RBE4 Cells Research Articles

Transient alterations of the blood–brain barrier tight junction and receptor potential channel gene expression by chlorpyrifos

The blood-brain barrier (BBB) is formed by specialized endothelial cells lining capillaries in the central nervous system (CNS). We previously demonstrated that exposure to very low concentrations of the organophosphorus insecticide chlorpyrifos (CPF) decreased electrical resistance across the BBB in vitro, indicating a loss of BBB integrity. The present study examined the transient effects of CPF on expression of genes contributing to tight junctions of the BBB. Rat brain endothelial cells (RBE4) were co-cultured with rat astrocytes on membrane inserts to form an in vitro BBB. The RBE4 cells in the BBB were then exposed to CPF for 2, 4 and 12 h. Total RNA was extracted from RBE4 cells and quantitative real-time PCR (qRT-PCR) was used to quantify levels of gene expression of tight junction proteins claudin5, scaffold proteins zona occludens (ZO1) and transient receptor potential (canonical) channels (TRPC4). Gene expression decreased 2 h after exposure to CPF, especially TRPC4, but the effects were reversed 12 h later. CPF exposure for only 15 min caused less effect than longer exposures, with TRPC4 gene expression above the control at 4 h. These results suggest that altering gene expression for claudin5, TRPC4 and ZO1 by CPF may directly contribute to BBB disruption, and that the alteration is reversible upon removal of CPF.

Evaluation of rifampicin and derivatives cytotoxic profile in RBE4 cells

Evaluation of rifampicin and derivatives cytotoxic profile in RBE4 cells

Rifampicin derivative, Red-Rif, protects against paraquat cytotoxicity in RBE4 cells

Rifampicin derivative, Red-Rif, protects against paraquat cytotoxicity in RBE4 cells

Mechanistic Studies of the Effect of Bile Salts on Rhodamine 123 Uptake into RBE4 Cells

To examine the ability of bile salts (BS) to act as permeation enhancers at the blood brain barrier, the effect of four BS (cholate, deoxycholate, monoketocholate and taurocholate) on accumulation of rhodamine 123 (R123) in rat brain endothelial (RBE4) cells was investigated. Experiments were performed using BS concentrations shown to be noncytotoxic to RBE4 cells. Uptake and efflux of R123 in the absence and presence of BS were studied by fluorescence spectroscopy and confocal microscopy. Changes in RBE4 cell membrane fluidity in the presence of BS were evaluated using fluorescence anisotropy. The direct interaction between BS and R123 (ion pairing) and the effect of BS on distribution of R123 into liposomes were studied by capillary electrophoresis. All BS influenced R123 uptake in a concentration-dependent manner and increased cell membrane fluidity. Monoketocholate produced the greatest increase in uptake and also significantly reduced R123 efflux probably by inhibition of P-glycoprotein (P-gp). Direct interaction of BS and R123 was weak, but distribution of R123 into liposomes was increased by BS. The results suggest that BS increase R123 uptake by increasing cell membrane fluidity and, in the case of MKC, by inhibiting P-gp.

Functionalization of liposomes with ApoE-derived peptides at different density affects cellular uptake and drug transport across a blood-brain barrier model

A promising strategy to enhance blood-brain barrier penetration by drugs is the functionalization of nanocarriers with uptake-facilitating ligands. We studied the cellular uptake, by cultured RBE4 brain capillary endothelial cells, of nanoliposomes (NLs) covalently coupled with monomer or tandem dimer of apolipoprotein E (ApoE)-derived peptides (residues 141-150), at various densities. NLs without functionalization did not show either relevant membrane accumulation or cellular uptake, as monitored by confocal microscopy and quantified by fluorescence-activated cell sorting. Functionalization with peptides mediated an efficient NLs uptake that increased with peptide density; NLs carrying monomeric peptide performed the best. Moreover, we studied the ability of ApoE-NLs to enhance the transport of a drug payload through a RBE4 cell monolayer. The permeability of a tritiated curcumin derivative was enhanced after its entrapment into ApoE-NLs, in particular those functionalized with the dimer (+83% with respect to free drug, P < 0.01). Thus, these NLs appear particularly suitable for implementing further strategies for drug brain targeting. From the Clinical Editor Re and her collaborators present a method for delivering nanoliposomes via the blood brain barrier by utilizing peptide fragments including monomers or tandem dimers ApoE. This method may enable enhanced nanoliposome associated drug delivery via the blood-brain barrier, which would have enormous significance in neurodegenerative and other CNS disorders.

Insights into the putative catechin and epicatechin transport across blood-brain barrier

The identification of mechanisms associated with phenolic neuroprotection is delayed due to a lack of information regarding the ability of phenolic compounds to enter the central nervous system (CNS). The aim of this work was to evaluate the transmembrane transport of catechin and epicatechin across blood-brain barrier (BBB). Two BBB cell lines, RBE-4 cells (immortalized cell line of rat capillary cerebral endothelial cells) and hCMEC/D3 (immortalized human cerebral microvessel endothelial cell line), were used. HPLC-DAD/MS was used to detect these compounds and their metabolites in the studied samples. The metabolites of the tested flavan-3-ols were synthesized to be used as standards. Catechin and epicatechin could cross both cells in a time-dependent manner. This transport was stereoselective (epicatechin ≫ catechin), involving one or more stereoselective entities. Additionally, these cells were capable of metabolizing these compounds, particularly by conjugation with glucuronic acid, since this metabolite was detected in the basolateral media. Several studies suggest that blood levels of catechin and epicatechin are far below the levels used in this study and that these compounds appeared mainly as methyl, sulfate and glucuronide metabolites. Nevertheless, the information obtained by this study is valuable for the new insights about flavan-3-ols transport. (i) catechin and epicatechin are capable of crossing the BBB; (ii) a stereoselective process was involved in the passage of these compounds across BBB cells; (iii) these endothelial cells have enzymes capable of metabolizing these compounds.

Uptake of folates by rat and human brain endothelial cell lines

The blood–brain barrier (BBB) is essential for central nervous system homeostasis. Folates are essential for the biosynthesis of purines, pyrimidines and methionine. Our aim was to characterize 3H‐folic acid (3H‐FA) and 14C‐methyltetrahydrofolic acid (14C‐MTHF) uptake by rat (RBE4) and human (hCMEC/D3) BBB endothelial cell lines. Uptake of 3H‐FA and 14C‐MTHF by RBE4 cells was time‐dependent and linear for the first 2 min of incubation. On the other hand, uptake of these substances by hCMEC/D3 cells showed less time‐dependency. Further experiments were performed in RBE4 cells. Uptake of 3H‐FA was found to be (1) stimulated at an acidic and at an alkaline pH, (2) Na+‐dependent and stimulated when F‐ substituted for Cl−, (3) energy‐independent, (4) inhibited by premetrexed (PTX) and stimulated by cytochalasin D (cyt D) and (5) not affected by MTHF, DIDS, SITS, methotrexate (MTX), monensin (MON) and FA. Uptake of 14C‐MTHF was found to be (1) pH‐independent, (2) Na+, Cl− and energy‐independent, (3) inhibited by PTX and MTX and stimulated by cyt D and (4) not affected by FA, DIDS, SITS, MON and MTHF. RT‐PCR analysis demonstrates mRNA expression of RFC (reduced folate carrier), but neither of FRα (folate receptor α) nor of PCFT (proton‐coupled folate transporter) in RBE4 cells and mRNA expression of both RFC and PCFT, but not of FRα, in hCMEC/D3 cells. Human and rat BBB cells show little capacity for folates uptake.

Characterization of uptake of folates by rat and human blood–brain barrier endothelial cells

This study aimed to characterize (3)H-folic acid ((3)H-FA) and (14)C-methyltetrahydrofolic acid ((14)C-MTHF) uptake by rat (RBE4) and human (hCMEC/D3) blood-brain barrier (BBB) endothelial cell lines. Uptake of (3)H-FA and (14)C-MTHF by RBE4 cells was time dependent and linear for the first 2 min of incubation; uptake by hCMEC/D3 cells showed a less marked time-dependency and a greater experimental variability. So, further experiments were performed in RBE4 cells only. Uptake of (3)H-FA was stimulated at acidic and alkaline pH, Na(+) dependent, stimulated when F(-) substituted for Cl(-), energy independent, inhibited by premetrexed, stimulated by cytochalasin D, and unaffected by MTHF, DIDS, SITS, methotrexate, monensin, and FA. Uptake of (14)C-MTHF was found to be pH-, Na(+)-, Cl(-)- and energy independent, inhibited by premetrexed and methotrexate, stimulated by cytochalasin D, and unaffected by FA, DIDS, SITS, monensin, and MTHF. RT-PCR analysis showed mRNA expression of reduced folate transporter (RFC), but neither of FRalpha nor of proton-coupled folate transporter (PCFT) in RBE4 cells, and mRNA expression of RFC and PCFT, but not of FRalpha, in hCMEC/D3 cells. In conclusion, both human and rat BBB endothelial cells show little capacity for (3)H-FA and (14)C-MTHF apical uptake. Hence, these cell lines do not appear to be a good model to study the transport of folates at the BBB.

Rat brain endothelial cells are a target of manganese toxicity

Manganese (Mn) is an essential trace metal; however, exposure to high Mn levels can result in neurodegenerative changes resembling Parkinson's disease (PD). Information on Mn's effects on endothelial cells of the blood–brain barrier (BBB) is lacking. Accordingly, we tested the hypothesis that BBB endothelial cells are a primary target for Mn-induced neurotoxicity. The studies were conducted in an in vitro BBB model of immortalized rat brain endothelial (RBE4) cells. ROS production was determined by F 2–isoprostane (F 2–IsoPs) measurement. The relationship between Mn toxicity and redox status was investigated upon intracellular glutathione (GSH) depletion with diethylmaleate (DEM) or l-buthionine sulfoximine (BSO). Mn exposure (200 or 800 μM MnCl 2 or MnSO 4) for 4 or 24 h led to significant decrease in cell viability vs. controls. DEM or BSO pre-treatment led to further enhancement in cytotoxicity vs. exposure to Mn alone, with more pronounced cell death after 24-h DEM pre-treatment. F 2–IsoPs levels in cells exposed to MnCl 2 (200 or 800 μM) were significantly increased after 4 h and remained elevated 24 h after exposure compared with controls. Consistent with the effects on cell viability and F 2–IsoPs, treatment with MnCl 2 (200 or 800 μM) was also associated with a significant decrease in membrane potential. This effect was more pronounced in cells exposed to DEM plus MnCl 2 vs. cells exposed to Mn alone. We conclude that Mn induces direct injury to mitochondria in RBE4 cells. The ensuing impairment in energy metabolism and redox status may modify the restrictive properties of the BBB compromising its function.

Comparison of Two Blood-Brain Barrier In Vitro Systems: Cytotoxicity and Transfer Assessments of Malathion/Oxon and Lead Acetate

Toxicity and integrity disruption in response to transport through the blood-brain barrier (BBB) of the organophosphates malathion and malaoxon and heavy metal lead acetate were assessed in two in vitro barrier systems. One system was constructed using bovine brain microvascular endothelial cells (BMEC), while the other system was constructed with rat brain microvascular endothelial cells (RBE4); both were cocultured with rat astrocytes. We hypothesized that these models would respond differently to neurotoxic compounds. Concentrations of malathion, malaoxon, and lead acetate between 0.01 microM and 1 mM were assessed for their capacity to cause cytotoxicity to the astrocytes and endothelial cells utilized to construct the BBB systems, with the least cytotoxic concentrations chosen for transfer assessments of neurotoxicants through the barrier systems. Concentrations of malathion at 10 microM, malaoxon at 1 microM, and lead acetate at 1 and 10 microM were selected. Lead concentrations were measured in media of the abluminal and luminal sides of both systems using graphite furnace atomic absorption at the beginning of the treatment (T0) and 14 h later (T14). Passage of organophosphate compounds was determined utilizing inhibition of acetylcholinesterase enzyme in a neuroblastoma cell line (SH-SY5Y) localized below the barrier system. Transendothelial electrical resistance was assessed as a measurement of integrity of the barrier systems, with baseline values higher with the RBE4-astrocyte system than with the BMEC-astrocyte system. Metabolic capability, as measured by esterase activity, was higher in BMECs, which were more likely to retain lead than RBE4 cells. Results suggest that differences in endothelial cell source can affect the outcome of studies on toxicant transfer through in vitro BBB systems.

Flavonoid transport across RBE4 cells: A blood-brain barrier model.

There is a growing interest in dietary therapeutic strategies to combat oxidative stress-induced damage to the Central Nervous System (CNS), which is associated with a number of pathophysiological processes, including Alzheimer’s and Parkinson’s diseases and cerebrovascular diseases. Identifying the mechanisms associated with phenolic neuroprotection has been delayed by the lack of information concerning the ability of these compounds to enter the CNS. The aim of this study was to evaluate the transmembrane transport of flavonoids across RBE-4 cells (an immortalized cell line of rat cerebral capillary endothelial cells) and the effect of ethanol on this transport. The detection and quantification of all of the phenolic compounds in the studied samples (basolateral media) was performed using a HPLC-DAD (Diode Array Detector). All of the tested flavonoids (catechin, quercetin and cyanidin-3-glucoside) passed across the RBE-4 cells in a time-dependent manner. This transport was not influenced by the presence of 0.1% ethanol. In conclusion, the tested flavonoids were capable of crossing this blood-brain barrier model.

Ammonia interferes with permeability and proliferation of rat brain endothelial cells in a RBE-4 cell line

Ammonia interferes with permeability and proliferation of rat brain endothelial cells in a RBE-4 cell line

Stimulatory Effects of Thyroid Hormone on Brain Angiogenesis in vivo and in vitro

Thyroid hormone is critical for the proper development of the central nervous system. However, the specific role of thyroid hormone on brain angiogenesis remains poorly understood. Treatment of rats from birth to postnatal day 21 (P21) with propylthiouracil (PTU), a reversible blocker of triiodothyronine (T3) synthesis, resulted in decreased brain angiogenesis, as indicated by reduced complexity and density of microvessels. However, when PTU was withdrawn at P22, these parameters were fully recovered by P90. These changes were paralleled by an altered expression of vascular endothelial growth factor A (Vegfa) and basic fibroblast growth factor (Fgf2). Physiologic concentrations of T3 and thyroxine (T4) stimulated proliferation and tubulogenesis of rat brain-derived endothelial (RBE4) cells in vitro. Protein and mRNA levels of VEGF-A and FGF-2 increased after T3 stimulation of RBE4 cells. The thyroid hormone receptor blocker NH-3 abolished T3-induced Fgf2 and Vegfa upregulation, indicating a receptor-mediated effect. Thyroid hormone inhibited the apoptosis in RBE4 cells and altered mRNA levels of apoptosis-related genes, namely Bcl2 and Bad. The present results show that thyroid hormone has a substantial impact on vasculature development in the brain. Pathologically altered vascularization could, therefore, be a contributing factor to the neurologic deficits induced by thyroid hormone deficiency.

Cerebral microvascular IL15 is a novel mediator of TNF action

The blood-brain barrier is a gatekeeper and modulatory interface for the CNS. Cerebral endothelial cells are the major component of the blood-brain barrier, and they modify inflammatory signals from the circulation to the CNS by production and secretion of secondary substances. The inflammatory mediators induced by tumor necrosis factor alpha (TNF) were determined by microarray analysis of RBE4 cerebral endothelial cells, at 0, 6, 12, or 24 h after TNF treatment. Interleukin (IL)-15 and its receptors were among the most robustly up-regulated genes. This was confirmed by real-time RT-PCR and western blotting. The three subunits of the IL15 receptor complex (IL15Ralpha, IL2Rbeta, and IL2Rgamma) showed differential regulation by TNF in their time course and amplitude of increased expression. Consistent with increased expression of the specific high affinity receptor IL15Ralpha, TNF increased cellular uptake of (125)I-IL15 and enhanced the fluorescent intensity of Alexa568-IL15 in RBE4 cells. TNF treatment in mice also increased the level of expression of IL15 receptors in enriched cerebral microvessels. We conclude that the cerebral microvascular IL15 system is a novel inflammatory mediator that transduces the actions of TNF.

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.

Uptake and efflux of methylmercury in vitro: Comparison of transport mechanisms in C6, B35 and RBE4 cells

Methylmercury (MeHg) is a neurotoxicant which enters the brain and may cause permanent change. Thus, the properties of MeHg transport across cell membranes are a key factor in designing an appropriate model for MeHg neurotoxicity. This study uses cell cultures to examine the uptake and efflux mechanisms of methylmercury in C6 glioma, B35 neuroblastoma and rat brain endothelial (RBE4) cells. The cellular uptake and efflux of MeHg was investigated using 14C-labeled MeHg. The uptake of MeHg-chloride was temperature-independent while the uptake of MeHg- l-cysteine was temperature-dependent in all the three cell types. This indicates that uptake of MeHg-chloride is due to passive diffusion and uptake of MeHg- l-cysteine is due to a protein carrier. Substrates of the amino acid transport system L inhibited uptake of MeHg- l-cysteine in C6 and RBE4 cells, but not B35 cells, indicating a role for system L in MeHg-uptake in the former two. Probenecid, Na +-free medium, MeHg and several l-amino acids did not alter the efflux of MeHg from C6 and RBE4 cells. The amino acids l-cysteine and cystine however, increased the efflux. Both cysteine and cystine are important in the generation of glutathione (GSH), suggesting the involvement of GSH in MeHg efflux. HgCl 2 at low concentrations (0.5 and 1.0 μM) decreased the MeHg efflux and at high concentrations (5.0 and 10.0 μM) increased the efflux. This inhibiting effect of HgCl 2 at low concentrations is possibly due to binding to GSH while the effect of high HgCl 2 concentrations is attributed to disrupted membrane integrity, as measured by Trypan blue. This study demonstrates differing transport mechanisms of MeHg in the cell lines C6, B35 and RBE4.

The colony formation assay as an indicator of carbon nanotube toxicity examined in three cell lines

The cytotoxicity of multiwall carbon nanotubes (MWCNT) was studied in three different cell lines; rat brain endothelial cells (RBE4), a rapidly dividing epithelial like human cell line originating from a cervix carcinoma (NHIK3025) and a human lung carcinoma cell line (A549). The colony formation assay was used as end point to avoid sample interference in colorimetric assays. It was possible to differentiate between four different types of MWCNTs in concentrations between 2 and 20 µg/ml with this method. There was a great difference in sensitivity between the three cell lines. RBE4 cells were best suited for this assay within this concentration range. The results show that the clonogenic assay has the potential to be a useful tool in the cytotoxicity testing of carbon based nanomaterials even at low concentrations provided a suitable cell line is used.

Antioxidants prevent the cytotoxicity of manganese in RBE4 cells

Manganese (Mn) is an essential trace element required for ubiquitous enzymatic reactions. Chronic overexposure to this metal may, however, promote potent neurotoxic effects. The mechanism of Mn toxicity is not well established, but several studies indicate that oxidative stress and mitochondria play major roles in the Mn-induced neurodegenerative processes that lead to dysfunction in the basal ganglia. The aim of this study was to address the toxic effects of MnCl 2 and MnSO 4 on the immortalized rat brain microvessel endothelial cell line (RBE4) and to characterize toxic mechanism associated with exposure to Mn. The cytotoxicity of Mn in RBE4 cells was evaluated using the LDH and the MTT assays. A significant increase was noted in LDH release from RBE4 cells exposed for 24 h to MnCl 2 at concentrations of 800 μM and MnSO 4 at concentrations ≥ 400 μM ( p < 0.05) when compared with control unexposed cells. The MTT assay established significant decrease in cellular viability upon exposure to MnCl 2 at concentrations ≥ 100 μM and to MnSO 4 at concentrations ≥ 50 μM ( p < 0.05). Thus, the cytotoxicity assays showed that the MTT assay was more sensitive than the LDH assay, suggesting that mitochondrial changes precede other toxic effects of Mn. In addition, upon exposure to MnCl 2 (200 and 800 μM), intracellular reduced glutathione (GSH) levels in RBE4 cells decreased as Mn exposure concentrations increased ( p < 0.05). To confirm the oxidative hypothesis of Mn cytotoxicity, co-exposure of MnCl 2 with antioxidant agents ( N-acetylcysteine [NAC] or Trolox) were carried out. The cellular viability was evaluated using the MTT assay. A significant decrease in Mn cytotoxicity was observed in co-exposed cells confirming that (1) oxidative stress plays a critical role in the mechanism of Mn toxicity, and (2) antioxidants may offer a useful therapeutic modality to reverse the aberrant effects of Mn.

Transport of valproate at intestinal epithelial (Caco-2) and brain endothelial (RBE4) cells: Mechanism and substrate specificity

To reach its target cells, the antiepileptic drug valproate has to cross both the intestinal epithelial barrier and the blood–brain barrier in intact form as well as in sufficient amounts. This study was performed to characterize the epithelial transport of valproate at intestinal (Caco-2) and at blood–brain barrier (RBE4) cells. At both cell types, uptake of [ 3H]valproate was independent of inwardly directed Na +, Ca 2+, Mg 2+, K + or Cl − gradients. Uptake was, however, strongly stimulated by an inwardly directed H + gradient. The cells accumulated valproate against a concentration gradient and the uptake rate of valproate was saturable with K t values of 0.6 and 0.8 mM. At Caco-2 cell monolayers, the total apical-to-basolateral flux of [ 3H]valproate exceeded the basolateral-to-apical flux 14-fold. Various monocarboxylic acids like salicylate, benzoate, acetate, propionate, butyrate, hexanoate, diclofenac and ibuprofen inhibited [ 3H]valproate uptake at both cell types. Lactate and pyruvate inhibited valproate uptake at RBE4 cells but not at Caco-2 cells. We conclude that valproate is accumulated in intestinal cells against a concentration gradient by the activity of a specific H +-dependent DIDS-insensitive transport system for monocarboxylates not identical with monocarboxylate transporter 1 (MCT1). The passage of valproate across the blood–brain barrier is very likely mediated by MCT1.

Regulation of brain endothelial cells migration and angiogenesis by P-glycoprotein/caveolin-1 interaction

We have investigated the involvement of P-glycoprotein (P-gp)/caveolin-1 interaction in the regulation of brain endothelial cells (EC) migration and tubulogenesis. P-gp overexpression in MDCK-MDR cells was correlated with enhanced cell migration whereas treatment with P-gp inhibitors CsA or PSC833 reduced it. Transfection of RBE4 rat brain endothelial cells with mutated versions of MDR1, in the caveolin-1 interaction motif, decreased the interaction between P-gp and caveolin-1, enhanced P-gp transport activity and cell migration. Moreover, down-regulation of caveolin-1 in RBE4 cells by siRNA against caveolin-1 stimulated cell migration. Interestingly, the inhibition of P-gp/caveolin-1 interaction increased also EC tubulogenesis. Furthermore, decrease of P-gp expression by siRNA inhibited EC tubulogenesis. These data indicate that the level of P-gp/caveolin-1 interaction can modulate brain endothelial angiogenesis and P-gp dependent cell migration.