The Significance of Chymase in the Progression of Abdominal Aortic Aneurysms in Dogs

349 Hypertens Res Vol.30 (2007) No.4 p.349-357 Original Article The Significance of Chymase in the Progression of Abdominal Aortic Aneurysms in Dogs Keiichi FURUBAYASHI1),2), Shinji TAKAI1), Denan JIN1), Michiko MURAMATSU1), Toshihiko IBARAKI1), Masayoshi NISHIMOTO3), Hitoshi FUKUMOTO3), Takahiro KATSUMATA2), and Mizuo MIYAZAKI1) In this study, we investigated the effect of a specific chymase inhibitor, NK3201, in the progression of abdominal aortic aneurysm in a dog experimental model. Abdominal aortic aneurysms were induced in dogs by injecting elastase into the abdominal aorta. NK3201 (1 mg/kg per day, p.o.) or a placebo was started 3 days before elastase injection and continued for 8 weeks after the injection. On abdominal ultrasound, the aortic diameter was seen to gradually expand in the placebo-treated group, but not in the NK3201-treated group. Eight weeks after elastase injection, the ratio of the medial area to the total area in the placebotreated group was significantly smaller than that in the normal group, but it was significantly larger than that in the NK3201-treated group. In addition to chymase activity, angiotensin II–forming and matrix metalloproteinase-9 activities were significantly higher in the placebo-treated group than in the normal group; in the NK3201-treated group, all of these activities were significantly decreased. On immunohistochemical analyses, there was a significantly greater number of chymase-positive cells in the placebo-treated group than in the normal group, but the number was significantly smaller in the NK3201-treated group than in the placebo-treated group. Thus, chymase inhibition may become a useful strategy for preventing abdominal aortic aneurysms. (Hypertens Res 2007; 30: 349–357) Key Words: abdominal aortic aneurysm, angiotensin II, chymase, inhibitor, matrix metalloproteinase-9 Introduction Abdominal aortic aneurysms (AAAs) are generally characterized by the widespread destruction of elastic lamellae in the media and by an inflammatory response in the vascular wall. The pathophysiology of AAAs includes aortic atherosclerosis, chronic inflammation within the outer aortic wall, and an imbalance between the production and degradation of structural extracellular matrix proteins (1–3). Ruptured AAAs have a high mortality rate unless the appropriate diagnosis is made, since only timely surgery can benefit patients with AAAs. However, aged patients with AAAs who have a poor quality of life due to a mental or physical handicap are not surgical candidates. Therefore, there is a need for effective pharmacotherapy for AAAs. The destruction of elastin is considered to be one of the major pathogenetic mechanisms of AAAs. Although elastic fibers normally maintain the structure of the vascular wall against hemodynamic stress, enzymatic degradation may induce remodeling of the extracellular matrix, resulting in aneurysmal development and finally rupture. Chymase is a chymotrypsin-like serine protease located in the secretory granules of mast cells. Dog chymase converts promatrix met- From the 1)Department of Pharmacology and 2)Department of Thoracic and Cardiovascular Surgery, Osaka Medical College, Takatsuki, Japan; and 3) Osaka Mishima Critical Care Medical Center, Takatsuki, Japan. This study was partly supported by the Novartis Foundation (Japan) and the Japanese Research Foundation for Clinical Pharmacology. This study was partly supported by a grant from the Novartis Foundation (Japan) to the Japanese Research Foundation for Clinical Pharmacology. Address for Reprints: Shinji Takai, Ph.D., Department of Pharmacology, Osaka Medical College, 2–7 Daigaku-machi, Takatsuki 569–8686, Japan. Email: Received August 25, 2006; Accepted in revised form December 4, 2006. Hypertens Res Vol. 30, No. 4 (2007) alloproteinase (proMMP)-9 to matrix metalloproteinase (MMP)-9 (4). Previous studies in mice have demonstrated that the various expressions of MMP-9 are associated with the development of AAAs through elastic fiber disruption (5, 6). For example, an MMP inhibitor limits the expansion of experimental AAAs, and targeted gene disruption of MMP-9 suppresses AAA development (5, 6). Thus, increased activity of MMP-9 may play an important role in the development of AAAs. Chymase converts angiotensin I to angiotensin II in cardiovascular tissues (7–9). Previously, we have demonstrated that chymase activity was significantly higher in AAAs than in normal aortas (10, 11). Daugherty et al. (12) suggested that angiotensin II is related to aortic aneurysms, because angiotensin II promotes aortic aneurysms in apolipoprotein E–deficient mice. In fact, angiotensin II blockade might have inhibitory effects on the aneurysmal formation (13). On the other hand, we demonstrated that a chymase inhibitor also prevents the development of AAA in hamsters (14). However, it has been unclear whether chymase is involved in MMP-9 activation in AAAs. In this study, we wanted to confirm whether chymase inhibition is involved in the suppression of MMP-9 in a dog AAA model. (A) Drugs 2-(5-Formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine1-yl)-N-[{3,4-dioxo-1-phenyl-7-(2-pyridyloxy)}-2-heptyl]acetamide (NK3201) was synthesized as a specific chymase inhibitor (Nippon Kayaku Co. Ltd., Tokyo, Japan) (15). Experimental Model Fifteen male beagle dogs weighing 9–11 kg were obtained from Japan SLC (Shizuoka, Japan) and were randomly divided into 3 groups before the operation. To create the AAA model, under pentobarbital anesthesia (35 mg/kg i.v.), the abdominal aorta from the infrarenal aorta to the bifurcation of the aorta was isolated using a retroperitoneal approach. Temporary forcep blocks were placed at the proximal and distal portions of the aorta (the distance between the 2 portions was 5 cm). From the lumbar artery, 400 units/ml of porcine pancreatic elastase (type I; SERVA Electrophoresis GmbH, Heidelberg, Germany) in saline were injected into the aorta and incubated for 2 h. Beginning 3 days before the elastase was given and continuing until the end of the experiment, each dog was given the chymase inhibitor NK3201 (1 mg/kg once daily, p.o., n= 5) or placebo (n= 5). In the placebotreated group, the placebo consisted of the same volume of 0.5% carboxymethylcellulose as was given to the treated dogs. Untreated dogs constituted the normal group (n= 5). We measured the external diameter of the AAAs using an abdominal ultrasound pre-operatively, postoperatively, and every 2 NK3201 12 8 4 0 Methods Placebo (B) Aortic diameter (mm) 350 Pre Post 2 4 6 8 Time (weeks) Fig. 1. A: Photographs of a typical ultrasound image of a placebo-treated dog and an NK3201-treated dog 8 weeks after elastase injection. B: Aortic diameters in the placebo (open circle)- and NK3201 (closed circle)-treated groups during the experiment. “Pre” and “Post” indicate data obtained before and just after elastase injection, respectively. **p< 0.01 vs. “Pre” in each group. ##p< 0.01 (...truncated)