Down-Regulation of a Profibrotic Transforming Growth Factor-β1/Cellular Communication Network Factor 2/Matrix...

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)-β1 was up-regulated primarily in glandular epithelium. Cellular communication network factor 2 (CCN2) was mainly up-regulated in stromal fibroblasts surrounding TGF-β1-positive glandular structures. SILSI improved iSGS by reducing fibroblast infiltration and increasing matrix remodeling. Mechanistically, SILSI counteracted the effects of TGF-β1 by inducing matrix metalloprotease 9 (MMP9) expression while repressing CCN2 expression, without affecting TGFβ1 levels. Treatment of primary iSGS-derived fibroblasts with TGF-β1 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-β1. 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-β1/CCN2/MMP9 axis involved in iSGS development.