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

Mol Pharm. 2013 Jan 7;10(1):289-96. doi: 10.1021/mp3004308. Epub 2012 Dec 11.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Transport
  • Blood-Brain Barrier / metabolism*
  • Cell Line
  • Cell Membrane / metabolism
  • Cerebrovascular Circulation
  • Cerebrum / blood supply*
  • Cerebrum / metabolism*
  • Endothelial Cells / metabolism*
  • Endothelium, Vascular / metabolism*
  • Human Umbilical Vein Endothelial Cells / metabolism
  • Humans
  • Membrane Proteins / metabolism*
  • Membrane Transport Proteins / metabolism
  • Microvessels / metabolism*
  • Models, Biological
  • Proteome / metabolism*
  • Proteomics / methods


  • Membrane Proteins
  • Membrane Transport Proteins
  • Proteome