Impact of cryotherapy on airway stenosis: In vivo, ex vivo, and in vitro studies

Cryotherapy is a promising technique for treating benign central airway stenosis (BCAS), but its underlying mechanisms remain unclear. This study investigated cryoablation effects on stenosis recurrence using in vivo, ex vivo, and in vitro models. Airway stenosis was induced in fourteen pigs, divided into balloon dilatation only (BD) and balloon dilatation with cryoablation (BD + C) groups (n = 7). Pigs were observed using bronchoscopy and computed tomography. Postmortem analyses included histopathology, Western blot, and quantitative RT-PCR (qPCR). In vitro studies using human lung fibroblasts (MRC-5 cells) assessed fibrosis-related markers and cellular responses using Western blot, qPCR, immunocytochemistry, and migration assays. In vivo observation revealed reduced stenosis progression in the BD + C group. Ex vivo analysis showed significantly lower histopathology scores for inflammation, epithelial denudation, and fibrosis, plus reduced fibrosis-related markers including collagen I and III, fibronectin, α-smooth muscle actin (α-SMA), and transforming growth factor-β1 (TGF-β1) in the BD + C group at protein and mRNA levels. In vitro studies using TGF-β1-treated MRC-5 cells exposed to freezing showed reduced collagen I and α-SMA expression and impaired migration compared to controls. Cryoablation may inhibit stenosis progression by reducing TGF-β1-regulated extracellular matrix deposition and fibrosis. These findings provide mechanistic insights into cryotherapy's therapeutic benefits for BCAS management.