Transcriptional Profiling of Human Brain Endothelial Cells Reveals Key Properties Crucial for Predictive In Vitro Blood-Brain Barrier Models

Brain microvascular endothelial cells (BEC) constitute the blood-brain barrier (BBB) which forms a dynamic interface between the blood and the central nervous system (CNS). This highly specialized interface restricts paracellular diffusion of fluids and solutes including chemicals, toxins and drugs from entering the brain. In this study we compared the transcriptome profiles of the human immortalized brain endothelial cell line hCMEC/D3 and human primary BEC. We identified transcriptional differences in immune response genes which are directly related to the immortalization procedure of the hCMEC/D3 cells. Interestingly, astrocytic co-culturing reduced cell adhesion and migration molecules in both BECs, which possibly could be related to regulation of immune surveillance of the CNS controlled by astrocytic cells within the neurovascular unit. By matching the transcriptome data from these two cell lines with published transcriptional data from freshly isolated mouse BECs, we discovered striking differences that could explain some of the limitations of using cultured BECs to study BBB properties. Key protein classes such as tight junction proteins, transporters and cell surface receptors show differing expression profiles. For example, the claudin-5, occludin and JAM2 expression is dramatically reduced in the two human BEC lines, which likely explains their low transcellular electric resistance and paracellular leakiness. In addition, the human BEC lines express low levels of unique brain endothelial transporters such as Glut1 and Pgp. Cell surface receptors such as LRP1, RAGE and the insulin receptor that are involved in receptor-mediated transport are also expressed at very low levels. Taken together, these data illustrate that BECs lose their unique protein expression pattern outside of their native environment and display a more generic endothelial cell phenotype. A collection of key genes that seems to be highly regulated by the local surroundings of BEC within the neurovascular unit are presented and discussed

Differences in expression levels of surface receptors between the cultured BECs and freshly isolated BECs.

<p>The RPL4 normalized mouse expression values (Mouse) (RPL4 normalized) are compared to the also RPL4 normalized expression values of hpBECs (Average P) and hCMEC/D3 (Average D3). The lower graph shows the absolute expression levels for each cell type. The upper graph shows the ratio between RPL4 normalized mouse BECs and hpBECs (Ratio M/P), the ratio between RPL4 normalized mouse BECs and hCMEC/D3 (Ratio M/D3) and the ratio between hpBECs and hCMEC/D3 (Ratio P/D3). Four genes that are expressed in much lower levels in both the hpBECs and the hCMEC/D3 cell line are Lrp8, IR, IGF1R and FcRn.</p

Induction of INF-stimulated genes in immortalized BECs.

<p><i>(</i><b><i>A</i></b><i>)</i> Expression levels of total IFN, IFNα/β and IFNγ signaling pathway genes were determined by microarray analysis for the two BEC lines. Each circle represents one gene of the indicated reactome pathway and the difference in expression between hCMEC/D3 and hpBEC cells. To compare IFN signaling reactomes between BEC lines, the values of the four replicates were averaged, and the probe-set with the highest value was used to represent each gene. The distributions of the genes within each gene set were compared with a one-sample t-test, to test whether the mean of the distributions were different from zero. <i>(</i><b><i>B</i></b><i>)</i> HLA class I, b2-microtublin and β actin gene expression in resting hCMEC/D3 (red bars) and hpBECs (blue bars) determined by microarray analysis. All displayed HLA class I and b2-microtublin genes were significantly (p<0.001) higher expressed in the hCMEC/D3 cells. <i>(</i><b><i>C–E</i></b><i>)</i> Flow cytometry analysis of surface expression of <i>(</i><b><i>C & D</i></b><i>)</i> HLA class I and <i>(</i><b><i>E</i></b><i>)</i> II molecules on hCMEC/D3 cells (red histograms, red bars) and hpBECs (blue histograms, blue bars). <i>(</i><b><i>C</i></b><i>)</i> The HLA class I expression levels and the mean fluorescence intensities (MFI) on both resting BEC lines, in one of four similar experiments, is presented. <i>(</i><b><i>D</i></b><i>)</i> The effect of IFNα upon HLA class I surface expression and <i>(</i><b><i>E</i></b><i>)</i> the effect of IFNγ upon HLA class II surface expression on the two BEC lines are displayed as average MFIs of three similar experiments. *p<0.05, **p<0.01 and ***p<0.001 (Student's <i>t</i>-test).</p

Astrocytic co-culturing reduced the expression of adhesion molecules.

<p>The expression of genes in <i>(</i><b><i>A</i></b><i>)</i> hCMEC/D3 and <i>(</i><b><i>B</i></b><i>)</i> hpBECs was compared in presence and absence of human astrocytes (HA). The data is represented as a dot plot on a log₂ scale, where each point represents a probe set on the gene chip. Red and blue dots indicate probe sets, which are differently expressed (adjusted p≤0.05) between culturing conditions. Blue dots represent lower and red dots higher expressed probes in the co-culturing conditions with HAs versus culturing the BECs alone. The mean expression values are averaged expression values for both cell lines. <i>(</i><b><i>C</i></b><i>)</i> Overlapping genes identified between the two BECs in the co-culturing conditions with HAs. The numbers of up-regulated (red numbers) or down-regulated (blue numbers) genes in the BECs with HA co-culture are displayed. <i>(</i><b><i>D</i></b><i>)</i> The enrichment map displays the differently expressed gene sets for hCMEC/D3 cells between culturing conditions. Blue node color represents lower expression in hCMEC/D3 + HA, whereas red represents higher expression in hCMEC/D3 control. Node size is proportional to the number of genes in the gene set and edge thickness represents the degree of overlap between two gene sets. Labels for the clusters of functionally related gene sets were manually assigned: 1) Regulation of Immune Cell Activation and Proliferation, 2) Regulation of Kinase Cascade, 3) Regulation of Inflammatory and Defense Response, 4) Response to Pathogens, 5) Regulation of the Immune System, 6) Response to Cytokines, 7) Regulation of Cell-Cell Adhesion, 8) Cell Migration, 9) Signal Transduction, 10) Antigen Processing and Presentation, 11) Miscellaneous.</p

Differences in expression levels of SLC transporter between cultured BECs and freshly isolated BECs.

<p>The RPL4 normalized mouse expression values (Mouse) are compared to the also RPL4 normalized expression values of hpBECs (Average P) and hCMEC/D3 (Average D3). The lower graph shows the absolute expression levels for each cell type. The upper graph shows the ratio between RPL4 normalized mouse BECs and hpBECs (Ratio M/P), the ratio between RPL4 normalized mouse BECs and hCMEC/D3 (Ratio M/D3) and the ratio between hpBECs and hCMEC/D3 (Ratio P/D3). For instance genes that are expressed in much lower levels in both hpBECs and the hCMEC/D3 cell line are GLUT1, MCT8 and OAT3.</p

Immortalization influences BEC phenotype, growth behavior and expression of cell division related genes.

<p><i>(</i><b><i>A–C</i></b><i>)</i> Flow cytometry analysis of confluent hCMEC/D3 (red population, red histograms) and hpBECs (blue population, blue histograms) seeded on collagen I coated inserts. Staining was done with indicated antibodies or relevant isotype controls (grey population, tinted histograms). The average population sizes and standard deviations of three similar experiments are shown in <b>A</b>, whereas one representative experiment of three is shown in <b><i>B & C</i></b> (MFI = mean fluorescence intensity). <i>(</i><b><i>D</i></b><i>)</i> Real time monitoring of adherent BECs cultured in Resting (RM) or Growth Medium (GM) by the xCeLLIgence System. The curves show the time-, attachment- and density-dependent cell growth and viability of the individual BEC lines respectively culturing conditions. <i>(</i><b><i>E</i></b><i>)</i> Comparison of gene expression between hCMEC/D3 cells and hpBECs. The data is represented as a dot plot on a log₂ scale, where each point represents a probe set on the gene chip. Red and blue dots indicate probe sets, which have higher expression in hCMEC/D3 (red) or higher expression in hpBECs (blue). The mean expression values are averaged expression values for both cell lines. <i>(</i><b><i>F</i></b><i>)</i> The enrichment map displays the differently expressed gene sets between the two BEC lines. Red node color represents higher expression in hCMEC/D3 cells, whereas blue represents higher expression in hpBECs. Node size is proportional to the number of genes in the gene set and edge thickness represents the degree of overlap between two gene sets. Labels for the clusters of functionally related gene sets were manually assigned: 1) Mitosis, 2) DNA Repair, 3) Anaphase Promoting Complex, 4) Immune and Virus Response, 5) RNA Processing, 6) Cell-Cell Adhesion, 7) Differentiation/Maturation/Development, 8) Metabolic Processes, 9) Miscellaneous.</p

Differences in expression levels of ABC transporter between cultured BECs and freshly isolated BECs.

<p>The RPL4 normalized mouse expression values (Mouse) are compared to the also RPL4 normalized expression values of hpBECs (Average P) and hCMEC/D3 (Average D3). The lower graph shows the absolute expression levels for each cell type. The upper graph shows the ratio between RPL4 normalized mouse BECs and hpBECs (Ratio M/P), the ratio between RPL4 normalized mouse BECs and hCMEC/D3 (Ratio M/D3) and the ratio between hpBECs and hCMEC/D3 (Ratio P/D3). Three genes that are expressed in much lower levels in both the hpBECs and the hCMEC/D3 cell line are Pgp1, MRP4 and MDRA1.</p

Key BBB genes are expressed at low levels in cultured human BECs.

<p><i>(</i><b><i>A</i></b><i>)</i> All genes for mouse BECs versus hCMEC/D3 are displayed as grey dots. Tight junction genes (red square), SLC members (blue dot), ABC members (yellow triangle) and the surface receptors (green diamond) are highlighted. The x-axis shows the ratio between mouse and hCMEC/D3 and the y-axis shows the ratio between mouse BECs and hCMEC/D3 multiplied by the expression levels in mouse BECs. Genes in the upper right corner are therefore highly expressed in mouse BECs and much more in comparison to the hCMEC/D3 cell line. The graph shows that all key BBB genes are expressed at lower levels in the human cell line hCMEC/D3 (enlarged symbols). A similar result was obtained when comparing the mouse expression data to the hpBECs. <i>(</i><b><i>B</i></b><i>)</i> A schematic representation of the key genes identified in this study to have low level of expression in the hCMEC/D3 cells. The picture illustrates the spatial location of the BBB genes on BECs and the fold reduction in expression compared to mouse BECs.</p

Differences in expression levels of TJ protein between cultured BECs and freshly isolated BECs.

<p>The RPL4 normalized mouse expression values (Mouse) are compared to the also RPL4 normalized expression values of hpBECs (Average P) and hCMEC/D3 (Average D3). The lower graph shows the absolute expression levels for each cell type. The upper graph shows the ratio between RPL4 normalized mouse BECs and hpBECs (Ratio M/P); the ratio between RPL4 normalized mouse BECs and hCMEC/D3 (Ratio M/D3) and the ratio between hpBECs and hCMEC/D3 (Ratio P/D3). Three genes that are expressed in much lower levels in both the hpBECs and the hCMEC/D3 cell line are claudin-5, occludin and JAM2. Two genes that are expressed at higher levels in the human cell lines are JAM3 and CD31.</p