Marie-Pierre Dehouck

An easier, reproducible, and mass-production method to study the blood-brain barrier in vitro.

Abstract: To provide an “in vitro” system for studying brain capillary function, we have developed a process ofcoculture that closely mimics the “in vivo” situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. Under these conditions, endothelial cells retain all the endothelial cell markers and the characteristics of the blood–brain barrier, including tight junctions and γ‐glutamyl transpeptidase activity. The average electric resistance for the monolayers was 661 Ω cm2. The system is impermeable to inulin and sucrose but allows the transport of leucine. Arabinose treatment increases transcellular transport flux by 70%. The relative ease with which such monolayers can be produced in large quantities would facilitate the “in vitro” study of brain capillary functions.

Modelling of the blood–brain barrier in drug discovery and development

The market for neuropharmaceuticals is potentially one of the largest sectors of the global pharmaceutical market owing to the increase in average life expectancy and the fact that many neurological disorders have been largely refractory to pharmacotherapy. The brain is a delicate organ that can efficiently protect itself from harmful compounds and precisely regulate its microenvironment. Unfortunately, the same mechanisms can also prove to be formidable hurdles in drug development. An improved understanding of the regulatory interfaces that exist between blood and brain may provide novel and more effective strategies to treat neurological disorders.

In vitro model for evaluating drug transport across the blood-brain barrier

The passage of substances across the blood-brain barrier (BBB) is regulated in the cerebral capillaries, which possess certain distinct different morphological and enzymatic properties compared with the capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies some characteristics of the in vivo BBB are lost. To provide an in vitro system for studying brain capillary functions, we have developed a process of coculture that closely mimics the in vivo situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. In order to assess the drug transport across the blood-brain barrier, we compared the extraction ratios in vivo to the permeability of the in vitro model. The in vivo and the in vitro values showed a strong correlation. The relative ease with which such cocultures can be produced in large quantities facilitates the screening of new centrally active drugs. This model provides an easier, reproducible and mass-production method to study the blood-brain barrier in vitro.

Receptor-mediated Transcytosis of Lactoferrin through the Blood-Brain Barrier

Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation; it accumulates in the brain during neurodegenerative disorders. Before determining Lf function in brain tissue, we investigated its origin and demonstrate here that it crosses the blood-brain barrier. An in vitro model of the blood-brain barrier was used to examine the mechanism of Lf transport to the brain. We report that differentiated bovine brain capillary endothelial cells exhibited specific high (K d = 37.5 nm; n = 90,000/cell) and low (K d = 2 μm;n = 900,000 sites/cell) affinity binding sites. Only the latter were present on nondifferentiated cells. The surface-bound Lf was internalized only by the differentiated cell population leading to the conclusion that Lf receptors were acquired during cell differentiation. A specific unidirectional transport then occurred via a receptor-mediated process with no apparent intraendothelial degradation. We further report that iron may cross the bovine brain capillary endothelial cells as a complex with Lf. Finally, we show that the low density lipoprotein receptor-related protein might be involved in this process because its specific antagonist, the receptor-associated protein, inhibits 70% of Lf transport.

Drug Transfer Across the Blood-Brain Barrier: Correlation Between in Vitro and in Vivo Models

Abstract: To assess the drug transport across the blood‐brain barrier (BBB), we compared the maximal brain extraction values at time 0 [E(0) values] obtained using either in vitro or in vivo methods. The in vitro BBB model consisted of a coculture of brain capillary endothelial cells growing on one side of a filter and astrocytes on the other. The in vivo model used intracarotid injection in anesthetized rats. Eleven compounds were tested. They were selected because they exhibit quantitatively different brain extraction rates: very low for inulin and sucrose, low for oxicam‐related nonsteroidal antiinflammatory drugs and diclofenac, and high for propranolol and diazepam. As these compounds are apparently transferred by a passive diffusion mechanism, two others, glucose and leucine, were added that cross the BBB by a known carrier‐mediated process. The in vivo and in vitro E(0) values showed a strong correlation as indicated by the Spearmans correlation coefficient (r= 0.88, p < 0.01). The relative ease with which such cocultures can be produced in large quantities could facilitate the screening of new centrally acting drugs.

Bovine Brain Endothelial Cells Express Tight Junctions and Monoamine Oxidase Activity in Long-Term Culture

Abstract: The passage of substances across the blood‐brain barrier is regulated by cerebral capillaries which possess certain distinctly different morphological and enzymatic properties compared to capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies a number of characteristics of the in vivo system are lost. To provide an in vitro system for studies of brain capillary functions, we developed a method of isolating and producing a large number of bovine brain capillary endothelial cells. These cells, absolutely free of pericyte contamination, are subcultured, at the split ratio of 1:20 (20‐fold increase of the cultured surface), with no apparent changes in cell morphology up to the fiftieth generation (10 passages). Retention of endothelial‐specific characteristics (factor V11I‐related antigen, angiotensin‐converting enzyme, and nonthrombogenic surface) is shown for brain capillary‐derived endothelial cells up to passage 10, even after frozen storage at passage 3. Furthermore, we showed that bovine brain capillary endothelial cells retain, up to the fiftieth generation, some of the characteristics of the blood‐brain barrier: occurrence of tight junctions, paucity of pinocytotic vesicles, and monoamine oxidase activity

Prediction of Drug Transport Through the Blood-Brain Barrier in Vivo: A Comparison Between Two in Vitro Cell Models

AbstractPurpose. Studies were conducted to evaluate whether the use of an in vitro model of the blood-brain barrier (BBB) resulted in more accurate predictions of the in vivo transport of compounds compared to the use of a human intestinal cell line (Caco-2). Methods. The in vitro BBB model employs bovine brain capillary endothelial cells co-cultured with primary rat astrocytes. The Caco-2 cells originate from a human colorectal carcinoma. The rat was used as experimental animal for the in vivo studies. Results. Strong correlations (r = 0.93-0.95) were found between the results generated by the in vitro model of the BBB and two different methodologies to measure the permeability across the BBB in vivo. In contrast, a poor correlation (r = 0.68) was obtained between Caco-2 cell data and in vivo BBB transport. A relatively poor correlation (r = 0.74) was also found between the two in vitro models. Conclusion. The present study illustrates the limitations of the Caco-2 model to predict BBB permeability of compounds in vivo. The results emphasize the fact that the BBB and the intestinal mucosa are two fundamentally different biologic barriers, and to be able to make accurate predictions about the in vivo CNS penetration of potential drug candidates, it is important that the in vitro model possesses the main characteristics of the in vivo BBB.

Astrocyte mediated modulation of blood-brain barrier permeability does not correlate with a loss of tight junction proteins from the cellular contacts

In the central nervous system (CNS) complex endothelial tight junctions (TJs) form a restrictive paracellular diffusion barrier, the blood-brain barrier (BBB). Pathogenic changes within the CNS are frequently accompanied by the loss of BBB properties, resulting in brain edema. In order to investigate whether BBB leakiness can be monitored by a loss of TJ proteins from cellular borders, we used an in vitro BBB model where brain endothelial cells in co-culture with astrocytes form a tight permeability barrier for 3H-inulin and 14C-sucrose. Removal of astrocytes from the co-culture resulted in an increased permeability to small tracers across the brain endothelial cell monolayer and an opening of the TJs to horseradish peroxidase as detected by electron microscopy. Strikingly, opening of the endothelial TJs was not accompanied by any visible change in the molecular composition of endothelial TJs as junctional localization of the TJ-associated proteins claudin-3, claudin-5, occludin, ZO-1 or ZO-2 or the adherens junction-associated proteins β-catenin or p120cas did not change. Thus, opening of BBB TJs is not readily accompanied by the complete loss of the junctional localization of TJ proteins.

Contribution of glial cells and pericytes to the mRNA profiles of P-glycoprotein and multidrug resistance-associated proteins in an in vitro model of the blood-brain barrier.

P-glycoprotein (P-gp) and the multidrug resistance-associated proteins (MRP), whose expression is associated with multidrug resistance, have been recently located in the brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB), without taking into account a possible influence or contribution of glial cells and pericytes. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), the present study analysed the transcriptional expression of P-gp and the seven homologues of MRP transporters in BCECs in solo culture or in an in vitro model of the BBB consisting of a co-culture of BCECs and glial cells. Pericytes, glial cells, isolated brain capillaries and bovine grey matter extracts were also tested. P-gp mRNA, absent in glial cells, was found in brain capillaries and in co-cultured BCECs with an increased signal compared to the in solo culture. No amplification was observed in pericytes or grey matter. While MRP2, MRP3 and MRP7 remained undetected, MRP1, absent in capillaries or grey matter, was amplified in BCECs, glial cells and pericytes. MRP4 gave a low signal in most cultures. MRP5 was ubiquitously expressed, displaying a potent signal in all conditions. In spite of its presence in cultured glial cells, MRP6 mRNA expression appeared to be restricted to BCECs, with the same upregulation in the co-cultured condition as observed with P-gp. Moreover, MRP6 was the only transporter whose endothelial mRNA expression was influenced by the presence of pericytes. The tissue distribution of the expression of these transporters and the contribution of the different cell populations are discussed.

Receptor-Mediated Transcytosis of Transferrin through Blood-Brain Barrier Endothelial Cells

A cell culture model of the blood-brain barrier consisting of a coculture of bovine brain capillary endothelial cells (BBCECs) and astrocytes has been used to examine the mechanism of iron transport to the brain. Binding experiments showed that BBCECs express 35,000 high-affinity (concn at 50% receptor saturation = 11.3 +/- 2.1 nM) transferin (Tf) receptors per cell. In contrast to apo-transferrin (apoTf) we observed a specific transport of holo-transferrin (holoTf) across BBCECs. This transport was inhibited completely at low temperature. Moreover, the anti-Tf receptor antibody (OX-26) competitively inhibited holoTf uptake by BBCECs. Pulse-chase experiments demonstrated that only 10% of Tf was recycled to the luminal side of the cells, whereas the majority of Tf was transcytosed to the abluminal side; double-labeling experiments clearly demonstrated that iron crosses BBCECs bound to Tf. No intraendothelial degradation of Tf was observed, suggesting that the intraendothelial pathway through BBCECs bypasses the lysosomal compartment. These results clearly show that the iron-Tf complex is transcytosed across brain capillary endothelial cells by a receptor-mediated pathway without any degradation.