Inhibition of Matrix Metalloproteinase-9 Activity by Trandolapril after Middle Cerebral Artery Occlusion in Rats

469 Hypertens Res Vol.30 (2007) No.5 p.469-475 Original Article Inhibition of Matrix Metalloproteinase-9 Activity by Trandolapril after Middle Cerebral Artery Occlusion in Rats Hidekazu TANAKA1), Shinji TAKAI2), Denan JIN2), Keiichi FURUBAYASHI2), Nao INOUE2), Yoshinaga KAJIMOTO1), Shin-Ichi MIYATAKE1), Toshihiko KUROIWA1), and Mizuo MIYAZAKI2) We investigated whether an angiotensin-converting enzyme (ACE) inhibitor could inhibit matrix metalloproteinase (MMP) activities in cerebral infarct lesions after middle cerebral artery occlusion (MCAO) in rats. After placebo or trandolapril (5 mg/kg per day) was administered orally for 7 days, we permanently occluded the right middle cerebral artery. ACE activity in extracts from the infarct side of placebo-treated rats was significantly higher than that in extracts from the non-infarct side from 5 days after MCAO, though they did not differ at 1 day. ACE activities in extracts from both hemispheric segments in the trandolapril-treated group were significantly decreased compared with those in the placebo-treated group before MCAO, and this significant reduction persisted even at 7 days after MCAO. In the placebo-treated group, MMP-9 and MMP-2 activities in the infarct side were significantly increased at 12 h and at 1 day after MCAO, respectively. Trandolapril treatment significantly reduced MMP-9 and MMP-2 activities to 68.5% and 53.2%, respectively. Seven days after MCAO, the ratios of infarct areas to the hemispheric sectional areas in placebo- and trandolapril-treated rats were 55.4 ± 2.1% and 30.9 ± 2.9%, respectively, and this difference was significant. Neurological severity scores were significantly improved from 1 to 7 days after MCAO in trandolapril-treated rats. Cumulative survival in trandolapril-treated rats was significantly increased compared with that in placebo-treated rats. Thus, the inhibition of MMP-9 by trandolapril might be part of the mechanism that prevents cerebral damage after cerebral ischemia. (Hypertens Res 2007; 30: 469–475) Key Words: angiotensin converting enzyme, cerebral infarction, macrophage, inhibitor Introduction Angiotensin-converting enzyme (ACE) is a Zn2+-dependent endopeptidase expressed in endothelial cells, macrophages and smooth muscle cells that converts angiotensin I to angiotensin II (1). Angiotensin II plays a crucial role in tissue remodeling via increases in growth factors, cytokines, chemokines and extracellular matrix, as well as in the regulation of blood pressure (2, 3). In cerebral infarction after middle cerebral artery occlusion (MCAO), macrophages accumulate at the border between infarct and non-infarct areas and might induce the expansion of cerebral infarction (4, 5). We reported that brain ACE activity is significantly increased in the infarct area after MCAO in rats (6). In that study, trandolapril significantly reduced not only brain ACE activity but also the ratio of the infarct to the total area 7 days after MCA occlusion. However, whether trandolapril has functional merit such as reducing neurological severity scores or increasing survival rates remains obscure. Matrix metalloproteinases (MMPs) belong to a family of Zn2+-dependent endopeptidases. Extracellular matrix mole- From the 1)Department of Neurosurgery and 2)Department of Pharmacology, Osaka Medical College, Takatsuki, Japan. Address for Reprints: Shinji Takai, Ph.D., Department of Pharmacology, Osaka Medical College, 2–7 Daigaku-cho, Takatsuki 569–8686, Japan. E-mail: Received November 9, 2006; Accepted in revised form December 20, 2006. Hypertens Res Vol. 30, No. 5 (2007) cules such as type IV collagen, laminin and fibronectin, which support the structure of the vascular basement membrane, are degraded by MMP-2 and MMP-9 (7). Their enzymatic functions play an important role in the progression of inflammation and atherosclerosis (8, 9). The vascular basement membrane in the brain plays a crucial role in maintaining the blood-brain barrier (BBB) (10), which protects the central nervous system. However, focal stroke damages the BBB, resulting in neuronal cell death (11). Other studies have shown that MMP-2 and MMP-9, but not MMP-1 and MMP3, are increased in brain infarcts after MCAO in rats (12). After cerebral ischemia and reperfusion, levels of type IV collagen, laminin and fibronectin decrease (13). These reports suggest that MMP-2 and MMP-9 are involved in the cerebral damage after focal ischemia. In fact, MMP-9 inhibition results in a decrease of the infarct area after rat MCAO (12). The activities of MMP-2 and MMP-9 are inhibited by ACE inhibitors in extracts of rat renal and human cardiac tissues (14, 15). However, whether ACE inhibitors affect MMP-2 and MMP-9 activities after focal ischemia remains unknown. Here, we examined the effects of ACE inhibitors on MMP-2 and MMP-9 activities after MCAO in rats. Methods Animals Male Wistar Kyoto (WKY) rats weighing 230–260 g were purchased from Japan SLC (Shizuoka, Japan). All rats were housed individually in metabolic cages for measurement of urinary albumin at room temperature (23–26°C) under a 12-h light-dark cycle and had free access to standard food (F-2; Funahashi Co., Tokyo, Japan) and water. The rats were divided into 2 groups, the vehicle group and the trandolapril (5 mg/kg per day)-treated group. The experimental protocols involving the animals proceeded in accordance with the Guide for the Care and Use of Laboratory Animals (Animal Research Laboratory, Osaka Medical College, Takatsuki, Japan). Systolic blood pressure (mmHg) 470 120 ** ** 80 40 0 Day -7 Day 0 Pretreatment Day 7 MCAO Fig. 1. Systolic blood pressure 7 days before (day − 7), immediately before (day 0) and 7 days (day 7) after MCAO in placebo- and trandolapril-treated rats (each group, n= 6). Open circles represent placebo-treated rats. Closed circles represent trandolapril-treated rats. **p< 0.01 vs. placebotreated rats. Blood Pressure Systolic blood pressure was monitored by tail-cuff plethysmography (BP-98; Muromachi Co., Kyoto, Japan). Neurological Severity Score The rats underwent behavioral tests before and 1, 3 and 7 days after MCAO as described previously (16). A modified neurological severity score (mNSS) (16) was used to grade the aspects of neurological function. Each test in mNSS has been used to grade various aspects of neurological function. mNSS is a composite of the motor (muscle status, abnormal movement), sensory (visual, tactile, and proprioceptive), and reflex tests. A higher score reflects more severe injury. Tissue Extract MCAO Model Focal cerebral ischemia was accomplished by using the intraluminal filament model (4-0 nylon monofilament suture) of proximal MCAO as previously described (6, 16). Rats were anesthetized initially with 3.5% halothan and maintained with 1.0% to 2.0% halothan in 70% N2O and 30% O2 using a face mask. The right common carotid artery was exposed through a lateral incision, separated from the vagus nerve, and li (...truncated)