Eiichi Kakizoe

Skin mast cell promotion of matrix remodeling in burn wound healing in mice: relevance of chymase

Abstract Inflammation, granulation, and collagen accumulation, which are observed in the wound healing process, occasionally lead to hypertrophic scarring. Several in vitro reports have suggested that skin mast cells (MCs) and their major protease, chymase, participate in the healing process as well as in fibrotic skin diseases. The present study examined the potential involvement of MCs and MC chymase in the healing of burns in mouse dorsal skin. The size of the burn wounds, density of the capillaries, collagen accumulation, MC number, and chymase activity were measured before and 1, 3, 7, and 14 days after burning. The healing process corresponded strongly with MC density and chymase activity in both acute and subacute phases. The maximum decrease in MC number and chymase activity occurred on day 3 when tissue loss due to necrosis was maximal. From day 7 to 14, the burn wounds retracted rapidly accompanied by increases in capillaries and collagen fibers, in correspondence with fast increments in MC numbers and chymase activity at the wound edges. The present results combined with previous in vitro results strongly support the contention that skin MC chymase plays a role in the normal wound healing process, and presumably in dermal fibrotic disorders.

Pathophysiological Role of Skin Mast Cells in Wound Healing after Scald Injury: Study with Mast Cell-Deficient W/WV Mice

Background: The major role of mast cells in wound healing process has not been identified. In this study, we used mast cell-deficient W/WV mice and their congenic control (+/+) mice to examine the role of mast cells in scald wound healing. Methods: The size of the scald wound, thickness of the dermis, collagen deposition, vascularization, number of mast cells and chymase activity were measured before and at 3, 7, 14 and 21 days after inducing scald injury. Results: Although the process of wound closure and re-epithelialization was not markedly different between W/WV mice and +/+ mice, the degree of fibrous proliferation at the wound edge and wound vascularization in the proliferative phase was significantly lower in W/WV mice than in +/+ mice, and no vascular regression in the late remodeling phase was observed in W/WV mice. Mast cells producing chymase, FGF2, TGF-β1 and VEGF were highly accumulated at the edge of scald wound in +/+ mice during the proliferative and remodeling phases at days 14 and 21. Chymase activity in the injured tissues of +/+ mice decreased in the acute phase, but recovered to no-injury level at days 14 and 21. The number of mast cells and chymase activity were very low in the injured tissues of W/WV mice throughout the experiment. Conclusions: Wound healing after skin scald injury was partially impaired in mast cell-deficient mice. Mast cells may contribute to the wound healing process, especially in the proliferative and remodeling phases after scald injury.

Effect of mast cell chymase inhibitor on the development of scleroderma in tight-skin mice

1 Although the pathogenesis of scleroderma is not fully understood, activation of connective‐tissue‐type mast cells (CTMCs) has been implicated in various fibrotic diseases. 2 Our previous study showed that the number of CTMCs was markedly increased during fibrous proliferation in the skin of a scleroderma model, namely tight‐skin (Tsk) mice. Because mast cells express numerous bioactive factors, such as cytokines, growth factors, proteases, and others, it is crucial to identify the primary factors that may be involved in the pathogenesis of scleroderma. Our previous study also showed that a CTMC‐specific protease, chymase‐4, was selectively upregulated in accordance with the development of skin fibrosis in Tsk mice. 3 To further elucidate the role of chymase secreted from CTMCs, we evaluated the therapeutic effects of a synthetic chymase‐specific inhibitor, SUN‐C8257, on the development of skin fibrosis in Tsk mice. SUN‐C8257 (50 mg kg−1 day−1) was administered via intraperitoneal injection in 13‐week‐old Tsk mice for a period of 2 weeks. 4 Treatment with SUN‐C8257 significantly reduced chymase activity by 43% and the chymase‐4 mRNA level by 47%, and also decreased the thickness of the subcutaneous fibrous layer of Tsk mice by 42% compared with that of Tsk mice injected with vehicle. 5 Furthermore, immunohistochemical analysis revealed that transforming growth factor (TGF)‐beta1 staining in the fibrous layer of Tsk skin was markedly reduced by the treatment with SUN‐C8257. This chymase inhibitor may prevent the chymase‐dependent pathway that activates the latent TGF‐beta1 in fibrous tissue, and may exhibit beneficial effects that inhibit the development of fibrosis. 6 In conclusion, our results strongly support the assumption that CTMC‐derived chymase may play a key role in the pathogenesis of scleroderma.

COMPARISON OF VASOPRESSOR EFFECTS OF NITRO ARGININE IN STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RATS AND WISTAR-KYOTO RATS

1. NG‐nitro‐L‐arginine (NO2Arg) is a guanidine nitro arginine derivative and an inhibitor of endothelium‐dependent vascular relaxation. Significant rise of the systolic blood pressure was observed after 1 week administration of NO2Arg in food (0.023% in weight, about 2.8 mg of NO2Arg/rat per day) in female rats of stroke‐prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar‐Kyoto rats (WKY). The rises were not different between SHRSP (21 mmHg) and WKY (23 mmHg).

Spectrophotometric study of α-adrenoceptors affecting microcirculation of rat skin

1. To determine the α‐adrenergic receptor subtypes that affect the microcirculation of skin, the relative absorption (RA) spectra of the skin on the backs of rats were measured using reflectance spectrophotometric methods. We injected α‐adrenergic agonists, noradrenaline (NA), phenylephrine (PE) and clonidine (CL), intravenously and determined changes in the RA value at 569 nm, one of the isosbestic points of the oxyhaemoglobin and deoxyhaemoglobin absorption.

ACETYLCHOLINE‐INDUCED SYSTEMIC VASODILATION RESISTANT TO NG‐NITRO‐L‐ARGININE IN ANAESTHETIZED RATS

1. The effects of NG‐nitro‐L‐arginine (l‐NNA; 20mg/kg bodyweight (BW), i.v.) and metyrapone (300 mg/kg BW, s.c.) on acetylcholine (ACh)‐induced depressor responses were investigated in anaesthetized rats.

CARDIOTOXIC INTERACTION OF METABOLITES FROM A PRODRUG SEGMENT CILEXETIL (CYCLOHEXYLOXY-CARBONYLOXY-ETHYL) WITH DIGOXIN IN THE CANINE FAILING HEART

Potential risks of cyclohexanol (CH) and cyclohexanediol (CHD) isomers, which are the metabolites derived from cilexetil ester side-chain of several prodrugs such as antibiotics (e.g. cefotiam hexetil) and an antihypertensive agent (candesartan cilexetil), were examined in beagles that were made congestive heart failure (CHF) by rapid ventricular pacing. The following three experiments tested the cardiac effects of i.v. doses of: (1) the metabolites alone, (2) the metabolites under the digoxin-induced bradycardia, and (3) the metabolites given concomitantly with digoxin (0.02 mg kg(-1)). Experiment 1: t-1,2- or 1,4-CHD alone (0.1-12 mg kg(-1)) exerted transient yet reproducible supraventricular or ventricular arrhythmia dose-dependently, whereas CH and 1,3-CHD at 12 mg kg(-1) showed no cardiac effect at all. Experiment 2: t-1,2-CHD (0.1-4 mg kg(-1)), but not CH or 1,3-CHD, induced the additive arrhythmia dose-dependently; t-1,2-CHD (12 mg kg(-1)) caused frequent premature supraventricular contractions and/or irreversible paroxysmal supraventricular tachycardia. Experiment 3: t-1,2-CHD, not CH or 1,3-CHD, caused fatal arrhythmia: one dog showed torsade de pointes followed by ventricular fibrillation, while another showed 3rd degree atrioventricular block and eventually cardiac arrest. In both Experiments 2 and 3, saline vehicle added onto digoxin never caused the irreversible, fatal arrhythmia. In a separate study using healthy dogs without CHF, none of these metabolites did produce cardiac effect. Given the potential risk of generating cardiotoxic metabolites from cilexetil-bearing prodrugs, the use of such prodrugs should be avoided from the patients with CHF, particularly from those who are receiving cardiac glycosides.

EFFECT OF NG-M0N0METHYL-L-ARGININE ON THE MICROCIRCULATION OF RAT SKIN

1. The effects of NG‐monomethyl‐L‐arginine (L‐NMMA), a nitric oxide (NO) synthase inhibitor, on the microcirculation of rat dorsal skin were studied to examine the role of NO in the regulation of regional haemodynamics.