Mechanisms of fibrosis in iatrogenic laryngotracheal stenosis: New discoveries and novel targets.

Iatrogenic laryngotracheal stenosis (iLTS) is a pathological condition characterized by the narrowing of the laryngeal and tracheal structures due to the formation of abnormal scar tissue. The core of iLTS lies in the fibrosis of the laryngotracheal tissue, and recent research has unveiled novel discoveries regarding the underlying mechanisms of fibrosis. This review provides an overview of the recent advancements in understanding the mechanisms of fibrosis in iLTS. It encompasses various aspects, such as immune system dysregulation, changes in the extracellular matrix (ECM), metabolic alterations, and the role of microbial flora. The review also explores the interplay and relationships between these new mechanisms, establishing a theoretical foundation for the development of multi-target therapies and combination therapies for iLTS.

Sirolimus-eluting airway stent reduces profibrotic Th17 cells and inhibits laryngotracheal stenosis.

Laryngotracheal stenosis (LTS) is pathologic fibrotic narrowing of the larynx and trachea characterized by hypermetabolic fibroblasts and CD4+ T cell-mediated inflammation. However, the role of CD4+ T cells in promoting LTS fibrosis is unknown. The mTOR signaling pathways have been shown to regulate the T cell phenotype. Here we investigated the influence of mTOR signaling in CD4+ T cells on LTS pathogenesis. In this study, human LTS specimens revealed a higher population of CD4+ T cells expressing the activated isoform of mTOR. In a murine LTS model, targeting mTOR with systemic sirolimus and a sirolimus-eluting airway stent reduced fibrosis and Th17 cells. Selective deletion of mTOR in CD4+ cells reduced Th17 cells and attenuated fibrosis, demonstrating CD4+ T cells' pathologic role in LTS. Multispectral immunofluorescence of human LTS revealed increased Th17 cells. In vitro, Th17 cells increased collagen-1 production by LTS fibroblasts, which was prevented with sirolimus pretreatment of Th17 cells. Collectively, mTOR signaling drove pathologic CD4+ T cell phenotypes in LTS, and targeting mTOR with sirolimus was effective at treating LTS through inhibition of profibrotic Th17 cells. Finally, sirolimus may be delivered locally with a drug-eluting stent, transforming clinical therapy for LTS.

Molecular Mechanisms and Physiological Changes behind Benign Tracheal and Subglottic Stenosis in Adults.

Laryngotracheal stenosis (LTS) is a complex and heterogeneous disease whose pathogenesis remains unclear. LTS is considered to be the result of aberrant wound-healing process that leads to fibrotic scarring, originating from different aetiology. Although iatrogenic aetiology is the main cause of subglottic or tracheal stenosis, also autoimmune and infectious diseases may be involved in causing LTS. Furthermore, fibrotic obstruction in the anatomic region under the glottis can also be diagnosed without apparent aetiology after a comprehensive workup; in this case, the pathological process is called idiopathic subglottic stenosis (iSGS). So far, the laryngotracheal scar resulting from airway injury due to different diseases was considered as inert tissue requiring surgical removal to restore airway patency. However, this assumption has recently been revised by regarding the tracheal scarring process as a fibroinflammatory event due to immunological alteration, similar to other fibrotic diseases. Recent acquisitions suggest that different factors, such as growth factors, cytokines, altered fibroblast function and genetic susceptibility, can all interact in a complex way leading to aberrant and fibrotic wound healing after an insult that acts as a trigger. However, also physiological derangement due to LTS could play a role in promoting dysregulated response to laryngo-tracheal mucosal injury, through biomechanical stress and mechanotransduction activation. The aim of this narrative review is to present the state-of-the-art knowledge regarding molecular mechanisms, as well as mechanical and physio-pathological features behind LTS.

Down-Regulation of a Profibrotic Transforming Growth Factor-ß1/Cellular Communication Network Factor 2/Matrix Metalloprotease 9 Axis by Triamcinolone Improves Idiopathic Subglottic Stenosis.

Idiopathic subglottic stenosis (iSGS) is a progressive fibrotic disease characterized by life-threatening airway narrowing. Although the molecular underpinnings are unknown, previous reports showing that subglottic serial intralesional steroid injections (SILSIs) improve clinical outcomes suggest a steroid-sensitive pathway in iSGS. Herein, a prospective study was conducted to determine the changes in profibrotic markers during SILSI to identify steroid-sensitive profibrotic drivers. Seven newly diagnosed patients with iSGS were recruited for SILSI. Subglottic biopsies before and after SILSI treatments were evaluated for histologic and molecular markers by confocal microscopy and RT-qPCR. At baseline, iSGS subglottises contained abundant vimentin-positive/α-smooth muscle actin-negative fibroblasts, intermingled with a matrix of fibronectin and types I and VI collagen. Transforming growth factor (TGF)-ß1 was up-regulated primarily in glandular epithelium. Cellular communication network factor 2 (CCN2) was mainly up-regulated in stromal fibroblasts surrounding TGF-ß1-positive glandular structures. SILSI improved iSGS by reducing fibroblast infiltration and increasing matrix remodeling. Mechanistically, SILSI counteracted the effects of TGF-ß1 by inducing matrix metalloprotease 9 (MMP9) expression while repressing CCN2 expression, without affecting TGFß1 levels. Treatment of primary iSGS-derived fibroblasts with TGF-ß1 recapitulated aspects of the disease in vivo, demonstrating that the induction in CCN2 and repression of MMP9 are caused by changes in histone acetylation induced by TGF-ß1. Triamcinolone counteracted the coregulation of these genes by impairing SMAD2/3 binding to promoter regions, and not through histone acetylation. In conclusion, this study shows that SILSI counteracts a dysregulated TGF-ß1/CCN2/MMP9 axis involved in iSGS development.

Proteomic and Genomic Methylation Signatures of Idiopathic Subglottic Stenosis.

OBJECTIVE: Idiopathic subglottic stenosis (iSGS) is a chronic inflammatory condition that causes dyspnea and affects middle-aged women of White race and non-Latino or Hispanic ethnicity. To better characterize its phenotype and pathogenesis, we assessed the proteomic and genomic methylation signatures of subglottic tissue collected from iSGS patients compared to controls. STUDY DESIGN: Molecular analysis of clinical biospecimens. METHODS: We collected subglottic tissue biopsies from 12 patients during direct laryngoscopy, immediately prior to surgical treatment of iSGS; as well as from 4 age-, sex-, and race/ethnicity-matched control patients undergoing other direct laryngoscopic procedures. We isolated protein and genomic DNA, acquired proteomic data using label-free quantitative mass spectrometry techniques, and acquired genome-wide methylation data using bisulfite conversion and a microarray platform. We compared molecular profiles across the iSGS and control groups, and with respect to clinical course in the iSGS group. Eight of the 12 iSGS patients underwent subsequent blood collection and plasma isolation for further assessment. RESULTS: Proteomic analysis revealed 42 differentially abundant proteins in the iSGS biopsies compared to controls, inferring enrichment of biological pathways associated with early wound healing, innate immunity, matrix remodeling, and metabolism. Proteome-based hierarchical clustering organized patients into two iSGS and one control subgroups. Methylation analysis revealed five hypermethylated genes in the iSGS biopsies compared to controls, including the biotin recycling enzyme biotinidase (BTD). Follow-up analysis showed elevated plasma BTD activity in iSGS patients compared to both controls and published normative data. CONCLUSION: iSGS exhibits distinct proteomic and genomic methylation signatures. These signatures expand current understanding of the iSGS phenotype, support the possibility of disease subgroups, and should inform the direction of future experimental studies. LEVEL OF EVIDENCE: Not applicable Laryngoscope, 131:E540-E546, 2021.

Increased Expression of Estrogen Receptor Beta in Idiopathic Progressive Subglottic Stenosis.

BACKGROUND/OBJECTIVES: Idiopathic progressive subglottic stenosis (IPSS) predominantly affects females in perimenopause. It has, therefore, been hypothesized that estrogen is involved in its pathogenesis. There are two main types of estrogen receptors: ER-α and ER-ß. Abnormal variants of ER-ß have previously been shown to be associated with poor wound healing. Estrogen receptors have recently been identified in subglottic tissue samples, with elevated levels of ER-α and progesterone receptors, and no expression of ER-ß, in stenotic specimens reported in one study. The objective of this study was to confirm the presence of estrogen receptors in the subglottis and investigate levels of expression and types of estrogen receptors in normal and stenotic subglottic tissue. METHODS: Subglottic tissue was obtained from three female and one male cadaver without laryngotracheal pathology to serve as controls. Subglottic tissue specimens from five female patients with IPSS were also analysed. Immunofluorescence stains for ER-α and ER-ß were performed on specimens. Staining patterns were compared qualitatively and semi-qualitatively between control and IPSS specimens. RESULTS: Immunofluorescence stains demonstrated the presence of both ER-α and ER-ß in subglottic tissue. IPSS specimens demonstrated significantly greater staining intensity of ER-α in the epithelium and ER-ß in glands and ducts compared to controls. CONCLUSIONS: This study confirms the presence of estrogen receptors in the subglottis. Increased expression of ER-α in the epithelium and ER-ß in glands and ducts in IPSS compared to controls may help to explain the predisposition to stenosis in these individuals. LEVEL OF EVIDENCE: 3b Laryngoscope, 130:2186-2191, 2020.

Laryngeal Diffuse Large B-Cell Lymphoma Presenting as Laryngeal Stenosis.

BACKGROUND: Laryngeal stenosis is challenging for treatment due to uncertain etiology. Primary laryngeal lymphoma as the initial clinical manifestation of laryngeal stenosis has been rarely reported. Primary diffuse large B-cell lymphoma as an underlying etiology has not been reported. CASE REPORT: A 79-year-old male presented with dyspnea, stridor and dysphonia of 6 months' duration. Computed tomography scans and flexible laryngoscopic examination revealed vocal cord mobility with bilaterally limited abduction and a subglottic stenosis up to 50%. The laryngeal mucosa was smooth. Laryngeal biopsy showed atypical lymphoid infiltrates, predominantly large sized B-cells, in the submucosa with crush/cauterized artifacts. The tumor cells were positive for B-lymphocyte antigen CD20, paired-box 5 (PAX5), B-cell lymphoma 2 (BCL2), BCL6 and multiple myeloma oncogene 1 (MUM1). They were negative for CD10, CD30, cyclin D1 (CCND1), SRY-box 11 (SOX11), activin-receptor like kinase 1 (ALK1), CD138 and c-MYC, and negative for kappa/lambda light chain and Epstein-Barr virus-encoded small RNA by in situ hybridization. The pathologic diagnosis was diffuse large B-cell lymphoma. Fluorescent in situ hybridization (FISH) for MYC was negative. Next-generation sequencing using a 175-gene panel was performed and no pathologic mutations were identified. No lymphadenopathy elsewhere was identified. The patient was treated with chemotherapy and was doing well at the 5-month follow-up. CONCLUSION: To the best of our knowledge, this is the first documented case of primary laryngeal diffuse large B-cell lymphoma presenting as increasing laryngeal stenosis. The rarity, diagnosis and treatment of this entity are discussed.

Targeting metabolic abnormalities to reverse fibrosis in iatrogenic laryngotracheal stenosis.

OBJECTIVE: Management of laryngotracheal stenosis (LTS) remains primarily surgical, with a critical need to identify targets for adjuvant therapy. Laryngotracheal stenosis scar fibroblasts exhibit a profibrotic phenotype with distinct metabolic shifts, including an increased glycolysis/oxidative phosphorylation ratio. This study examines the effects of the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) on collagen production, gene expression, proliferation, and metabolism of human LTS-derived fibroblasts in vitro. METHOD: Paired normal and scar-derived fibroblasts isolated from subglottic and proximal tracheal tissue in patients with iatrogenic laryngotracheal stenosis (iLTS) were cultured. Proliferation rate, gene expression, protein production, and cellular metabolism were assessed in two conditions: 1) fibroblast growth medium, and 2) fibroblast growth medium with 1 × 10-4 M DON. RESULTS: DON treatment reduced cellular proliferation rate (n = 7, P = 0.0150). Expression of genes collagen 1 and collagen 3 both were reduced (n = 7, P = 0.0102, 0.0143, respectively). Soluble collagen production decreased (n = 7, P = 0.0056). As measured by the rate of extracellular acidification, glycolysis and glycolytic capacity decreased (n = 7, P = 0.0082, 0.0003, respectively). adenosine triphosphate (ATP) production and basal respiration decreased (n = 7, P = 0.0045, 0.0258, respectively), determined by measuring the cellular rate of oxygen consumption. CONCLUSION: The glutamine antagonist DON reverses profibrotic changes by inhibiting both glycolysis and oxidative phosphorylation in iLTS scar fibroblasts. In contrast to untreated iLTS scar fibroblasts, collagen gene expression, protein production, metabolic rate, and proliferation were significantly reduced. These results suggest DON and/or its derivatives as strong candidates for adjuvant therapy in the management of iatrogenic laryngotracheal stenosis. Enzymes involved in glutamine metabolism inhibited by DON offer targets for future investigation. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E59-E67, 2018.

Hormone receptors analysis in idiopathic progressive subglottic stenosis.

OBJECTIVE: Idiopathic subglottic stenosis predominantly affects fertile and perimenopausal women. Estrogens and/or progesterone have been proposed as mediators of its pathogenesis by stimulating collagen deposition within the upper airway. We evaluated the presence and expression of estrogen-alpha (ER-α), estrogen-beta (ER-ß), and progesterone receptors (PR) in idiopathic stenotic patients. STUDY DESIGN: A retrospective analysis on 42 surgical specimens from idiopathic stenosis female patients (mean age, 52.4; age range, 31-79) and 28 gender- and age-matched controls. METHODS: Immunoreactivity of ER-α, ER-ß, and PR was calculated as the product of intensity (1 = weak, 2 = moderate, 3 = strong) and positive cell percentage (1-4, for < 10/10-50/50-80/ > 80%). This score was calculated on the stenotic and peristenotic tissues. Influence of menopausal status on hormonal expression and stenotic grade was tested. RESULTS: Stenosis showed ER-α overexpression versus peristenotic tissue and controls (score 6.6 ± 4.4, 0.3 ± 0.5, and 2.2 ± 1.5, respectively; P < 0.001). Overexpression was even more marked for progesterone receptors (score 8.3 ± 3.6, 0.8 ± 0.6, and 1.0 ± 0.7, respectively; P < 0.001). There was no expression of ER-ß in stenosis (score 0), whereas it was normally expressed in peristenotic tissue and controls (score 0.7 ± 0.5 and 0.5 ± 0.5; P < 0.001 vs. stenosis). Expression of ER-α was higher in postmenopausal stenotic patients (P < 0.01). This subgroup included a higher proportion of Cotton-Myer grade III stenosis than in premenopausal subjects (P < 0.001). CONCLUSION: An imbalance between ER-α, ER-ß, and PR is present in idiopathic stenosis patients. The hormonal background may be involved in inappropriate inflammation and increased stenosis susceptibility. Menopausal changes seem to play a role in both stenosis grade and receptor patterns. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E72-E77, 2018.

Fibroblasts in Hypoxic Conditions Mimic Laryngotracheal Stenosis.

Objective To elucidate the role of hypoxia and inflammatory pathways in the pathogenesis of iatrogenic laryngotracheal stenosis (iLTS). Study Design (1) Examination of mucosal surface gene expression in human iLTS. (2) In vitro comparison of normal and scar laryngotracheal fibroblasts under normoxic and hypoxic conditions. Setting Tertiary care hospital in a research university (2012-2016). Subjects and Methods Brush biopsies were obtained from normal laryngotracheal tissue and scar in iLTS patients; gene expression was compared. Fibroblasts were isolated from normal and scarred trachea and grown in vitro in either a 1% O2 or normoxic environment. Cell growth and gene and protein expression were compared. Statistical analysis utilized a multilevel mixed effects model. Results Expression of IL-6 (fold change = 2.8, P < .01), myofibroblast marker αSMA (fold change = 3.0, P = .01), and MMP13 (fold change = 5.4, P = .02) was significantly increased in scar biopsy samples as compared to normal. Under hypoxic conditions in vitro, normal laryngotracheal fibroblasts proliferated significantly faster (n = 8, P < .01 each day). Expression of IL-6 (n = 8, fold change = 2.6, P < .01) increased significantly after 12 hours under hypoxia. Expression of αSMA (n = 8, fold change= 2.0, P = .03), COL1 (n = 8, fold change = 1.1, P = .03), and MMP13 (n = 8, fold change = 1.6, P = .01) increased significantly after 48 hours under hypoxia. Scar fibroblasts also proliferated significantly faster under hypoxic conditions but did not display the same expression profile. Conclusion Human iLTS scar has a myofibroblast phenotype. Under hypoxic conditions in vitro, normal laryngotracheal fibroblasts can transdifferentiate into a similar phenotype. These changes may be mediated by IL-6, a fibrosis-related cytokine.

Metabolic variations in normal and fibrotic human laryngotracheal-derived fibroblasts: A Warburg-like effect.

OBJECTIVES/HYPOTHESIS: Laryngotracheal stenosis (LTS) is a chronic fibrotic disease characterized by fibroblast proliferation, collagen deposition, and matrix remodeling in the lamina propria of the larynx and/or trachea. Current medical therapies are limited by a poor understanding of the effector cell's (fibroblasts) cellular biology and metabolism. The purpose of this study was to compare cellular proliferation, function, and metabolism between normal and LTS-derived fibroblasts in vitro. We hypothesize that LTS-derived fibroblasts will demonstrate aberrant behavior with faster proliferation, increased collagen production, and altered metabolic allocation compared with normal fibroblasts. STUDY DESIGN: In vitro comparative analysis. METHODS: Human biopsies of normal and iatrogenic LTS tissue (n = 7) were obtained, and fibroblasts were isolated and cultured in vitro. Cellular proliferation, cellular histology, gene expression, and metabolic analyses were performed. Statistical analyses comparing normal and scar-derived fibroblasts were performed. RESULTS: LTS fibroblast proliferation rate, cellular surface area, and collagen-1 expression were increased compared to normal fibroblasts. Cellular metabolic analysis of LTS-derived fibroblasts demonstrated reduced oxidative phosphorylation and increased glycolysis/oxidative phosphorylation ratio compared with normal fibroblasts. CONCLUSIONS: Human iatrogenic LTS-derived fibroblasts demonstrated aberrant behavior when compared with normal fibroblasts. A Warburg-like effect was revealed, suggesting human iatrogenic LTS fibroblasts drive their proliferation with aerobic glycolysis. The distinct metabolism suggests metabolic inhibitors could reduce fibroblast hyperplasia and hypertrophy in LTS and fibrosis in general. LEVEL OF EVIDENCE: NA Laryngoscope, 127:E107-E113, 2017.

[The influence of endogenous intoxication on the clinical picture of various forms of acute stenosinglaryngotracheitis in the children].

The objective of the present study was to evaluate the influence of endogenous intoxication on the clinical picture of various forms of acute stenosinglaryngotracheitis in the children. The clinical and laboratory examination involved 275 patients presenting with this pathology. Special emphasis was laid on diagnostics of the character and severity of intoxication syndrome. To this effect, we carried out a dynamic study of variations in the blood levels of medium molecular weight peptides, the toxic blood factor, and circulating immune complexes (CIC). It is concluded that the parameters of endogenous intoxication in the children with acute stenosinglaryngotracheitis are directly related to the specific clinical features and severity of this disease.

IL-1 Receptor Antagonist Inhibits Early Granulation Formation.

PURPOSE: Using a functional model of airway granulation tissue in laryngotracheal stenosis, we investigated changes in histopathology and inflammatory markers within granulation tissue in response to an interleukin-1 receptor antagonist (IL-1Ra). This study allows us to further delineate the immune response to wound healing and potentially identify treatment markers. METHODS: Laryngotracheal complexes (LTCs) of donor mice underwent direct airway injury. The LTCs were transplanted into subcutaneous tissue of recipient mice in 2 groups: IL-1Ra treated and untreated. The IL-1Ra-treated arm received daily intraperitoneal injections of IL-1Ra for 3 weeks. The LTCs were then harvested. Granulation formation was measured. The mRNA expression of transforming growth factor (TGF) beta and IL-1 was quantified using real-time reverse transcript polymerase chain reaction. RESULTS: There were statistically significant differences in lamina propria thickness. There were no statistically significant changes in mRNA expression of TGF-ß and IL-1ß between the treated and untreated specimens. CONCLUSIONS: Using a previously described murine model, we delineate inflammatory markers that can be targeted for potential therapy. While the levels of inflammatory markers do not change significantly, the lamina propria thickness shows that the effects of IL-1 have been inhibited. The early use of the IL-1Ra will inhibit the efficacy of IL-1 in the inflammatory cascade and can prevent early granulation formation.

Inflammatory protein expression in human subglottic stenosis tissue mirrors that in a murine model.

OBJECTIVES: We undertook to describe the genetic and protein composition of subglottic stenosis (SGS) by measuring an array of protein expression and messenger RNA levels within human SGS tissue. We also sought to compare this human array to cytokine expression from a murine model of SGS in order to confirm the effective translational nature of our animal model. METHODS: Human granulation tissue from 10 patients with early symptomatic SGS was compared to control bronchus. The expression levels of 24 different cytokines were measured by a Luminex protein assay and real-time polymerase chain reaction. RESULTS: The protein expression in human SGS mirrors that seen in murine SGS. Transforming growth factor ß1, interleukin 1ß, and matrix metalloproteinase 9 were markedly elevated in both human and mouse SGS tissues. The protein array showed a statistically significant elevation in the proinflammatory cytokines tumor necrosis factor α, interleukin 1, granulocyte macrophage colony-stimulating factor, and interferon γ. CONCLUSIONS: This is the first study, to our knowledge, to measure an array of protein expression within human SGS tissue. The expression profile suggests that symptomatic tracheal granulation tissue is mostly within the early inflammatory phase of wound healing and has only begun fibrotic and angiogenic remodeling. This study validates our murine model of SGS, and also helps to define the exact pathways of tissue injury, in the hope of leading to new treatments for this difficult condition.

Histopathologic and immunophenotypic profile of subglottic hemangioma: multicenter study.

OBJECTIVE: Subglottic hemangioma (SGH) is a rare tumor of childhood often associated with airway compromise. Recently, the possibility that many SGH may be congenital hemangiomas rather than classical infantile hemangiomas (IH) has been raised, with important diagnostic and therapeutic implications. We present the largest, multi-institutional study, to date, with immunophenotypic characterization of SGH. METHODS: Biopsy proven SGH samples were retrieved from the archived files of three tertiary referral children's hospitals. Relevant demographic and clinical data corresponding to each of the specimen was collected. Standard hematoxylin and eosin staining (H&E) and glucose transporter protein isoform 1 (GLUT 1) immunohistochemical staining was used to confirm the histological diagnosis and evaluate the immunophenotypic profile of the specimens. RESULTS: 19 cases of SGH were reviewed, 18 of these with tissue blocks available for histological and GLUT1 immunohistochemical staining, and the remaining case with GLUT1 immunostaining previously reported. There was a female to male ratio of 3.75:1. Stridor was the presenting feature of all the patients. Concomitant cutaneous lesions were noted in 9 of 19 patients; with three of the PHACES variety. 16/19 specimens were histologically diagnostic of classical IH by H&E staining and also demonstrated strong GLUT 1 positivity; 1/19 was too small for histological diagnosis, but contained GLUT1-positive capillaries; 1/19 was inflamed granulation tissue by histology and was GLUT1-negative; the final 1/19 was inflamed fibrous tissue, negative for GLUT1. CONCLUSIONS: This multi-institutional pathologic assessment confirms that most subglottic hemangiomas examined were histologically and immunohistochemically confirmed to be IH and similar to IH located in other anatomic locations. GLUT-1 immunohistochemical staining is a valuable tool for confirmatory diagnosis in these patients.

Role of vascular endothelial growth factor A in children with acquired airway stenosis.

OBJECTIVES: Vascular endothelial growth factor A (VEGF-A) is important in the angiogenic response for wound healing. This study investigated whether VEGF-A may play a role in the pathogenesis of acquired airway stenosis. METHODS: Eight lesions from 5 pediatric patients with subglottic stenosis after airway reconstruction (N = 4) or prolonged intubation (N = 1) and normal laryngeal tissue from 5 autopsy patients were included. Formalin-fixed sections of subglottic tissue from each patient were examined by in situ hybridization for the presence of messenger RNA (mRNA) for VEGF-A, vascular endothelial growth factor receptor 1 (VEGFR-1), and vascular endothelial growth factor receptor 2 (VEGFR-2). RESULTS: Strong expression of VEGF-A mRNA was noted in hyperplastic squamous epithelium overlying granulation tissue. Strong expression of VEGFR-1 and VEGFR-2 was noted in the endothelial cells within granulation tissue. No strong labeling of VEGF-A mRNA or its receptors was noted in 2 specimens with mature scar tissue or in the control specimens. CONCLUSIONS: The angiogenic growth factor VEGF-A is strongly expressed in hyperplastic epithelium overlying granulation tissue in airway stenosis. Also, VEGFR-1 and VEGFR-2 mRNAs are strongly expressed in the endothelial cells of granulation tissue. This finding suggests an important role of VEGF-A in the pathogenesis of airway scar formation and stenosis.

Transforming growth factor-beta1, myofibroblasts, and tissue remodeling in the pathogenesis of tracheal injury: potential role of gastroesophageal reflux.

Subglottic stenosis (SGS) is characterized by the obliteration of the tracheal lumen due to excessive deposition of connective tissue. We hypothesize that tracheal injury triggers the early production of transforming growth factor-beta1 (TGF-beta1), a factor implicated in fibroproliferative disorders. In turn, TGF-beta1 stimulates the transformation of tracheal fibroblasts into myofibroblasts with increased matrix production and scar contraction that might be influential in laying the foundation for the development of SGS. Consistent with this hypothesis, histologic analysis of tracheas from humans with SGS and from rats with experimental tracheal injury revealed increased alpha-smooth muscle actin-positive cells as compared to control tracheas, suggesting increased myofibroblast differentiation. Rat tracheal fibroblasts exposed to TGF-beta1 or gastric juice in vitro showed increased expression of alpha-smooth muscle actin, alterations in the expression of matrix molecules, and increased contraction of collagen gels. These findings suggest that gastric juice or other agents of tracheal injury promote tissue remodeling through the stimulation of the differentiation of fibroblasts into myofibroblasts.

Transforming growth factor and neutralizing antibodies in subglottic stenosis.

Transforming growth factor beta 1 (TGF-beta1), which is implicated in the pathogenesis of fibrotic diseases such as interstitial fibrosis, may be associated with subglottic stenosis. To study this hypothesis, we measured TGF-beta1 expression sequentially in 28 rats after posterior cricoid injury, using both standard immunohistochemistry and reverse transcriptase-polymerase chain reaction. In addition, an osmotic pump infused TGF-beta1 in 18 rats, normal saline solution in 9 rats, and neutralizing antibodies in 9 rats. Specimens were stained for fibronectin and procollagen at 1, 7, and 21 days and underwent optical density analysis. In the injured airway, TGF-beta1 expression peaked at 1 day and returned to baseline by 21 days. The TGF-beta1 infusion led to an increase in the expression of extracellular matrix proteins relative to controls. In contrast, neutralizing antibodies led to a decrease in extracellular matrix protein expression. These findings suggest that TGF-beta1 may possibly play a role in the pathogenesis of subglottic stenosis.

Pharmacologic inhibition of collagen in an experimental model of subglottic stenosis.

Subglottic stenosis occurs as a complication of prolonged endotracheal intubation secondary to inflammation with development of scar tissue and subsequent fibrosis. Collagen I and III levels increase during the healing process. Steroids alter the inflammatory response, decreasing recruitment of macrophages and fibroblasts. Beta-aminopropionitrile (betaAPN) inhibits the development of collagen cross-linking. A mechanism that would minimize hypertrophic scarring was sought. Eighteen dogs were anesthetized, had tracheostomies performed, and later had cautery of the mucosa and inner layer of the cricoid cartilage. Of 18 survivors, 6 animals were used as controls, 6 animals received oral Decadron, 2 mg/d, and 6 animals received oral betaAPN, 40 mg/d. There were 9 early deaths--5 in the steroid group. Animals were painlessly sacrificed, and the specimens were sectioned at the cricoid cartilage level and were stained immunohistochemically for antibodies to collagen types I to VI. Analysis of the area of scar and the intensity of stain was performed with Mocha image analysis software. Collagen III increased in control animals to 14.38 +/- 1.85 (intensity stain index), but this reaction was reduced by betaAPN (5.77 +/- 1.78, p < .01). Steroids had no significant effect on formation of any type of collagen. Lathyrogens (betaAPN) may offer a pharmacologic tool to reduce scar tissue.