Characterization of microvesicles released from human red blood cells

Nguyen, Duc Bach, Thuy Ly, Thi Bich, Wesseling, Mauro Carlos, Hittinger, Marius, Torge, Afra, Devitt, Andrew, Perrie, Yvonne and Bernhardt, Ingolf (2016). Characterization of microvesicles released from human red blood cells. Cellular Physiology and Biochemistry, 38 (3), pp. 1085-1099.

Abstract

Background/Aims: Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. Conclusion: An increase of intracellular Ca2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs.

Publication DOI: https://doi.org/10.1159/000443059
Divisions: Life & Health Sciences
Life & Health Sciences > Chronic and Communicable Conditions
Life & Health Sciences > Biosciences
Life & Health Sciences > Cellular and Molecular Biomedicine
Life & Health Sciences > Cell & Tissue Biomedical Research
Life & Health Sciences > Pharmacy
Additional Information: © 2016 The Author(s). Published by S. Karger AG, Basel. This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution for commercial purposes as well as any distribution of modified material requires written permission.
Uncontrolled Keywords: extracellular vesicles,microvesicles,red blood cells,phosphatidylserine,fluorescence imaging,cell adhesion,Physiology
Full Text Link: http://www.karg ... FullText/443059
Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
Published Date: 2016-04-03
Authors: Nguyen, Duc Bach
Thuy Ly, Thi Bich
Wesseling, Mauro Carlos
Hittinger, Marius
Torge, Afra
Devitt, Andrew ( 0000-0002-4651-6761)
Perrie, Yvonne
Bernhardt, Ingolf

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