Excessive Stretch of Airway Smooth Muscle (but Not Epithelial) Cells Activates Paracrine TGF-β1 to Induce Smad2-Mediated G1 Arrest in Airway Epithelial Cells.

BACKGROUND

Although mechanical ventilation (MV)-associated excessive stretch directly damages airway epithelial cells (AECs) and contributes to ventilator-induced lung injury (VILI), the contribution of paracrine factors from airway cells, particularly airway smooth muscle cells (ASMCs) and AECs, remains poorly understood. As stretch-sensitive cells, ASMCs and AECs may respond to excessive stretch by secreting paracrine factors such as transforming growth factor β1 (TGF-β1), thereby disrupting airway epithelial integrity. This study compared the sensitivity of cultured ASMCs and AECs to MV-associated excessive stretch and their ability to induce paracrine factors, particularly TGF-β1, that inhibit AEC proliferation in vitro.

METHODS

Human ASMCs and AECs (16HBE14o-) were cultured under static, physiological stretch (5% strain), or pathological excessive stretch (13% strain) conditions for 72 h. Conditioned media were collected and used to culture 16HBE14o- cells for 48 hours. Active and total TGF-β1 levels in the conditioned media were measured using an enzyme-linked immunosorbent assay. Cell viability, proliferation, migration, stress fiber formation, cell cycle distribution, and Smad2 signaling were assessed using the Cell Counting Kit-8 assay, cell counting, wound healing assay, immunofluorescence, flow cytometry, quantitative reverse transcription polymerase chain reaction, and Western blotting, respectively.

RESULTS

Conditioned media derived from ASMCs, but not 16HBE14o- cells, cultured under 13% excessive stretch inhibited 16HBE14o- cell proliferation. In addition, excessive stretch enhanced TGF-β1 activation in ASMCs in an integrin αV-dependent manner. Experiments using a TGF-β1-neutralizing antibody and transforming growth factor β receptor I inhibitors demonstrated that inhibition of 16HBE14o- cell proliferation by conditioned media derived from excessively stretched ASMCs was dependent on TGF-β1 activation and Smad2 signaling.

CONCLUSION

Conditioned media derived from ASMCs, but not AECs, exposed to MV-associated excessive stretch induced G1 cell cycle arrest in AECs via TGF-β1/Smad2 signaling. These findings suggest that ASMCs are more sensitive than AECs to excessive stretch-induced TGF-β1 activation and may thereby contribute to disruption of airway epithelial integrity. Targeting this paracrine pathway may warrant further investigation as a potential strategy for preventing or treating VILI.