Abstract
BackgroundIn vitro models based on brain capillary endothelial cells (BCECs) are among the most versatile tools in blood–brain barrier research for testing drug penetration into the brain and how this is affected by efflux transporters such as P-glycoprotein (Pgp). However, compared to freshly isolated brain capillaries or primary BCECs, the expression of Pgp in immortalized BCEC lines is markedly lower, which prompted us previously to transduce the widely used human BCEC line hCMEC/D3 with a doxycycline-inducible MDR1-EGFP fusion plasmid. The EGFP-labeled Pgp in these cells allows studying the localization and trafficking of the transporter and how these processes are affected by drug exposure. Here we used this strategy for the rat BCEC line RBE4 and performed a face-to-face comparison of RBE4 and hCMEC/D3 wild-type (WT) and MDR1-EGFP transduced cells.MethodsMDR1-EGFP-transduced variants were derived from WT cells by lentiviral transduction, using an MDR1-linker-EGFP vector. Localization, trafficking, and function of Pgp were compared in WT and MDR1-EGFP transduced cell lines. Primary cultures of rat BCECs and freshly isolated rat brain capillaries were used for comparison.ResultsAll cells exhibited typical BCEC morphology. However, significant differences were observed in the localization of Pgp in that RBE4-MDR1-EGFP cells expressed Pgp primarily at the plasma membrane, whereas in hCMEC/D3 cells, the Pgp-EGFP fusion protein was visible both at the plasma membrane and in endolysosomal vesicles. Exposure to doxorubicin increased the number of Pgp-EGFP-positive endolysosomes, indicating a lysosomotropic effect. Furthermore, lysosomal trapping of doxorubicin was observed, likely contributing to the protection of the cell nucleus from damage. In cocultures of WT and MDR1-EGFP transduced cells, intercellular Pgp-EGFP trafficking was observed in RBE4 cells as previously reported for hCMEC/D3 cells. Compared to WT cells, the MDR1-EGFP transduced cells exhibited a significantly higher expression and function of Pgp. However, the junctional tightness of WT and MDR1-EGFP transduced RBE4 and hCMEC/D3 cells was markedly lower than that of primary BCECs, excluding the use of the cell lines for studying vectorial drug transport.ConclusionsThe present data indicate that MDR1-EGFP transduced RBE4 cells are an interesting tool to study the biogenesis of lysosomes and Pgp-mediated lysosomal drug trapping in response to chemotherapeutic agents and other compounds at the level of the blood–brain barrier.
Highlights
In vitro models based on brain capillary endothelial cells (BCECs) are among the most versatile tools in blood–brain barrier research for testing drug penetration into the brain and how this is affected by efflux transporters such as P-glycoprotein (Pgp)
In RBE4 cells, Pgp-Enhanced green fluorescent protein (EGFP) was predominantly expressed at the cell surface (Additional file 4A), consistent with its function as an efflux transporter, whereas, as reported previously [13, 31], in hCMEC/D3 cells, the Pgp-EGFP fusion protein was visible both at the plasma membrane and in intracellular vesicles, most likely presenting endolysosomal compartments (Additional file 4B)
Endolysosomal vesicles were positive for Pgp-EGFP fluorescence signal in hCMEC/D3 cells, whereas intracellular Pgp-EGFP localization was hardly observed in RBE4 cells (Fig. 1A). These results further substantiated findings from our previous studies with endolysosomal markers in hCMEC/D3-multidrug resistance 1 (MDR1)-EGFP cell cultures that the Pgp-EGFP-positive intracellular vesicles are of endolysosomal origin [13, 31]
Summary
In vitro models based on brain capillary endothelial cells (BCECs) are among the most versatile tools in blood–brain barrier research for testing drug penetration into the brain and how this is affected by efflux transporters such as P-glycoprotein (Pgp). At the blood–brain barrier (BBB), which tightly regulates the movement of ions, molecules, and cells between the blood and the brain, Pgp is mainly located at the apical (luminal) surface of brain capillary endothelial cells (BCECs) that primarily form the BBB [2] Through this localization, Pgp restricts or prevents brain entry of a wide variety of small lipophilic drugs, which presents a significant hurdle to the treatment of various central nervous system (CNS) diseases [1, 3,4,5]. This so-called “drug safe house” effect of lysosomes is thought to contribute to the chemoresistance of cancer cells, but, as shown by us recently, occurs in other cell types such as BCECs that form the BBB [13]
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