Microvesicle Formation Induced by Oxidative Stress in Human Erythrocytes

Abstract

Extracellular vesicles (EVs) released by different cell types play significant role in many physiological and pathophysiological processes. In physiological conditions red blood cells (RBCs) derived EVs compose 4 - 8% of all circulating EVs, and oxidative stress (OS) as a consequence of different pathophysiological conditions significantly increases the amount of circulated RBC-derived EVs, however the mechanisms of EV formation are not fully defined yet. To analyze OS-induced EV formation and RBCs transformations we used flow cytometry to evaluate cell esterase activity, caspase-3 activity, and band 3 clustering. Band 3 clustering was additionally analyzed by confocal microscopy. Two original laser diffraction-based approaches were used for analysis of cell deformability and band 3 activity. Hemoglobin species were characterized spectrophotometrically. We showed that cell viability in tert-butyl hydroperoxide-induced OS directly correlated with oxidant concentration to cell count ratio, RBCs-derived EVs contained hemoglobin oxidized to hemichrome (HbChr). OS induced caspase-3 activation and band 3 clustering in cells and EVs. Importantly, we showed that OS-induced EV formation is independent from calcium. Presented data indicated that during OS RBCs eliminate HbChr by vesiculation, in order to sacrifice the cell itself thereby prolonging lifespan and delaying the untimely clearance of in all other respects healthy RBCs.

Extracellular Vesicles from Red Blood Cells and Their Evolving Roles in Health, Coagulopathy and Therapy

“…Therefore, Ca 2+ dependent vesicle release is a raft-based process. These studies do stress an important role for Ca 2+ in the liberation of MVs or NVs from RBCs; however, non Ca 2+ dependent pathways are also observed [ 65 , 70 , 71 ]. Specifically, limiting oxidative stress by the addition of small molecule antioxidants (e.g., ascorbic acid) during blood banking conditions reduces MV release over the time course of storage and also attenuates alloimmunogenicity in murine models of transfusion [ 72 ].…”

Low-Dose Ammonium Preconditioning Enhances Endurance in Submaximal Physical Exercises

“…Therefore, our results indicated the beneficial effects of AMP on maintaining the acid-base balance thus promoting endurance during exercise. As we [ 50 , 60 , 65 ] and others [ 56 , 57 , 58 , 59 ] showed previously, human RBCs can import AM thus possibly lowering the metabolic stress and preventing Am-induced damage of muscle cells by ROS. Therefore, we assume RBCs to play an important role in AM homeostasis and maintenance of the acid-base balance during the PE, however, this idea merits future examinations.…”

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

“…An additional red blood cell lysis step following isolation of the stromalvascular fraction may be necessary to eliminate the effects of erythrocytes on adipocytes. This is important because erythrocyte-derived EVs can impact the cellular function of other cells 17 and erythrocyte-derived EVs are elevated in the presence of oxidative stress 18 and in patients with metabolic syndrome 19 , 20 . Therefore, adipose tissue derived from metabolic disease patients may contain elevated levels brief vortex must be used, as vigorous vortexing may disrupt the EV membranes.…”