Inhibition of extracellular signal-regulated kinase pathway suppresses tracheal stenosis in a novel mouse model.

Tracheal stenosis is a refractory and recurrent disease induced by excessive cell proliferation within the restricted tracheal space. We investigated the role of extracellular signal-regulated kinase (ERK), which mediates a broad range of intracellular signal transduction processes in tracheal stenosis and the therapeutic effect of the MEK inhibitor which is the upstream kinase of ERK. We histologically analyzed cauterized tracheas to evaluate stenosis using a tracheal stenosis mouse model. Using Western blot, we analyzed the phosphorylation rate of ERK1/2 after cauterization with or without MEK inhibitor. MEK inhibitor was intraperitoneally injected 30 min prior to cauterization (single treatment) or 30 min prior to and 24, 48, 72, and 96 hours after cauterization (daily treatment). We compared the stenosis of non-inhibitor treatment, single treatment, and daily treatment group. We successfully established a novel mouse model of tracheal stenosis. The cauterized trachea increased the rate of stenosis compared with the normal control trachea. The phosphorylation rate of ERK1 and ERK2 was significantly increased at 5 min after the cauterization compared with the normal controls. After 5 min, the rates decreased over time. The daily treatment group had suppressed stenosis compared with the non-inhibitor treatment group. p-ERK1/2 activation after cauterization could play an important role in the tracheal wound healing process. Consecutive inhibition of ERK phosphorylation is a potentially useful therapeutic strategy for tracheal stenosis.

A rat tracheal model to investigate stent-induced tissue hyperplasia: a pilot study.

PURPOSE: To evaluate the feasibility of stent placement and the formation of stent-induced tissue hyperplasia and to verify matrix metalloproteinase (MMP) expression in stent-induced tissue hyperplasia in a rat tracheal model. MATERIALS AND METHODS: In a pilot study, four rats were used to verify MMP expression in stent-induced tracheal tissue hyperplasia based on zymography and Western blot analysis. In the main experiment, 12 rats were divided into two groups with different stent-mesh gap sizes (group I, 1.23 × 1.92 mm; group II, 0.80 × 1.80 mm). The stent was 4 mm in diameter and 8 mm long. Follow-up tracheogram was performed before sacrifice 3 weeks after stent placement. The gross and the microscopic findings were evaluated. RESULTS: Stent placement was technically successful in all rats. During follow-up, two rats (one from each group) died within 4 days after stent placement, and one stent in group II was placed outside the trachea. MMP-9 expression was significantly higher in tissue hyperplasia than in control normal tissue. The excised specimens showed tissue hyperplasia through the mesh, and all of the stents had become incorporated into the wall of the trachea. The papillary projection thickness, granulation tissue area, percentage of granulation tissue area, and degree of inflammatory cell infiltration were higher in group II than in group I, but there was no statistically significant difference. CONCLUSIONS: In this study, stent placement was feasible, and the formation of stent-induced tissue hyperplasia was evident in a rat tracheal model. MMP-9 overexpression was evident in the stent-induced tracheal tissue hyperplasia.

Increased matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase activity and expression in heterotopically transplanted murine tracheas.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) is the most common long-term cause of morbidity and mortality after heart-lung or lung transplantation. One pathologic feature of BOS is infiltration of fibroblasts and connective tissue products into the airway lumen, which form a fibrous, collagen-rich occlusion. Heterotopically transplanted allogeneic murine tracheal stenosis resemble BOS in the development of obliterans airway disease. Matrix metalloproteinases (MMPs) are key enzymes involved in tissue remodeling and, clinically, have several roles in pulmonary diseases. Among the MMP family, type IV collagenases, MMP-2 and MMP-9, have high gelatinolytic activity and are thought to play a role in several pulmonary diseases. Membrane type 1 MMP (MT1-MMP) activates the zymogen of MMP-2 (proMMP-2, 72 kd), and activated MMP-2 (active MMP-2, 62 kd) degrades type IV collagen and plays an important role in clinical pulmonary disease. In this study, we examine the expression of MMP-2, its activator MT1-MMP and MMP-9 in BOS using murine trachea transplantation models. METHODS: Rats were divided into 5 experimental groups (n = 10 in each group). Group I was a control group with intact tracheas. Animals with tracheal grafts underwent heterotopically syngeneic (Groups II and III) or allogeneic (Groups IV and V) transplantation. The recipient rats were killed 7 days (Groups II and IV) or 28 days (Groups III and V) after transplantation. The harvested tracheal grafts were examined histologically. MMP activity was assessed using gelatin zymography analysis, and MMP-2 and MT1-MMP gene expression was examined by quantitative real-time polymerase chain reaction analysis. Distribution of gelatinolytic activity was studied using in situ zymography. RESULTS: There was little histologic change in the intact trachea (Group I) and in all isografts (Groups II and III). Fibrotic tissues in Group V significantly occluded the tracheal lumen, and there was severe lymphocyte infiltration in Group IV. According to gelatin zymography, proMMP-9 was faint at 7 days, but activated MMP-9 was not present in all groups. The MMP-2 gelatinolytic bands were predominant; the activation in Group V was significantly greater than that in Group IV, and in Group III it was significantly greater than that in Group II. Gene expression of both MMP-2 and MT1-MMP were significantly higher in Group V than in the other groups (p < 0.01), and MMP-2 was clearly activated. Gelatinolytic activity was localized in the fibrotic tissues or lymphocytes of thickening lumen after destruction of the epithelium by stenosis. CONCLUSIONS: These results demonstrate that MMP-2, together with its activator MT1-MMP, may have an important role in the development of BOS, which is associated with destruction of the tracheal epithelium, leading to fibrosis.