Abstract
We have used an in vitro model system to probe the iron transport pathway across the brain microvascular endothelial cells (BMVEC) of the blood-brain barrier (BBB). This model consists of human BMVEC (hBMVEC) and C6 glioma cells (as an astrocytic cell line) grown in a transwell, a cell culture system commonly used to quantify metabolite flux across a cell-derived barrier. We found that iron efflux from hBMVEC through the ferrous iron permease ferroportin (Fpn) was stimulated by secretion of the soluble form of the multi-copper ferroxidase, ceruloplasmin (sCp) from the co-cultured C6 cells. Reciprocally, expression of sCp mRNA in the C6 cells was increased by neighboring hBMVEC. In addition, data indicate that C6 cell-secreted hepcidin stimulates internalization of hBMVEC Fpn but only when the end-feet projections characteristic of this glia-derived cell line are proximal to the endothelial cells. This hepcidin-dependent loss of Fpn correlated with knock-down of iron efflux from the hBMVEC; this result was consistent with the mechanism by which hepcidin regulates iron efflux in mammalian cells. In summary, the data support a model of iron trafficking across the BBB in which the capillary endothelium induce the underlying astrocytes to produce the ferroxidase activity needed to support Fpn-mediated iron efflux. Reciprocally, astrocyte proximity modulates the effective concentration of hepcidin at the endothelial cell membrane and thus the surface expression of hBMVEC Fpn. These results are independent of the source of hBMVEC iron (transferrin or non-transferrin bound) indicating that the model developed here is broadly applicable to brain iron homeostasis.
Highlights
Dysregulation of iron homeostasis has been associated with a variety of neurodegenerative disorders
The upper chambers of the transwells were filled with media containing serum to mirror the luminal surface of the human BMVEC (hBMVEC) capillary milieu while the lower chamber contained media without serum to mirror the abluminal context. hBMVEC grown on the upper-surface of the transwell membrane formed tight-junctions after 5 days as demonstrated quantitatively via transendothelial electrical resistance (TEER)
Co-culture model of the blood-brain barrier (BBB) we have obtained evidence for the regulatory interactions between brain microvascular endothelial cells (BMVEC) and C6 cells that may be central to the mechanism underlying the established physiologic developmental pattern of brain iron accumulation
Summary
Dysregulation of iron homeostasis has been associated with a variety of neurodegenerative disorders. Progressive accumulation of iron in a normal aging brain [1] or pathologic alterations of its homeostasis can be the cause of or contribute to the cellular degeneration observed in many neurologic disorders [1,2,3,4]. The primary regulator of brain iron is the layer of brain microvascular endothelial cells (BMVEC) which, together with underlying astrocytes form the blood-brain barrier (BBB). BMVEC lack the fenestrations common to the endothelial cells in peripheral capillaries; in contrast, they form tight-junctions and regulate the transport of polar molecules across the BBB [6,7]. In this report we provide experimental evidence in support of the mechanism by which the iron accumulated by BMVEC is exported from the basal (brain; abluminal) surface of these cells, trafficking plasma iron across the BBB and into the brain interstitium
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