Effect of exercise on the plasma vesicular proteome: a methodological study comparing acoustic trapping and centrifugation

Abstract

a Extracellular vesicles (EVs) are a heterogeneous group of actively released vesicles originating from a wide range of cell types. Characterization of these EVs and their proteomes in the human plasma provides a novel approach in clinical diagnostics, as they reflect physiological and pathological states. However, EV isolation is technically challenging with the current methods having several disadvantages, requiring large sample volumes, and resulting in loss of sample and EV integrity. Here, we use an alternative, non-contact method based on a microscale acoustic standing wave technology. Improved coupling of the acoustic resonator increased the EV recovery from 30% in earlier reports to 80%, also displaying long term stability between experiment days. We report a pilot study, with 20 subjects who underwent physical exercise. Plasma samples were obtained before and 1 h after the workout. Acoustic trapping was compared to a standard high-speed centrifugation protocol, and the method was validated by flow cytometry (FCM). To monitor the device stability, the pooled frozen plasma from volunteers was used as an internal control. A key finding from the FCM analysis was a decrease in CD62E+ (E-selectin) EVs 1 h after exercise that was consistent for both methods. Furthermore, we report the first data that analyse differential EV protein expression before and after physical exercise. Olink-based proteomic analysis showed 54 significantly changed proteins in the EV fraction in response to physical exercise, whereas the EV-free plasma proteome only displayed four differentially regulated proteins, thus underlining an important role of these vesicles in cellular communication, and their potential as plasma derived biomarkers. We conclude that acoustic trapping offers a fast and efficient method comparable with high-speed centrifugation protocols. Further, it has the advantage of using smaller sample volumes (12.5 μL) and rapid contact-free separation with higher yield, and can thus pave the way for future clinical EV-based diagnostics.

Platelets, endothelial cells and leukocytes contribute to the exercise‐triggered release of extracellular vesicles into the circulation

“…Yet, we did not find evidence that muscle cells or hepatic cells contribute to the ExerV pool in our exercise setting. In a recent study using acoustic trapping, ExerVs have been studied 1 h after exercise [53]. Similar to us, the group reported the release of protein cargo of the respective ExerVs that mostly reflect proteins known to be released or expressed by either blood cells, or the endothelium.…”

Proteomic profiling of extracellular vesicles reveals additional diagnostic biomarkers for myocardial infarction compared to plasma alone

“…We have previously shown that the proteomic composition of EVs analyzed with mass spectrometry is highly similar comparing acoustic seed trapping and high-speed centrifugation 26 but wanted to confirm that there was no impact of EV isolation technique on the results of the proximity extension assay. To this end, we re-analyzed a previously published data set 27 comprising a total of 30 acoustically trapped and 30 centrifuged EV samples from 10 healthy donors assayed with the Olink CVD II PEA Panel. 59 and 55 proteins were detected (i.e.…”

The mechanisms and treatments for sarcopenia: could exosomes be a perspective research strategy in the future?

“…Exosomes, an important group of extracellular vehicles (EVs), are membrane‐contained vesicles that carry protein, nucleic acid, and lipid cargoes released from a wide range of cell types . According to their size, membrane composition and content, exosomes are defined as a homogenous population of endogenous vesicles with a diameter of 30–200 nm, which are derived from inward budding of the multivesicular body membrane and secreted into the outside of the cells upon fusion of multivesicular body and plasma membrane .…”