Dysregulation of extracellular matrix (ECM) is implicated in the pathogenesis of multiple lung diseases including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and asthma. Although alterations in ECM composition, structure and stiffness have been documented in these conditions, a significant knowledge gap remains in our mechanistic understanding of how airway basal cells (BCs) interpret and respond to these mechanical cues in ways that influence functional airway regeneration. The objective of this study is to elucidate the mechanisms by which ECM stiffness regulates airway BC phenotype and to determine how these changes impact airway function. We hypothesize that increased ECM stiffness enhances the expression of Ca 2+ -dependent mechanosensitive channels in airway BCs inducing squamous genes programs that drive pathological airway repair rather than functional regeneration. To test this hypothesis, we generated polydimethylsiloxane (PDMS) blends with elastic moduli (E) of 5 kPa, 50 kPa, and 1.34 MPa, mimicking the stiffness of distinct anatomical regions of the airway including muscle (dorsal), intermediate and cartilaginous (ventral) membranes, respectively. Donor-derived human airway BCs (N >3 biological replicates) were seeded on these stiffness-defined substrates coated with growth factor-free matrigel and cultured for six days prior to analysis. For comparison, BCs were also cultured in 3D hydrogels to mimic ‘soft’ (106 kPa) conditions. Our preliminary data demonstrate increased expression of squamous genes, including involucrin (IVL), small proline-rich (SPR) proteins and cytokeratin 13 (KRT13), as substrate stiffness increased. Interestingly, these stiffness-dependent transcriptional changes were more pronounced in male donors compared to female donors. To assess the reversibility of this phenotype, we conducted reciprocal culture experiments involving expansion in 3D ‘soft’ matrix followed by transition to 2D ‘stiff’ matrix for analysis and vice versa. These experiments revealed that ‘stiff’ 2D substrates promote a squamous BC profile, whereas 3D matrices reverse this phenotype. To investigate the mechanistic drivers of this phenotype, we measured expression of mechanosensitive Piezo channels and transient receptor potential vanilloid 4 (TRPV4) channels. We observed a significant increase in TRPV4 expression with increasing substrate stiffness. In conclusion, our findings support a critical role for ECM stiffnesses defining BC phenotype, with stiffer substrates promoting a squamous program in airway BCs. The observed upregulation of TRPV4 on the stiff substrates suggests that this channel may mediate key intracellular signaling events that shape BC behavior in chronically stiff microenvironments. These studies will help identify fundamental regulators of airway epithelial mechanotransduction and clarify how ECM stiffness influences BC stemness and tissue regeneration. This project was supported by NSF: ENG-BIOTECH #15265800 and the Cystic Fibrosis Foundation (CFF) #RYAN21XX0, both awarded to ALR This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Pathmanathan et al. (Fri,) studied this question.