title: Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model.
abstract: Human cerebral microvascular endothelial cell line hCMEC/D3 is an established model of the human blood-brain barrier (BBB). The purpose of the present study was to determine, by means of quantitative targeted absolute proteomics, the protein expression levels in hCMEC/D3 cells of multiple transporters, receptors and junction proteins for comparison with our previously reported findings in isolated human brain microvessels. Among 91 target molecules, 12 transporters, 2 receptors, 1 junction protein and 1 membrane marker were present at quantifiable levels in plasma membrane fraction of hCMEC/D3 cells. ABCA2, MDR1, MRP4, BCRP, GLUT1, 4F2hc, MCT1, ENT1, transferrin and insulin receptors and claudin-5 were detected in both hCMEC/D3 cells and human brain microvessels. After normalization based on Na(+)/K(+) ATPase expression, the differences in protein expression levels between hCMEC/D3 cells and human brain microvessels were within 4-fold for these proteins, with the exceptions of ENT1, transferrin receptor and claudin-5. ABCA8, LAT1, LRP1 and γ-GTP were below the limit of quantification in the cells, but were found in human brain microvessels. ABCA3, ABCA6, MRP1 and ATA1 were found only in hCMEC/D3 cells. Furthermore, compared with human umbilical vein endothelial cells (HUVECs) as reference nonbrain endothelial cells, MDR1 was found only in hCMEC/D3 cells, and GLUT1 expression was 15-fold higher in hCMEC/D3 cells than in HUVECs. In conclusion, this is the first study to examine the suitability and limitations of the hCMEC/D3 cell line as a BBB functional model in terms of quantitative expression levels of transporters, receptors and tight junction proteins.
authors.affiliation: Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
authors.forename: Sumio
authors.initials: S
authors.lastname: Ohtsuki
authors.forename: Chiemi
authors.initials: C
authors.lastname: Ikeda
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Uchida
authors.forename: Yumi
authors.initials: Y
authors.lastname: Sakamoto
authors.forename: Florence
authors.initials: F
authors.lastname: Miller
authors.forename: Fabienne
authors.initials: F
authors.lastname: Glacial
authors.forename: Xavier
authors.initials: X
authors.lastname: Decleves
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Scherrmann
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Couraud
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kubo
authors.forename: Masanori
authors.initials: M
authors.lastname: Tachikawa
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Terasaki
mesh_terms: D001692:Biological Transport; D001812:Blood-Brain Barrier / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002560:Cerebrovascular Circulation; D054022:Cerebrum / Q000098:blood supply* / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D055806:Microvessels / Q000378:metabolism*; D008954:Models, Biological; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods
chemical_list: D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D020543:Proteome