Matrix Stiffness Promotes Cigarette Smoke-induced Pro-inflammatory Secretion in Small Airway Epithelial Cells via PIEZO1 Activation

Background: Persistent airway inflammation following smoking cessation drives irreversible airflow limitation in COPD. Small airway remodeling in COPD establishes a stiffened extracellular matrix surrounding airway epithelium. However, whether ECM stiffness promotes cigarette smoking extracts (CSE)-induced epithelial inflammatory responses via PIEZO1-mediated mechanotransduction remains unclear. Methods: Primary human small airway epithelial cells (hSAECs) were cultured on substrates with defined stiffness (8, 32, and 64kPa) under 6% CSE stimulation for 48 h. Piezo1 was silenced using siRNA or activated with Yoda1 (1 μM). RNA-sequencing of cell lysates was performed, with differential gene/protein expression validated by qPCR/WB and cytokines secretion detected via ELISA. Results: Piezo1 knockdown attenuated, whereas PIEZO1 activation exacerbated, cigarette smoke extract (CSE)-induced IL-1β and CXCL5 expression in hSAECs at both mRNA and protein levels. Increased substrate stiffness amplified CSE-triggered inflammatory responses (IL-1β/CXCL5), while Piezo1 knockdown disrupted the mechanical amplification of inflammatory responses. RNA-sequencing analysis reveals that Piezo1 knockdown induced enrichment of AGE-RAGE, Focal adhesion and Hippo signaling etc. We then validated by WB that YAP (Hippo signaling) is up-regulated by Piezo1 knockdown in CSE-treated hSAECs. Conclusion: Elevated matrix stiffness by small airway remolding might synergize with cigarette smoke exposure to amplify pro-inflammatory cytokine secretion in small airway epithelium via PIEZO1-dependent mechanotransduction, thus contributes to persistent airway inflammation in COPD.