Naotaka Shiota

Activation of paracrine TGF-β1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase

As a source of transforming growth factor β1 (TGF‐β1), mast cells have been implicated as potential effector cells in many pathological processes. However, the mechanisms by which mast cells express, secrete, and activate TGF‐β1 have remained vague. We show here by means of RT‐PCR, immunoblotting, and immunocytochemistry that isolated rat peritoneal mast cells synthesize and store large latent TGF‐β1 in their chymase 1‐containing secretory granules. Mast cell stimulation and degranulation results in rapid secretion of the latent TGF‐β1, which is converted by chymase 1 into an active form recognized by the type II TGF‐β serine/ threonine kinase receptor (TβRII). Thus, mast cells secrete active TGF‐β1 by a unique secretory mechanism in which latent TGF‐β1 and the activating enzyme chymase 1 are coreleased. The activation of latent TGF‐β1 specifically by chymase was verified using recombinant human latent TGF‐β1 and recombinant human chymase. In isolated TβRI‐ and TβRII‐expressing peritoneal macrophages, the activated TGF‐β1 induces the expression of the plasminogen activator inhibitor 1 (PAI‐1), whereas in the mast cells, the levels of TβRI, TβRII, and PAI‐1 expression were below detection. Selective stimulation of mast cells in vivo in the rat peritoneal cavity leads to rapid overexpression of TGF‐β1 in peritoneal mast cells and of TβRs in peritoneal macrophages. These data strongly suggest that mast cells can act as potent paracrine effector cells both by secreting active TGF‐β1 and by enhancing its response in target cells.—Lindstedt, K. A., Wang, Y., Shiota, N., Saarinen, J., Hyytiäinen, M., Kokkonen, J. O., Keski‐Oja, J., Kovanen, P. T. Activation of paracrine TGF‐β1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase. FASEB J. 15, 1377–1388 (2001)

Induction of chymase that forms angiotensin II in the monkey atherosclerotic aorta

Chymase shows a catalytic efficiency in the formation of angiotensin (Ang) II. In the present study, the characterization and primary structure of monkey chymase were determined, and the pathophysiological role of chymase was investigated on the atherosclerotic monkey aorta. Monkey chymase was purified from cheek pouch vascular tissue using heparin affinity and gel filtration columns. The enzyme rapidly converted Ang I to Ang II (K m=98 μM, k cat=6203/min) but did not degrade several peptide hormones such as Ang II, substance P, vasoactive intestinal peptide and bradykinin. The primary structure, which was deduced from monkey chymase cDNA, showed a high homology to that of human chymase (98%). The mRNA levels of the aorta chymase were significantly increased in the atherosclerotic aorta of monkeys fed a high‐cholesterol diet. These results indicate that monkey chymase has a highly specific Ang II‐forming activity and may be related to the pathogenesis of atherosclerosis.

Purification and characterization of angiotensin II-generating chymase from hamster cheek pouch

Hamster cheek pouch vascular tissues contain an angiotensin II-forming enzyme which is inhibited by chymostatin but not by any angiotensin-converting enzyme inhibitors. The enzyme was purified to apparent homogeneity by gel filtration and heparin-Sepharose affinity chromatography. The molecular mass estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis was 28 kDa and the optimum pH was between 7.5 and 9.0. The angiotensin II-forming activity was inhibited by chymostatin, soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, but not by aprotinin. The N-terminal sequence showed high homology with chymases from various species. Thus, the angiotensin II-generating enzyme obtained from hamster cheek pouch vessels is a chymase.

Characterization of chymase from human vascular tissues

A chymostatin-sensitive angiotensin II-generating enzyme was found in human gastroepiploic arteries. The enzyme was purified using heparin affinity and gel filtration columns. The molecular mass of the purified enzyme was 30 kDa, and the optimum pH was between 7.5 and 9.0. Enzyme activity was inhibited by soybean trypsin inhibitor, phenylmethylsulfonyl fluoride and chymostatin, but not by ethylenediaminetetraacetic acid, pepstatin and aprotinin. The enzyme rapidly converted angiotensin I to angiotensin II (K(m), 67 mumol/l; Vmax, 43 pmol/s, kcat, 65/s), but did not hydrolyse angiotensin II, substance P, bradykinin, vasoactive intestinal peptide, luteinizing hormone-releasing hormone, somatostatin and alpha-melanocyte-stimulating hormone. The N-terminal sequence was identical to the sequence for human skin/heart chymase. Thus, the chymostatin-sensitive angiotensin II-generating enzyme in human vascular tissues is identified as chymase.

Activation of two angiotensin-generating systems in the balloon-injured artery

Participation of angiotensin II in the myointimal proliferation following a vascular injury was postulated. This study assessed the potential involvement of the local angiotensin II‐forming enzymes in injured arteries of dogs. The potential angiotensin II‐forming enzymes are angiotensin‐converting enzyme (ACE) and chymostatin‐sensitive angiotensin II‐generating enzyme (CAGE) which is highly homologous to or could be identical to the mast cell chymase. Both ACE and CAGE catalyze the conversion of angiotensin I to angiotensin II. We found that the enzymatic activities of ACE and CAGE, and the mRNA levels of ACE and chymase were increased in the injury‐induced hypertrophied vessels. The results suggest that ACE and CAGE participate in the hypertrophy through the production of angiotensin II which is a growth promoter for vascular smooth muscle cells.

Tranilast Suppresses Vascular Chymase Expression and Neointima Formation in Balloon-Injured Dog Carotid Artery

BACKGROUND Activation of vascular chymase plays a major role in myointimal hypertrophy after vascular injury by augmenting the production of angiotensin (ANG) II. Because chymase is synthesized mainly in mast cells, we assumed that the chymase-dependent ANG II formation could be downregulated by tranilast, a mast cell-stabilizing antiallergic agent. We have assessed inhibitory effects of tranilast on neointima formation after balloon injury in the carotid artery of dogs, which share a similar ANG II-forming chymase with humans, and further explored the pathophysiological significance of vascular chymase. METHODS AND RESULTS Either tranilast (50 mg/kg BID) or vehicle was orally administered to beagles for 2 weeks before and 4 weeks after balloon injury. Four weeks after the injury, remarkable neointima was formed in the carotid arteries of vehicle-treated dogs. Chymase mRNA levels and chymaselike activity of vehicle-treated injured arteries were increased 10.2- and 4.8-fold, respectively, those of uninjured arteries. Angiotensin-converting enzyme (ACE) activity was slightly increased in the injured arteries, whereas ACE mRNA levels were not. Tranilast treatment completely prevented the increase in chymaselike activity, reduced the chymase mRNA levels by 43%, and decreased the carotid intima/media ratio by 63%. In vehicle-treated injured arteries, mast cell count in the adventitia showed a great increase, which was completely prevented by the tranilast treatment. Vascular ACE activity and mRNA levels were unaffected by tranilast. CONCLUSIONS Tranilast suppressed chymase gene expression, which was specifically activated in the injured arteries, and prevented neointima formation. Suppression of the chymase-dependent ANG II-forming pathway may contribute to the beneficial effects of tranilast.

Activation of angiotensin II-forming chymase in the cardiomyopathic hamster heart

Background Angiotensin (ANG) II plays crucial roles in promoting cardiovascular tissue remodeling. Human chymase catalyzes ANG II formation, whereas rat chymase (rat mast cell protease I) degrades ANG I to inactive fragments. Such species differences should be considered when the functions of chymase in human cardiovascular diseases are investigated assuming an analogy with animal models. Objective To further characterize the recently identified ANG II-forming hamster chymase, and to analyze pathophysiologic roles played by chymase in the cardiomyopathy of the hamster. Methods The gene organization and the primary structure of hamster chymase were determined through molecular cloning. Chymase and angiotensin converting enzyme messenger RNA levels, and chymase-like and angiotensin converting enzyme activities were measured in the heart of BIO 14.6 cardiomyopathic hamsters aged 4, 12, and 25 weeks. Results The hamster chymase gene is 3 kb long. It has five exons and four introns, and the deduced amino-acid sequence was homologous to other mammalian chymases. The chymase messenger RNA levels and chymase-like activities in the BIO 14.6 hamster hearts were increased significantly at the ages of 12 weeks (the fibrotic stage) and 25 weeks (the hypertrophic stage), but not at age 4 weeks (the premyolytic stage). Conclusions These results indicate that heart chymase is activated concurrently with the development of cardiomyopathy. Thus, we conclude that heart chymase could play the primary role in accelerating ANG II formation, thereby causing deleterious changes in the cardiomyopathic heart.

Increase of angiotensin converting enzyme gene expression in the hypertensive aorta

To investigate the possible role of vascular angiotensin converting enzyme (ACE) in the development and maintenance of hypertension, we examined aortic ACE messenger RNA (mRNA) levels in two-kidney, one clip (2K1C) hypertensive rats. The blood pressure was increased remarkably at 4 weeks (early stage) after clipping and remained elevated at 12 weeks (chronic stage). The aorta ACE mRNA levels were significantly elevated in both early and chronic stages concurrently with the increases in aortic ACE activity and blood pressure. The plasma renin activity rose markedly at 4 weeks, but returned to the normal level at 12 weeks. Neither ACE activity in the lung and plasma, nor ACE mRNA level in the lung was altered at either stage. The aorta and liver angiotensinogen mRNA levels and renal renin mRNA level were increased at 4 weeks but decreased at 12 weeks. These results indicate that the acceleration of all components in the renin-angiotensin system may contribute to the development of 2K1C hypertension in the early stage. In the chronic stage, the increased vascular ACE induced by the elevated ACE mRNA levels in the aorta may play the primary role in the acceleration of local angiotensin II formation and thus may sustain the hypertension.

Induction of angiotensin converting enzyme and angiotensin II receptors in the atherosclerotic aorta of high-cholesterol fed Cynomolgus monkeys

Antiatherogenic effects of imidapril and involvement of renin angiotensin system were examined in experimental atherosclerosis induced by feeding a high-cholesterol diet to Cynomolgus monkeys. Eighteen male monkeys were divided into three groups and placed under (1) normal diet (normal group), (2) high-cholesterol diet (control group), (3) high-cholesterol diet with imidapril (20 mg/kg body wt/day, orally) treatment (imidapril group). At the end of the experiment, the normal group showed no apparent atherosclerosis in their aorta evaluated by oil red-O staining, while the control group exhibited marked atherosclerotic involvement of the intimal surface of the aorta (58.4 +/- 9.3%, P < 0.01). Imidapril reduced systolic blood pressure and atherosclerotic involvement (24.1 +/- 5.5%, P < 0.05). Total cholesterol content of the descending thoracic aorta was also significantly reduced in the imidapril group. In the atherosclerotic vessels, angiotensin converting enzyme (ACE) activity evaluated by quantitative in vitro autoradiography was significantly increased in the intimal lesion. Further evaluation revealed angiotensin II (Ang II) type I (AT1) receptor density was significantly increased in the medial lesion and type II (AT2) receptor density in the adventitia. When the progression of atherosclerosis was impeded by imidapril treatment, the ACE activity level as well as the AT1 and AT2 receptor density remained at normal. Expression of mRNA for fibronectin, TGF-beta1, types I and III collagen was studied by Northern blot analysis. No significant differences in types I and III collagen mRNA levels were found between the control and imidapril group. On the other hand, mRNA expression for fibronectin and TGF-beta1 were much lower in the imidapril group than in the control group. These results suggest that increased production of Ang II and activated receptors may be involved in atherosclerotic process in this model and also antiatherogenic effect of imidapril may be derived from reduction of local Ang II production as well as its hypotensive action.

Chymase is activated in the hamster heart following ventricular fibrosis during the chronic stage of hypertension

Chronic pressure overload induces cardiac tissue remodeling. Chymase is known to regulate matrix metabolism and angiotensin II formation. In the present study, we investigated the pathophysiological functions of chymase in the pressure‐overloaded hamster heart induced by a two‐kidney, one‐clip (2K1C) hypertension procedure. Fibrosis and apoptosis were observed in the pressure‐overloaded hearts of 2K1C hamsters 32 weeks after clipping, but these histological changes were not detected at 16 weeks. Heart chymase‐like activity of 2K1C hamsters at 32 weeks increased 5.2‐fold compared with that at 16 weeks, while angiotensin‐converting enzyme was not activated. Chymase might be involved in cardiac tissue remodeling during the chronic stage of hypertension.