Cellular senescence increases alveolar epithelial vulnerability to mechanical stress: implications for lung injury

Mechanical ventilation is essential for patients with respiratory failure, but mechanical stretch can promote ventilator-induced lung injury, characterized by epithelial damage and inflammatory responses. Aging is a major risk factor for severe respiratory disease; however, how cellular aging influences the response of the alveolar epithelium to mechanical stretch remains poorly understood. We aimed  to investigate whether aging alters the response of primary alveolar epithelial cells (AECs) to cyclic mechanical stretch, focusing on senescence, viability, cytoskeletal integrity, and inflammatory mediators. AECs were isolated from the lungs of young (2–3 months) and aged (20–22 months) male Wistar rats (CEUA 031/21). Senescence was assessed by senescence-associated β-galactosidase (SA-β-gal) flow cytometry, and p16, p21, and p53 expression by quantitative polymerase chain reaction (qPCR). AECs were exposed to cyclic mechanical stretch for 4 h at 0.25 Hz, while non-stretched cells served as controls. Viability was assessed by lactate dehydrogenase (LDH) assay, cytoskeletal organization by phalloidin immunostaining, tight junctions by zonula occludens-1 (ZO-1) immunostaining, and expression of interleukin-6 (IL-6), amphiregulin (AREG), and monocyte chemoattractant protein-1 (MCP-1) by qPCR. Aged AECs exhibited higher p53 and SA-β-gal expression than young cells (p=0.03 for both). Stretch increased LDH release in young (0.15±0.08) and aged (0.13±0.06) AECs compared with static controls (0.08±0.06 and 0.06±0.04). Stretched aged AECs displayed reduced and disorganized actin filaments. No significant differences in IL-6, AREG, or MCP-1 expression were detected. These findings indicate that aging establishes a senescent phenotype associated with increased epithelial vulnerability to mechanical stress. The absence of significant changes in the inflammatory mediators evaluated highlights the complex interplay between aging, mechanical stress, and inflammatory lung injury.