Actin, gelsolin and filamin‐A are dynamic actors in the cytoskeleton remodelling contributing to the blood brain barrier phenotype

Abstract

The brain vascular endothelium operates as a dynamic regulatory interface to maintain the cell environment of the nervous system. In the vicinity of astrocytes, brain endothelial cells develop characteristic features conferring a strong cellular impermeability which limits the penetration of various compounds. The aim of our study was to determine by differential proteomic analysis the changes occurring in bovine brain capillary endothelial cells (BBCEC) differentiated in co-culture with astrocytes compared with endothelial cells cultured alone. In order to obtain reproducible and meaningful protein profiles of in vitro blood-brain barrier models, three sample preparation procedures were carried out to provide the first 2-D comparative proteomic study of BBCEC. Our study highlights advantages and drawbacks of each procedure. The cellular proteins prepared from mechanical scraping of collagen-seeded BBCEC were strongly contaminated by serum proteins. Enzymatic dissociation of BBCEC by trypsin or collagenase solved this problem. A comparative 2-DE profile study of collagenase-harvested BBCEC revealed that cytoskeleton-related proteins (actin, gelsolin and filamin-A) show the most significant quantitative changes in the Triton soluble protein fraction from BBCEC that exhibit characteristics closest to the in vivo situation.

Proteomic Analysis of Plasma Membrane Proteins in an In Vitro Blood-Brain Barrier Model

“…However, for the first time, we report a few PM proteins that had not previously been identified as such in BCECs (notably integrin alpha-D, nitric oxide synthase 3, dysferlin, myoferlin, transmembrane 9 superfamily member 4) and confirmed the presence of previously reported PM proteins (notably ATP-binding cassette sub-family C member 8, platelet endothelial cell adhesion molecule and Na + /K + ATPase) [Uchida et al, 2011]. Although moesin is not a PM protein, it appears to be associated with the PM in BCECs with re-induced BBB functions (as previously reported [Pottiez et al, 2009b]). …”

“…Re-induction of BBB properties was confirmed by the fact that Pe LY for bovine BCECs with re-induced BBB functions (0.6 x10 -3 cm/min) was just over half that for cells with limited BBB functions (1.0 x10 -3 cm/min). Immunostaining also confirmed the presence and localization of the main tight junction proteins occludin and claudin-5 and the associated protein ZO-1, as described elsewhere by our group [Gosselet et al, 2009;Pottiez et al, 2009b].…”

“…Excess quenching buffer was removed by washing the cells twice in CMF-PBS. The cells were harvested by adding collagenase type XI (Clostridium histolyticum, Sigma, Lyon, France) as described previously [Pottiez et al, 2009b]. Briefly, bovine BCECs were incubated for 15 min with 1.5 mL of a 0.1% w/v collagenase solution.…”

“…It concerns mid-to high-throughput proteomics initiated with in vivo or in vitro cells and that seek to answer a well-defined question (e.g. to identify the broadest possible protein expression profile in the brain microvascular endothelium [Haseloff et al, 2003;Lu Q. et al, 2008;Pottiez et al, 2010]; investigate cerebral ischemia [Haqqani et al, 2007;Haqqani et al, 2005;Haseloff et al, 2006] or evaluate a differential solubility approach for the characterization of EC proteins [Lu L. et al, 2007;Murugesan et al, 2011;Pottiez et al, 2009b]. Nevertheless, some PM proteins have been identified in the course of these high-throughput studies.…”

Gelsolin in Cerebrospinal Fluid as a Potential Biomarker of Epilepsy

“…In addition, through differential proteomic analysis of in vitro blood-brain barrier (BBB) models, gelsolin was reported as a dynamic actor in the cytoskeleton remodeling contributing to BBB leakage [24,44]. Previous studies Fig.…”