Exosomes are involved in iron transport from human blood–brain barrier endothelial cells and are modified by endothelial cell iron status

Iron is essential for normal brain development and function. Hence, understanding the mechanisms of iron efflux at the blood–brain barrier and their regulation are critical for the establishment of brain iron homeostasis. Here, we have investigated the role of exosomes in mediating the transfer of H-ferritin (FTH1)- or transferrin (Tf)-bound iron across the blood–brain barrier endothelial cells (BBBECs). Our study used ECs derived from human-induced pluripotent stem cells that are grown in bicameral chambers. When cells were exposed to (55)Fe-Tf or (55)Fe-FTH1, the (55)Fe activity in the exosome fraction in the basal chamber was significantly higher compared to the supernatant fraction. Furthermore, we determined that the release of endogenous Tf, FTH1, and exosome number is regulated by the iron concentration of the endothelial cells. Moreover, the release of exogenously added Tf or FTH1 to the basal side via exosomes was significantly higher when ECs were iron loaded compared to when they were iron deficient. The release of exosomes containing iron bound to Tf or FTH1 was independent of hepcidin regulation, indicating this mechanism by-passes a major iron regulatory pathway. A potent inhibitor of exosome formation, GW4869, reduced exosomes released from the ECs and also decreased the Tf- and FTH1-bound iron within the exosomes. Collectively, these results indicate that iron transport across the blood–brain barrier is mediated via the exosome pathway and is modified by the iron status of the ECs, providing evidence for a novel alternate mechanism of iron transport into the brain.