Background: The major role of mast cells in wound healing process has not been identified. In this study, we used mast cell-deficient W/W^V 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/W^V mice and +/+ mice, the degree of fibrous proliferation at the wound edge and wound vascularization in the proliferative phase was significantly lower in W/W^V mice than in +/+ mice, and no vascular regression in the late remodeling phase was observed in W/W^V 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/W^V 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.

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.

1 Although the pathogenesis of scleroderma is not fully understood, activation of connective-tissuetype 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 À1 day À1) was administered via intraperitoneal injection in 13-weekold 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.

The involvement of connective-tissue-type mast cells and chymase, a protease unique to their secretory granules, has been implicated in fibrotic diseases. To elucidate the role of chymase in fibroproliferative inflammation, in this study we examined the enzymatic activity and mRNA expression of chymase in the sclerotic skin of tight-skin mice; syngeneic Pallid mice served as the control. Dorsal skin specimens from mice aged 5, 10, and 20 wk were evaluated by morphometric and biochemical analyses. At ages 10 and 20 wk, the hydroxyproline concentration in tight-skin dermis was higher than that in Pallid. At any age, the subcutaneous fibrous layer was thicker in tight-skin than in Pallid. In accordance with these fibrous changes, both connective-tissue-type mast cell counts and chymase activity were higher in tight-skin skin than in Pallid skin up to 20 wk of age. Age-matched (10-wk-old) tight-skin and Pallid were quantified for their mRNA of connective-tissue-type mast-cell-specific chymase, mouse mast cell protease-4, by the competitive reverse transcriptase polymerase chain reaction technique, which revealed its higher level in tight-skin than Pallid. In contrast, the mRNA level of mouse mast cell protease-5, the chymase isoform of undifferentiated mast cells, in tight-skin skin was only a tenth that of mouse mast cell protease-4 and no different from the mouse mast cell protease-5 mRNA level of Pallid mice. An in situ hybridization study confirmed the higher expression of mouse mast cell protease-4 by connective-tissue-type mast cells in tight-skin skin than Pallid skin. These results strongly support the contention that the connective-tissue-type mast cell chymase plays a crucial role in fibroproliferative remodeling of the skin.

The present results demonstrate increases in MCs and chymase in fibroproliferative paws of collagen-induced arthritic mice.