PhD Scientific Days 2024

Budapest, 9-10 July 2024

Poster Session A - Molecular Medicine 1.

Characterization of human red blood cell-derived extracellular vesicles (RBC-EVs)

Author(s)

Zsuzsanna Adamecz1, Edit Buzás1, Evelyn Orsó1
1: Institute of Genetics, Cell- and Immunbiology, Semmelweis University, Budapest

Text of the abstract

Introduction: Extracellular vesicles (EVs) are cell derived membrane particles. Red blood cells (RBCs) are anucleated cells without an extended intramembranous system. Formation of RBC-EVs occur under different circumstances, for example during eryptosis, an apoptosis-like process of RBCs. Previous studies have demonstrated the association of RBC-EVs with cardiovascular diseases.
Aims: The major purpose of our study is to characterise RBC-EVs, induced by different in vitro stimuli.
Methods: Human RBCs were isolated from EDTA-anticoagulated whole peripheral blood of healthy volunteers. Formation of RBC-EVs were induced in vitro by A23187 (ionophore) or cold stress (4°C), modulated by the administration of Ca2+, glucose, adenosine or 2-deoxy-D-glucose. Characterization of RBC-EVs were performed by flow cytometry, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM).
Results: Spontaneous formation of RBC-EVs is a time-dependent process and it can be dramatically enhanced by the administration of A23187 and/or Ca2+. EV formation could be reversed by glucose and adenosine. Glucose treatment was more effective in inhibiting EV formation than adenosine, but the two substances acted synergistically. This effect of inhibiting EV formation could not be seen when using the glucose analogue 2-deoxy-D-glucose, suggesting the important role of glucose in RBC-EV release.
The released RBC-EVs were annexin-V positive, showing the externalized phosphatidylserine on their surface, and were also positive for CD235a (glycophorine-A). On the other hand they did not carry any of the three classical tetraspanin markers CD9, CD63 and CD81. In flow cytometry the particles were sensitive to Triton-X 100 detergent, indicating their vesicular nature.
The average diameter of the RBC-EVs were 200-210 nm (measured by NTA), which is in line with previously published data. TEM imaging revealed at least two different subsets of RBC-EVs: (i) „standard” vesicles of low electron density and (ii) other vesicular structures with high electron density. TEM images also show the budding of RBC-EVs, thus producing even smaller EVs.
Conclusion: We have established a well-managed in vitro system to study the formation of RBC-EVs. In future experiments we plan to study the interaction of RBC-EVs with different cell lines.
Funding: National Cardiovascular Laboratory