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Allergic granulomatous angitis (AGA) is characterized by bronchial asthma, eosinophilia and systemic necrotizing vasculitis involving medium and small-sized vessels with or without granulomas.[1-3] AGA causes serious organ damages, including skin, nerves, digestive canals, lungs and so on. To date, an effective therapy has not been established despite many clinical trials.
The mechanism of AGA is not completely understood. Eosinophils are the most dominant cells in the blood and extravascular tissues in AGA, and are known to release cytotoxic products such as major basic proteins, eosinophil-derived neurotoxins and oxygen radicals.[4, 5] In this regard, endothelial cell injury triggered by eosinophils has been considered to be the initial step toward the vasculitis of AGA.
We previously reported a murine model of pulmonary allergic vasculitis, which was induced by repeated inhalation of ovalbumin (OVA) in C57BL/6 mice sensitized with OVA. We observed that small pulmonary arteries were occluded with accumulated myofibroblasts and collagen deposition on the seventh day.
Imatinib mesylate (IM) is a potent and specific tyrosine kinase inhibitor against the tyrosine kinases c-ABL, BCR-ABL and c-KIT. IM has been demonstrated to be highly active in chronic myeloid leukemia and gastrointestinal stromal tumors.[7-10] The reported data regarding the specificity of IM for various tyrosine kinases show that IM also specifically inhibits platelet-derived growth factor receptor (PDGFR) tyrosine kinase. It is known that PDGF acts as a chemotactic factor and growth factors for vascular smooth muscle and fibroblasts.[12, 13]
In this regard, we examined the effects of IM on the histological changes of allergic vasculitis in this model. The result of this study may contribute to finding a therapy for allergic vasculitis.
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- Methods and Materials
- Declaration of Interest
The present study demonstrated that IM suppressed the histological changes of allergic vasculitis in the pulmonary arteries of OVA-exposed mice and reduced the number of eosinophils in BALF without changing IL-4 or IL-5 concentrations in the BALF.
IM is a potent and specific tyrosine kinase inhibitor against Abl, Bcr/Abl, Kit, and PDGF receptor-α (PDGFRA) and -β (PDGFRB) tyrosine kinases. Recently, IM has been demonstrated to be highly effective for the treatment of a subgroup of patients with hypereosinophilic syndrome (HES) or clonal eosinophilia, including systemic mast cell disease (SMCD).[16-20]
In the present study, we used the murine model of pulmonary allergic vasculitis which we previously reported as an animal model of AGA. The following histopathological changes in our murine model resembled allergic granulomatous angiitis in humans: (i) infiltration of mononuclear cells and eosinophils, and granuloma with multinuclear giant cells in the arterial wall; (ii) disruption of internal elastic layer; and (iii) obliteration of pulmonary arteries by mesenchymal cells. On the other hand, the fibrinoid degeneration of arterial walls observed in the human cases was not found in the murine model. As described above, the histopathological features of this model mouse are not completely the same as those of human AGA. However, our AGA mouse model is thought to be a useful animal model for analyzing human disease on the basis of its granulomatous pulmonary vasculitis accompanied by eosinophils infiltration with eosinophilia.
In order to elucidate the pathogenesis of vasculitis in this model, we tried to detect myeloperoxidase – antineutrophil cytoplasmic antibodies (MPO-ANCA) in the serum of our murine model against the recombinant murine MPO, but we could not detect an antibody against the recombinant murine MPO in the serum of the murine model. Therefore, to date, there is no evidence of ANCA-associated vasculitis in our murine model.
The proliferation and differentiation of eosinophils are known to be regulated by IL-5.[22, 23] As shown in the results, OVA exposure to sensitized mice induced marked increases in the number of eosinophils and IL-5 concentration in BALF, suggesting that IL-5 was a major inducer of eosinophil accumulation in BALF in our murine model of pulmonary allergic vasculitis. On the other hand, activation of c-kit also induces eosinophil activation and degranulation and proliferation that may be synergistic with IL-3, granulocyte macrophage–colony-stimulating factor (GM-CSF) and IL-5, and increased adhesion that could contribute to tissue localization. In addition, platelet-derived growth factor activates eosinophils. The present study demonstrated that the number of eosinophils in BALF was significantly reduced in OVA-exposed mice treated with IM compared to those not treated with IM. These results suggest that PDGF-A or B might have played a role in the pulmonary accumulation of eosinophils in our murine model of pulmonary allergic vasculitis. However, the reduction of the eosinophil number in BALF by IM was limited. This result suggested that eosinophilia was caused mostly by IL-5 and PDGF might have played a partial role in eosinophil accumulation in the lung in the present murine model. The pathways of PDGF and IL-5 were independent of each other. Therefore, IM is thought to have a limited role in suppressing the IL-5 pathway.
Although it has been known that neutrophils were thought to play a critical role in AGA, we could find very few neutrophils in the bronchoalveolar lavage fluid in our murine model. In addition, IM did not significantly suppress the number of alveolar macrophages. We believe the PDGF pathway was not strongly involved in the increase of alveolar macrophages in our murine model. Further study to elucidate the role of neutrophils and alveolar macrophages in this murine model is needed.
Drastic obstructive remodeling of small-sized pulmonary arteries was observed on the seventh day in the OVA-sensitized mice stimulated with repetitive OVA inhalation. The intraluminally accumulated cells were myofibroblasts which are spindle-shaped cells, and were positively stained with anti-actin antibody. These cells expressed Ki-67, suggesting that they were proliferating cells. Several growth factors have been reported to be involved in vascular smooth muscle cell proliferation. Among them, PDGF plays a critical role in chemotaxis and proliferation of vascular smooth muscle cells and myofibroblasts.[28-31] As shown in the results, OVA exposure to sensitized mice induced an increase of the PDGF concentration in the BALF, suggesting that the increased PDGF might be involved in the intraluminal myofibroblast proliferation. Danal et al. demonstrated that IM exerted suppressive effects on vascular smooth muscle cell proliferation in hypoxia-induced pulmonary hypertension in mice. In this case, IM was thought to inhibit tyrosine phosphorylation of the PDGF receptor, resulting in attenuation of the vascular smooth muscle cell proliferation.
IM has been also reported to be a possible therapeutic molecule for pulmonary fibrosis. Abdollahi et al. reported the increased PDGF molecules (PDGF-A, B, C, D) induced exaggerated fibroblast proliferation in radiation-induced pulmonary fibrosis. They also demonstrated that SU9518, as a PDGF receptor tyrosine kinase inhibitor, inhibited radiation-induced pulmonary fibrosis and reduced PDGF-β receptor phosphorylation.
The effects of IM were also evaluated in bleomycin-induced pulmonary fibrosis in mice. Aono et al. demonstrated that IM attenuated bleomycin-induced pulmonary fibrosis on days 7 and 14 without affecting the number of inflammatory cells in the BALF. They suggested that IM prevented the proliferation of mesenchymal cells, including murine lung fibroblasts, by inhibiting the autophosphorylation of PDGFR-β induced by PDGF. As shown in our results, a higher percentage of intraluminal myofibroblasts were positive for Ki-67 in our murine model of pulmonary allergic vasculitis and the treatment with IM reduced the percentage of Ki-67-positive cells which indicated proliferating cells.
TGF-β is also an important molecule that is involved in pulmonary fibrosis. In the present murine model of pulmonary allergic vasculitis, the concentration of TGF-β in BALF was strikingly high in OVA-exposed mice regardless of IM treatment. In this regard, TGF-β might also have played a role in the vascular remodeling in the present model. Concerning myofibroblast proliferation, Daniels et al. reported that fibroblasts respond to TGF-β by stimulating c-ABL kinase activity independently of Smad2/3 phosphorylation or PDGFR activation, and that inhibition of c-ABL by IM prevented TGF-β -induced ECM gene expression, morphologic transformation, and cell proliferation independently of any effect on Smad signaling. These findings suggest that in the present study, the inhibition of c-ABL by IM might have been involved in the inhibition of vascular remodeling in the mice treated with IM.
In conclusion, IM suppressed the vascular remodeling in a murine model of pulmonary allergic vasculitis by inhibiting the proliferation of vascular myofibroblasts.