Small extracellular vesicles as mediators of paracrine senescence in cellular models of aging

Senescent cells accumulate during both physiological and pathological processes, including aging. They are characterized by the expression of the cell cycle inhibitor p16^INK4A and the senescence-associated secretory phenotype (SASP). However, fibroblasts ectopically expressing p16^INK4A do not exhibit a canonical SASP, leaving the mechanisms by which they communicate with their microenvironment largely unknown. Here, we show that human primary fibroblasts ectopically expressing p16^INK4A release significantly more small extracellular vesicles (sEVs) than proliferating cells. Furthermore, we demonstrate that sEVs isolated from p16^INK4A-expressing fibroblasts are biologically active and can mediate paracrine senescence in proliferating cells. Specifically, treatment with these sEVs induces proliferative arrest, upregulates p16^INK4A expression, and activates a DNA damage response in recipient cells. Notably, sEVs from p16^INK4A -expressing cells do not induce the expression of the canonical SASP factors IL-6 and IL-8, suggesting that they promote senescence independently of a classical inflammatory response. We further demonstrate sEV-mediated transmission of paracrine senescence in two cellular models of aging: progerin expression and telomere dysfunction induced by a dominant-negative telomere mutant. In addition, sEVs isolated from fibroblasts derived from aged donors induce paracrine senescence in fibroblasts from young donors. Collectively, these findings identify sEVs released by senescent and aged cells as an important mechanism of intercellular communication and a key mediator of paracrine senescence.