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.

Quantitative Targeted Proteomics for Membrane Transporter Proteins: Method and Application

“…A series of investigations demonstrated that the expression pattern of ATP-binding cassette transporter messenger RNAs in hCMEC/D3 cells is, at least qualitatively, similar to that of human brain microvessels (72,73). To understand the suitability and limitations of the hCMEC/D3 cell line being a BBB functional model, Ohtsuki et al (74) applied quantitative targeted proteomics to determine the protein expression levels of multiple transporters, receptors, and junction proteins in hCMEC/D3 cells and compared the results with isolated human brain microvessels. They found that hCMEC/D3 cells retain protein expression of most transporters including MDR1, BCRP, multidrug resistanceassociated protein 4 and receptors such as transferrin and insulin receptors expressed in vivo at the human BBB (74).…”

“…To understand the suitability and limitations of the hCMEC/D3 cell line being a BBB functional model, Ohtsuki et al (74) applied quantitative targeted proteomics to determine the protein expression levels of multiple transporters, receptors, and junction proteins in hCMEC/D3 cells and compared the results with isolated human brain microvessels. They found that hCMEC/D3 cells retain protein expression of most transporters including MDR1, BCRP, multidrug resistanceassociated protein 4 and receptors such as transferrin and insulin receptors expressed in vivo at the human BBB (74). The quantitative analysis of functionally important transport proteins provides fundamental information in extrapolation of in vitro results to in vivo (74).…”

“…They found that hCMEC/D3 cells retain protein expression of most transporters including MDR1, BCRP, multidrug resistanceassociated protein 4 and receptors such as transferrin and insulin receptors expressed in vivo at the human BBB (74). The quantitative analysis of functionally important transport proteins provides fundamental information in extrapolation of in vitro results to in vivo (74).…”

Diet-Induced Obesity Suppresses Expression of Many Proteins at the Blood–Brain Barrier

“…[9][10][11] To determine how BBB functions are modulated by obesity, we performed comparative proteomic studies to determine the…”

Cell Surface Membrane Proteins as Personalized Biomarkers: Where we Stand and Where we are Headed

“…With this technology, by using appropriate standards and controlled procedures, a large number of membrane spanning proteins, including recep tors and transporters, became easily measurable [46,47]. In spite of promising new data, the rela tively low abundance and the inaccessibility of the hydrophobic regions of the membrane proteins to proteolysis hampers this type of proteomic analysis, especially in a quantitative diagnostic approach [48].…”