Mechanosensitive Ca²⁺signaling and lung endothelial cell senescence

Cell senescence is an irreversible fate in which the cell stops replicating. Senescent cells accumulate in all organs and tissues with advanced aging, while aging-associated senescence mainly occurs in endothelial cells and macrophages. Upregulation of the cell-cycle arrest genes, including p53, CDKN1A (p21 WAF1/Cip1 ) and CDKN2A (p16 INK4A ), along with the increased activity of senescence-associated β-galactosidase (SA-βgal), are the hallmarks of senescent cells. In the lungs, the percentage of senescent cells (revealed by p16 expression) of total cells increased from 0.016% in young mice (1.6-2.3 months) to 6.208% in aged mice (18-19 months) and 6.926% in old mice (24-25 months). Senescent cells in the lungs release the senescence-associated secretory phenotype (SASP) factors (including inflammatory and mitogenic cytokines and chemokines, extracellular matrix proteases, and circular and mitochondrial DNAs) that can cause vascular inflammation/remodeling, contributing to the development and progression of pulmonary vascular disease. In this study, we aimed to investigate the potential role of mechanosensitive Ca 2+ signaling in lung endothelial cell senescence via the Ca 2+ -activated AKT signaling cascade. Human lung vascular ECs were treated with Doxorubicin to induce cell senescence. The expression levels of p53, p21 and p16 were examined by Western blot and the activity of βgal was examined by X-Gal assay. Extracellular EGTA, a chelator of free Ca 2+ , was used to remove or decrease extracellular free Ca 2+ , and GsMTx4 was used to block mechanosensitive Ca 2+ channels in lung vascular ECs. Doxorubicin, a chemotherapy drug that interacts with DNA, increased p53 and p21 expression levels in lung vascular ECs in a dose-dependent (0.1 to 1 µM) and a time-dependent (6 to 48 hrs) manner. The increase of SA-βgal activity was detected in Doxorubicin-treated lung vascular ECs in a time-dependent manner. Doxorubicin increased the p16 level later in comparison to its augmenting effect on p53 and p21. Doxorubicin increased phosphorylation of AKT at S473 in lung vascular ECs. More importantly, chelation of extracellular Ca 2+ with EGTA significantly inhibited Doxorubicin-induced upregulation of p53 and p21. Blockade of mechanosensitive Ca 2+ influx with GsMTx4, an inhibitor of the mechanosensitive cation channels Piezo1 and TRPC6, also markedly inhibited Doxorubicin-mediated increases in p53 and p21 in lung vascular ECs. These results suggest that Ca 2+ influx through the GsMTX4-sensitive mechanosensitive cation channels (e.g., Piezo1, TRPC6) may contribute to the Doxorubicin-induced senescence, possibly through activation of the AKT/mTOR signaling pathway in lung vascular endothelial cells. This study is supported in part by a grant from the NHLBI/NIH (HL171538) This abstract was presented at the American Physiology Summit 2025 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.