An Ovine Model Yields Histology and Gene Expression Changes Consistent with Laryngotracheal Stenosis.

OBJECTIVES: Animal models for laryngotracheal stenosis (LTS) are critical to understand underlying mechanisms and study new therapies. Current animal models for LTS are limited by small airway sizes compared to human. The objective of this study was to develop and validate a novel, large animal ovine model for LTS. METHODS: Sheep underwent either bleomycin-coated polypropylene brush injury to the subglottis (n = 6) or airway stent placement (n = 2) via suspension microlaryngoscopy. Laryngotracheal complexes were harvested 4 weeks following injury or stent placement. For the airway injury group, biopsies (n = 3 at each site) were collected of tracheal scar and distal normal regions, and analyzed for fibrotic gene expression. Lamina propria (LP) thickness was compared between injured and normal areas of trachea. RESULTS: No mortality occurred in sheep undergoing airway injury or stent placement. There was no migration of tracheal stents. After protocol optimization, LP thickness was significantly increased in injured trachea (Sheep #3: 529.0 vs. 850.8 um; Sheep #4: 933.0 vs. 1693.2 um; Sheep #5: 743.7 vs. 1378.4 um; Sheep #6: 305.7 vs. 2257.6 um). A significant 62-fold, 20-fold, 16-fold, 16-fold, and 9-fold change of COL1, COL3, COL5, FN1, and TGFB1 was observed in injured scar specimen relative to unaffected airway, respectively. CONCLUSION: An ovine LTS model produces histologic and transcriptional changes consistent with fibrosis seen in human LTS. Airway stent placement in this model is safe and feasible. This large airway model is a reliable and reproducible method to assess the efficacy of novel LTS therapies prior to clinical translation. LEVEL OF EVIDENCE: N/A Laryngoscope, 2024.

Dysfunctional Epithelial Barrier Is Characterized by Reduced E-Cadherin in Idiopathic Subglottic Stenosis.

OBJECTIVES: To aim of the study was to characterize the molecular profile and functional phenotype of idiopathic subglottic stenosis (iSGS)-scar epithelium. METHODS: Human tracheal biopsies from iSGS scar (n = 6) and matched non-scar (n = 6) regions were analyzed using single-cell RNA sequencing (scRNA-seq). Separate specimens were used for epithelial cell expansion in vitro to assess average growth rate and functional capabilities using transepithelial-electrical resistance (TEER), fluorescein isothiocyanate-dextran flux permeability assay, ciliary coverage, and cilia beating frequency (CBF). Finally, epithelial tight junction protein expression of cultured cells was quantified using immunoblot assay (n = 4) and immunofluorescence (n = 6). RESULTS: scRNA-seq analysis revealed a decrease in goblet, ciliated, and basal epithelial cells in the scar iSGS cohort. Furthermore, mRNA expression of proteins E-cadherin, claudin-3, claudin-10, occludin, TJP1, and TJP2 was also reduced (p < 0.001) in scar epithelium. Functional assays demonstrated a decrease in TEER (paired 95% confidence interval [CI], 195.68-890.83 Ω × cm2 , p < 0.05), an increase in permeability (paired 95% CI, -6116.00 to -1401.99 RFU, p < 0.05), and reduced epithelial coverage (paired 95% CI, 0.1814-1.766, fold change p < 0.05) in iSGS-scar epithelium relative to normal controls. No difference in growth rate (p < 0.05) or CBF was found (paired 95% CI, -2.118 to 3.820 Hz, p > 0.05). Immunoblot assay (paired 95% CI, 0.0367-0.605, p < 0.05) and immunofluorescence (paired 95% CI, 13.748-59.191 mean grey value, p < 0.05) revealed E-cadherin reduction in iSGS-scar epithelium. CONCLUSION: iSGS-scar epithelium has a dysfunctional barrier and reduced structural protein expression. These results are consistent with dysfunctional epithelium seen in other airway pathology. Further studies are warranted to delineate the causality of epithelial dysfunction on the downstream fibroinflammatory cascade in iSGS. LEVEL OF EVIDENCE: NA Laryngoscope, 134:374-381, 2024.

Murine Model of Airway Fibrosis has Anatomic, Physiologic, and Molecular Congruency to Human iSGS.

OBJECTIVE: To narrow knowledge gaps in the pathophysiology of idiopathic subglottic stenosis (iSGS) through comparison of a murine subglottic stenosis model with iSGS. STUDY DESIGN: In vivo animal study. SETTING: Academic institution. METHODS: Murine samples/measurements were obtained from mice that underwent chemomechanical injury with a wire brush and bleomycin. Human samples/measurements were obtained from iSGS patients. Anatomic, physiologic, and epithelial molecular data were collected using histology, human peak expiratory flow (PEF) and murine airway conductance, gene expression analysis with quantitative polymerase chain reaction, and protein analysis with quantitative immunohistochemistry. RESULTS: Anatomic patterns of scars at the subglottis and proximal trachea seen in the murine model are similar to iSGS patients. Subglottic stenosis (SGS) mice had a decrease (P = .0194) in airway conductance compared to healthy controls, similar to a decrease (P = .0001) in predilation PEF versus postdilation in iSGS patients. There was decreased epithelial gene expression of E-cadherin (ECAD) (P < 0.01), occludin (OCLN) (P < .01), and cytokeratin-5 (CK5) (P < .05) and protein expression of ECAD (H/M: P < .001), OCLN (H: P < 0.05, M: P < .001), and CK5 (H: P < .001, M: P < .01) in murine SGS and iSGS versus controls. CONCLUSION: The murine SGS model shows anatomic, physiologic, and molecular congruency with human iSGS, making it a reasonable model to investigate iSGS. The molecular similarities in epithelial barrier dysfunction suggest it may best be suited to explore epithelial mechanisms of iSGS and therapies directed at epithelial reconstitution. This model provides a foundation to collect data that will improve understanding of iSGS, and, ultimately, translate into more accurate animal models for future use.

Characterizing the Macrophage Population in Patients With Idiopathic Subglottic Stenosis.

OBJECTIVES: Idiopathic subglottic stenosis (iSGS) is characterized by progressive fibrosis and subglottic luminal narrowing. Currently, immune characterization has focused on T-cells; however, macrophages remain largely unexplored. The goals of this study are to characterize the transcriptome of iSGS macrophages and the fibrogenic nature of identifed biomarkers. STUDY DESIGN: Bioinformatics and in vitro. METHODS: Human tracheal biopsies from iSGS scar (n = 4), and matched non-scar (n = 4) regions were analyzed using single-cell RNA-seq (scRNA-seq). Immunofluorescence (IF) was performed on rapidly processed autopsies (RPA) and iSGS tracheal resections (n = 4) to co-localize S100A8/9 and CD11b. Collagen gene/protein expression was assessed in iSGS fibroblasts (n = 4) treated with protein S100A8/9 (1000 ng/ml). Macrophages were subclustered to identify distinct subpopulations. RESULTS: scRNA-seq analysis revealed S100A8/S100A9 (fold change (FC) = 4.1/1.88, p < 0.001) as top differentially expressed genes in iSGS macrophages. IF exhibited increased CD11b+/S100A8/9+ cells in tracheal samples of iSGS versus RPA (26.75% ± 7.08 vs. 0.594% ± 0.974, n = 4, p = 0.029). iSGS fibroblasts treated with S100A8/9 demonstrated increased gene expression of COL1A1 (FC = 2.30 ± 0.45, p = 0.03, n = 4) and COL3A1 (FC = 2.44 ± 0.40, p = 0.03, n = 4). COL1A1 protein assays revealed an increase in the experimental group, albeit not significant, (p = 0.12, n = 4). Finally, macrophage sub clustering revealed one subpopulation as a predominant source of S100A8/S100A9 expression (FC = 7.94/5.47, p < 0.001). CONCLUSIONS: S100A8/9 is a key biomarker in iSGS macrophages. Although S100A8/9 demonstrates profibrotic nature in vitro, the role of S100A8/9+ macrophages in vivo warrants further investigation. LEVEL OF EVIDENCE: NA Laryngoscope, 133:2308-2316, 2023.