Expression and deposition of basement membrane proteins by brain capillary endothelial cells in a primary murine model of the blood-brain barrier

The blood-brain barrier (BBB) represents the interface between the blood and the brain parenchyma and consists of endothelial cells which are tightly sealed together by tight junction proteins. The endothelial cells are in addition supported by pericytes, which are embedded in the vascular basement membrane, and astrocyte endfeet. To study the interaction of the different cells of the BBB, construction of in vitro BBB models is valuable. However, the modulation and contribution of the cells of the BBB to the synthesis of basement membrane proteins in vitro is not fully elaborated. Thus, the aim of the present study was to create four different in vitro constructs of the murine BBB to characterise if the expression and secretion of basement membrane proteins by the murine brain capillary endothelial cells (mBCECs) was affected by co-culturing with pericytes, mixed glial cells, or both. Primary mBCECs and pericytes were isolated from brains of adult mice. Mixed glial cells were
prepared from cerebral cortices of newborn mice. The mBCECs were grown as mono-culture, or co-cultured with pericytes, mixed glial cells, or both. To study the expression of basement membrane proteins RT-qPCR, mass spectrometry, and immunofluorescent labelling were used. The mBCECs were found to express major basement membrane proteins in vitro and increased expression of laminin α5 and collagen IV α1 was correlated to the addition of BBB inducing factors (hydrocortisone, Ro20-1724, pCPT-cAMP). Co-culturing of the mBCECs with pericytes, mixed glial cells or both did not influence the gene expression of the investigated basement membrane proteins.