Elevated red blood cell‑derived extracellular vesicles under hyperglycemic conditions are associated with CD47 expression and production of intracellular reactive oxygen species

Diabetes mellitus (DM), particularly type 2 DM, is characterized by chronic hyperglycemia, which has notable effects on red blood cells (RBCs), including alterations in membrane integrity, morphology and function. The present study aimed to assess the effect of oxidative stress under hyperglycemic conditions on RBC-derived extracellular vesicle (REV) production using an <i>in vitro</i> model. RBCs from healthy participants were cultured in several glucose concentrations (5, 45 and 100 mM) to mimic euglycemic, intermediate and severe hyperglycemic conditions. The levels of intracellular reactive oxygen species (ROS) and CD47-expressing RBCs were measured using flow cytometry. REVs were purified using centrifugation and then their levels were measured using CountBright and flow cytometry. The results revealed a significantly increased level of intracellular ROS and REV production under hyperglycemic conditions. Additionally, hyperglycemia was demonstrated to reduce CD47 expression in RBCs, particularly at higher glucose concentrations. Elevated ROS production was also significantly associated with REV production in the early phase of hyperglycemic conditions, whereas the significant decrease in CD47 expression was observed later. Furthermore, pretreatment with N-acetylcysteine effectively reduced ROS production and REV formation, highlighting the role of oxidative stress in hyperglycemia-induced RBC alterations and REV production. The findings of the present study suggest that prolonged hyperglycemia facilitates intracellular oxidative stress and RBC membrane vesiculation, which markedly contributed to the understanding of the pathophysiology of diabetes-related complications.