Extracellular vesicles from blood plasma as mediators of anti-inflammatory effects, oxidative stress and angiogenesis in HUVEC

Abstract

Introduction: Blood plasma is used in regenerative medicine for more than 30 years as it was found to have impact on angiogenesis and proliferation of endothelial cells. This effect was initially ascribed to growth factors and cytokines, however, particularly in the last decade researchers are focusing on platelet-derived extracellular vesicles (PEVs) that are also present in the platelet rich plasma. Methods: We prepared platelet and extracellular vesicles-rich plasma (PVRP) by centrifugation of the human blood, and differential (ultra) centrifugation of PVRP to isolate PEVs. We exposed Human Umbilical Vein Endothelial Cells (HUVEC) to 5% PEVs for 24 hours and assessed inflammation markers (Interleukin(IL)-1beta, IL-6 and Tumor necrosis factor (TNF)-alpha using ELISA tests), oxidative stress markers (Cholinesterase (ChE) and glutathione S-transferase (GST) activity by spectrophotometry, as well reactive oxygen species (ROS) and lipid droplets (LD) by flow cytometry). We observed morphological changes in HUVEC indicating angiogenesis by using optical microscopy. Results: We found that after 24 hours caused a decrease of the concentration of IL-6, IL-1beta and TNF-alpha. Also, ChE and GST activity and ROS count were decreased. LD production, which is triggered in need to protect the cells from free radicals and oxidative stress damage, was however higher. Treatment of the cells had impact on cell morphology with progressed formation of the tubes and cell connecting, which is regarded as the beginning of HUVEC angiogenesis process. Discussion: Beneficial effect of PVRP in healing and regeneration may include suppression of inflammation and oxidative stress by PEVs.