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

Sumio Ohtsuki, Chiemi Ikeda, Yasuo Uchida, Yumi Sakamoto, Florence Miller, Fabienne Glacial, Xavier Decleves, Jean Michel Scherrmann, Pierre Olivier Couraud, Yoshiyuki Kubo, Masanori Tachikawa, Tetsuya Terasaki

Research output: Contribution to journalArticlepeer-review

131 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)289-296
Number of pages8
JournalMolecular pharmaceutics
Volume10
Issue number1
DOIs
Publication statusPublished - 2013 Jan 7

Keywords

  • ATP-binding cassette transporter
  • breast cancer resistant protein
  • claudin-5
  • human blood-brain barrier
  • multidrug resistant protein 1
  • protein quantification
  • quantitative targeted absolute proteomics
  • receptor, junction protein
  • transporter

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmaceutical Science
  • Drug Discovery

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