Exercise-triggered release of extracellular vesicles and circulating cell-free DNA in healthy athletes

Physical exercise induces acute physiological changes leading to enhanced whole body cross-talk. These acute alterations cause systemic adaptations which promote health on a long-term. Extracellular vesicles (EVs), which transport DNA and RNA, as well as cell-free DNA (cfDNA) are elevated in blood plasma in response to various exercise stimuli. EVs are considered to be involved in cardiovascular and inflammatory signaling leading to adaptation processes induced by regular physical exercise. However, the triggers of cfDNA and EV release upon exercise remain elusive.

cfDNA and EV dynamics and possible origin were evaluated in incremental cycling involving 21 healthy male athletes. Venous blood collected pre, during, and post exercise, was used for the estimation of cfDNA concentrations and semi-quantitative analysis of EV elevations. Increasing levels of EV markers were detected in a load-responsive fashion (~1.5-fold during, and ~2.5-fold post) estimated by western blot analysis of EV specific proteins (CD9, CD63, CD81, and Syntenin) in isolated EVs. Multiplexed phenotyping revealed a panel of cellular marker proteins present on EVs and indicated origin from leukocytes, including lymphocytes (CD4⁺EVs, CD8⁺EVs), monocytes (CD14⁺EVs) and antigen presenting cells (MHCI⁺EVs, MHCII⁺EVs), endothelial cells (CD105⁺EVs, CD146⁺EVs) and platelets (CD41b⁺EVs, CD62P⁺EVs). Increasing concentrations of venous cfDNA (25 ±11.9 ng/ml pre exercise, 45.2 ±25 ng/ml at a RQ 0.9 and 163.5 ±90.2 ng/ml post exercise) were recorded. cfDNA changes correlated with leukocyte (ρ = 0.637, p-value < 0.001), lymphocyte (ρ = 0.546, p-value < 0.001), neutrophil (ρ = 0.507, p-value < 0.001), and monocyte (ρ = 0.489, p-value < 0 .001) blood counts, as well as with performance diagnostic parameters like oxygen uptake (ρ = 0.815, p‑value < 0.001) and lactate levels (ρ = 0.819, p-value < 0.001).

Conclusively, blood cfDNA and EV concentrations both reflect physical exertion in cycling exercise testing and their release can be associated with cells of the circulation and the inflammatory system.