PhD Scientific Days 2025

Molecular Medicine I.

Comparison of spontaneously released and experimentally induced red blood cell-derived extracellular vesicles

Name of the presenter

Adamecz Zsuzsanna

Institute/workplace of the presenter

Semmelweis University, Department of Genetics, Cell- and Immunobiology

Authors

Zsuzsanna Adamecz¹, Mária Péter², Gábor Balogh², Zsolt Török², Attila Tiba³, Edit Buzás^1,4,5, Evelyn Orsó¹

1: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest
2: Laboratory of Molecular Stress Biology, Institute of Biochemistry, HUN-REN Biological Research Centre, Szeged
3: Department of Data Science and Visualization, Faculty of Informatics, University of Debrecen, Debrecen
4: HCEMM SU Extracellular Vesicles Research Group
5: HUN-REN-SU Translational Extracellular Vesicle Research Group

Text of the abstract

Introduction: Red blood cells (RBCs) are anucleated cells that lack a complex endomembrane system. Extracellular vesicles (EVs) are membrane-bound particles released by various cell types. RBC-derived EVs (RBC-EVs) form under various conditions, including eryptosis, an apoptosis-like process in RBCs.
Aims: This study aimed to compare spontaneously released and experimentally induced RBC-EV populations, and to investigate the mechanisms underlying RBC-EV biogenesis through lipidomic profiling.
Methods: RBCs were isolated from EDTA-anticoagulated peripheral blood from healthy volunteers. Although erythrocytes undergo spontaneous vesicle formation, this process can be enhanced in vitro by exposure to A23187 calcium ionophore. In our system, calcium ions and glucose were applied to modulate vesicle formation. RBC-EVs were isolated using differential centrifugation and characterized by nanoparticle tracking analysis (NTA), flow cytometry, transmission electron microscopy (TEM) combined with bioinformatic image analysis, and electrospray ionization mass spectrometry (ESI-MS)-based lipidomics.
Results: RBC-EVs were positive for Annexin V and CD235a, and were sensitive to Triton-X 100 detergent, confirming their vesicular nature. TEM images revealed vesicular structures with heterogeneous size distribution. Bioinformatic analysis of TEM images further supported differences between spontaneously released and A23187-induced RBC-EVs. Lipidomic profiling indicated that A23187-induced RBC-EVs were enriched in membrane curvature-promoting lipids, including phosphatidic acid and ceramide, compared to intact RBCs. Furthermore, increased levels of sphingomyelin in RBC-EVs indicated that raft membrane microdomains are preferentially sorted in the vesicles. We hypothesize that phospholipase-mediated free fatty acid release may contribute to the maintenance of RBC vesiculation.
In addition, exposure to A23187 and calcium ions significantly increased RBC-EV release, while glucose partially inhibited vesicle formation, indicating a regulatory role of glucose in RBC-EV production.
Conclusion: Our findings highlight differences between spontaneously released and experimentally induced RBC-EVs, which should be considered in future research. Lipidomic analysis provides insights into the membrane remodeling processes, contributing to EV biogenesis.
Funding: RRF-2.3.1-21-2022-00003