Tracheobronchomalacia (TBM) is a common congenital disorder in which the cartilaginous rings of the trachea are weakened or missing. Despite the high prevalence and clinical issues associated with TBM, the etiology is largely unknown. Our previous studies demonstrated that Wntless (Wls) and its associated Wnt pathways are critical for patterning of the upper airways. Deletion of Wls in respiratory endoderm caused TBM and ectopic trachealis muscle. To understand mechanisms by which Wls mediates tracheal patterning, we performed RNA sequencing in prechondrogenic tracheal tissue of Wls;Shh embryos. Chondrogenic Bmp4, and Sox9 were decreased, while expression of myogenic genes was increased. We identified Notum, a deacylase that inactivates Wnt ligands, as a target of Wls induced Wnt signaling. Notum's mesenchymal ventral expression in prechondrogenic trachea overlaps with expression of Axin2, a Wnt/β-catenin target and inhibitor. Notum is induced by Wnt/β-catenin in developing trachea. Deletion of Notum activated mesenchymal Wnt/β-catenin and caused tracheal mispatterning of trachealis muscle and cartilage as well as tracheal stenosis. Notum is required for tracheal morphogenesis, influencing mesenchymal condensations critical for patterning of tracheal cartilage and muscle. We propose that Notum influences mesenchymal cell differentiation by generating a barrier for Wnt ligands produced and secreted by airway epithelial cells to attenuate Wnt signaling.

Tracheobronchomalacia and Complete Tracheal Rings are congenital malformations of the trachea associated with morbidity and mortality for which the etiology remains poorly understood. Epithelial expression of Wls (a cargo receptor mediating Wnt ligand secretion) by tracheal cells is essential for patterning the embryonic mouse trachea's cartilage and muscle. RNA sequencing indicated that Wls differentially modulated the expression of BMP signaling molecules. We tested whether BMP signaling, induced by epithelial Wnt ligands, mediates cartilage formation. Deletion of Bmp4 from respiratory tract mesenchyme impaired tracheal cartilage formation that was replaced by ectopic smooth muscle, recapitulating the phenotype observed after epithelial deletion of Wls in the embryonic trachea. Ectopic muscle was caused in part by anomalous differentiation and proliferation of smooth muscle progenitors rather than tracheal cartilage progenitors. Mesenchymal deletion of Bmp4 impaired expression of Wnt/β-catenin target genes, including targets of WNTsignaling: Notum, and Axin2. In vitro, rBMP4 rescued the expression of Notum in Bmp4 deficient tracheal mesenchymal cells and induced Notum promoter activity via SMAD1/5. RNA sequencing of Bmp4 deficient tracheas identified genes essential for chondrogenesis and muscle development co-regulated by BMP and WNT signaling. During tracheal morphogenesis, WNT signaling induces Bmp4 in mesenchymal progenitors to promote cartilage differentiation and restrict trachealis muscle. In turn, Bmp4 differentially regulates the expression of Wnt/β-catenin targets to attenuate mesenchymal WNT signaling and to further support chondrogenesis.

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Objectives/Hypothesis To develop a reproducible and consistent chronic subglottic stenosis (SGS) in an endoscopic animal model. Study Design Prospective study. Methods We conducted a prospective study using New Zealand white rabbits. Chronic SGS was induced endoscopically by Bugbee electrocautery to 50% to 75% of the subglottic area's circumference, followed by 4‐hour endotracheal intubation. The rabbit airways were endoscopically assessed and sized with uncuffed endotracheal tubes (ETTs) before the injury, during follow‐up, and at the endpoints. There were four endpoints: 2, 4, 6, and 8 weeks post SGS induction. Animals were humanely euthanized for histopathological examination of the subglottic injury site and microscopic measurement of the cricoid lumen. Results Twenty‐two rabbits reached the endpoints, and 18 rabbits developed chronic SGS. ETT size significantly decreased by 0.5 from preinjury to the endpoint in all groups, P < .001. Control median cricoid lumen measurements were 20.48 mm2, the median cricoid lumen measurement for the 2 weeks endpoint was 14.3 mm2, 4 weeks 11.69 mm2, 6 weeks 16.03 mm2, and 8 weeks endpoint median was 16.33 mm2. Histopathological examination showed chronic scar tissue and new cartilage formation at the cricoid level, mainly at the posterior subglottic injury site starting from 4 weeks postinjury. Collagen staining revealed substantial amounts of organized collagen and different collagen orientation starting 4 weeks postinjury lasting until 8 weeks postinjury. Conclusion We developed an animal model to study chronic SGS. This model will be utilized to compare different endoscopic treatment interventions in acute SGS versus chronic SGS and further define the molecular basis of SGS. Level of Evidence NA Laryngoscope, 132:1909–1915, 2022