In vitro, in silico and in vivo study challenges the impact of bronchial thermoplasty on acute airway smooth muscle mass loss

Abstract

Bronchial thermoplasty is a treatment for asthma. It is currently unclear whether its histopathological impact is sufficiently explained by the proportion of airway wall that is exposed to temperatures necessary to affect cell survival.Airway smooth muscle and bronchial epithelial cells were exposed to media (37-70°C) for 10 s to mimic thermoplasty. In silico we developed a mathematical model of airway heat distribution post-thermoplasty. In vivo we determined airway smooth muscle mass and epithelial integrity pre- and post-thermoplasty in 14 patients with severe asthma.In vitro airway smooth muscle and epithelial cell number decreased significantly following the addition of media heated to ≥65°C. In silico simulations showed a heterogeneous heat distribution that was amplified in larger airways, with <10% of the airway wall heated to >60°C in airways with an inner radius of ∼4 mm. In vivo at 6 weeks post-thermoplasty, there was an improvement in asthma control (measured via Asthma Control Questionnaire-6; mean difference 0.7, 95% CI 0.1-1.3; p=0.03), airway smooth muscle mass decreased (absolute median reduction 5%, interquartile range (IQR) 0-10; p=0.03) and epithelial integrity increased (14%, IQR 6-29; p=0.007). Neither of the latter two outcomes was related to improved asthma control.Integrated in vitro and in silico modelling suggest that the reduction in airway smooth muscle post-thermoplasty cannot be fully explained by acute heating, and nor did this reduction confer a greater improvement in asthma control.

FIGURE 1

Response of in vitro heated airway smooth muscle (ASM) and human bronchial epithelial (hBEC) cells. a, b) Representative cell morphology for cultures following addition of media heated to 65°C; note the incomplete recovery of ASM (a) compared to hBEC (b) cells over 2 weeks. Scale bar, 0.1 mm. c, d) Longitudinal viability of ASM (c) and hBEC (d) cells following addition of media heated to specified temperatures (mean±se). e, f) Total cell count relative to 37°C-matched control 1 week after the addition of heated media for ASM (e) and hBEC (f) cells. g, h) Proportion of apoptotic and necrotic ASM (g) and hBEC (h) cells determined by flow cytometry 24 h after the addition of media heated to specified temperatures (mean, 95% CI). *p<0.05, **p<0.01, ***p<0.001 versus 37°C controls.

FIGURE 2

Characterisation of bronchial thermoplasty (BT) heating patterns. a) Reference model geometry (inner wall radius of 2.2 mm, outer radius of 3.3 mm). b) Heat map at the end of a single BT activation (10 s). c) Temporal dynamics of the applied voltage (red), electrode temperature (solid blue) and temperature at the midpoint between two electrodes (dashed green; marked by a white dot in b). d) Distribution of heated wall area fractions, corresponding to b.

FIGURE 3

Airway temperature heterogeneity across bronchial generations and heating scenarios. Heating patterns a) at the lowest end of bronchial thermoplasty (BT) applicability (luminal radius of 1.5 mm); b) for a midrange airway (luminal radius of 2.2 mm, corresponding to figure 2b, d) with impeded luminal evaporative cooling (e.g. occluded with a bronchoscope); and c) for a larger airway (luminal radius of 4.4 mm). d) Thermal dynamics of an airway wall after the end of a single BT activation (marked by vertical dashed line) for the reference case of figure 2 (solid and dashed) and for the case of absent tissue perfusion and evaporative cooling (dotted lines). e, f) Temperature distributions at 10  s (e) and 12 s (f), corresponding to the case of absent volumetric tissue cooling.

FIGURE 4

Histology analysis of airway smooth muscle (ASM) content and epithelial integrity in bronchial biopsies (at baseline and at about 1 month post-bronchial thermoplasty (BT)). a) Example endobronchial biopsy stained for α-smooth muscle actin (E: epithelium; LP: lamina propria; G: gland). b) ASM mass % pre- and post-BT (p<0.05). c) Epithelial integrity pre- and post-BT (p<0.01). In b and c, the horizontal line represents the median, the box represents the interquartile range (IQR) and the whiskers represent the minimum and maximum. d) Detailed breakdown of epithelial structure at baseline and post-BT (mean). The total percentage for the baseline does not equal 100% owing to rounding. e) Change in ASM mass versus the number of myofibroblasts per mm² of lamina propria following BT (Spearman's rank correlation r= −0.55, p=0.046), with change in Asthma Control Questionnaire-6 (ACQ6) score (mean (IQR)) reported for each response subgroup (quadrants).