Abstract
Although it is widely accepted that cancer-derived extracellular vesicles (EVs) carry DNA cargo, the association of cell-free circulating DNA (cfDNA) and EVs in plasma of healthy humans remains elusive. Using a physiological exercise model, where EVs and cfDNA are synchronously released, we aimed to characterize the kinetics and localization of DNA associated with EVs. EVs were separated from human plasma using size exclusion chromatography or immuno-affinity capture for CD9+, CD63+, and CD81+ EVs. DNA was quantified with an ultra-sensitive qPCR assay targeting repetitive LINE elements, with or without DNase digestion. This model shows that a minute part of circulating cell-free DNA is associated with EVs. During rest and following exercise, only 0.12% of the total cfDNA occurs in association with CD9+/CD63+/CD81+EVs. DNase digestion experiments indicate that the largest part of EV associated DNA is sensitive to DNase digestion and only ~20% are protected within the lumen of the separated EVs. A single bout of running or cycling exercise increases the levels of EVs, cfDNA, and EV-associated DNA. While EV surface DNA is increasing, DNAse-resistant DNA remains at resting levels, indicating that EVs released during exercise (ExerVs) do not contain DNA. Consequently, DNA is largely associated with the outer surface of circulating EVs. ExerVs recruit cfDNA to their corona, but do not carry DNA in their lumen.
Highlights
The results demonstrate that only a minute part of cellfree DNA (cfDNA) is associated with extracellular vesicles (EVs) in human plasma
Using a highly sensitive quantitative real-time PCR (qPCR), we found a small amount of DNA in the lumen of immuno-affinity captured CD9+ /CD63+ /CD81+ EVs, which was protected from DNase digestion
CfDNA and EVs both increase during exercise, whereas the cfDNA increase appears to occur completely independent of EVs and DNA is attached to the EV surface
Summary
All cells of the human body are constantly and actively releasing a large amount of molecular material into the extracellular space. Or indirectly, those extracellular molecules can interact in signaling pathways having crucial roles in the development of homeostasis and the coordination of physiological processes [1]. Acute physical exercise is a relevant stressor to disrupt homeostasis and trigger the release of a plethora of molecules into the circulation. A single bout of exercise affects thousands of molecules which orchestrate biological processes including energy metabolism, oxidative stress, inflammation, growth factor response, as well as other regulatory pathways [2]. To the release of proteins, collectively referred as secretome [3], nucleic acids including DNA [4,5,6], and RNA [7]
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