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(Chest. 2006;129:1298-1304.)
© 2006 American College of Chest Physicians

High-Level Expression of Matrix-Associated Transforming Growth Factor-ß1 in Benign Airway Stenosis*

Christian Karagiannidis, MD; Viorica Velehorschi; Barbara Obertrifter; Hans-Nicol Macha, MD; Albert Linder, MD and Lutz Freitag, MD, FCCP

* From the Lungenklinik Hemer, Center for Pulmonary Medicine and Thoracic Surgery, Hemer, Germany.

Correspondence to: Lutz Freitag, MD, FCCP, Lungenklinik Hemer, Center for Pulmonary Medicine and Thoracic Surgery, Theo-Funccius Str 1, 58675 Hemer, Germany; e-mail: freitag-hemer{at}t-online.de

Abstract

Study objectives: Acquired tracheal and subglottic stenosis frequently leads to severe airway narrowing, which requires repeated interventions, such as dilatation, laser resection, stent implantation, or surgery. To get a more detailed insight into the pathogenesis of this condition, we investigated the expression of profibrotic cytokines and the proliferation of the airway wall in benign human airway stenoses.

Methods: Specimens from patients with subglottic and tracheal stenosis and stent-related stenoses were obtained (n = 20) for reverse transcription (RT) polymerase chain reaction (PCR) analysis and immunohistochemistry testing.

Results: Transforming growth factor (TGF)-ß1 messenger RNA expression was significantly increased in biopsy specimens from stent-related stenoses compared to nonstenotic control sections. In contrast, TGF-ß3 and interleukin-1ß showed no such differences in messenger RNA expression. Immunohistochemistry revealed a strong matrix-associated, subepithelial expression of TGF-ß1 in tracheal stenosis. Proliferating Ki-67-positive cells were mainly localized in the basal epithelial layer. Only 2 of 16 patients with tracheal stenoses and 3 of 4 patients with stent-related stenoses showed a weak expression of Ki-67-positive cells in the subepithelium. Furthermore, TGF-ß1 dose-dependently enhanced the proliferation of human lung fibroblasts in vitro, even in the presence of mitomycin-C.

Conclusion: While a weak subepithelial proliferation occurs in stent-related stenoses, the dominant factor in late stages of untreated tracheal stenoses seems to be the high-level expression of TGF-ß1 and the deposition of extracellular matrix.

Key Words: Ki-67 • proliferation • stent • tracheal stenosis • transforming growth factor-ß

Acquired tracheal and subglottic stenosis most commonly results from endotracheal intubation and long-term ventilation. The resulting airway narrowing frequently leads to symptomatic airway obstruction, which requires repeated interventions such as dilatation, laser resection, stent implantation, or surgery.1234 Airway stents, which are supposed to counteract strictures, also seem to promote the development of secondary stenoses.56 Granulation tissue formation at stent edges is a common endoscopic finding.7 Microscopic examination of the narrowed tissue reveals the deposition of extracellular matrix (ECM).8 Members of the transforming growth factor (TGF)-ß superfamily, which include TGF-ßs, activin, and bone morphogenetic proteins, are pluripotent cytokines, which mediate their biological effects in a large variety of cell types.9 TGF-ß and activin are known to have modifying roles in the growth and differentiation of several cells, especially in lung fibroblasts.10 Previous studies have suggested that TGF-ß plays a central role in fibrosis by regulating the deposition of ECM components such as collagen, fibronectin, and proteoglycans.11 TGF-ß exists in the following three isoforms: TGF-ß1, TGF-ß2, and TGF-ß3. TGF-ß1 appears to be the most common isoform associated with disorders that are characterized by inflammation and fibrosis. In contrast to TGF-ß3, which is known to improve laryngeal healing at least in animal models,12 TGF-ß1 is one of the strongest inducers of myofibroblasts13 and is a mitogen to immature fibroblasts.14 Compared to normal fibroblasts, myofibroblasts have been reported to have higher collagen synthesis activity, especially in type I and type III collagen.15 It is reasonable to assume that myofibroblasts may be responsible for the structural alterations causing tracheal and stent-related stenosis, but there is only little evidence that TGF-ß1 plays a crucial role in the pathogenesis of subglottic, tracheal, and stent stenoses.16

The principal aim of the present study was to determine whether the proliferation of the tissue and/or the deposition of the ECM is one of the key events in the occurrence of benign airway stenosis, with special regard to potential therapeutic intervention. Therefore, the immunohistochemical localization and expression pattern of TGF-ß and Ki-67, which is a marker for proliferation in sections and biopsy specimens obtained from benign stenotic airways, was determined.

Materials and Methods

Patients
Specimens consisted of segments of human trachea that had been excised for the surgical repair of intubation injury-related subglottic and tracheal stenoses. Furthermore, biopsy specimens from stent stenosis and restenosis were obtained by rigid bronchoscopy during interventional bronchoscopy. The study population consisted of 16 patients with subglottic and tracheal stenoses occurring after they had undergone endotracheal intubation and 7 patients with stenoses at the edges of airway stents. Biopsy specimens from prospective resection margins of patients with non-small cell lung cancer were obtained during routine bronchoscopy. Only samples that were definitely tumor-free were used as controls for this study. All patients had given their consent, and the study had been approved by the internal review board.

Immunohistochemistry
Archival tissue material from patients who had undergone sleeve resections for the treatment of tracheal and subglottic stenoses were investigated by means of immunohistochemical analysis for Ki-67, {alpha}-smooth-muscle actin, and TGF-ß1. Specimens were fixed in 4% formaldehyde and were embedded in paraffin blocks. Five-micrometer sections were deparaffined and rehydrated before staining with hematoxylin-eosin and elastica-van Gieson stain or immunohistochemistry. Slides were incubated with a rabbit polyclonal antibody against TGF-ß1 (Santa Cruz Biotechnology; Santa Cruz, CA) at a dilution of 1:100, a mouse monoclonal antibody against Ki-67 (clone MIB-1; Dako; Glostrup, Denmark), and a mouse monoclonal antibody against {alpha}-smooth muscle actin (clone 1A4; Dako) at a dilution of 1:100. Matched isotype controls were used at the same protein concentration as the test antibodies. The reaction was revealed using the amino ethyl carbazol (AEC)-horseradish peroxidase method (Envision+ system; Dako) for TGF-ß1, and with an automated system (NexES; Ventana Medical Systems; Tucson, AZ) for Ki-67. In short, slides were deparaffined and rehydrated. After antigen demasking by means of microwaving in a citrate buffer (pH 6) and background blocking with 2% bovine serum albumin, slides were incubated for 1 h with the primary antibody in the dilution mentioned above. Afterward, the prediluted secondary antibody was administered for 30 min, and sections were stained using an horseradish-AEC substrate and were counterstained with hematoxylin for 1 min. Ki-67 was stained using an automated immunohistochemical stainer (NexES; Ventana Medical Systems) on 5-µm-thick tissue sections following routine deparaffinization, rehydration, and appropriate antigen retrieval. Staining was done by AEC-chromogenic detection.

Reverse Transcription Polymerase Chain Reaction
Total RNA was extracted from biopsy specimens obtained during rigid bronchoscopy (RNeasy midi kit; Qiagen; Hilden, Germany) according to the instructions of the manufacturer. The integrity was checked with 1.2% agarose-gel electrophoresis. Reverse transcription (RT) was performed with an RT kit (Omniscript RT; Qiagen) and oligo d(T). Polymerase chain reaction (PCR) [22 to 32 cycles] was performed (Taq PCR Master Mix; Qiagen). Amplification was optimized for each primer set. The following primers were used, as reported by Jakowlew et al17: ß-actin 5' CGC GAG AAG ATG ACC CAG ATC ATG T; 3' CGA CGT AGC ACA GCT TCT CCT TAA TG; interleukin (IL)-1ß 5' TAC GAA TCT CCG ACC ACC ACT ACA GC; 3' TAT CAT CTT TCA ACA CGC AGG ACA GG; TGF-ß1; and TGF-ß3.

Cell Cultures
All cell cultures were performed in a humidified atmosphere containing 5% CO2 at 37°C. Human lung fibroblasts were purchased (Clonetics; Cambrex, Belgium), incubated, and cultured in the recommended medium (FGM-2; Clonetics). For proliferation experiments, a basal medium with 0.5% fetal calf serum without antibiotics was used. Recombinant TGF-ß (RnD Systems; Abingdon, UK) was activated by HCl before use, and the pH was adjusted to 7.4.

Evaluation of Expression
Microscopic fields with the highest degree of immunoreactivity were chosen for analysis. At least 1,000 cells were analyzed in each case. A numeric intensity score was set from 0 to 3 (0, no staining; 1 [+], weak staining; 2 [++], moderate staining; and 3 [+++], strong staining).

Statistical Analysis
The nonparametric Mann-Whitney U test was used to test the non-Gauss-distributed data. The error bars in each figure indicate the SD of one experiment. Differences with probability values of p ≤ 0.05 were considered to be significant.

Results

Increased Expression of TGF-ß1 But Not TGF-ß3 and IL-1ß in Stent-Related Stenoses
The expression of TGF-ß1 and TGF-ß3 was measured by RT-PCR in biopsy specimens from stent stenoses (Fig 1 , top left, A) and were compared to biopsy specimens from control subjects with nonnarrowed airways. Agarose gel electrophoresis revealed the PCR products shown in Figure 1. ß-actin was used as an internal standard. Patients with stent stenosis showed a significantly higher expression of TGF-ß1 (p ≤ 0.5) [Fig 1, top right, B]. No significant differences were observed in TGF-ß3 and IL-1ß expression between nonstenotic mucosa and tissue samples from patients with stent stenosis (Fig 1, bottom left, C, and bottom right, D). The IL-1ß expression showed a tendency toward a higher expression level in the stent stenosis, but these findings were not always seen and revealed no statistical significance.


Figure 1
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  		Figure 1.. Increased TGF-ß1 expression, but not TGF-ß3 and IL-1-ß expression in biopsy specimens from stent stenoses in relation to healthy control subjects. Top left, A: representative agarose gel electrophoresis showing RT-PCR products for TGF-ß1, TGF-ß3, and IL-1ß from biopsy specimens of stent stenoses and healthy control subjects. ß-actin served as an internal control. Lane 1, marker (M); lanes 2 to 8, biopsy specimens from stent stenosis; lanes 9 to 12, healthy control subjects; and lane 13, water control (W). Top right, B: increased TGF-ß1 expression in stent stenosis in relation to healthy control subjects. Bottom left, C, and bottom right, D: no significant difference in TGF-ß3 and IL-1ß expression in stent stenosis in relation to healthy control subjects. A densitometric measurement was used to analyze the data from the agarose gel (top left, A). * = p ≤ 0.05. mRNA = messenger RNA; n.s. = not significant.

 
Strong Subepithelial Expression of TGF-ß1 Causing Enhanced Proliferation of Lung Fibroblasts But Weak Ki-67 Expression in Subglottic Fibrosis
No significant immunostaining was detected in control sections with isotype sera. In biopsy specimens from stent stenoses, and sections of subglottic and tracheal stenoses, immunoreactive TGF-ß1 was principally localized extracellularly in association with the subepithelial connective tissue (Fig 2 , bottom left, C, and bottom right, F, Table 1 ). The subepithelium showed a moderate-to-strong expression of TGF-ß1. In contrast, TGF-ß1 was rarely expressed in epithelial cells. A weak epithelial expression of TGF-ß1 could be detected in only 2 of 11 patients (Table 1). Of note, 4 of 11 patients showed an infiltration of CD4+ T-cells (not shown), expressing TGF-ß1 (Table 1, Fig 2, middle right, E), one of the key mediators of regulatory T-cells, mediating suppression and tolerance.1819 Control sections from unaffected mucosa showed only a weak expression of TGF-ß1 in the subepithelium (data not shown).


Figure 2
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  		Figure 2.. Specimens from stent and tracheal stenosis show a large deposition of ECM (top left, A, and top right, D) [elastica-van Gieson staining]. Ki-67-positive cells (brown), indicating proliferation mainly occur in the basal layer of the epithelium in stent and tracheal stenoses (middle left, B, and middle right, E). Subepithelial and ECM-associated expression of TGF-ß1 in stent stenosis, without epithelial expression pattern (bottom left, C). Infiltration of TGF-ß1 expressing lymphocytes in the subepithelium (bottom right, F). All panels, original x 20.

 

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  		Table 1.. TGF-ß1*

 
In contrast to TGF-ß1, Ki-67, which is a crucial marker for proliferation, could be detected mainly in the epithelium but not in the subepithelium (Table 2 ). Only 2 of 16 patients with tracheal stenosis showed Ki-67-positive cells in the subepithelium, whereas 13 of 16 showed a weak-to-moderate expression of Ki-67 in the epithelium with strong expression in the basal layer (Table 2, Fig 2, middle left, B, and middle right, E). In three of four patients with stent-related stenoses, a weak expression of Ki-67-positive subepithelial cells could be detected. Furthermore, 75% of the specimens showed an epithelial metaplasia with basal layer hyperplasia (Fig 2, top right, D, middle left, B, and bottom right, F). In only one patient with stent stenosis was an epithelial dysplasia found (image not shown). Furthermore, one section revealed tracheal bone development. All specimens showed a thickening of the airways with a large deposition of ECM and subepithelial fibrosis (Fig 2, top left, A, and top right, D). Furthermore, subepithelial fibroblasts showed a differentiation into myofibroblasts, indicated by the expression of {alpha}-smooth muscle actin in fibroblasts (Fig 3 , bottom, B). Regarding the function of TGF-ß1, we observed enhanced TGF-ß1-mediated proliferation of human lung fibroblasts in a dose-dependent manner in the presence of mitomycin-C, which is a drug that is commonly used to treat airway stenosis (Fig 4 , bottom, B). Mitomycin-C levels dose-dependently decreased the proliferation of lung fibroblasts (Fig 4, top, A), whereas TGF-ß1 increased their proliferation (Fig 4, bottom, B).


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  		Table 2.. Ki-67*

 

Figure 3
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  		Figure 3.. Top, A: typical metal stent-related stenoses of a main bronchus. Bottom, B: a representative section of a biopsy specimen from a stent stenosis with subepithelial fibrosis, showing fibroblasts expressing {alpha}-smooth muscle actin (bright green).

 

Figure 4
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  		Figure 4.. Dose-dependent inhibition of proliferation of lung fibroblasts (IMR-90 cell line) in response to mitomycin-C (MMC) under serum-free conditions in Dulbecco minimal essential medium without supplements. Increased dose-dependent proliferation of lung fibroblasts (IMR-90 cell line) in response to increasing concentrations of TGF-ß1 in serum-free Dulbecco minimal essential medium with 100 ng/mL mitomycin-C. The results represent three independent experiments. Proliferation was measured by [3H]-thymidine incorporation for 12 h. Error bars indicate the SD of each experiment. cpm = counts per minute.

 
Discussion

The current study shows a high messenger RNA expression and a strong matrix-associated protein expression of TGF-ß1 in specimens from benign tracheal and stent stenoses. Furthermore, only a very weak proliferation, as indicated by Ki-67, could be obtained in the fibrotic subepithelium of patients with subglottic and tracheal stenoses, in contrast to the proliferating epithelium. Patients with stent-related stenoses showed a more pronounced proliferation of the subepithelium.

The current data are in line with the known role of TGF-ß1 in fibrosis2021 and provide evidence that TGF-ß1 also plays a key role in benign human airway stenosis, as was previously shown in animal models.16 In contrast to TGF-ß1, TGF-ß3 was reported to improve laryngeal healing in an animal model.12 However, we observed no difference in TGF-ß3 messenger RNA expression in stenotic lesions compared to healthy control subjects. This suggests the disequilibrium of profibrotic TGF-ß1 and protective TGF-ß3 in benign human airway stenoses. Furthermore, the high-level expression of IL-1ß, one of the major proinflammatory cytokines involved in many acute and chronic diseases,21 leads in animal models to an increased expression of profibrotic platelet-derived growth factor and TGF-ß1 in the lung.22 Although our data revealed no statistically significant difference in IL-1ß messenger RNA expression between stenotic lesions and controls, a clear tendency toward a higher expression in stenoses could be observed. At least in some patients, IL-1ß may enhance the TGF-ß1–mediated fibrosis of stenotic airways. The significance of this and other cytokines should therefore be tested in a larger group of patients.

Local radiation therapy, such as high-dose intraluminal brachytherapy or the local administration of mitomycin-C, are common approaches to treat recurrent scar stenoses.723 Surprisingly, we observed almost no subepithelial proliferation in subglottic and tracheal stenoses, but a huge ECM deposition. This may be part of the underlying pathogenic mechanism without a crucial proliferation of the airway wall tissue. It may also be that subepithelial proliferation occurs early in the pathogenesis, and we just observed the end stage of the disease. There is growing evidence that antiproliferating drugs such as the antineoplastic antibiotic mitomycin-C are less efficient than originally assumed.2425 We can demonstrate that the inhibiting effect of mitomycin-C on fibroblast proliferation in vitro could at least be in part abolished by the addition of TGF-ß1. The local milieu with high-level, matrix-associated expression of TGF-ß1 in stenoses could therefore be one reason for the limited treatment effect of mitomycin-C.

Although the mucosal trauma from intubation or dilatation occurred weeks and months before specimens were obtained, epithelial metaplasia and a high proliferation of the basal epithelial layer still could be observed. Of note, proliferating Ki-67-positive cells are normally very rare in adult airways.2627 This suggests that the first or repeated mechanical attraction after intubation leads to an independent proliferation of the epithelium and epithelial metaplasia, as is known to occur in patients with epithelial metaplasia caused by airway stents.28 In contrast, the subepithelium of patients with stent-related stenoses showed a weak subepithelial proliferation in addition to the deposition of ECM and fibrosis. Therefore, additional pharmacologic therapy should target both the proliferation of fibroblasts and their differentiation into myofibroblasts, as well as ECM deposition and the imbalance of TGF-ß1 and TGF-ß3.

Although TGF-ß1 is a strong profibrotic cytokine, it is on the other hand one of the main cytokines of regulatory T cells, mediating suppression and peripheral tolerance.181929 The observed infiltration of TGF-ß1-expressing T cells (Fig 2, bottom right, F) in some patients with subglottic and tracheal stenoses may be part of an immunologic attempt to control the disease, especially to mediate tolerance towards airway stents.

The current study provides evidence, that the high-level expression of TGF-ß1 and the deposition of ECM is one of the key events in benign stent and tracheal stenoses. At least in patients who experience late-stage tracheal and subglottic stenoses after intubation, the proliferation of the fibrotic subepithelium is not the main mechanism in the development of airway narrowing. Therefore, we conclude that additional pharmacologic therapy should target more the inhibition of profibrotic cytokines such as TGF-ß1 and the deposition of ECM in subglottic and tracheal stenoses, than the subepithelial proliferation of fibroblasts. Patients with stent stenoses may benefit from additional antiproliferative therapy, as well as from drugs targeting the TGF-ß1/TGF-ß3 imbalance. Furthermore, current research should focus on widespread gene-expression analysis to find further fibrosis-inducing genes, which could be a target for new biological drugs.

Footnotes

Abbreviations: AEC = amino ethyl carbazol; ECM = extracellular matrix; IL = interleukin; PCR = polymerase chain reaction; RT = reverse transcription; TGF = transforming growth factor

This work was supported by the Verein zur Förderung der Lungenklinik Hemer e.V.

Received for publication September 25, 2005. Accepted for publication October 20, 2005.

References

  1. Kocyildirim, E, Kanani, M, Roebuck, D, et al (2004) Long-segment tracheal stenosis: slide tracheoplasty and a multidisciplinary approach improve outcomes and reduce costs. J Thorac Cardiovasc Surg 128,876-882[Abstract/Free Full Text]
  2. de Mello-Filho, FV, Antonio, SM, Carrau, RL Endoscopically placed expandable metal tracheal stents for the management of complicated tracheal stenosis. Am J Otolaryngol 2003;24,34-40[CrossRef][ISI][Medline]
  3. Backer, CL, Mavroudis, C, Gerber, ME, et al Tracheal surgery in children: an 18-year review of four techniques. Eur J Cardiothorac Surg 2001;19,777-784[Abstract/Free Full Text]
  4. Ciccone, AM, De Giacomo, T, Venuta, F, et al Operative and non-operative treatment of benign subglottic laryngotracheal stenosis. Eur J Cardiothorac Surg 2004;26,818-822[Abstract/Free Full Text]
  5. Saad, CP, Murthy, S, Krizmanich, G, et al Self-expandable metallic airway stents and flexible bronchoscopy: long-term outcomes analysis. Chest 2003;124,1993-1999[Abstract/Free Full Text]
  6. Ernst, A Airway obstruction and stenting: more complex than we thought? Am J Respir Crit Care Med 2004;169,1081-1082[Free Full Text]
  7. Freitag, L Interventional bronchoscopy. 2000 Karger. Basel, Switzerland:
  8. Liu, H, Chen, JC, Holinger, LD, et al Histopathologic fundamentals of acquired laryngeal stenosis. Pediatr Pathol Lab Med 1995;15,655-677[Medline]
  9. Blobe, GC, Schiemann, WP, Lodish, HF Role of transforming growth factor ß in human disease. N Engl J Med 2000;342,1350-1358[Free Full Text]
  10. Coker, RK, Laurent, GJ, Shahzeidi, S, et al Transforming growth factors-ß1, -ß2, and -ß3 stimulate fibroblast procollagen production in vitro but are differentially expressed during bleomycin-induced lung fibrosis. Am J Pathol 1997;150,981-991[Abstract]
  11. Xu, YD, Hua, J, Mui, A, et al Release of biologically active TGF-ß1 by alveolar epithelial cells results in pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 2003;285,L527-L539[Abstract/Free Full Text]
  12. Loewen, MS, Walner, DL, Caldarelli, DD Improved airway healing using transforming growth factor beta-3 in a rabbit model. Wound Repair Regen 2001;9,44-49[ISI][Medline]
  13. Zhang, HY, Phan, SH Inhibition of myofibroblast apoptosis by transforming growth factor ß(1). Am J Respir Cell Mol Biol 1999;21,658-665[Abstract/Free Full Text]
  14. Hashimoto, S, Gon, Y, Takeshita, I, et al Transforming growth factor-ß1 induces phenotypic modulation of human lung fibroblasts to myofibroblast through a c-Jun-NH2-terminal kinase-dependent pathway. Am J Respir Crit Care Med 2001;163,152-157[Abstract/Free Full Text]
  15. Evans, RA, Tian, YC, Steadman, R, et al TGF-ß1-mediated fibroblast-myofibroblast terminal differentiation-the role of Smad proteins. Exp Cell Res 2003;282,90-100[CrossRef][ISI][Medline]
  16. Dillard, DG, Gal, AA, Roman-Rodriguez, J, et al Transforming growth factor and neutralizing antibodies in subglottic stenosis. Ann Otol Rhinol Laryngol 2001;110,393-400[Medline]
  17. Jakowlew, SB, Moody, TW, Mariano, JM Transforming growth factor-beta receptors in human cancer cell lines: analysis of transcript, protein and proliferation. Anticancer Res 1997;17,1849-1860[Medline]
  18. Levings, MK, Roncarolo, MG T-regulatory 1 cells: a novel subset of CD4 T cells with immunoregulatory properties. J Allergy Clin Immunol 2000;106,S109-112[CrossRef][Medline]
  19. Groux, H Type 1 T-regulatory cells: their role in the control of immune responses. Transplantation 2003;75,8S-12S[CrossRef][Medline]
  20. Kenyon, NJ, Ward, RW, McGrew, G, et al TGF-ß1 causes airway fibrosis and increased collagen I and III mRNA in mice. Thorax 2003;58,772-777[Abstract/Free Full Text]
  21. Leask, A, Abraham, DJ TGF-ß signaling and the fibrotic response. FASEB J 2004;18,816-827[Abstract/Free Full Text]
  22. Kolb, M, Margetts, PJ, Anthony, DC, et al Transient expression of IL-1ß induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest 2001;107,1529-1536[ISI][Medline]
  23. Rahbar, R, Shapshay, SM, Healy, GB Mitomycin: effects on laryngeal and tracheal stenosis, benefits, and complications. Ann Otol Rhinol Laryngol 2001;110,1-6[ISI][Medline]
  24. Eliashar, R, Gross, M, Maly, B, et al Mitomycin does not prevent laryngotracheal repeat stenosis after endoscopic dilation surgery: an animal study. Laryngoscope 2004;114,743-746[Medline]
  25. Perepelitsyn, I, Shapshay, SM Endoscopic treatment of laryngeal and tracheal stenosis: has mitomycin C improved the outcome? Otolaryngol Head Neck Surg 2004;131,16-20[Medline]
  26. Fjellbirkeland, L, Cambier, S, Broaddus, VC, et al Integrin alphavbeta8-mediated activation of transforming growth factor-ß inhibits human airway epithelial proliferation in intact bronchial tissue. Am J Pathol 2003;163,533-542[Abstract/Free Full Text]
  27. Boers, JE, den Brok, JL, Koudstaal, J, et al Number and proliferation of neuroendocrine cells in normal human airway epithelium. Am J Respir Crit Care Med 1996;154,758-763[Abstract]
  28. Coppit, G, Perkins, J, Munaretto, J, et al The effects of mitomycin-C and stenting on airway wound healing after laryngotracheal reconstruction in a pig model. Int J Pediatr Otorhinolaryngol 2000;53,125-135[CrossRef][ISI][Medline]
  29. Chen, W, Jin, W, Hardegen, N, et al Conversion of peripheral CD4+CD25-naive T cells to CD4+CD25+ regulatory T cells by TGF-ß induction of transcription factor Foxp3. J Exp Med 2003;198,1875-1886[Abstract/Free Full Text]




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