Impact of cigarette smoke on vascular smooth muscle cell senescence

Aging and smoking are major risk factors for cardiovascular diseases. One characteristic of aging is the accumulation of senescent cells (e.g. cells that permanently lose the ability to divide). Senescent vascular smooth muscle cells (VSMCs) are present in atherosclerotic plaques, inducing an inflammatory environment and contributing to plaque instability and atherosclerotic complications. Traditional cigarette (TC) smoke is a known exacerbator of age-related diseases and the combustion of tobacco releases thousands of toxic chemicals. To reduce the harm associated with TC, alternative products, such as tobacco heating-not-burning products (THPs), have been developed. In our study, we compared, in the context of aging, the effects of aqueous extracts (AEs) from TC and THP on senescent VSMCs. Doxorubicin-induced senescent (DIS) VSMCs have been used as a positive control. VSMCs passaged 5 to 7 times (non-senescent cells) were incubated for 48h with 10% TC and THP AEs or doxorubicin 100 nM. Then, we measured several typical senescence markers, such as: senescence associated-β-galactosidase (SA-β-gal) activity, cell proliferation, cell cycle, senescence-associated genes and proteins expression, reactive oxygen species (ROS) production, and morphological changes. TC increased SA-β-gal positive cells, slowed down cell proliferation, and down-regulated proliferation cell nuclear antigen (PCNA) expression, similar to what we observed in the DIS VSMCs. Instead, THP did not affect cell proliferation or PCNA expression. Expression of cell cycle inhibitors was upregulated in both TC and THP, but only TC arrested cells at the G2/M phase. Moreover, only TC significantly induced the expression of inflammatory markers (IL1, IL6, IL8, and MMP3) and increased mitochondrial and intracellular ROS production. Finally, TC, but not THP, increased the number of large and regular nuclei, a typical feature of senescent cells. In conclusion, traditional cigarette smoke induces senescence in VSMCs similar to what we observed in doxorubicin-treated VSMCs. On the contrary, THP lacked most TC and doxorubicin-related functional, structural, and molecular effects on senescence, demonstrating a significantly reduced impact on VSMC senescence.