Aging impact on canine extracellular vesicles production, size, and miRNA content.

Increasing life expectancy has been accompanied by a higher prevalence of age-associated conditions, underscoring the need to better understand biological mechanisms of aging and to identify accessible biomarkers. Extracellular vesicles (EVs) are lipid-bilayer vesicles that mediate intercellular communication by transporting proteins, lipids, and regulatory RNAs, including microRNAs (miRNAs). Here, we investigated whether the abundance, morphology, and molecular cargo of plasma-derived EVs differ between young and senior dogs. Blood samples were obtained from 24 clinically healthy dogs in Gama (Federal District, Brazil), and plasma EVs were isolated by sequential centrifugation, filtration through a 0.22 μm filter, and ultracentrifugation. EVs concentration and size distribution were assessed by nanoparticle tracking analysis (NTA), morphology by transmission electron microscopy (TEM), and cargo by quantification of total protein and sterols using commercial assays. In addition, we quantified miR-19b, miR-29c, miR-7, miR-155, and miR-21 by Real Time Quantitative Polymerase Chain Reaction (RT-qPCR). Senior dogs exhibited a lower plasma EV yield and greater size heterogeneity, with a higher proportion of larger EVs. Total protein and sterol content per starting plasma volume were reduced in the senior group; however, sterol normalized per EV was increased, consistent with compositional remodeling of circulating vesicles with age. Finally, EV-associated miRNA levels were reduced in senior dogs, particularly miR-19b and miR-29c. Collectively, these findings indicate that canine aging is associated with marked changes in plasma EV abundance, morphology, and cargo, indicating that EVs could represent a promising tool for investigating age-related disorders in dogs.