Culture-Induced Changes in Blood—Brain Barrier Transcriptome: Implications for Amino-Acid Transporters in vivo

Abstract

Tight homeostatic control of brain amino acids (AA) depends on transport by solute carrier family proteins expressed by the blood-brain barrier (BBB) microvascular endothelial cells (BMEC). To characterize the mouse BMEC transcriptome and probe culture-induced changes, microarray analyses of platelet endothelial cell adhesion molecule-1-positive (PECAM1 + ) endothelial cells (ppMBMECs) were compared with primary MBMECs (pMBMEC) cultured in the presence or absence of glial cells and with b.End5 endothelioma cell line. Selected cell marker and AA transporter mRNA levels were further verified by reverse transcription real-time PCR. Regardless of glial coculture, expression of a large subset of genes was strongly altered by a brief culture step. This is consistent with the known dependence of BMECs on in vivo interactions to maintain physiologic functions, for example, tight barrier formation, and their consequent dedifferentiation in culture. Seven (4F2hc, Lat1, Taut, Snat3, Snat5, Xpct, and Cat1) of nine AA transporter mRNAs highly expressed in freshly isolated ppMBMECs were strongly downregulated for all cultures and two (Snat2 and Eaat3) were variably regulated. In contrast, five AA transporter mRNAs with low expression in ppMBMECs, including y + Lat2, xCT, and Snat1, were upregulated by culture. We hypothesized that the AA transporters highly expressed in ppMBMECs and downregulated in culture have a major in vivo function for BBB transendothelial transport.

Transport of Biogenic Amine Neurotransmitters at the Mouse Blood–Retina and Blood–Brain Barriers by Uptake1 and Uptake2

“…While functional Oct1 and/or Oct2 have been suggested at the BBB using in vitro models (Liou et al, 2007;Dickens et al, 2012) and in vivo by extracellular fluid microdialysis (Lin et al, 2010), these results may not be conclusive. The isolation and culture of endothelial cells for use in in vitro BBB models often leads to the dysregulation of transporters (Lyck et al, 2009). Moreover, brain extracellular fluid microdialysis sampling experiments do not discriminate between the kinetic processes at the BBB and those at the other brain extracellular fluid/ intracellular fluid interfaces where these transporters are present (e.g., neurons and ventricles) (Amphoux et al, 2006;Vialou et al, 2004).…”

Comparison of Immortalized bEnd5 and Primary Mouse Brain Microvascular Endothelial Cells as in vitro Blood–Brain Barrier Models for the Study of T Cell Extravasation

“…Coisne et al (2005) recently described an in vitro model using primary mouse brain microvascular endothelial cells (pMBMECs) in coculture with primary mouse glial cells. Primary mouse brain microvascular endothelial cells form differentiated endothelial monolayers that retain numerous phenotypic properties of the CNS microvasculature in vitro, such as complex tight junctions and barrier formation (Coisne et al, 2005(Coisne et al, , 2006Lyck et al, 2009).…”

Differential axial localization along the mouse brain vascular tree of luminal sodium-dependent glutamine transporters Snat1 and Snat3

“…On the basis of our previous RNA study, we expected a low expression in the vasculature (Lyck et al, 2009). However, Figure 5 shows a representative staining in which vascular Snat1 can be seen colocalized with Glut1 in two larger ( > 15 mm diameter) cortical microvessels at a branch point (Figures 5A and 5C).…”

“…We identified a subset of AA transporters with high in vivo mRNA levels that were strongly decreased by culture and were suggested to be involved in the differentiated BBB transport function (4F2hc/Slc3a2, Lat1/Slc7a5, Snat3/Slc38a3, Snat5/Slc38a5, and Cat1/Slc7a1). In contrast, culture induced a robust increase in Snat1/Slc38a1, y + Lat2/Slc7a6, and xCT/Slc7a11 mRNAs, thus indicating their potential involvement in supplying AAs for endogenous cellular metabolism (Lyck et al, 2009).…”

“…The distribution of Na + -dependent transporters, which use the Na + electrochemical gradient to actively transport substrates potentially against their concentration gradient, is of particular interest. Therefore, we focused on two Na + -dependent AA transporters (namely Snat1/ Slc38a1 and Snat3/Slc38a3) hypothesized to have different roles in endothelial nutrient supply versus transendothelial AA transport (Lyck et al, 2009). To investigate BBB membrane localization, we examined immunofluorescent-labeled mouse brain tissue sections by confocal microscopy focusing on endothelial cell nuclear regions where luminal and abluminal membranes can be distinguished.…”