Keiko Shimoura

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.

Role of nitric oxide from the endothelium on the neurogenic contractile responses of rabbit pulmonary artery

The effects of L-NG-nitro arginine (L-NO2Arg), a stereospecific inhibitor of nitric oxide formation, on the responsiveness of rabbit pulmonary artery to transmural electrical stimulation were studied. The contractile response evoked by electrical stimulation at 4 Hz was abolished by tetrodotoxin (10(-7) M) and depressed to approximately 10% by bunazosin (10(-6) M), an alpha 1-antagonist. Pretreatment with L-NO2Arg (10(-5) M) significantly potentiated the response to electrical stimulation without changing the resting tension. D-NO2Arg (10(-5) M) did not show such a potentiating action. In endothelium-denuded arteries, L-NO2Arg did not potentiate the response to electrical stimulation. The effect of L-NO2Arg on endogenous noradrenaline release in response to electrical stimulation was also examined by HPLC with electrochemical detection; L-NO2Arg did not affect noradrenaline release. The contractions induced by exogenous noradrenaline (10(-6)-10(-5) M) were enhanced by L-NO2Arg, but not by D-NO2Arg. These results suggest that the vasoconstriction induced by sympathetic nerve stimulation in the rabbit pulmonary artery is modulated by endogenous nitric oxide or nitric oxide-like substances released from endothelial cells.

Endothelium-dependent and -independent relaxation by dopamine in the rabbit pulmonary artery

1. The relaxing response of dopamine (DA) was studied in the rabbit pulmonary artery. DA caused concentration‐related relaxation in helically cut strips of the artery contracted with prostaglandin F2α in the presence of prazosin.

The anti-allergic compound tranilast attenuates inflammation and inhibits bone destruction in collagen-induced arthritis in mice

Background and purpose:  Recent findings suggest the importance of mast cells in the pathogenesis of rheumatoid arthritis and their potential as a therapeutic target. Tranilast is an anti‐allergic compound with a potent membrane‐stabilizing effect on mast cells and a wide range of anti‐inflammatory effects, thus may be advantageous in the treatment of arthritis. Here, we have evaluated the effects of tranilast on the progression of collagen‐induced arthritis in mice.

AGE-RELATED CHANGES IN AORTIC SENSITIVITY TO NORADRENALINE AND ACETYLCHOLINE IN RATS

1. The purpose of the present study was to determine the relationship between plasma and tissue lipid levels and the effects of age on vascular responses to noradrenaline (NA) and acetylcholine (ACh).

Evidence for Elevated Levels of Dopamine in the Rabbit Pulmonary and Carotid Artery

Catecholamine levels in six arteries, two aortae, and atria of rabbits were determined by high-performance liquid chromatography (HPLC) coupled with electrochemical detection. An appreciable concentration of dopamine (DA) was found in the pulmonary and carotid arteries. The content in these two arteries was >200 ng/g, although the mesenteric, celiac, renal, and femoral arteries had <55 ng/g. The ratio of DA to norepinephrine (NE) in the pulmonary artery was 32.2 ± 6.3%, and that in the carotid artery was 9.0 ± 3.8%. These values are equivalent to the ratio in the canine paw pad and kidney, which are considered to be innervated by a dopaminergic system among peripheral tissues. The presence of DA in the pulmonary artery was confirmed by gas chromatography and mass spectrometry. In addition, uptake and distribution of [3H]DA and significant enhancement of [3H]DA release by transmural stimulation in the pulmonary artery were observed. These results suggested the possibility of dopaminergic innervation in the rabbit pulmonary and, possibly, carotid arteries.

Effects of thyrotropin-releasing hormone and its analogue, NS-3, on blood pressure, heart rate, and serum catecholamine levels in rats

1. Thyrotropin-releasing hormone (TRH) and its metabolically stable analog NS-3 (montirelin hydrate; [3R, 6R]-6-methyl-5-oxo-3-thiomorpholinyl carbonyl-L-histidyl-L-prolinamide tetrahydrate) produced dose-dependent increases in blood pressure, heart rate, and the levels of serum noradrenaline and adrenaline in rats. 2. The pressor and tachycardiac effects and increases in serum catecholamine levels caused by NS-3 were longer lasting, but not more potent than those caused by TRH. 3. NS-3 did not change the blood pressure, heart rate, or the serum catecholamine levels in either pithed or hexamethonium-pretreated rats.

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.