Mechanistic analysis of iron accumulation by endothelial cells of the BBB

Themechanism(s) bywhich iron in blood is transported across the blood-brain barrier (BBB) remains controversial. Here we have examined the first step of this trans-cellular pathway, namely the mechanism( s) of iron uptake into human brain microvascular endothelial cells (hBMVEC). We show that hBMVEC actively reduce non-transferrin bound FeIII (NTBI) and transferrin-bound FeIII (TBI); this activity is associated with one or more ferrireductases. Efficient, exo-cytoplasmic ferri-reduction from TBI is dependent upon transferrin receptor (TfR), also. Blocking holo-Tf bindingwith an anti-TfR antibody significantly decreases the reduction of iron from transferrin by hBMVEC, suggesting that holo-Tf needs to bind to TfR in order for efficient reduction to occur. Ferri-reduction from TBI significantly decreases when hBMVEC are pre-treated with PtII, an inhibitor of cell surface reductase activity. Uptake of 59Fe from 59Fe-Tf by endothelial cells is inhibited by 50 %when ferrozine is added to solution; in contrast, no inhibition occurs when cells are alkalinized with NH4Cl. This indicates that the iron reduced from holo-transferrin at the plasmamembrane accounts for at least 50 % of the iron uptake observed. hBMVECdependent reduction and uptake of NTBI utilizes a PtII- insensitive reductase. Reductase-independent uptake of FeII by hBMVEC is inhibited up to 50 %by ZnII and/or MnII by a saturable process suggesting that redundant FeII transporters exist in the hBMVEC plasma membrane. These results are the first to demonstratemultiple mechanism(s) ofTBI andNTBI reduction and uptake by endothelial cells (EC) of the BBB.