Hideki Okunishi

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.

Effects of montelukast, a cysteinyl-leukotriene type 1 receptor antagonist, on the pathogenesis of bleomycin-induced pulmonary fibrosis in mice.

Cysteinyl-leukotrienes are potent mediators involved in various inflammatory diseases and lung disorders such as asthma. However, their precise role in the pathogenesis of pulmonary fibrosis is unknown. In the present study, we investigated the effect of montelukast, a cysteinyl-leukotriene type 1 receptor antagonist, on bleomycin-induced pulmonary fibrosis in mice. Montelukast (10mg/kg/day) was orally administered to the bleomycin-induced pulmonary fibrosis mice for 3days before and 14days after intratracheal instillation of bleomycin. We evaluated the effects of montelukast on the development of pulmonary fibrosis in these mice and investigated the expression of various cytokines and two cysteinyl-leukotriene receptors. Treatment with montelukast significantly attenuated the increased fibrotic area and hydroxyproline content in the fibrotic lungs of bleomycin-instilled mice. Montelukast treatment also decreased mRNA levels of IL-6, IL-10, IL-13, and TGF-β1, all of which were elevated in fibrotic lungs. In fibrotic lungs, TNF-α and IL-1β mRNA levels were increased and IFN-γ mRNA levels were decreased, but montelukast did not affect these mRNA levels. Furthermore, cysteinyl-leukotriene type 1 receptor mRNA levels were increased, whereas cysteinyl-leukotriene type 2 receptor mRNA levels were decreased in fibrotic lungs. Montelukast treatment induced the recovery of cysteinyl-leukotriene type 2 receptor mRNA levels to normal control levels but did not change cysteinyl-leukotriene type 1 receptor mRNA levels. These results suggest that montelukast exhibits its beneficial effects by inhibiting the overexpression of IL-6, IL-10, IL-13, and TGF-β1 and by modulating the homeostatic balance between the cysteinyl-leukotriene type 1 and type 2 receptors.

Proteomic analysis of calcified abdominal and thoracic aortic aneurysms

Aortic aneurysm is a complex multifactorial disease with genetic and environmental risk factors. It is often accompanied by aortic calcification. Here, to uncover proteins that are significantly changed in calcified abdominal aortic aneurysms (CAAs) and calcified thoracic aortic aneurysms (CTAs) compared with those in adjacent normal aorta tissues, comprehensive analysis of differentially expressed proteins in their tissues was performed by a quantitative proteomic approach with iTRAQ labeling in combination with nanoLC-MALDI-TOF/TOF-MS/MS followed by ProteinPilot analysis. The proteomic analysis revealed 138 and 134 proteins differentially expressed in CAAs and CTAs in contrast to neighboring normal aorta tissues with high confidence, respectively. Significantly increased expression (≥1.3-fold) was found in 41 and 28 proteins, whereas decreased expression (<0.77-fold) was found in 4 and 60 proteins in CAAs and CTAs, respectively. Among them, we identified already known proteins involved in aneurysm formation and vascular calcification, such as type I and III collagen, matrix Gla protein, and α-2-HS-glycoprotein in CAAs and fibrinogen α, β and γ chains and α-2-HS-glycoprotein in CTAs with increased expression and mimecan in CAAs and fibulin-5 in CTAs with decreased expression. Based on the Panther pathway and Genesis clustering analyses, some of the proteins could be linked to corresponding biochemical pathways, such as the integrin signaling pathway with increased expression in CAAs, the blood coagulation pathway with increased expression in CTAs, and the inflammation mediated by chemokine and cytokine signaling pathway and the glycolysis pathway with decreased expression in CTAs. Interestingly, it was found by clustering analysis that samples from CAAs of patients with both CAAs and CTAs were clustered outside the samples of patients with CAAs and were clustered with samples of patients with CTAs. Our results provide a comprehensive patient-based proteomic analysis for the identification of potential biomarkers for CAAs and CTAs.

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.

A disynaptic pathway from the central amygdaloid nucleus to the paraventricular hypothalamic nucleus via the parastrial nucleus in the rat

The organization of projections from the central amygdaloid nucleus (CeA) to the paraventricuilar hypothalamic nucleus (PVH) has been studied in order to understand the anatomical substrates of amygdaloid modulation of endocrine and autonomic functions, and a hypothesis that the bed nucleus of the stria terminalis (BST) may act as a relay site between the CeA and PVH has been proposed. Using anterograde and retrograde tract-tracing methods, in the rat, we first indicated that neurons in the parastrial nucleus (PS), where projection fibers from the central amygdaloid nucleus (CeA) terminated, sent their axons to the paraventricular hypothalamic nucleus (PVH). We further demonstrated that the CeA terminals formed symmetrical synaptic contacts with somata and dendrites of the PVH-projecting PS neurons, and that the PS received CeA fibers predominantly from the lateral part and sent large numbers of projection fibers to almost all the subdivisions of the PVH. Using anterograde tracing combined with the postembedding immunogold method, we finally revealed that nearly all the CeA terminals in the PS were immunoreactive for gamma-aminobutyric acid. The present data suggest that output signals from the CeA are transmitted disynaptically to the PVH neurons via the PS neurons and modulate PVH neuron activity by way of disinhibition.

Pranlukast, a cysteinyl leukotriene type 1 receptor antagonist, attenuates the progression but not the onset of silica-induced pulmonary fibrosis in mice.

Background: Although cysteinyl leukotrienes (CysLTs) have been implicated in the etiology of acute inflammatory diseases, recent studies have suggested that they also directly stimulate fibroblasts. However, their precise role in the pathogenesis of pulmonary fibrosis is unclear. Methods: In this study, we evaluated the effect of both short- and long-term treatment with pranlukast, a CysLT type 1 (CysLT1) receptor antagonist, on silica-induced pulmonary fibrosis in mice, which is characterized by persistent progression of fibrosis in the chronic phase. Pranlukast (30 mg/kg/day) was administered orally to mice for 2 or 10 weeks after intratracheal silica instillation. Results: Pranlukast treatment for 10 weeks significantly attenuated the progression of pulmonary fibrosis, and decreased the content of CysLTs and LTB4, which were markedly increased in the bronchoalveolar lavage fluid (BALF) and lung tissues of silica-instilled mice in the chronic phase. However, pranlukast treatment for 2 weeks neither affected the acute inflammatory response induced by silica instillation nor inhibited the onset of fibrosis. The expression of TGF-β1 and TNF-α was not affected by pranlukast treatment for either 2 or 10 weeks. Conclusions: Pranlukast attenuates the progression of pulmonary fibrosis in the chronic phase but has no effect on the acute inflammatory response or on the onset of pulmonary fibrosis. The antifibrotic effect of pranlukast may be exhibited by antagonizing the direct profibrotic effect of CysLTs, without affecting the expression of other profibrotic cytokines such as TGF-β1 and TNF-α, and also by decreasing the production of CysLTs and LTB4.

Involvement of leukotrienes in the pathogenesis of silica-induced pulmonary fibrosis in mice

ABSTRACT The authors investigated the role of leukotrienes (LTs) in the pathogenesis of silica-induced pulmonary fibrosis in mice during the progression from acute to chronic phases. Intratracheal instillation of silica particles induced progressive pulmonary fibrosis. The tissue content of cysteinyl (Cys) LTs and LTB4 was markedly increased in the acute phase after silica instillation, concurrently with the up-regulation of LTB4 receptor, transforming growth factor (TGF)-β1, and tumor necrosis factor (TNF)-α, along with down-regulation of the CysLT type 2 receptor. Importantly, the tissue content of CysLTs and mRNA levels of TGF-β1 and TNF-α were increased in the fibrotic lung in the chronic phase. Furthermore, strong immunohistochemical staining for the CysLT type 1 receptor, TNF-α, and TGF-β1, but not for the CysLT type 2 receptor, was codetected in the pathological lesions during both acute and chronic phases. These findings suggest that an increase in LT production in the lung and modulation of homeostatic balance among LT receptors may contribute to the progression of pulmonary fibrosis.

Proteomic comparison between abdominal and thoracic aortic aneurysms

The pathogenesis of abdominal aortic aneurysms (AAAs) and that of thoracic aortic aneurysms (TAAs) is distinct. In this study, to reveal the differences in their biochemical properties, we performed quantitative proteomic analysis of AAAs and TAAs compared with adjacent normal aorta (NA) tissues. The proteomic analysis revealed 176 non-redundant differentially expressed proteins in the AAAs and 189 proteins in the TAAs which were common in at least 5 samples within 7 samples of each. Among the identified proteins, 55 and 68 proteins were unique to the AAAs and TAAs, respectively, whereas 121 proteins were identified in both the AAAs and TAAs. Panther overrepresentation analysis of the unique proteins in the AAAs and TAAs revealed a significant downregulation of the blood coagulation pathway in the AAAs and that of the integrin signaling pathway in the TAAs. On the other hand, Genesis analysis revealed distinct expression patterns of 58 proteins among the 121 proteins. Panther overrepresentation analysis of these 58 proteins revealed that the expression of these proteins in the blood coagulation and the plasminogen activating cascade was decreased in the AAAs, whereas it was increased in the TAAs compared with the NA tissues. On the other hand, the protein expression in the integrin signaling pathway was increased in the AAAs, whereas it was decreased in the TAAs compared with the NA tissues. Thus, the data presented in this study indicate that the proteins that show differential expression patterns in AAAs and TAAs may be involved in the distinct pathogenesis of AAAs and TAAs.