Characterization of replicative and doxorubicin-induced senescence models in vascular smooth muscle cells

Background: Cellular senescence is characterized by progressive exhaustion proliferation, senescence-associated secretory phenotype (SASP), high level of oxidative stress and dysfunctional mitochondria. Accumulation of senescent vascular smooth muscle cells (VSMCs) contributes to aging as well as cardiovascular disease. Senescent VSMCs are present in atherosclerotic plaques and contribute to their instability. Aim: Since there are no unanimously agreed senescence markers in human VSMCs, we aimed at establishing the molecular signatures of replicative senescence (RS) and doxorubicin-induced senescence in human VSMCs by means of multi-biomarkers approaches, performing an in-deep cellular morphological analysis and evaluating the expression of manually selected senescence- associated genes. Materials and methods: Human aortic VSMCs were serially passaged to represent different stages of RS and used from 5th to 7th passages (young cells) and from 15th to 17th passages (old cells), and to perform doxorubicin-induced senescence model we treated young cells with 100nM of doxorubicin for 48 hours. We measured SA-β-gal activity (a marker of senescence), genes and proteins expression by qPCR and western blot analysis, cell proliferation by cell counting, cell cycle, reactive oxygen species (ROS) production and mitochondrial membrane potential by flow cytometry, mitochondrial function by seahorse, mitochondrial morphology by confocal microscopy and morphological and nuclear changes by immunofluorescence. Results: Both RS and doxorubicin-induced senescence cells showed more than 50% of SA-β-gal positive cells compared to 20% of young cells, a flattened appearance, and enlarged and irregular nuclei, with LMNB1 and HMGB1 expression downregulated, indicating an altered nuclear membrane. The expression of cell cycle inhibitors (p21/p16) was upregulated in both models, in particular we observed a G1-cycle arrest in RS instead a G2-cycle arrest in doxorubicin-induced senescence model. Then, RS significantly induced SASP molecules (e.g. IL1β, IL6, IL8, MMP-3) expression, instead in doxorubicin-induced senescence model we observed higher expression of inflammatory markers only after 3 days of recovery. Finally, the two senescence models showed mitochondrial dysfunction characterized by a decrease in respiratory capacity together with a decreased mitochondrial membrane potential, accompanied by increased production of mitochondrial ROS. Furthermore, both senescence models showed altered mitochondria morphology with a high percentage of fragmented mitochondria compared to the high proportion of elongated mitochondria in young VSMCs . Conclusions and Future Perspectives: We performed a detailed characterization of RS and doxorubicin-induced senescence model in VSMCs underling their similarities and differences. Then, these models will be suitable to identify potential anti-aging factors (e.g. senolytics or senomorphics) as a therapeutic approach in aging and aging-related diseases, or analyze the effects of pro-atherosclerotic risk factors(i.e. cigarette and tobacco products smoke) on senescence.