Monolayers Of Brain Capillary Endothelial Cells Research Articles

Selenomethionine as alternative label to the fluorophore TAMRA when exploiting cell-penetrating peptides as blood-brain barrier shuttles to better mimic the physicochemical properties of the non-labelled peptides

The cell-penetrating peptides (CPPs) Tat and penetratin are frequently explored as shuttles for drug delivery across the blood-brain barrier (BBB). CPPs are often labelled with fluorophores for analytical purposes, with 5(6)-carboxytetramethylrhodamine (TAMRA) being a popular choice. However, TAMRA labelling affects the physicochemical properties of the resulting fluorophore-CPP construct when compared to the CPP alone. Selenomethionine (MSe) may be introduced as alternative label, which, due to its small size and amino acid nature, likely results in minimal alterations of the peptide physicochemical properties. With this study we compared, head-to-head, the effect of MSe and TAMRA labelling of Tat and penetratin with respect to their physicochemical properties, and investigated effects hereof on brain capillary endothelial cell (BCEC) models. TAMRA labelling positively affected the ability of the peptides to adhere to the cell membranes as well being internalized into the BCECs when compared to MSe labelling. TAMRA labelling of penetratin added toxicity to the BCECs to a higher extent than TAMRA labelling of Tat, whereas MSe labelling did not affect the cellular viability. Both TAMRA and MSe labelling of penetratin decreased the barrier integrity of BCEC monolayers, but not to an extent that improved transport of the paracellular marker 14C-mannitol. In conclusion, MSe labelling of Tat and penetratin adds minimal alterations to the physicochemical properties of these CPPs and their resulting effects on BCECs, and thereby represents a preferred alternative to TAMRA for peptide quantification purposes.

Efflux at the Blood-Brain Barrier Reduces the Cerebral Exposure to Ochratoxin A, Ochratoxin α, Citrinin and Dihydrocitrinone.

Recent studies have implied that environmental toxins, such as mycotoxins, are risk factors for neurodegenerative diseases. To act directly as neurotoxins, mycotoxins need to penetrate or affect the integrity of the blood-brain barrier, which protects the mammalian brain from potentially harmful substances. As common food and feed contaminants of fungal origin, the interest in the potential neurotoxicity of ochratoxin A, citrinin and their metabolites has recently increased. Primary porcine brain capillary endothelial cells were used to investigate cytotoxic or barrier-weakening effects of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone. The transfer and transport properties of the mycotoxins across the barrier formed by porcine brain capillary endothelial cell monolayers were analysed using HPLC-MS/MS. High levels of Ochratoxin A caused cytotoxic and barrier-weakening effects, whereas ochratoxin α, citrinin and dihydrocitrinone showed no adverse effects up to 10 µM. Likely due to efflux transporter proteins, the transfer to the brain compartment was much slower than expected from their high lipophilicity. Due to their slow transfer across the blood-brain barrier, cerebral exposure of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone is low and neurotoxicity is likely to play a subordinate role in their toxicity at common physiological concentrations.

Menthol-modified BSA nanoparticles for glioma targeting therapy using an energy restriction strategy

Current chemotherapeutics for glioma are not sufficiently effective due to their low tolerance and poor blood–brain barrier (BBB) permeability. Energy restriction based on co-delivery of albendazole (Abz) and nano-silver has been proven to suppress tumor growth without adverse effects. Accordingly, menthol was conjugated to BSA (MeB) to overcome the BBB-permeability issue. In this study, Abz-loaded MeB-silver nanoparticles (MBS-Abz) were developed by self-assembly of MeB, albendazole and nano silver for glioma targeting therapy. The nanoparticle entered the brain across the BBB and specifically accumulated in the glioma region. MeB delivered the nanoparticles across the brain capillary endothelial cell monolayer by promoting internalization and BBB disruption. MBS-Abz hindered ATP generation via the inhibition of glycolytic and mitochondrial pathways both in vitro and in vivo. The anti-glioma mechanisms of energy restriction were related to the cytotoxicity, proliferation inhibition, cell cycle arrest, and apoptosis induced by ATP exhaustion, and the effects of MBS-Abz were significantly better than those induced by monotherapy nanoparticles or unmodified nanoparticles. These results demonstrated that by combining the energy restriction effect of albendazole and nano silver, as well as the BBB penetration ability of menthol, MBS-Abz achieves superior anti-glioma efficacy and can be an effective strategy for glioma therapy.

Peptide Derived from HIV-1 TAT Protein Destabilizes a Monolayer of Endothelial Cells in an in Vitro Model of the Blood-Brain Barrier and Allows Permeation of High Molecular Weight Proteins

Most chemotherapeutic agents are blood-brain barrier (BBB) impermeants. HIV-1-derived TAT protein variants contain a transmembrane domain, which may enable them to cross the BBB and reach the brain. Here we synthesized CAYGRKKRRQRRR, a peptide containing a cysteine moiety attached to the N terminus of the transmembrane domain (C-TAT peptide), and studied its effects in an in vitro BBB model, which we found to reflect penetration by a receptor-independent pathway. Incubation of the brain capillary endothelial cell monolayer with 0.3-0.6 μmol/ml of this C-TAT peptide, for a period of 1-2 h, destabilizes brain capillary endothelial cell monolayer and introduces the ability of impermeant therapeutic agents including high molecular weight proteins to penetrate it substantially. The cysteinyl moiety at position 1 of the C-TAT peptide contributes largely to the destabilizing potency and the penetration efficacy of impermeant substances. The destabilizing effect was reversed using heparin. In summary, experimental conditions allowing a significant increase in entry of impermeant low and high molecular weight substances from the luminal (blood) to the abluminal side (brain) were found in an in vitro BBB model reflecting in vivo protein penetrability by a receptor-independent pathway.

Homocysteine Enhances Transmigration of Rat Monocytes through a Brain Capillary Endothelial Cell Monolayer via ICAM-1

Increased homocysteine (Hcy) levels contribute to a variety of cardiovascular and cerebrovascular diseases including stroke and Alzheimer's disease. Recent data has shown that elevated levels of Hcy can lead to the blood-brain barrier (BBB) dysfunction and activation. However, the mechanism for Hcy-mediated dysfunction remains unclear. The aim of this study is to characterize the effects of moderate Hcy administration in rat brain capillary endothelial cells (BCECs), which serve as a simple model to study blood-brain barrier (BBB) functions. This present study shows that addition of 20 microM Hcy for 6 days did not significantly affect BCEC survival, as measured by acridine orange staining, propidium iodide staining, and nitrite content. However, addition of 20 microM Hcy for 6 days did elevate lactate dehydrogenase (LDH) activity released into the supernatant of BCECs, as well as significantly enhance the transmigration of monocytes across the BCEC in a time-dependent manner. In addition, TNFalpha levels in BCEC were also elevated by Hcy, whereas inflammatory markers MIP3alpha and RANTES were significantly reduced. Finally, this study also shows that intercellular adhesion molecule-1 (ICAM-1) expression is significantly enhanced by 20 microM Hcy treatment compared to control conditions. These results suggest that moderate levels of homocysteine can affect proinflammatory patterns expressed by BCECs ultimately leading to BBB activation and dysfunction through enhanced monocyte transmigration and ICAM-1 expression.

Monocytes deliver bioactive nerve growth factor through a brain capillary endothelial cell-monolayer in vitro and counteract degeneration of cholinergic neurons

Alzheimer's disease is an age-dependent brain disorder, characterized by progressive memory deficits and cognitive decline and loss of cholinergic neurons. Nerve growth factor (NGF) is the most potent protein to protect cholinergic neurons against degeneration. However, problems of delivery to the brain limit the therapeutical use of NGF. The aim of the present study was to test, if primary rat monocytes can be loaded with recombinant NGF and pass an in vitro monolayer of brain capillary endothelial cells (BCEC), release NGF, and support the cholinergic neurons in an organotypic brain slice model. Monocytes were isolated from rat blood by negative magnetic selection, loaded with recombinant NGF using Bioporter™. The monocytes adhered and migrated through an in vitro rat BCEC-monolayer. NGF released at the basolateral side counteracted degeneration of cholinergic basal nucleus of Meynert neurons. In conclusion, our present study shows a proof-of-principle, that primary monocytes secreting NGF might be useful tools to deliver NGF into the brain, however, further in vivo studies are necessary.

Uptake of apolipoprotein E fragment coupled liposomes by cultured brain microvessel endothelial cells and intact brain capillaries

The suitability of surface modified liposomes as drug carriers for brain-specific targeting was investigated using apolipoprotein E fragments as brain-directed vectors. Liposomes coated with polyethylene glycol-2000 (sterically stabilized, PEGylated liposomes) were prepared from hydrogenated egg phosphatidylcholine, cholesterol, and a PEG-derivatized phospholipid. Liposomes were covalently coupled to a peptide of 26 amino acids length, derived from the binding site of human apolipoprotein E4 (ApoE4) and a peptide of random amino acid sequence, respectively. Rhodamine-labeled dipalmitoylphosphatidylethanolamine was incorporated into the lipid bilayer in order to visualize the liposomal interaction with brain capillary endothelial cell monolayers. The interaction of the liposomes with monolayers of porcine brain capillary endothelial cells (BCEC), the rodent cell line RBE4, and freshly isolated porcine brain capillaries was studied by means of confocal laser scanning fluorescence microscopy. In contrast to random peptide coupled liposomes, the ApoE4-fragment coupled liposomes were rapidly taken up by cultured BCECs and RBE4 cells. Uptake could be inhibited by ApoE4, free peptide, and antibodies against the LDL receptor in a concentration-dependent manner. The results indicate that the liposomes are internalized via the LDL receptor, which is expressed at the blood−brain barrier. In conclusion, liposomes coupled to ApoE4 fragments are taken up into brain endothelium via an endocytotic pathway and may therefore be a suitable carrier for drug delivery to the brain.

Dendritic Cell Transmigration through Brain Microvessel Endothelium Is Regulated by MIP-1α Chemokine and Matrix Metalloproteinases

Dendritic cells (DCs) accumulate in the CNS during inflammatory diseases, but the exact mechanism regulating their traffic into the CNS remains to be defined. We now report that MIP-1alpha increases the transmigration of bone marrow-derived, GFP-labeled DCs across brain microvessel endothelial cell monolayers. Furthermore, occludin, an important element of endothelial tight junctions, is reorganized when DCs migrate across brain capillary endothelial cell monolayers without causing significant changes in the barrier integrity as measured by transendothelial electrical resistance. We show that DCs produce matrix metalloproteinases (MMP) -2 and -9 and GM6001, an MMP inhibitor, decreases both baseline and MIP-1alpha-induced DC transmigration. These observations suggest that DC transmigration across brain endothelial cell monolayers is partly MMP dependent. The migrated DCs express higher levels of CD40, CD80, and CD86 costimulatory molecules and induce T cell proliferation, indicating that the transmigration of DCs across brain endothelial cell monolayers contributes to the maintenance of DC Ag-presenting function. The MMP dependence of DC migration across brain endothelial cell monolayers raises the possibility that MMP blockers may decrease the initiation of T cell recruitment and neuroinflammation in the CNS.

Bilirubin Efflux By Brain Capillary Endothelial Cell Monolayers in Vitro: Role of P-Glycoprotein.

Background: The passage of bilirubin across the blood-brain barrier into the CNS is central to the development of kernicterus. Indirect in vivo evidence suggests that P-glycoprotein (Pgp), a multidrug transporter expressed on brain capillary endothelial cells, may limit the influx and CNS retention of unconjugated bilirubin (Pediatr Res 44:763–766, 1998). This phenomenon has not been studied in brain capillary endothelial cell monolayers in vitro. Objective: To test the hypothesis that Pgp mediates bilirubin transport across brain capillary endothelial cell monolayers in vitro. Design/Methods: Bovine brain capillary endothelial cells (Cell Systems Corp.) were grown in confluent monolayers at a density of 5 × 104 cells/insert in 1 cm2 Transwell dishes. [3H]-bilirubin (100nM) transport (pnol/cm2/min) was tested in both the apical to basolateral [A→B] and basolateral to apical [B→A] directions in the presence and absence of a Pgp inhibitor (cyclosporin A[5uM]). Involvement of a Pgp mediated efflux mechanism is suggested by a B→A/A→B ratio of greater than 1.5 (Pharm Res 16:1206, 1999). Brain capillary endothelial cell Pgp expression was confirmed by Western immunoblotting techniques. Results: Brain capillary endothelial cells express Pgp as seen on Western immunoblots. Bilirubin transport in the B→A direction (0.67±0.05 pmol/cm2/min) was 6.4 fold higher than the rate for A→B direction (0.11±0.02) suggesting active efflux of bilirubin across brain capillary endothelial cell monlolayers. B→A bilirubin transport decreased (0.60±0.07) and A→B bilirubin transport was enhanced (0.17±0.03) in the presence of the Pgp inhibition, with an overall decrease in bilirubin efflux of 27% suggesting that bilirubin transport by brain capillary endothelial cells is mediated in part by Pgp. Conclusions: We conclude that i) bilirubin is transported by brain capillary endothelial cell monolayers in vitro in a net B→A direction (i.e. activie efflux); ii) Pgp plays an active role in this barrier function, and iii) unconjugated bilirubin is a substrate for Pgp. We speculate that i) brain capillary endothelial Pgp limits the CNS passage and retention of unconjugated bilirubin and ii) that low brain capillary endothelial cell Pgp expression, as reported in premature neonates, may enhance brain bilirubin levels during hyperbilirubinemia. Disclosure: Supported by NINDS (038993), the 25 Club of Magee-Womens Hospital and the Mario Lemieux Centers for Patient Care and Research.

In Vitro Models of the Blood-Brain Barrier: When to Use Which?

In this paper the various BBB systems have been described including the various isolation procedures of brain capillary endothelial cells (BCEC), the culture of monolayers of BCEC and endothelial cell lines, and the coculture of BCEC with various types of astrocytes. The transference of results between BBB culture systems has been discussed together with the application of the various BCEC co-culture systems in research. It is concluded that there is a need for interlaboratory validation of BBB data, particularly BBB transport data.

HIV protease inhibitor ritonavir: a more potent inhibitor of P-glycoprotein than the cyclosporine analog SDZ PSC 833

The effect of P-glycoprotein inhibition on the uptake of the HIV type 1 protease inhibitor saquinavir into brain capillary endothelial cells was studied using porcine primary brain capillary endothelial cell monolayers as an in vitro test system. As confirmed by polymerase chain reaction and Western blot analysis, this system functionally expressed class I P-glycoprotein (pgp1A). P-Glycoprotein isoforms pgp1B or pgp1D could not be detected. The uptake of saquinavir into endothelial cells could be described as the result of a diffusional term of uptake and an oppositely directed saturable extrusion process. Net uptake of saquinavir into cultured brain endothelial cells could be increased significantly up to 2-fold by SDZ PSC 833 in a dose-dependent manner, with an ic 50 of 1.13 μM. In addition, the HIV protease inhibitor ritonavir inhibited p-glycoprotein-mediated extrusion of saquinavir with an ic 50 of 0.2 μM, indicating a high affinity of ritonavir for p-glycoprotein. In conclusion, we showed that the HIV protease inhibitor ritonavir is a more potent inhibitor of P-glycoprotein than the multidrug resistance (MDR)-reversing agent SDZ PSC 833. The inclusion of this drug in combination regimens may greatly facilitate brain uptake of HIV protease inhibitors, which is especially important in patients suffering from AIDS dementia complex.

Sodium and chloride-dependent high and low-affinity uptakes of GABA by brain capillary endothelial cells

The mechanisms of carrier-mediated transport of γ-aminobutyric acid (GABA) at the blood–brain barrier (BBB) were examined by investigating [ 3 H ]GABA uptake by isolated bovine brain capillaries, monolayers of primary cultured brain capillary endothelial cells (BCECs) attached to plates or suspended BCECs. The uptake of [ 3 H ]GABA was concentration-dependent and saturable. Nonlinear regression analysis of the original data indicated the existence of two distinct high and low-affinity GABA transporters on isolated brain capillaries or suspended BCECs, with K m1, K m2, V m1 and V m2 equal to 25.3 μM, 485.2 μM, 3.6 and 8.4 nmol/5 min/mg protein, respectively, for the capillaries, and 21.3 μM, 322.0 μM, 6.1 and 15.7 nmol/5 min/mg protein, respectively, for the suspended BCECs. In contrast, a single low-affinity transporter was found for monolayers of BCECs attached to plates with K m and V m equal to 338.7 μM and 18.8 nmol/5 min/mg protein, respectively. Subcellular location of the two distinct transporters on BCECs is discussed, suggesting that the low-affinity GABA transporter is probably localized to the luminal membrane of BCECs, and the high-affinity GABA transporter is probably localized to the antiluminal membrane. Low temperature (4°C) and metabolic inhibitors markedly diminished both high and low-affinity uptakes of [ 3 H ]GABA by isolated brain capillaries. The substitution of Na + with choline +, K + or Li + with the counter anion Cl − almost completely abolished both uptakes. Substitution of Cl − with Br −, I −, F − or NO 3 − in the presence of Na + significantly reduced both uptakes to different extents. Alanine, leucine, phenylalanine, arginine, glutamate and pyruvate had no obvious effect on either uptake. Probenecid, amino-oxyacetic acid, β-alanine, taurine, betaine, and nipecotic acid significantly reduced both uptakes. These data suggested that both the GABA transporters at the BBB were temperature, metabolic energy, Na + and Cl −-dependent, and may be specific and different from the known monocarboxylic acid, GABA and other amino acid transporters, which may play a role in the disposition of GABA in the brain.

Effects of tumor necrosis factor on receptor-mediated endocytosis and barrier functions of bovine brain capillary endothelial cell monolayers

Tumor necrosis factor α (TNF- α) plays a crucial role in the pathogenesis of the central nervous system infections. In an in vitro reconstructed blood–brain barrier model, a significant dysregulation of receptor mediated endocytosis of low density lipoproteins (LDL) and transferrin (Tf) is demonstrated at delayed phase of direct TNF- α activation. Concomitant with the increase in LDL uptake, we demonstrate a decrease of Tf-receptor mediated endocytosis. The potential role of TNF action in the differential or opposite routing of macromolecules is also characterized by a stimulation of their transcytosis. These findings may provide a new insight into the inflammatory effect of TNF- α on brain capillary endothelial cells.

The mechanism of free radical generation in brain capillary endothelial cells after anoxia and reoxygenation.

We studied the mechanism of reoxygenation injury of cerebral microvessels in cultured rat brain capillary endothelial cells (BCECs). BCECs were isolated from rat cerebral cortices by a two step enzymatic treatment. The monolayers of BCECs were subjected to anoxia for 20 minutes followed by reoxygenation for 3 hours. Cell damage was assessed by measuring the leakage of intracellular lactic dehydrogenase (LDH). The control group was anoxia/reoxygenated BCECs without any protective reagents. To study the protective effect of free radical scavengers and antioxidants, superoxide dismutase, catalase, deferoxamine, oxypurinol, indomethacin, or NG-nitro-L-arginine methyl ester (L-NAME) was applied during anoxia/reoxygenation. Thus 7 experimental conditions were established. Lactic dehydrogenase (LDH) leaked from reoxygenated BCECs due to cell membrane damage. This leakage was almost totally suppressed by superoxide dismutase, indicating that reoxygenation injury of BCECs is mediated by superoxide generation. The other scavengers and antioxidants partially suppressed LDH leakage. Reduction of Ca2+ in the culture medium from 1.6 mM to 0.016 mM also suppressed LDH leakage. These results indicate that BCECs subjected to anoxia/reoxygenation become potent generators of superoxide anion, which is thought to be responsible for reoxygenation injury. The superoxide generation partially depends on the xanthine oxidase and cyclooxygenase pathways. As L-NAME partially suppressed LDH leakage peroxynitrite may contribute to reoxygenation injury of BCECs. The extracellular Ca2+ concentration also plays a critical role in the reoxygenation injury of BCECs.

Penetration of small molecular weight substances through cultured bovine brain capillary endothelial cell monolayers: the early effects of cyclic adenosine 3',5'-monophosphate.

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.

Transport of cyclosporin A across the brain capillary endothelial cell monolayer by P-glycoprotein

P-glycoprotein, a multidrug transporter protein, exists in the brain capillary endothelium. To study the function of P-glycoprotein in brain capillary endothelium as a barrier against cyclosporin A, we examined the interaction of cyclosporin A with P-glycoprotein expressed in cultured brain capillary endothelial cells (MBEC4). P-glycoprotein of MBEC4 specifically bound [ 125I]iodoaryl azidoprazosin, and the binding was inhibited by cyclosporin A and vincristine. Intracellular accumulation of cyclosporin A in MBEC4 was about one-third the amount accumulated in mouse aortic endothelial cells (MAEC3), a cell line that did not express P-glycoprotein. The reduced accumulation of cyclosporin A in MBEC4 was increased by verapamil, a competitive inhibitor of transport function of P-glycoprotein. Cyclosporin A was preferentially transported from basal to apical side when the cell monolayer of MBEC4 was formed; however this transendothelial transport was not observed across cell monolayer of MAEC3. Verapamil inhibited the transendothelial transport of cyclosporin A across the MBEC4 monolayer. Thus P-glycoprotein in brain capillary endothelium could transport cyclosporin A across the endothelium from the basal to the apical side. These observations suggest that P-glycoprotein is involved in the complex function of the blood-brain barrier as a secretory detoxifying transporter of cyclosporin A.

The effect of leukotriene C4 on the permeability of brain capillary endothelial cell monolayer.

The role of leukotrienes as mediator of brain edema is still controversial. Recently, the ability of gamma-GTP to act as enzymatic barrier and to inactivate leukotrienes in normal brain capillaries was pointed out. A hypothesis tested in our experiments was that Leukotriene C4 (LTC4) increases permeability of a cerebral capillary endothelial monolayer which lacks gamma-GTP activity. Brain capillary endothelial cells were obtained of 10 rats from cerebral cortex by an enzymatic isolation procedure. The cells have an intact function, however, lack gamma-GTP activity. The endothelial cells were cultured on an optically clear collagen membrane mounted on a plastic frame. Effects of bradykinin (1 x 10(-5) M) and LTC4 (1 x 10(-7) M, 1 x 10(-6) M, 5 x 10(-6) M, 1 x 10(-5) M) were tested on permeability of the endothelial cell monolayer by measuring leakage of 14C-sucrose. The effect of LTC4 and bradykinin on intracellular calcium was studied by laser scanning confocal microscopy. LTC4 did not increase permeability of the brain capillary endothelial cell monolayer which lacked gamma-GTP activity. LTC4 did neither increase the concentration of intracellular calcium. Differences of LTC4 receptor function in normal brain capillaries and tumor capillaries remain to be studied.