Modulated Fibrosis and Mechanosensing of Fibroblasts by SB525334 in Pediatric Subglottic Stenosis

Objective: Subglottic stenosis (SGS) may result from prolonged intubation where fibrotic scar tissue narrows the airway. The scar forms by differentiated myofibroblasts secreting excessive extracellular matrix (ECM). TGF-beta 1 is widely accepted as a regulator of fibrosis; however, it is unclear how biomechanical pathways co-regulate fibrosis. Therefore, we phenotyped fibroblasts from pediatric patients with SGS to explore how key signaling pathways, TGF-beta and Hippo, impact scarring and assess the impact of inhibiting these pathways with potential therapeutic small molecules SB525334 and DRD1 agonist dihydrexidine hydrochloride (DHX). Methods: Laryngeal fibroblasts isolated from subglottic as well as distal control biopsies of patients with evolving and maturing subglottic stenosis were assessed by alpha-smooth muscle actin immunostaining and gene expression for alpha-SMA, FN, HGF, and CTGF markers. TGF-beta and Hippo signaling pathways were modulated during TGF-beta 1-induced fibrosis using the inhibitor SB525334 or DHX and analyzed by RT-qPCR for differential gene expression and atomic force microscopy for ECM stiffness. Results: SGS fibroblasts exhibited higher alpha-SMA staining and greater inflammatory cytokine and fibrotic marker expression upon TGF-beta 1 stimulation (p < 0.05). SB525334 restored levels to baseline by reducing SMAD2/3 nuclear translocation (p < 0.0001) and pro-fibrotic gene expression (p < 0.05). ECM stiffness of stenotic fibroblasts was greater than healthy fibroblasts and was restored to baseline by Hippo pathway modulation using SB525334 and DHX (p < 0.01). Conclusion: We demonstrate that distinct fibroblast phenotypes from diseased and healthy regions of pediatric SGS patients respond differently to TGF-beta 1 stimulation, and SB525334 has the superior potential for subglottic stenosis treatment by simultaneously modulating TGF-beta and Hippo signaling pathways.