Extracellular vesicles derived by hypoxic preconditioned EPCs attenuated atherosclerosis by suppressing ferroptosis via regulating arginine metabolism

mice and ox-LDL treated macrophages. N-EVs administration attenuated atherosclerosis progression, Hy-EVs further decreased the plaque area, increased collagen content, reduced lipid deposition and improved inflammatory response. Proteomics and in vitro data suggested that Hy-EVs inhibited macrophages ferroptosis and improved oxidative stress via increasing GPX4 and xCT level. Hy-EVs proteomics revealed that argininosuccinate synthetase1 (ASS1) levels were significantly higher in Hy-EVs than N-EVs, ASS1 knockdown in EPCs blocked the protective effects of Hy-EVs on macrophage ferroptosis and AS progression, whereas ASS1 supplementation restored the effect. Mechanistically, increased ASS1 in Hy-EVs suppressed GCN2 activation by enhancing arginine generation in macrophages. GCN2 agonist or arginine consumption significantly attenuated the protective effect of Hy-EVs on macrophage ferroptosis and AS progression. Furthermore, LC/MS indicated that GCN2 bonded to NRF2, while arginine generated by ASS1 enhanced NRF2 nuclear translocation and increased levels of its downstream target gene GPX4/xCT by reducing GCN2/NRF2 interaction. Our study suggested that Hy-EVs further attenuated atherosclerosis progression by inhibiting macrophage ferroptosis via enhancing arginine generation and inhibiting GCN2/NRF2 interaction. The data provides strong evidence for the translational application of EPCs derived Hy-EVs as atherosclerotic plaques.