Large extracellular vesicles derived from red blood cells in coronary artery disease patients with anemia promote endothelial dysfunction.

Endothelial dysfunction (ED) is a hallmark of cardiovascular disease (CVD). We recently showed that anemia is associated with the progression of ED after acute myocardial infarction (AMI), which is partly mediated by red blood cells (RBCs). Extracellular vesicles (EVs) are efficient communicators between cells and can functionally contribute to various CVDs, including AMI. The potential role of RBC-derived large extracellular vesicles (REVs) in anemia-associated ED in stable coronary artery disease (CAD) patients has not yet been investigated. We hypothesize that REVs, but not plasma-derived EVs (PLEVs), mediate ED in anemic CAD patients. In this study, we demonstrated an increased release of REVs, but not PLEVs, in anemic patients compared to non-anemic patients. These REVs showed enhanced nitric oxide (NO) consumption in anemic patients. REVs and PLEVs were dose-dependently taken up by endothelial cells (ECs) in vitro. Aortic rings co-incubated with REVs, but not PLEVs, from anemic patients showed an attenuated endothelial NO-dependent (EDNO) relaxation. Mice injected with REVs from anemic patients showed impaired flow-mediated dilation responses in vivo, accompanied by reduced NO bioavailability. Proteomic analysis of REVs from anemic patients revealed increased myeloperoxidase (MPO) abundance. Co-incubation of ECs with REVs but not PLEVs from anemic patients increased reactive oxygen species (ROS) production. Pre-treatment of anemic REVs with an MPO inhibitor AZD-5904, followed by co-incubation with aortic rings, improved EDNO relaxation. These findings suggest that anemia increases the release of REVs with enhanced NO consumption. Additionally, anemic REVs promote ED in ECs by delivering oxidative stress-promoting MPO and increasing ROS production.