[Show abstract][Hide abstract] ABSTRACT: By culturing Caco-2 cells according to a new and optimized protocol, it has been possible to accelerate the cell culture process in such a way that the cells can be used for experiments after only 6days. The accelerated Caco-2 model has been compared to the traditional model (requiring 21-25days of culture) in terms of tightness of the junctions, ability to rank chemical compounds for apparent permeability, active efflux and to discriminate P-gp substrates.
In the new protocol, Caco-2 cells were cultured with the classical Caco-2 medium supplemented with puromycin. The initial cell seeding density was increased two times compared to the traditional procedure and the presence of a low concentration of puromycin in the culture medium reduced the Caco-2 permeability of mannitol. Bi-directionally studies were performed with known P-gp substrates (rhodamine 123, digoxin and saquinavir) and with a total of 20 marketed drugs covering a wide range of physicochemical characteristics and therapeutic indications. Strong correlations were obtained between the apparent permeability in absorptive (Papp A→B) or secretory (Papp B→A) of the drugs in the accelerated model and in the traditional models and comparable efflux ratios were observed in the two studied models.
The new protocol reduces costs for screening and leads to higher throughput compared to traditional Caco-2 cell models. This accelerated model provides short time-feedback to the drug design during the early stage of drug discovery.
Journal of pharmacological and toxicological methods 08/2013; · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of the current study was to investigate whether blood-brain barrier (BBB) permeability studies in vitro could be accelerated by running several compounds together in the same experiment.
To address this question, we compared the transport of six compounds run separately with the results of the same compounds run together (cocktails).
The study clearly demonstrated that the outcome of the experiments were totally different depending on the strategy used. Furthermore, the study highlights the importance of having the resistance to drug transport offered by filters without cells under control, as the filter membrane itself can be the rate-limiting step for some compounds; in addition, there is always a potential risk of interactions between molecules in cocktails as well as drug-drug interaction at the level of BBB transporters. In this study, the presence of several P-glycoprotein substrates in the drug cocktail was found to cause breakdown of the BBB.
The results demonstrate that unless a strategy that involves running several compounds in the same experiment is properly validated, the results are of little predictive value.
Pharmaceutical Research 06/2004; 21(5):756-60. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite remarkable advances in our understanding of mechanisms involved in the development of the brain diseases, drug delivery to the brain remains a challenge. The efflux transporter P-glycoprotein (P-gp) is a key element that confers permeability properties to the blood–brain barrier (BBB). Using an in vitro model, we provided evidence of the P-gp expression and functionality at the BBB level. Furthermore, tissue distribution of multidrug resistance-associated proteins, recently detected at the BBB site, and the contribution of the different cell populations in their expression level will be discussed.
Journal of Neurochemistry 06/2003; 85:7-7. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tetanus neurotoxin reaches the CNS by axonal retrograde transport and thus becomes inaccessible to current treatments. A possible strategy to improve current therapy for tetanus disease would be the vectorization of Fab'2 fragments, allowing their delivery into the CNS. The purpose of this study was to investigate whether after cationization anti-tetanus Fab'2 fragments are able to cross the blood-brain barrier, the first obstacle to CNS delivery. We used primary cocultures of bovine brain capillary endothelial cells and newborn rat astrocytes as an in vitro model to study the binding and transport of cationized Fab'2 (cFab'2) fragments across the brain endothelium. We first show that cationization does not alter Fab'2 affinity for tetanus toxin. Then we demonstrate that after cationization Fab'2 fragments are able to bind to the negative charges on the surface of endothelial cells and subsequently to be transported across the endothelial cell monolayer without any modification of affinity. Finally, using fluorescence microscopy, we show that cFab'2 fragments are transported through endocytotic vesicles. The present study demonstrates that cationization allows Fab'2 directed against tetanus toxin to be transported through brain endothelium by adsorptive-mediated transcytosis. We suggest that this vectorization way could be a promising delivery strategy for carrying anti-tetanic immunoglobulin fragments across the blood-brain barrier to improve tetanus treatment.
Journal of Neurochemistry 12/1999; 73(5):2002-8. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Advanced drug delivery reviews 05/1999; · 11.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 (Kd = 37.5 nM; n = 90,000/cell) and low (Kd = 2 microM; 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.
Journal of Biological Chemistry 04/1999; 274(11):7011-7. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The passage of either unesterified docosahexaenoic acid (DHA) or lysophosphatidylcholine-containing DHA (lysoPC-DHA) through an in vitro model of the blood-brain barrier was investigated. The model was constituted by a brain capillary endothelial cell monolayer set over the medium of an astrocyte culture. Cells were incubated for 4 h with a medium devoid of serum, then the endothelial cell medium was replaced by the same medium containing labeled DHA or lysoPC-DHA and incubations were performed for 2 h. DHA uptake by cells and its transfer to the lower medium (astrocyte medium when they were present) were measured. When the lower medium from preincubation and astrocytes were maintained during incubation, the passage of lysoPC-DHA was higher than that of unesterified DHA. The passage of both forms decreased when astrocytes were removed. The preference for lysoPC-DHA was not seen when the lower medium from preincubation was replaced by fresh medium, and was reversed when albumin was added to the lower medium. A preferential lysoPC-DHA passage also occurred after 2 h with brain endothelial cells cultured without astrocytes but not with aortic endothelial cells cultured and incubated under the same conditions. Altogether, these results suggest that the blood-brain barrier cells released components favoring the DHA transfer and exhibit a preference for lysoPC-DHA.
Journal of Neurochemistry 02/1999; 72(1):338-45. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine the respective roles of endothelial cells from brain capillaries and astrocytes in the conversion of circulating 18:2n-6 and 18:3n-3 into 20:4n-6 and 22:6n-3, respectively, a coculture of the two cell types mimicking the in vivo blood-brain barrier was used. During the culture period, endothelial cells cultured on an insert were set above the medium of a Petri dish containing or not a stabilized culture of astrocytes. Five days after confluence, labeled 18:2n-6 and 18:3n-3 (10 microM each) were added to the endothelial cells and incubated for 48 h. Analogous experiments were also performed by using each cell type cultured alone in the culture device. The distribution of radioactivity in lipids and fatty acids was studied in all the compartments of the culture device. Endothelial cells cultured alone weakly converted the precursor fatty acids into 20:4n-6 and 22:6n-3. When endothelial cells were cocultured with astrocytes, their content of polyunsaturated fatty acids increased dramatically. This effect was associated with the uptake of polyunsaturated fatty acids from the lower medium (astrocyte medium). These fatty acids were released by astrocytes after they were synthesized from the precursor fatty acids that passed through the endothelial cell monolayer into the lower medium. Polyunsaturated fatty acids were released by astrocytes as unesterified fatty acids and as phospholipids (mainly phosphatidylcholine and lysophosphatidylcholine) even when the medium was devoid of serum. These results suggest that astrocytes could play a major role in the delivery of essential polyunsaturated fatty acids to the barrier itself and to the brain.
The Journal of Lipid Research 10/1998; 39(9):1816-24. · 4.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of this work was to assess, in vitro, the passage of P-glycoprotein dependent drugs across brain capillary endothelial cells, when these drugs are associated with a reversing agent.
An in vitro model of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes was used.
We demonstrate that P-glycoprotein expression is upregulated by the presence of astrocytes. Uptake in the cells and transport across endothelial cell monolayers of vincristine, cyclosporin A and doxorubicin were studied. Using S9788 or verapamil as reversing agents, we found an increase in vincristine transport across the endothelial cell monolayers. On the other hand, the association of S9788 or verapamil with cyclosporin A failed to increase the transport of this drug. An increase in the transport of doxorubicin from luminal to abluminal compartment was also observed, due to endothelial cell monolayer breakdown.
Using this model, it is possible to predict the passage of a P-glycoprotein dependent drug to the brain or its sequestration in brain capillary endothelial cells when this drug is associated with a reversing agent, or its toxicity on the blood-brain barrier integrity.
Pharmaceutical Research 08/1998; 15(7):993-1000. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously demonstrated that full-length heparin stimulates the synthesis and secretion of beta-amyloid precursor protein (APP) through an amyloidogenic pathway in neuroblastoma cells. In the present study, heparin was chemically depolymerized, and the effect of low-molecular-weight (LMW) heparin on APP secretion was investigated. In contrast to full-length heparin, LMW heparin had no significant effect on APP secretion. However, LMW heparin fragments, especially heparin disaccharides, were able to inhibit efficiently the stimulatory effect of heparin on APP secretion. LMW heparin derivatives also inhibit the binding of heparin to the beta-amyloid peptide (1-28). Using an in vitro model, we further demonstrated the passage of LMW heparin derivatives through the blood-brain barrier. This study suggests that LMW heparin derivatives or analogues may be effective as therapeutic agents to prevent or slow the process of amyloidogenesis in Alzheimer's disease.
Journal of Neurochemistry 03/1998; 70(2):736-44. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lipoprotein transport across the blood-brain barrier (BBB) is of critical importance for the delivery of essential lipids to the brain cells. The occurrence of a low density lipoprotein (LDL) receptor on the BBB has recently been demonstrated. To examine further the function of this receptor, we have shown using an in vitro model of the BBB, that in contrast to acetylated LDL, which does not cross the BBB, LDL is specifically transcytosed across the monolayer. The C7 monoclonal antibody, known to interact with the LDL receptor-binding domain, totally blocked the transcytosis of LDL, suggesting that the transcytosis is mediated by the receptor. Furthermore, we have shown that cholesterol-depleted astrocytes upregulate the expression of the LDL receptor at the BBB. Under these conditions, we observed that the LDL transcytosis parallels the increase in the LDL receptor, indicating once more that the LDL is transcytosed by a receptor-mediated mechanism. The nondegradation of the LDL during the transcytosis indicates that the transcytotic pathway in brain capillary endothelial cells is different from the LDL receptor classical pathway. The switch between a recycling receptor to a transcytotic receptor cannot be explained by a modification of the internalization signals of the cytoplasmic domain of the receptor, since we have shown that LDL receptor messengers in growing brain capillary ECs (recycling LDL receptor) or differentiated cells (transcytotic receptor) are 100% identical, but we cannot exclude posttranslational modifications of the cytoplasmic domain, as demonstrated for the polymeric immunoglobulin receptor. Preliminary studies suggest that caveolae are likely to be involved in the potential transport of LDL from the blood to the brain.
The Journal of Cell Biology 09/1997; 138(4):877-89. · 9.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endothelial cells and pericytes are closely associated in brain capillaries. Together with astrocytic foot processes, they form the blood-brain barrier. Capillaries were isolated from bovine brain cortex. Pure populations of endothelial cells and pericytes were isolated and cultured in vitro. Polarized monolayers of endothelial cells preferentially secreted immunoreactive endothelin-1 (Et-1) at their abluminal (brain-facing) membrane. They did not express receptors for Et-1. Pericytes expressed BQ-123-sensitive ETA receptors for endothelins as evidenced by 125I-Et-1 binding experiments. These receptors were coupled to phospholipase C as demonstrated by intracellular calcium measurements using indo-1-loaded cells. Addition of Et-1 to pericytes induced marked changes in the cell morphology that were associated with a reorganization of F-actin and intermediate filaments. It is concluded that Et-1 is a paracrine mediator at the bovine blood-brain barrier and that capillary pericytes are target cells for endothelium-derived Et-1.
[Show abstract][Hide abstract] ABSTRACT: gamma-Glutamyl transpeptidase (gamma-GT), primarily described as a kidney enzyme, is also expressed in several cell types of the central nervous system (CNS). It is involved in the glutathione cycle and in cysteine transport. Here we report that the specific activity of this enzyme is transiently increased in the rat brain, following a treatment with 1,25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D. In vitro experiments showed that this positive regulatory effect does not affect endothelial cells of the brain microvessels, but does affect pericytes and parenchymal astrocytes. Changes in the specific activity of gamma-GT were not correlated with any important modification of brain amino acid concentrations. Since gamma-GT is though to participate in the scavenging of reactive oxygen species, these data suggest that 1,25-D3 could be an effector controlling detoxification processes in the brain.
[Show abstract][Hide abstract] ABSTRACT: 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.
The American journal of physiology 05/1996; 270(4 Pt 2):H1149-58. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using a cell culture model of the blood-brain barrier (BBB), we investigated the brain capillary endothelial cell (EC) response to hypoxia. The activities of antioxidant enzymes such as glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase and the GSH level of brain capillary ECs alone or in coculture with astrocytes, as well as those of pericytes, were compared with those obtained with freshly isolated microvessels. These results demonstrated that brain capillary ECs cocultured with astrocytes and used in the presence of a coculture-conditioned medium provided a relevant in vitro model for studying the effect of hypoxia-reoxygenation at the BBB level. The effect of hypoxia on antioxidant enzymes, GSH, and ATP levels was studied, as well as the modification of the permeability to small weight molecules. A decrease in all enzymes and the GSH level could explain an increase in the susceptibility of the brain capillary ECs to further oxidant injury. Second, profound rearrangements of F-actin filaments of the ECs and a decrease in the ATP level could be associated with an increase in the permeability of the monolayer. Furthermore, an apoptotic process was detected by in situ end labeling of DNA. These results indicate that hypoxia distorts the function of ECs and that these cells in culture provide a valuable tool for exploring mechanisms after hypoxia-reoxygenation.
Journal of Neurochemistry 12/1995; 65(5):2138-45. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have investigated the fatty acid composition of brain capillary endothelial cells cultured alone or in coculture with astrocytes, using an in vitro model in which endothelial cells and astrocytes were grown from one part of a filter to another. We found that the fatty acid composition of the cocultured cerebral endothelial cells was markedly different from that of non-cocultivated endothelial cells. The most striking difference was the increase of arachidonic acid (20:4n-6) at the expense of its precursor, linoleic acid (18:2n-6). Similar modifications were found for the n-3 family of fatty acids with an increase of docosahexaenoic acid (22:6n-3) at the expense of its precursors, but the differences were less than within the n-6 fatty acids. These changes induced by the coculture were observed only in endothelial cell phospholipids, especially the phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine classes, but were not detected in phosphatidylinositols and in other lipid classes. Only the composition of the n-3 series fatty acids was altered in another capillary endothelial cell type (from adrenal cortex) cocultured with astrocytes under the same conditions. The fatty acid changes observed might be biologically relevant as they tended to make the fatty acid composition of the brain capillary endothelial cells more closely resemble that of brain microvessels.
The Journal of Lipid Research 12/1995; 36(11):2311-9. · 4.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, has long been known to be involved in the pathogenesis of central nervous system infections and of certain neurodegenerative diseases. However, the possible role of the blood-brain barrier (BBB), the active interface between the blood circulation and brain tissue, remained unknown during these pathological conditions. In our in vitro reconstructed BBB model, 1-hr exposure of recombinant human TNF-alpha (in concentrations of 50, 250, and 500 U/ml, respectively) to the luminal membrane of bovine brain capillary endothelial cells (BBCEC) did not change significantly the transendothelial flux of either sucrose (m.w. 342 Da), or inulin (m.w. 5 kDa) up to 4 hr (early phase), except for a slight decrease (P < 0.05) in sucrose permeation at 2-4 hr with the highest dose of TNF-alpha. On the other hand, at 16 hr after the 1-hr challenge with TNF-alpha (delayed phase) at all 3 concentrations, significant increase was induced in the permeability of BBCEC monolayers for both markers. These changes of permeability were accompanied by a selective reorganization of F-actin filaments into stress fibers, while the intracellular distribution of vimentin remained similar to the control. These results suggest that BBCEC can respond directly to TNF-alpha by a delayed increase of permeability and reorganization of actin filaments.
Journal of Neuroscience Research 08/1995; 41(6):717-26. · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Second messengers, such as cyclic adenosine 3',5'-monophosphate (cAMP), have been shown to take part in the regulation of blood-brain barrier permeability. In the present study, elevation of cAMP levels decreased sucrose (mol. wt, 342) and inulin (mol. wt, 5000) permeability across monolayers of bovine brain capillary endothelial cells as early as 1 h after exposure. Since both tracers use predominantly a paracellular pathway, we assume that cAMP may increase the tightness of the tight junctions through protein phosphorylation.