title: Afamin is synthesized by cerebrovascular endothelial cells and mediates alpha-tocopherol transport across an in vitro model of the blood-brain barrier.
abstract: Alpha-tocopherol (alphaTocH), a member of the vitamin E family, is essential for normal neurological function. Despite the importance of alphaTocH transport into the CNS, transfer mechanisms across the blood-brain barrier (BBB) are not entirely clear. We here investigate whether afamin, a known alphaTocH-binding protein, contributes to alphaTocH transport across an in vitro model of the BBB consisting of primary porcine brain capillary endothelial cells (BCEC) and basolaterally cultured astrocytoma cells. Exogenously added afamin had no adverse effects on BCEC viability or barrier function and was transported across BCEC Transwell cultures. Furthermore, alphaTocH transport across polarized BCEC cultures to astrocytoma cells is facilitated by afamin, though to a lesser extent than by high-density lipoprotein-mediated transport, an essential and in vivo operating alphaTocH import pathway at the cerebrovasculature. We also demonstrate that porcine BCEC endogenously synthesize afamin. In line with these in vitro findings, afamin was detected by immunohistochemistry in porcine, human postmortem, and mouse brain, where prominent staining was observed almost exclusively in the cerebrovasculature. The demonstration of afamin mRNA expression in isolated brain capillaries suggests that afamin might be a new family member of binding/transport proteins contributing to alphaTocH homeostasis at the BBB in vivo.
authors.affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Graz, Austria.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Kratzer
authors.forename: Eva
authors.initials: E
authors.lastname: Bernhart
authors.forename: Andrea
authors.initials: A
authors.lastname: Wintersperger
authors.forename: Astrid
authors.initials: A
authors.lastname: Hammer
authors.forename: Sabine
authors.initials: S
authors.lastname: Waltl
authors.forename: Ernst
authors.initials: E
authors.lastname: Malle
authors.forename: Günther
authors.initials: G
authors.lastname: Sperk
authors.forename: Georg
authors.initials: G
authors.lastname: Wietzorrek
authors.forename: Hans
authors.initials: H
authors.lastname: Dieplinger
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Sattler
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D001254:Astrocytoma / Q000378:metabolism; D001693:Biological Transport, Active; D001812:Blood-Brain Barrier / Q000502:physiology*; D015153:Blotting, Western; D016466:CHO Cells; D002196:Capillaries / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis*; D002560:Cerebrovascular Circulation / Q000502:physiology*; D018920:Coculture Techniques; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000096:biosynthesis*; D006801:Humans; D008075:Lipoproteins, HDL / Q000096:biosynthesis / Q000302:isolation & purification; D051379:Mice; D008810:Mice, Inbred C57BL; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012709:Serum Albumin / Q000096:biosynthesis*; D000075462:Serum Albumin, Human; D013552:Swine; D013778:Tetrazolium Salts; D013844:Thiazoles; D024502:alpha-Tocopherol / Q000378:metabolism*
chemical_list: C088246:AFM protein, human; D002352:Carrier Proteins; D006023:Glycoproteins; D008075:Lipoproteins, HDL; D011994:Recombinant Proteins; D012709:Serum Albumin; D013778:Tetrazolium Salts; D013844:Thiazoles; D012313:RNA; C022616:thiazolyl blue; D024502:alpha-Tocopherol; D000075462:Serum Albumin, Human