Abstract
BackgroundThe human cerebral microvascular endothelial cell line, hCMEC/D3, has been used extensively to model the blood–brain barrier (BBB) in vitro. Recently, we reported that cytokine-treatment induced loss of brain endothelial barrier properties. In this study, we further determined the gene expression pattern of hCMEC/D3 cells in response to activation with TNFα and IFNγ.FindingsUsing a microarray approach, we observed that expression of genes involved in the control of barrier permeability, including inter-brain endothelial junctions (e.g. claudin-5, MARVELD-2), integrin-focal adhesions complexes (e.g. integrin β1, ELMO-1) and transporter systems (e.g. ABCB1, SLC2A1), are altered by pro-inflammatory cytokines.ConclusionsOur study shows that previously-described cytokine-induced changes in the pattern of gene expression of endothelium are reproduced in hCMEC/D3 cells, suggesting that this model is suitable to study inflammation at the BBB, while at the same time it has provided insights into novel key molecular processes that are altered in brain endothelium during neuroinflammation, such as modulation of cell-to-matrix contacts.
Highlights
The human cerebral microvascular endothelial cell line, hCMEC/D3, has been used extensively to model the blood–brain barrier (BBB) in vitro
Our study shows that previously-described cytokine-induced changes in the pattern of gene expression of endothelium are reproduced in hCMEC/D3 cells, suggesting that this model is suitable to study inflammation at the BBB, while at the same time it has provided insights into novel key molecular processes that are altered in brain endothelium during neuroinflammation, such as modulation of cell-to-matrix contacts
Culture conditions hCMEC/D3 cells were cultured in EGM-2 MV medium (Lonza, Slough Wokingham, UK) and supplemented with the following components obtained from the manufacturer: 0.025% (v/v) rhEGF, 0.025% (v/v) VEGF, 0.025% (v/v) IGF, 0.1% (v/v) rhFGF, 0.1% (v/v) gentamycin, 0.1% (v/v) ascorbic acid, 0.04% (v/v) hydrocortisone and 2.5% (v/v) foetal bovine serum (FBS)
Summary
Culture conditions hCMEC/D3 cells were cultured in EGM-2 MV medium (Lonza, Slough Wokingham, UK) and supplemented with the following components obtained from the manufacturer: 0.025% (v/v) rhEGF, 0.025% (v/v) VEGF, 0.025% (v/v) IGF, 0.1% (v/v) rhFGF, 0.1% (v/v) gentamycin, 0.1% (v/v) ascorbic acid, 0.04% (v/v) hydrocortisone and 2.5% (v/v) foetal bovine serum (FBS). RNA extraction and mRNA microarray analysis hCMEC/D3 cells were grown on collagen-coated six-well plates and treated with 10 ng/ml of TNFα and IFNγ (R&D systems, Abingdon, Oxon, UK) for 24 h, while control cells received the vehicle solution. Total RNA from three biological replicates was isolated using miRNeasy mini kit (Qiagen, Crawley, West Sussex, UK) according to the manufacturer’s protocols. For each biological sample 100 ng of total RNA was used, obtained from approximately 1.5 ×106 cells. A quantile normalization was performed and a number of quality plots were generated to assess the quality of the data. Differences between control and treated cells were estimated when a gene signal in two or more replicates is at the background level. Instead of using numerical values, we indicated these genes as upregulated (+) or downregulated (-) in cytokine-treated cells. Detailed procedures and complete data are available at the Gene Expression Omnibus (www. ncbi.nlm.nih.gov/geo) under accession number GSE45880
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