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

Endothelial progenitor cells (EPCs) derived extracellular vesicles (EVs) are involved in maintaining vascular homeostasis, while effects of hypoxic preconditioned EPCs-derived EVs (Hy-EVs) on atherosclerosis (AS) remain unclear. Hy-EVs and normal oxygen EVs (N-EVs) were pretreated for ApoE −/− 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.