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

The brain vascular basement membrane is important for both blood-brain barrier (BBB) development, stability, and barrier integrity and the contribution hereto from brain capillary endothelial cells (BCECs), pericytes, and astrocytes of the BBB is probably significant. The aim of this study was to analyse four different in vitro models of the murine BBB for expression and possible secretion of major basement membrane proteins from murine BCECs (mBCECs). mBCECs, pericytes and glial cells (mainly astrocytes and microglia) were prepared from brains of C57BL/6 mice. The mBCECs were grown as monoculture, in co-culture with pericytes or mixed glial cells, or as a triple-culture with both pericytes and mixed glial cells. The integrity of the BBB models was validated by measures of transendothelial electrical resistance (TEER) and passive permeability to mannitol. The expression of basement membrane proteins was analysed using RT-qPCR, mass spectrometry and immunocytochemistry. Co-culturing mBCECs with pericytes, mixed glial cells, or both significantly increased the TEER compared to the monoculture, and a low passive permeability was correlated with high TEER. The mBCECs expressed all major basement membrane proteins such as laminin-411, laminin-511, collagen [α1(IV)]2 α2(IV), agrin, perlecan, and nidogen 1 and 2 in vitro. Increased expression of the laminin α5 subunit correlated with the addition of BBB-inducing factors (hydrocortisone, Ro 20-1724, and pCPT-cAMP), whereas increased expression of collagen IV α1 primarily correlated with increased levels of cAMP. In conclusion, BCECs cultured in vitro coherently form a BBB and express basement membrane proteins as a feature of maturation. The vascular basement membrane is important for development, stability, and the maintenance of the integrity of the blood-brain barrier (BBB). The BBB can be modelled in vitro by co-culturing isolated brain capillary endothelial cells (BCECs) with pericytes and mixed glial cells. Four different in vitro models of the murine BBB were successfully developed in this study and it is shown that murine BCECs (mBCECs) synthesise several basement membrane proteins. The gene expression pattern of the two major basement membrane proteins laminin α5 and collagen IV α1 positively correlated with the addition of hydrocortisone and elevation of cAMP levels, both factors known to increase the expression of tight junction proteins and the trans-endothelial electrical resistance (TEER).