Thrombin Preconditioning Boosts Biogenesis of Extracellular Vesicles from Mesenchymal Stem Cells and Enriches Their Cargo Contents via Protease-Activated Receptor-Mediated Signaling Pathways.

Dong Kyung Sung,So Yoon Ahn,Yun Sil Chang,Won Soon Park,Se In Sung

doi:10.3390/ijms20122899

Dong Kyung Sung, So Yoon Ahn + Show 3 more

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https://doi.org/10.3390/ijms20122899

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Abstract

We investigated the role of protease-activated receptor (PAR)-mediated signaling pathways in the biogenesis of human umbilical cord blood-derived mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) and the enrichment of their cargo content after thrombin preconditioning. Immunoblot analyses showed that MSCs expressed two PAR subtypes: PAR-1 and PAR-3. Thrombin preconditioning significantly accelerated MSC-derived EV biogenesis more than five-fold and enriched their cargo contents by more than two-fold via activation of Rab5, early endosomal antigen (EEA)-1, and the extracellular signal regulated kinase (ERK)1/2 and AKT signaling pathways. Blockage of PAR-1 with the PAR-1-specific antagonist, SCH79797, significantly suppressed the activation of Rab5, EEA-1, and the ERK1/2 and AKT pathways and subsequently increased EV production and enriched EV cargo contents. Combined blockage of PAR-1 and PAR-3 further and significantly inhibited the activation of Rab5, EEA-1, and the ERK1/2 and AKT pathways, accelerated EV production, and enriched EV cargo contents. In summary, thrombin preconditioning boosted the biogenesis of MSC-derived EVs and enriched their cargo contents largely via PAR-1-mediated pathways and partly via PAR-1-independent, PAR-3-mediated activation of Rab5, EEA-1, and the ERK1/2 and AKT signaling pathways.

Highlights

Extracellular vesicles (EVs), which have a diameter of 40–150 nm, are secretory membrane vesicles released by a variety of cells
Preconditioning-induced increases in ERK1/2 and AKT phosphorylation were more significantly inhibited by SCH79797 than by protease-activated receptor (PAR)-3-specific siRNA, but were most significantly inhibited by the combination of the two (Figure 3G,H). These results suggest that PAR-1 signaling is more strongly involved, whereas PAR-3 signaling is partially involved, in the thrombin preconditioning-induced activation of Rab5, early endosomal antigen (EEA)-1, ERK1/2, and AKT
Increases in cargo protein by thrombin treatment were more significantly inhibited by SCH79797 treatment than by PAR-3 siRNA transfection and were most significantly inhibited by the combination of SCH79797 treatment and PAR-3 siRNA transfection (Figure 4E,F). These results suggest that PAR-1 signaling is strongly involved and PAR-3 signaling is partially involved in thrombin preconditioning-stimulated EV production and cargo protein enrichment

Summary

Introduction

Extracellular vesicles (EVs), which have a diameter of 40–150 nm, are secretory membrane vesicles released by a variety of cells. The major advantage of using cell-free MSC-derived EV therapy over transplantation of live MSCs is that EVs can overcome the concerns associated with live cell therapy. Compared to their parent MSCs, EVs can be stored without losing their biological function, and are more suitable for use as an “off the shelf” drug [17]. In our previous study [11], we observed that thrombin preconditioning of MSC-derived EVs accelerated cutaneous wound healing, which was better than that in hypoxia, H2O2, or lipopolysaccharide exposure, by boosting EV biogenesis and enriching their cargo contents. The precise molecular mechanisms underlying the positive effects of thrombin preconditioning on EV biogenesis and their cargo contents have not yet been elucidated

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