmal aorta was characterized as atherosclerosis. A remarkable hyperplasia of the intimal area in the aneurysmal aorta was observed compared with that in the normal
Fig. 3. Typical photographs of the aneurysmal aorta using immunostaining for ACE (A and C), macrophage (D) and chymase (B and E). Magnification 36 (A and B). Magnification 180 (C and D). Magnification 90 (E).
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aorta. In contrast, remarkable thinned and fibrous media were observed in the aneurysmal aorta. The media of normal aorta contained an abundance of elastic fibers, while that of the aneurysmal aorta did not. The adventitial area in the aneurysmal aorta was thicker than that in the normal aorta. Thickened lesions of intimal and adventitial areas were observed along with inflammatory cells. Typical photographs of the aneurysmal aorta using immunostaining for ACE and macrophages are shown in Fig. 3 (A, C and D). The ACE-positive cells were predominantly observed in the intimal and adventitial regions and were identified with macrophages. On the other hand, typical photographs of the aneurysmal aorta using immunostaining for chymase are shown in Fig. 3 (B and E). Chymase-positive cells are predominantly observed in the medial and adventitial areas. All chymase-positive cells in the normal and aneurysmal aortas were identified as cells stained by toluidine blue.
Discussion This is the first study to demonstrate the levels of the angiotensin II-forming enzymes, ACE and chymase, in human aneurysmal and normal aortas. The activity levels of both ACE and chymase were significantly increased in the aneurysmal aorta. The ACE and chymase activities in the aneurysmal aorta were about 5.9 and 17.5 times higher, respectively, than those in the normal aorta. Although ACEpositive cells in normal vessels are usually detected only in endothelial cells, the ACE-positive cells in the aneurysmal aorta of the present study were detected diffusely in the intimal and adventitial areas and these cells were identified as macrophages. Therefore, the increased ACE activity in the aneurysmal aorta may be dependent on an increase of ACE expression in macrophages. Chymase-positive cells were hardly detected in the normal vessels, and they were detected only in the adventitial area. These observations have been reported previously [24,25]. However, in the aneurysmal aorta, chymase-positive cells were detected in the medial area in addition to the adventitial area, and the number of mast cells was obviously increased in comparison with the normal aorta. All chymase-positive cells in the normal and aneurysmal aortas were identified as cells stained by toluidine blue. The increased chymase activity in the aneurysmal aorta is thought to be dependent on the accumulation of mast cells. These findings suggested that the local angiotensin II-forming ability in aneurysmal aorta was activated by increases of ACE and chymase via accumulations of macrophages and mast cells, respectively. Aneurysmal aorta has been reported to be closely associated with atherosclerosis [1,2]. Angiotensin II has also been reported to correlate with the development of atherosclerosis, and in fact, blockage of angiotensin II suppresses the development of atherosclerosis in various animal models [15–20]. In invitro experiments, the oxidation of LDL is accelerated by superoxide anion induced by angiotensin II, and oxidized LDL is taken up in macrophages [26–28]. Angiotensin II also induces lectin-like oxidized receptors, which are oxidized LDL receptors in endothelial cells [11]. These reports suggest that angiotensin II induces to uptake of oxidized LDL into vascular tissues, resulting in the development of atherosclerosis. In fact, in human atherosclerosis, increased expressions of ACE, chymase and angiotensin II receptors were also observed [29,30]. Therefore, the increase of local angiotensin II formation via activation of ACE and chymase may play a crucial role in developing atherosclerosis including aneurysmal lesions. It is widely recognized that aneurysmal aorta is closely associated with chronic inflammation and destruction of connective tissues in the aortic wall [1,2]. In the present study, the numbers of macrophages and mast cells both of which are closely connected with inflammation were increased
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in the aneurysmal aorta. Accumulations of macrophages and mast cells were observed in vessels after coronary interventions and grafting [31]. In dog models of balloon catheter-injured arteries and grafted veins, intimal hyperplasia in the vessels was obvious and both ACE and chymase activities were increased significantly [23,32]. The intimal hyperplasia in these models was significantly suppressed by angiotensin II antagonist [33,34]. Recent papers suggest that angiotensin II activates inflammatory cells such as macrophages and mast cells via the activation of chemokines and cytokines [35,36]. The increase of ACE expressed in macrophages is thought to activate macrophages when angiotensin II formed by ACE stimulates angiotensin II type 1 receptor on the surface of macrophages [37]. The activated macrophages induce nuclear factor-kB, and this in turn induces an inflammatory cytokine, interleukin-1, and a chemokine, monocyte chemoattractant protein (MCP)-1 [38,39]. Mabuchi et al. [40] reported that interleukin-1 produced by activated macrophages induced tissue damage. On the other hand, MCP-1 is known to induce the activation and migration of monocytes, resulting in an accumulation of macrophages [41]. Angiotensin II type 1 receptor antagonists and ACE inhibitors were found to reduce the gene expression of MCP-1, and these drugs also reduced the accumulation of macrophages [42,43]. The accumulation of macrophages activated by angiotensin II may contribute to the development of aneurysm. In fact, Daugherty et al. [44] demonstrated that infusion of angiotensin II leads to development of aneurysmal aorta in apolipoprotein E-deficient mice. Liao et al. [45] also reported that ACE inhibitors may suppress the development of aneurysmal aorta in a rat model. These findings suggest that angiotensin II may play an important role in the devcelopment of aneurysmal aorta. In this study, we demonstrated the increased of chymase-positive mast cells in human aneurysmal aortas. Mast cells release a large number of inflammatory mediators such as histamine, serotonin, chemotactic factors, cytokines and serine proteases [46,47]. Chymase processes and activates pro-matrix metalloproteinase to matrix metalloproteinase (MMP), which is a strong tissue-degradative protease [48]. However, generally, MMP is balanced by the tissue inhibitor of metalloproteinase (TIMP), and tissue degradation is dependent on an imbalance between MMP and TIMP [49]. Chymase cleaves TIMP to inactive fragments, and it also cleaves complexes of MMP and TIMP, which have no MMP activity, to form active MMP [49]. MMP activation by chymase may contribute to the tissue degradation. Therefore, mast cell products including chymase may be also involved in anerysmal diseases. In conclusion, both angiotensin II-forming enzymes, chymase and ACE, were significantly increased in the aneurysmal aorta, and increased local angiotensin II formation may play a crucial role in development of aneurysmal aorta. However, in the present study, the ascending aorta was used as the normal material of blood vessels. Further studies are needed, in which tissues of the abdominal aorta are used as normal.
Acknowledgements This study was supported in part by Grant-in-Aid 12770048 for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture, Japan.
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Increased local angiotensin II formation in aneurysmal aorta Masayoshi Nishimoto a,b, Shinji Takai a, Hitoshi Fukumoto b, Koutaro Tsunemi a,c, Atsushi Yuda a,c, Yoshihide Sawada c, Mayumi Yamada a, Denan Jin a, Masato Sakaguchi a, Yasuhisa Nishimoto b, Shinjiro Sasaki c, Mizuo Miyazaki a,* a
Department of Pharmacology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki City, Osaka 569-8686, Japan b Osaka Mishima Critical Care Medical Center, Takatsuki City, Osaka 569-1124, Japan c Department of Thoracic and Cardiovascular Surgery, Osaka Medical College, Takatsuki City, Osaka 569-8686, Japan Received 22 August 2001; accepted 24 May 2002
Abstract We investigated the levels and locations of angiotensin II-forming enzymes, angiotensin converting enzyme (ACE) and chymase, in aneurysmal and normal aortas. Aneurysmal aortic specimens (n = 14) were obtained at the time of operative aneurysm repair from 14 patients ranging in age from 57 to 84 y. Normal aortic specimens (n = 16) were obtained from 16 patients (48 to 72 y) who underwent coronary artery bypass surgery. The ACE and chymase activities were determined using each specimen. Sections of each specimen were immunostained with antibodies for ACE and chymase. The ACE activities in the aneurysmal and normal aortas were 0.82 F 0.10 and 0.14 F 0.05 mU/mg protein, respectively, and this difference was significant. The chymase activities in the aneurysmal and normal aortas were 17.9 F 2.40 and 1.02 F 0.18 mU/mg protein, respectively, and this difference was also significant. In the aneurysmal aorta, ACE-positive cells were detected with macrophages in the intima and media and chymase-positive cells were detected with mast cells in the media and adventitia, whereas positive ACE and chymase cells in the normal aorta were located only in the endothelium and adventitia, respectively. Angiotensin IIforming enzymes, chymase and ACE, were significantly increased in the aneurysmal aorta, and increased angiotensin II may be associated with the development of aneurysmal formations. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Aneurysm; Angiotensin II; Angiotensin-converting enzyme; Chymase
*
Corresponding author. Tel.: +81-726-84-6418; fax: +81-726-84-6518. E-mail address: [email protected] (M. Miyazaki). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 9 9 8 - 7
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Introduction Aneurysmal aorta, which represents a chronic degenerative condition associated with atherosclerosis [1,2], is characterized by segmental weakening and dilatation of the aortic wall, and carries a lifethreatening risk of rupture. Although the immediate risk associated with small symptomatic aneurysmal aorta is quite low, the natural history of these lesions involves gradual expansion over a period of years with a substantial number of aneurysmal aorta eventually rupturing [3–5]. The pathophysiology of aneurysmal aorta includes aortic atheroscrelosis, chronic inflammation within the outer aortic wall, and an imbalance between the production and degradation of structural extracellular matrix proteins [6]. Angiotensin II is known to play crucial roles in the remodelling of vascular tissues via activation of growth factors and cytokines, in addition to the regulation of blood pressure [7,8]. Recent papers demonstrated that angiotensin II also activates macrophages, and such activated macrophages could substantially oxidize low-density lipoprotein (LDL) and possess additional atherogenic properties such as an increase of oxidized LDL receptors [9–11]. Such activated macrophages are thought to induce the development of atherosclerosis [12–14]. In fact, angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor antagonists decrease significantly the development of atherosclerosis in monkeys fed a high cholesterol diet, suggesting a role for angiotensin II in the development of atherosclerosis [15–20]. However, the relationship between local angiotensin II formation and aneurysmal aorta has remained unclear. Angiotensin II is thought to be generated from angiotensin I by ACE, however human vessels also contain an alternative angiotensin II-forming enzyme, chymase [21,22]. To clarify the relationship between aneurysmal aorta and local angiotensin II formation, we investigated the levels and locations of the angiotensin II-forming enzymes, ACE and chymase, in human aneurysmal aorta in the present study.
Methods Tissue procurement Aneurysmal aortic specimens (n = 14) (12 men and 2 women) were obtained at the time of operative aneurysm repair from patients ranging in age from 57 to 84 y. All aneurysmal tissues were obtained 3 to 5 cm below the renal arteries. As a control, normal aortic specimens (n = 16) were obtained from 16 patients (13 men and 3 women, 48 to 72 y) who underwent coronary artery bypass surgery. No patient received ACE inhibitors or angiotensin II antagonists before surgery. All subjects gave written consent after a full explanation of the purpose of this study. The protocol of this study complied with the principles of the Helsinki Declaration. Preparation of vascular tissue The aneurysmal and normal aortic specimens were minced and homogenized in 10 volumes (w/v) of 20 mM Na-phosphate buffer, pH 7.4. The homogenate was centrifuged at 20,000 g for 30 min. The supernatant was discarded, and the pellet was re-suspended and homogenized in 5 volumes (w/v) of 10
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mM Na-phosphate buffer, pH 7.4, containing 2 M KCl and 0.1% Nonidet P-40. The homogenate was stored overnight at 4 jC, and centrifuged at 20,000 g for 30 min. The supernatant was used for measurement of the ACE and chymase activities. Measurement of enzyme activities ACE activity was measured using a synthetic substrate, hippuryl-His–Leu, specifically designed for ACE (Peptide Institute Inc., Osaka, Japan). Tissue extract was incubated for 30 min at 37 jC with 5 mM hippuryl-His–Leu in 10 mM phosphate buffer, pH 8.3, containing 600 mM NaCl [23]. The reaction was terminated by addition of 3% metaphosphoric acid, and then the mixture was centrifuged at 20,000 g for 5 min at 4 jC. The supernatant was applied to a reversed-phase column (4 mm i.d. 250 mm, IRICA Instrument, Kyoto, Japan), which had been equilibrated with 10 mM KH2PO4 and CH3OH (1:1, pH 3.0), and eluted with the same solution at a rate of 0.3 ml/min. Hippuric acid was detected by ultraviolet absorbance at 228 nm. One unit of ACE activity was defined as the amount of enzyme that cleaved 1 Amol hippuric acid/min. Chymase activity was measured by incubating tissue extracts for 30 min at 37 jC with 4 mM angiotensin I in 150 mM borax–borate buffer, pH 8.5, containing 8 mM dipyridyl, 770 AM diisopropyl fluorophosphate and 5 mM ethylenediaminetetraacetic acid, as described previously [23]. The reaction was terminated by addition of 15% trichloroacetic acid, and then the mixture was centrifuged at 20,000 g for 5 min at 4 jC. For fluorometric quantitation of His–Leu formed from an angiotensin I substrate, 10% o-phthaldialdehyde (dissolved in methanol) was added to the supernatant in the alkaline state, and was incubated for 10 min at room temperature. The reaction was terminated by addition of 6 M HCl and the fluorescence was measured at 340 nm excitation and 455 nm emission. One unit of chymase activity was defined as the amount of enzyme that cleaved 1 Amol His–Leu/min. A blank was also included, with an addition of 500 AM chymostatin. Protein concentration was assayed with BCA Protein Assay Reagents (Pierce, Rockford, IL), using bovine serum albumin as a standard. Histological analysis The segments of aneurysmal or normal aortic specimens were fixed in 10% methanol-Carnoy’s fixative overnight and embedded in paraffin. Sections of 3 Am thickness were cut from each block. The sections were stained with hematoxylin–eosin and van Gieson’s elastin stain, respectively. Other sections were used for immunohistochemical staining. These sections were fixed in cold acetone ( 20 jC) and preincubated with 0.3% hydogen peroxide in Dulbecco’s phosphate-buffered saline (PBS) to block endogenous peroxidase activity. The sections were incubated with primary antibodies diluted in PBS containing 10% horse serum at room temperature for 60 min. After the sections were washed in PBS containing 2% horse serum, species-appropriate biotinylated secondary antibodies were applied, followed by reaction with an avidin–biotin–peroxidase kit (Dako LSAB kit, DAKO Corporation, Carpinteria, CA) with 3-amino-9-ethylcarbazole color development. As a negative control, the primary antibody was omitted and replaced by an equal concentration of nonimmune mouse monoclonal IgG. The primary antibodies used in the present study were the following: mouse antihuman chymase monoclonal antibody (1:1000 dilution, Chemicon International Inc., Temecula, CA);
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Table 1 Demographic characteristics of patients used as aneurysmal and normal aorta Age (y) Male:Female Hypertention Diabetes Hyperlipidemia Smoking
Aneurysmal aorta patients (n = 9)
Normal aorta patients (n = 10)
66 F 4 12:2 9 2 (2) 7 (7) 6 (6)
68 F 3 13:3 9 3 (2) 6 (5) 5 (4) (Hypertention)
mouse anti-human ACE monoclonal antibody, 9B9 (1:1000 dilution, Chemicon International Inc.). Mouse anti-human macrophage monoclonal antibody, HAM-56 (1:100 dilution, DAKO Corporation). Mast cells were detected by staining with 0.5% toluidine blue acidified with 100 mM citrate (pH 4.8). Statistical analysis All data indicated in the text are expressed as means F standard error (S.E.). Statistical significance was determined with Student’s t-test. Differences were considered significant for P < 0.05.
Results Table 1 shows the demographic characteristics of the 9 aneurysmal and 10 control patients enrolled in the study. Hyperlipidemia and hypertension were more frequently observed in the aneurysmal patients than in the control patients, while the number of other diseases was almost the same. The ACE activities in the aneurysmal and normal aortas were 0.82 F 0.10 and 0.14 F 0.05 mU/mg protein, respectively, and this difference was significant (Fig. 1). The chymase activity in the aneurysmal
Fig. 1. The ACE and chymase activities in the aneurysmal and normal aorta. * * P < 0.01 vs. normal aorta.
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aorta was 17.9 F 2.40 mU/mg protein, while that in the normal aorta was 1.02 F 0.18 mU/mg protein, and this difference was also significant (Fig. 1). The ACE and chymase activities in the aneurysmal aorta were about 5.9 and 17.5 times, respectively, greater than those in the normal aorta.
Fig. 2. Typical photographs of the aneurysmal (upper panels) and normal aorta (lower panels) stained with hematoxylin – eosin (left panels) and van Gieson’s elastin (right panels). I: intima, M: Media. Magnification 36.
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Fig. 2 shows sections of the aneurysmal and normal aortas stained with hematoxylin–eosin and van Gieson’s elastin. The wall of an aneurysmal aorta was characterized as atherosclerosis. A remarkable hyperplasia of the intimal area in the aneurysmal aorta was observed compared with that in the normal
Fig. 3. Typical photographs of the aneurysmal aorta using immunostaining for ACE (A and C), macrophage (D) and chymase (B and E). Magnification 36 (A and B). Magnification 180 (C and D). Magnification 90 (E).
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aorta. In contrast, remarkable thinned and fibrous media were observed in the aneurysmal aorta. The media of normal aorta contained an abundance of elastic fibers, while that of the aneurysmal aorta did not. The adventitial area in the aneurysmal aorta was thicker than that in the normal aorta. Thickened lesions of intimal and adventitial areas were observed along with inflammatory cells. Typical photographs of the aneurysmal aorta using immunostaining for ACE and macrophages are shown in Fig. 3 (A, C and D). The ACE-positive cells were predominantly observed in the intimal and adventitial regions and were identified with macrophages. On the other hand, typical photographs of the aneurysmal aorta using immunostaining for chymase are shown in Fig. 3 (B and E). Chymase-positive cells are predominantly observed in the medial and adventitial areas. All chymase-positive cells in the normal and aneurysmal aortas were identified as cells stained by toluidine blue.
Discussion This is the first study to demonstrate the levels of the angiotensin II-forming enzymes, ACE and chymase, in human aneurysmal and normal aortas. The activity levels of both ACE and chymase were significantly increased in the aneurysmal aorta. The ACE and chymase activities in the aneurysmal aorta were about 5.9 and 17.5 times higher, respectively, than those in the normal aorta. Although ACEpositive cells in normal vessels are usually detected only in endothelial cells, the ACE-positive cells in the aneurysmal aorta of the present study were detected diffusely in the intimal and adventitial areas and these cells were identified as macrophages. Therefore, the increased ACE activity in the aneurysmal aorta may be dependent on an increase of ACE expression in macrophages. Chymase-positive cells were hardly detected in the normal vessels, and they were detected only in the adventitial area. These observations have been reported previously [24,25]. However, in the aneurysmal aorta, chymase-positive cells were detected in the medial area in addition to the adventitial area, and the number of mast cells was obviously increased in comparison with the normal aorta. All chymase-positive cells in the normal and aneurysmal aortas were identified as cells stained by toluidine blue. The increased chymase activity in the aneurysmal aorta is thought to be dependent on the accumulation of mast cells. These findings suggested that the local angiotensin II-forming ability in aneurysmal aorta was activated by increases of ACE and chymase via accumulations of macrophages and mast cells, respectively. Aneurysmal aorta has been reported to be closely associated with atherosclerosis [1,2]. Angiotensin II has also been reported to correlate with the development of atherosclerosis, and in fact, blockage of angiotensin II suppresses the development of atherosclerosis in various animal models [15–20]. In invitro experiments, the oxidation of LDL is accelerated by superoxide anion induced by angiotensin II, and oxidized LDL is taken up in macrophages [26–28]. Angiotensin II also induces lectin-like oxidized receptors, which are oxidized LDL receptors in endothelial cells [11]. These reports suggest that angiotensin II induces to uptake of oxidized LDL into vascular tissues, resulting in the development of atherosclerosis. In fact, in human atherosclerosis, increased expressions of ACE, chymase and angiotensin II receptors were also observed [29,30]. Therefore, the increase of local angiotensin II formation via activation of ACE and chymase may play a crucial role in developing atherosclerosis including aneurysmal lesions. It is widely recognized that aneurysmal aorta is closely associated with chronic inflammation and destruction of connective tissues in the aortic wall [1,2]. In the present study, the numbers of macrophages and mast cells both of which are closely connected with inflammation were increased
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in the aneurysmal aorta. Accumulations of macrophages and mast cells were observed in vessels after coronary interventions and grafting [31]. In dog models of balloon catheter-injured arteries and grafted veins, intimal hyperplasia in the vessels was obvious and both ACE and chymase activities were increased significantly [23,32]. The intimal hyperplasia in these models was significantly suppressed by angiotensin II antagonist [33,34]. Recent papers suggest that angiotensin II activates inflammatory cells such as macrophages and mast cells via the activation of chemokines and cytokines [35,36]. The increase of ACE expressed in macrophages is thought to activate macrophages when angiotensin II formed by ACE stimulates angiotensin II type 1 receptor on the surface of macrophages [37]. The activated macrophages induce nuclear factor-kB, and this in turn induces an inflammatory cytokine, interleukin-1, and a chemokine, monocyte chemoattractant protein (MCP)-1 [38,39]. Mabuchi et al. [40] reported that interleukin-1 produced by activated macrophages induced tissue damage. On the other hand, MCP-1 is known to induce the activation and migration of monocytes, resulting in an accumulation of macrophages [41]. Angiotensin II type 1 receptor antagonists and ACE inhibitors were found to reduce the gene expression of MCP-1, and these drugs also reduced the accumulation of macrophages [42,43]. The accumulation of macrophages activated by angiotensin II may contribute to the development of aneurysm. In fact, Daugherty et al. [44] demonstrated that infusion of angiotensin II leads to development of aneurysmal aorta in apolipoprotein E-deficient mice. Liao et al. [45] also reported that ACE inhibitors may suppress the development of aneurysmal aorta in a rat model. These findings suggest that angiotensin II may play an important role in the devcelopment of aneurysmal aorta. In this study, we demonstrated the increased of chymase-positive mast cells in human aneurysmal aortas. Mast cells release a large number of inflammatory mediators such as histamine, serotonin, chemotactic factors, cytokines and serine proteases [46,47]. Chymase processes and activates pro-matrix metalloproteinase to matrix metalloproteinase (MMP), which is a strong tissue-degradative protease [48]. However, generally, MMP is balanced by the tissue inhibitor of metalloproteinase (TIMP), and tissue degradation is dependent on an imbalance between MMP and TIMP [49]. Chymase cleaves TIMP to inactive fragments, and it also cleaves complexes of MMP and TIMP, which have no MMP activity, to form active MMP [49]. MMP activation by chymase may contribute to the tissue degradation. Therefore, mast cell products including chymase may be also involved in anerysmal diseases. In conclusion, both angiotensin II-forming enzymes, chymase and ACE, were significantly increased in the aneurysmal aorta, and increased local angiotensin II formation may play a crucial role in development of aneurysmal aorta. However, in the present study, the ascending aorta was used as the normal material of blood vessels. Further studies are needed, in which tissues of the abdominal aorta are used as normal.
Acknowledgements This study was supported in part by Grant-in-Aid 12770048 for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture, Japan.
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