TNAP and EHD1 Are Over-Expressed in Bovine Brain Capillary Endothelial Cells after the Re-Induction of Blood-Brain Barrier Properties

<div><p>Although the physiological properties of the blood-brain barrier (BBB) are relatively well known, the phenotype of the component brain capillary endothelial cells (BCECs) has yet to be described in detail. Likewise, the molecular mechanisms that govern the establishment and maintenance of the BBB are largely unknown. Proteomics can be used to assess quantitative changes in protein levels and identify proteins involved in the molecular pathways responsible for cellular differentiation. Using the well-established <em>in vitro</em> BBB model developed in our laboratory, we performed a differential nano-LC MALDI-TOF/TOF-MS study of Triton X-100-soluble protein species from bovine BCECs displaying either limited BBB functions or BBB functions re-induced by glial cells. Due to the heterogeneity of the crude extract, we increased identification yields by applying a repeatable, reproducible fractionation process based on the proteins' relative hydrophobicity. We present proteomic and biochemical evidence to show that tissue non-specific alkaline phosphatase (TNAP) and Eps15 homology domain-containing protein 1(EDH1) are over-expressed by bovine BCECs after the re-induction of BBB properties. We discuss the impact of these findings on current knowledge of endothelial and BBB permeability.</p> </div

List of proteins identified (in at least two out of three nano-LC MALDI-TOF/TOF-MS analyses and at least one peptide sequenced) in one type of BCECs and not in the other.

a<p>Identification score: the relevance of protein identities was judged according to the probability-based molecular weight search score <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048428#pone.0048428-Perkins1" target="_blank">[33]</a> (calculated with p<0.05)). The significance threshold of ions scores corresponds to the value of 31.</p>b<p>Protein identified in brain capillary endothelial cells displaying limited BBB (Lim. BBB) or re-induced BBB (Re-ind. BBB) functions.</p

RT-PCR and immunoblotting: confirmation of quantitative changes.

<p>(A) RT-PCR analysis: β-actin (<i>ACTB</i>), TNAP (<i>ALPL</i>) and EHD1 (<i>EHD1</i>) mRNA expression detected by RT-PCR analysis from bovine BCECs with limited BBB functions (Lim. BBB) or re-induced BBB functions (Re-ind. BBB). Analysis was performed using the primers and conditions described. (B) Western blot analysis: β-actin (ACTB), TNAP (ALPL) and EHD1 (EHD1) protein levels in Triton X-100 extracts from bovine BCECs. The Western blot analysis was performed using the antibodies and conditions described. Quantitative and statistical analyses were performed with Quantity One (RT-PCR) or TotalLab 100 (Western blots) and PRISM 5 software packages, respectively. The results correspond to the mean ± SEM from three distinct assays. * p<0.03; ** p<0.002; NS: non-significant (in an unpaired t-test for RT-PCR and a paired t-test for Western blots). The expression of β-actin was monitored as a sample quality control.</p

Alkaline phosphatase activity assay and levamisole-mediated inhibition.

<p>(A) Alkaline phosphatase activity, (B) monolayer permeability to Lucifer Yellow (Pe^LY) and (C) percentage of endothelial cell death (LDH leakage into the culture medium): determined after 4h of incubation of bovine BCECs with limited BBB functions (Lim. BBB) or re-induced BBB functions (Re-ind. BBB) with different concentrations of levamisole (0 to 5 mM). The extent of cell death in each experimental condition was expressed as percentage of full kill (where the latter corresponded to cells lysed with 9% Triton X-100 (w/v) buffer). The statistical analysis was carried out using PRISM 5 software. The results correspond to the mean ± SEM (N = 3, n = 9 BCEC monolayers per treatment). The significance of between-experiment differences were tested in a two-way analysis of variance, followed by a Bonferroni <i>post hoc</i> test. * p<0.05; *** p<0.001; NS: non-significant.</p

<i>In vitro</i> BBB model and assessment of protein fractionation.

<p>(A) A schematic drawing of the culture systems used in this study. (B) Gel image from silver-nitrate-stained 1D-PAGE of fractionated Triton X-100-extracted proteins of bovine brain capillary endothelial cells (BCECs) with either limited BBB functions (Lim. BBB) or re-induced BBB functions (Re-ind. BBB). Red arrows correspond to examples of proteins expressed differentially in the two conditions.</p

Overall evaluation of protein identities.

<p>(A) A Venn diagram showing the common proteins identified in bovine BCECs with limited BBB functions (Lim. BBB) or re-induced BBB functions (Re-ind. BBB) and the proteins identified only in each type of BCEC preparation. (B) Classification by biological processes of the proteins (as identified by nano-LC MALDI-TOF/TOF-MS) found in both BCEC preparations. After conversion to the homologous human genes, proteins were classified using the PANTHER classification system (<a href="http://www.pantherdb.org" target="_blank">http://www.pantherdb.org</a>).</p