Characterization of Microvesicles Released from Human Red Blood Cells

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.

Tumor-Derived Exosomal Long Noncoding RNAs as Promising Diagnostic Biomarkers for Prostate Cancer

“…They are released into the extracellular space upon fusion of multivesicular bodies with the plasma membrane [20, 21] and play important roles as cellular messengers to trigger diverse physiological and pathological responses and achieve cross-talk without direct contact with one another [22]. They have been found in a variety of biological fluids, such as blood [19], urine [23], saliva [24], milk [25] and bronchoalveolar lavage fluid [26] and are secreted by platelet [27], normal [28, 29] and tumor cells [30, 31]. Analyzing exosomes in the peripheral blood can therefore provide important information on the characteristics of parental cancer cells.…”

Spectrin and Other Membrane-Skeletal Components in Human Red Blood Cells of Different Age

“…Because at the time of the work of Morrison et al, the study of RBC vesiculation, a common phenomenon that can be induced in vitro by a number of treatments, and that also occurs in RBC storage, was in its infancy, no hypothesis was made about the composition of the membrane that was thought to be lost during RBC ageing in vivo . However, It became soon clear through the study of many, that the micro- and nano-vesicles that are released in vitro under various conditions are devoid of components of the membrane skeleton, so that they were very early qualified as spectrin-free vesicles [4-9]. For this reason, it is now generally accepted that the age-dependent loss of RBC membrane also occurs through the shedding of spectrin-free vesicles.…”

Endothelial Cell-Derived Microparticles from Patients with Obstructive Sleep Apnea Hypoxia Syndrome and Coronary Artery Disease Increase Aortic Endothelial Cell Dysfunction

“…Small membranous vesicles shed from cells in response to various stimuli [21], MPs have various roles in the pathogenesis of CAD [22, 23]. In acute coronary syndrome (ACS) patients, procoagulant endothelial MPs are found in greater circulatory concentration compared to control patients, and may contribute to intracoronary thrombus generation [22].…”