SMSCs-derived sEV overexpressing miR-433-3p inhibits angiogenesis induced by sEV released from synoviocytes under triggering of ferroptosis

Ferroptosis is characterized by accumulation of lipid peroxides that leads to oxidative stress. In progressive rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) suffered from oxidative stress induced by generation of excess reactive oxygen species (ROS) and survived from elevated lipid oxidation. However the phenomenon of abnormal synovial fibroblasts proliferation under ferroptotic stress remain to be explained and the effects of this event on disease progression of RA need to be investigated.FLS from RA patients (RA-FLS) were stimulated with LPS as an inflammatory model in vitro, and simultaneously treated with ferroptosis inducer Erastin/RSL3 or inhibitor ferrostatin-1. Besides, small extracellular vesicles (sEV) from the supernatant of RA-FLS culture under Erastin/RSL3 management were isolated. The degree of ferroptosis in cells were evaluated by Lipid-ROS detection via flowcytometry and ferroptosis marker protein expression determined by western bloting. The expression of core component of ESCRT-III CHMP4A and CHMP5 was determined by western bloting, and knockdown of CHMP4A was further performed to detect the influence of ESCRT-III complex on ferroptosis as well as LPS/Erastin induced sEV (LPS/Erastin-sEV) releasing. Moreover, miR-433-3p level in the isolated sEV was evaluated by RT-qPCR and interaction of miR-433-3p with FOXO1/VEGF axis were evaluated. MiR-433-3p was overexpressed in synovial mesenchymal stem cells (SMSCs) via miR-433-3p mimics transfection. RA-FLS was co-cultured with human dermal microvascular endothelial cells (HDMECs). LPS/Erastin-sEV or sEV derived from miR-433-3p-overexpressing SMSCs (miR-433-3p-SMSCs-sEV) were added to the co-culture system, and supernatants from co-culture without sEV were given to HDMECs. Angiogenic activity of HDMECs were identified by transwell test and endothelial tube formation analysis. Erastin-sEV and miR-433-3p-SMSCs-sEV were also administrated in collagen-induced arthritis (CIA) mouse model respectively, and progression of arthritis were evaluated.Ferroptosis of RA-FLS was triggered by LPS/Erastin and accompanied with increased expression of ESCRT-III core components as well as elevated release of sEV from RA-FLS. HDMECs' migration and tube formation in vitro was significantly induced/suppressed by supernatants from co-culture under management of Erastin-sEV/miR-433-3p-SMSCs-sEV due to varied VEGF expression regulated by miR-433-3p targeting FOXO1. MiR-433-3p-SMSCs-sEV could inhibit the Erastin-sEV promoted VEGF expression and mitigated arthritis severity.Erastin-sEV could aggravate synovial angiogenesis and promote arthritis progression. Administration of miR-433-3p-SMSCs-sEV may be a potential novel therapeutic method as significant antagonism to Erastin-sEV for RA treatment.