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

Background and purpose: Endothelial dysfunction (ED) is a hallmark of cardiovascular disease (CVD). We recently showed that anemia is associated with worsening of endothelial function after acute myocardial infarction (AMI). Extracellular vesicles (EVs) are efficient communicators between cells and can functionally contribute to different CVD, including, AMI. However, their specific role of EVs in stable coronary artery disease (CAD)-associated with anemia, particularly their contribution to ED, has not yet been investigated systematically. Experimental approach: Red blood cell-derived EVs (REVs) and plasma-derived EVs (PLEVs) from all blood cells and endothelium were isolated from patients with stable CAD. The isolated large REVs and PLEVs were characterized using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting. Uptake assays were performed by co-incubating with fluorescently-labeled REVs and PLEVs with human umbilical vein endothelial cells (ECs). Nitric oxide (NO) consumption ability of REVs was analyzed using a chemiluminescence detector (CLD). After co-incubation of aortic rings explanted from wild-type (WT) mice with REVs and PLEVs from anemic and non-anemic CAD patients, endothelial function was assessed using a wire myograph system. To investigate differences in the content of REVs and PLEVs between anemic and non-anemic CAD patients, proteomic analysis was performed. Key results: DLS analysis showed that both REVs and PLEVs were within the size distribution range of 100-1000 nm. NTA analysis revealed increased release of REVs in anemic patients compared to non-anemic patients. Co-incubation of labeled REVs and PLEVs with ECs demonstrated their uptake by ECs in vitro which was similar between anemic patients compared to non-anemic patients. REVs from anemic patients showed increased NO consumption compared to those from non-anemic patients. Aortic rings co-incubated with REVs from anemic patients showed attenuated endothelial NO-dependent relaxation responses compared to non-anemic patients. Proteomics analysis of REVs from anemic patients revealed numerous differentially expressed proteins, including decreased abundance of antioxidant proteins such as catalase 1 (CAT1), superoxide dismutase 1 (SOD1) and increased oxidative stress-promoting myeloperoxidase (MPO). Co-incubation of ECs with REVs from anemic patients demonstrated increased ROS production. Conclusion: Anemia is associated with increased release of RBC-derived large extracellular vesicles and enhanced NO consumption, which promotes ED. This is further exacerbated by an altered redox balance and increased ROS production, implicating therapeutic importance in anemic patients with CAD.