Role of Chymase-Dependent Angiotensin II Formation in Monocrotaline-Induced Pulmonary Hypertensive Rats

0031-3998/06/6001-0077 PEDIATRIC RESEARCH Copyright © 2006 International Pediatric Research Foundation, Inc. Vol. 60, No. 1, 2006 Printed in U.S.A. Role of Chymase-Dependent Angiotensin II Formation in Monocrotaline-Induced Pulmonary Hypertensive Rats KANTA KISHI, DENAN JIN, SHINJI TAKAI, MICHIKO MURAMATSU, HIROSHI KATAYAMA, HIROSHI TAMAI, AND MIZUO MIYAZAKI Department of Pharmacology [K.K., D.J., S.T., M.Mu., M.Mi.], Department of Pediatrics [H.K., H.T.], Osaka Medical College, 2-7 Daigaku-machi, Takatsuki City, Osaka 569-8686, Japan hymase is a chymotrypsin-like serine protease that is thought to be present in the secretory granules of mast cells. Chymase is able to cleave the bonds of angiotensin I between phenylalanine and histidine (1,2). Chymase purified from human cardiovascular tissues easily converts angiotensin I to angiotensin II (1,2), and chymase-dependent angiotensin II may have a role in human cardiovascular tissue function. For example, in human heart extracts, over 75% of total angiotensin II-forming activity depended on chymase, and the remaining depended on angiotensin-converting enzyme (ACE) (1). In human vascular tissue extracts, about 90% of total angiotensin II-forming activity was inhibited by a chymase inhibitor, and the remaining activity was inhibited by an ACE inhibitor (3). These findings suggest that chymase pri- marily produces angiotensin II compared with ACE in human cardiovascular tissues. However, both ACE inhibitor and angiotensin II receptor blocker (ARB) lower systemic blood pressure, suggesting that ACE plays a dominant role in regulating angiotensin II formation. Although chymase converts angiotensin I to angiotensin II in the extracts of hearts and arteries, chymase-dependent angiotensin II formation may be irrelevant for the regulation of blood pressure in vivo (4). Chymase is synthesized as an inactive prochymase, and dipeptidylpeptidase I (DPPI) is necessary for chymase activation in the secretory granules (5). DPPI is a thiol proteinase with a pH optimum of 6.0 and the pH optimum of DPPI is consistent with its proposed function as a prochymase-activating enzyme, since the pH within the granule is regulated at pH 5.5 (5). On the other hand, the optimum pH of chymase in human vascular tissues is between 7 and 9, and chymase has no enzymatic activity at pH 5.5 (2). Chymase shows enzymatic activity immediately upon release into the interstitial tissues after strong stimulation in cardiovascular tissues (4). Therefore, chymase may be released and have enzymatic activity only after the activation of mast cells in cardiovascular tissues. Two rat chymases, rat mast cell protease (RMCP) I and RMCP II, both of which are expressed in mast cells, are known. Since these chymases only weakly convert angiotensin I to angiotensin II (6), it has been thought that rat cardiovascular tissues hardly contain angiotensin II-forming chymases. However, recently, Guo et al. (7) cloned a novel chymase, rat vascular chymase (RVCH) from rat vascular smooth muscle cells (VSMCs). The cDNA encodes 247 amino acids and exhibits 74% and 80% homology for the amino acid sequence of RMCP I and RMCP II, respectively. RVCH, like human chymase, converts angiotensin I to angiotensin II (7). Over-expression of RVCH in VSMCs of transgenic mice induces systemic hypertension and vascular remodeling (8). In monocrotaline-induced pulmonary hypertensive (PH) rats, RVCH mRNA and protein are increased in the pulmonary VSMCs, but it is unclear whether RVCH-dependent angio- Received November 15, 2005; accepted February 28, 2006. Correspondence: Shinji Takai, Ph.D., Department of Pharmacology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki City, Osaka 569-8686, Japan; E-mail: This work was partly supported by a grant (16659068) from the Japanese Ministry of Education, Science, Sports, and Culture. Abbreviations: ACE, angiotensin-converting enzyme; ARB, angiotensin II receptor blocker; DPPI, dipeptidylpeptidase I; LV, left ventricle; MWT, medial wall thickness; PH rats, pulmonary hypertensive rats; RMCP, rat mast cell protease; RVCH, rat vascular chymase; RV, right ventricle; VSMCs, vascular smooth muscle cells ABSTRACT: Angiotensin II-forming chymase is expressed in the pulmonary arteries of the monocrotaline-induced pulmonary hypertensive rats, but its actual role is unclear. We studied chymasedependent angiotensin II formation in the pulmonary arteries of the monocrotaline-induced pulmonary hypertensive rats and observed the effects of an angiotensin II receptor blocker on vascular remodeling. Four weeks after the administration of monocrotaline (60 mg/kg, s.q.), echocardiographic, hemodynamic, morphometric and biochemical analyses were performed. Age-matched rats were used as controls. To evaluate the effects of an angiotensin II receptor blocker, 2 wk after beginning of monocrotaline treatment, the rats were given candesartan (10 mg/kg per day) or placebo for 2 wk. In the monocrotaline-induced pulmonary hypertensive rats, the elevated systolic pulmonary arterial pressure and right ventricular hypertrophy were observed. Medial hypertrophy of lung arterioles was also observed. Chymase activity and angiotensin II concentration, but not angiotensin-converting enzyme activity, were significantly increased in the lung. In the angiotensin II receptor blocker-treated group, both systolic pulmonary arterial pressure and right ventricular hypertrophy were significantly reduced, and arteriolar hypertrophy was also prevented. Thus, angiotensin II-forming chymase may play a role in the proliferation of the medial layer in the lung arterioles of monocrotaline-induced pulmonary hypertensive rats. (Pediatr Res 60: 77–82, 2006) C DOI: 10.1203/01.pdr.0000219431.45075.d9 77 78 KISHI ET AL. tensin II formation plays a pathophysiological role in vascular remodeling. In this study, we clarified the pathophysiological role of chymase-dependent angiotensin II formation in monocrotaline-induced PH rats. METHODS Materials. Monocrotaline were purchased from Sigma Chemical Co. (St. Louis, MO). An ARB candesartan was kindly provided by Takeda Chemical Industries (Osaka, Japan). An ACE inhibitor trandolaprilat was gifted from Hoechst Marion Roussel (Romainville, France). A chymase inhibitor NK3201 was gifted from Nippon Kayaku (Tokyo, Japan). NK3201 inhibited human, dog, rat and hamster chymases by IC50 at concentrations of 2.5, 1.2, 19 and 28 nM, respectively, but has no inhibitory activity to ACE, tryptase, thrombin, elastase, plasmin and plasminogen activator (9). Hemodynamics and medial thickness index. A total of 34 male SpragueDawley rats aged 6 wk, weighing 268 ⫾ 4 g at the beginning of the experiment, were obtained from Japan SLC Inc. (Shizuoka, Japan). The rats were fed a regular hamster chow diet, had free access to tap water, and were housed in a temperature-, humidity-, and light-controlled room. The experimental procedures were done in accordance with the Guide for the (...truncated)