Brain Capillary Endothelial Cell Line Research Articles

Naltrexone Transport by a Proton-Coupled Organic Cation Antiporter in hCMEC/D3 Cells, an in Vitro Human Blood-Brain Barrier Model.

Naltrexone is a mu-opioid receptor antagonist used in the treatment of opioid and alcohol dependence. The blood-brain barrier (BBB) transport characteristics of naltrexone was investigated by means of hCMEC/D3 cells, a human immortalized brain capillary endothelial cell line. In hCMEC/D3 cells, naltrexone is taken up in a concentration-dependent manner. Furthermore, naltrexone uptake significantly decreased in the presence of H+/organic cation (OC) antiporter substrates, during the little alteration exhibited by substrates of well-identified OC transporters classified into SLC22A family. Although naltrexone uptake by hCMEC/D3 cells was partially affected by changes of ionic conditions, it was markedly decreased in the presence of the metabolic inhibitor sodium azide. Furthermore, when treated by ammonium chloride, naltrexone uptake by hCMEC/D3 cells was altered by intracellular acidification and alkalization, suggesting the involvement of oppositely directed proton gradient in naltrexone transport across the BBB. The results obtained in the present in vitro study suggest the active transport of naltrexone from blood to the brain across the BBB by the H+/OC antiporter.

Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC2A3) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine.

The solute carrier L-type amino acid transporter 1 (LAT-1/SLC7A5) is a viable target for drug delivery to the central nervous system (CNS) and tumors due to its high abundance at the blood–brain barrier and in tumor tissue. LAT-1 is only localized on the cell surface as a heterodimer with CD98, which is not required for transporter function. To support future CNS drug-delivery development based on LAT-1 targeting, we established an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) assay for stable isotopically labeled leucine ([13C6, 15N]-L-leucine), with a dynamic range of 0.1–1000 ng/mL that can be applied for the functional testing of LAT-1 activity when combined with specific inhibitors and, consequently, the LAT-1 inhibition capacity of new compounds. The assay was established in a 96-well format, facilitating high-throughput experiments, and, hence, can support the screening for novel inhibitors. Applicable recommendations of the US Food and Drug Administration and European Medicines Agency for bioanalytical method validation were followed to validate the assay. The assay was applied to investigate the IC50 of two well-known LAT-1 inhibitors on hCMEC/D3 cells: the highly specific LAT-1 inhibitor JPH203, which was also used to demonstrate LAT-1 specific uptake, and the general system L inhibitor BCH. In addition, the [13C6, 15N]-L-leucine uptake was determined on two human brain capillary endothelial cell lines (NKIM-6 and hCMEC/D3), which were characterized for their expressional differences of LAT-1 at the protein and mRNA level and the surface amount of CD98. The IC50 values of the inhibitors were in concordance with previously reported values. Furthermore, the [13C6, 15N]-L-leucine uptake was significantly higher in hCMEC/D3 cells compared to NKIM-6 cells, which correlated with higher expression of LAT-1 and a higher surface amount of CD98. Therefore, the UPLC-MS/MS quantification of ([13C6, 15N]-L-leucine is a feasible strategy for the functional characterization of LAT-1 activity in cells or tissue.

Neuroprotective effects of an in vitro BBB permeable phenoxythiophene sulfonamide small molecule in glutamate-induced oxidative injury.

Reactive oxygen species (ROS) play a central role in oxidative stress-associated neuronal cell death during ischemia. Further investigation into the inhibition of excessive ROS generation post-stroke is urgently required for the treatment of ischemic stroke. In the present study, the neuroprotective properties of the blood-brain barrier (BBB) penetrant B355227 were investigated. B355227 is a chemical analogue of B355252, and the role of the phenoxythiophene sulfonamide compound B355227 was further investigated in a glutamate-induced oxidative injury model. An in vitro model of the BBB was established in the immortalized mouse brain capillary endothelial cell line, bEnd.3. Formation of barrier in Transwell inserts was confirmed using EVOM resistance meter and Caffeine, Imatinib and Axitinib were used to validate the efficacy of the model. The validated BBB assay in combination with high performance liquid chromatography were used to analyse and verify the permeability of B355227 through the barrier. The integrity of the cell junctions after the BBB assays were confirmed using immunofluorescence to visualize the expression of the barrier junction protein zonula occludens-1. Cell survival was measured with Resazurin, a redox indicator dye, in HT22, a hippocampal neuronal cell treated with 5 mM glutamate or co-treated with the B355227 recovered from the BBB permeability experiment. Changes in glutathione levels were detected using a glutathione detection kit, while analyses of ROS, calcium (Ca2+), and mitochondrial membrane potential (MMP) were accomplished with the fluorescent dyes 2',7'-dichlorofluorescein diacetate, Fura-2 AM and MitoTracker Red dyes, respectively. Immunoblotting was also performed to detect the expression and activation of Erk1/2, p-38, JNK, Bax and Bcl-2. The results of the present study demonstrated that B355227 crossed the BBB in vitro and protected HT22 from oxidative injury induced by glutamate exposure. Treatment of cells with B355227 blocked the glutamate-dependent depletion of intracellular glutathione and significantly reduced ROS production. Increased Ca2+ influx and subsequent collapse of the MMP was attenuated by B355227. Furthermore, the results of the present study demonstrated that B355227 protected against oxidative stress via the MAPK pathway, by increasing the activation of Erk1/2, JNK and P38, and restoring anti-apoptotic Bcl-2. Collectively, the results of the present study indicate that B355227 has potent antioxidant and neuroprotective attributes in glutamate-induced neuronal cell death. Further investigation into the role of B355227 in the modulation of glutamate-dependent oxidative stress is required.

Similarities and differences in the localization, trafficking, and function of P-glycoprotein in MDR1-EGFP-transduced rat versus human brain capillary endothelial cell lines

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.

Pretreatment Effect of Inflammatory Stimuli and Characteristics of Tryptophan Transport on Brain Capillary Endothelial (TR-BBB) and Motor Neuron Like (NSC-34) Cell Lines.

Tryptophan plays a key role in several neurological and psychiatric disorders. In this study, we investigated the transport mechanisms of tryptophan in brain capillary endothelial (TR-BBB) cell lines and motor neuron-like (NSC-34) cell lines. The uptake of [3H]l-tryptophan was stereospecific, and concentration- and sodium-dependent in TR-BBB cell lines. Transporter inhibitors and several neuroprotective drugs inhibited [3H]l-tryptophan uptake by TR-BBB cell lines. Gabapentin and baclofen exerted a competitive inhibitory effect on [3H]l-tryptophan uptake. Additionally, l-tryptophan uptake was time- and concentration-dependent in both NSC-34 wild type (WT) and mutant type (MT) cell lines, with a lower transporter affinity and higher capacity in MT than in WT cell lines. Gene knockdown of LAT1 (l-type amino acid transporter 1) and CAT1 (cationic amino acid transporter 1) demonstrated that LAT1 is primarily involved in the transport of [3H]l-tryptophan in both TR-BBB and NSC-34 cell lines. In addition, tryptophan uptake was increased by TR-BBB cell lines but decreased by NSC-34 cell lines after pro-inflammatory cytokine pre-treatment. However, treatment with neuroprotective drugs ameliorated tryptophan uptake by NSC-34 cell lines after inflammatory cytokines pretreatment. The tryptophan transport system may provide a therapeutic target for treating or preventing neurodegenerative diseases.

A face-to-face comparison of claudin-5 transduced human brain endothelial (hCMEC/D3) cells with porcine brain endothelial cells as blood\u2013brain barrier models for drug transport studies

BackgroundPredictive in vitro models of the human blood–brain barrier (BBB) are essential in early drug discovery and development. Among available immortalized human brain capillary endothelial cell lines (BCECs), the hCMEC/D3 cell line has become the most widely used in vitro BBB model. However, monolayers of hCMEC/D3 cells form only moderately restrictive barriers, most likely because the major tight junction protein, claudin-5, is markedly downregulated. Thus, hCMEC/D3 monolayers cannot be used for vectorial drug transport experiments, which is a major disadvantage of this model.MethodsHere we transduced hCMEC/D3 cells with a claudin-5 plasmid and compared the characteristics of these cells with those of hCMEC/D3 wildtype cells and primary cultured porcine BCECs.ResultsThe claudin-5 transduced hCMEC/D3 exhibited expression levels (and junctional localization) of claudin-5 similar to those of primary cultured porcine BCECs. The transduced cells exhibited increased TEER values (211 Ω cm2) and reduced paracellular mannitol permeability (8.06%/h), indicating improved BBB properties; however, the barrier properties of porcine BCECs (TEER 1650 Ω cm2; mannitol permeability 3.95%/h) were not reached. Hence, vectorial transport of a selective P-glycoprotein substrate (N-desmethyl-loperamide) was not observed in claudin-5 transduced hCMEC/D3 (or wildtype) cells, whereas such drug transport occurred in porcine BCECs.ConclusionsThe claudin-5 transduced hCMEC/D3 cells provide a tool to studying the contribution of claudin-5 to barrier tightness and how this can be further enhanced by additional transfections or other manipulations of this widely used in vitro model of the BBB.

UV irradiation of Type I collagen gels changed the morphology of the interconnected brain capillary endothelial cells on them.

We cultured mouse brain capillary endothelial cell line bEnd.3 on the UV-irradiated Type I collagen gel. Morphology of bEnd.3 cells on the Type I collagen gel was drastically changed if the gel was crosslinked by UV irradiation. The interconnecting network of bEnd.3 cells which have cord-like morphology on the soft collagen gels was converted to the monolayer of the flat cells, tightly-bound each other covering the gel surface, in a confluent state. The collagen gels were mechanically stiffened by UV irradiation for 15min with UV light at 254nm showing approximately two times higher value of Young's modulus E (1.51±0.58kPa) than the control gel (3.17±1.17kPa). AFM images of the collagen fibrils were not severely changed after irradiation. Collagen subunit proteins were crosslinked and degraded simultaneously under UV irradiation proved by results of SDS-PAGE and separation by centrifugation. Expression of Integrin gene was measured by quantitative real-time PCR. Expression of the integrin α2 gene, tight junction protein 1 gene, and claudin 5 gene were down-regulated in cells on the UV irradiated collagen gel in comparison with the unirradiated one while expression of the integrin β1 gene and Integrin α1 gene did not significantly change. Thick actin filaments were more clearly observed in the cells on the UV-irradiated collagen gel than the unirradiated one by fluorescent microscopy. We conclude that UV irradiation made the collagen gel stiffened and changed the physiological state of bEnd.3 cells including their adhesion, extension, and proliferation.

Polymer-coated nanoparticles and their effects on mitochondrial function in brain endothelial cells

Laser tissue soldering is a novel treatment method for injuries of hollow organs such as cerebrovascular aneurysms. Nanomaterials contained in the solder are foreign to the body. Hence, it is indispensable to carefully examine possible adverse effects prior to introducing this technique. The aim of this study was to characterize the impact of different concentrations of polymer-coated silica nanoparticles (NPs) on mitochondrial function and integrity of brain endothelial cells using the rat brain capillary endothelial cell line rBCEC4. At maximal capacity, NP exposure resulted in a decrease in the oxygen consumption rate whereas glycolysis was not affected. In combination with a stressor, i.e. lack of glucose in the medium, NP exposure interfered primarily with glycolytic ATP generation rather than oxidative phosphorylation. Furthermore, NPs caused a metabolic shift towards a stressed phenotype, exhibiting increased levels of the oxygen consumption rate and the extracellular acidification rate compared to untreated controls. Overall, mitochondrial mass, distribution and morphology as well as intracellular ATP content were not altered. The mitochondrial membrane potential was increased after exposure to the highest NP concentration and the content of proteins involved in mitochondrial dynamics was changed slightly, indicating possible modifications of the fusion / fission balance.In conclusion, PCL-NP exposure changed mitochondrial respiration, especially under glucose deprivation, but did not affect mitochondrial morphology and distribution. Further studies are needed to investigate whether the functional effects are transient or long-term as this will be crucial for the use of these NPs in laser tissue soldering.

Involvement of a Novel Organic Cation Transporter in Paeonol Transport Across the Blood-Brain Barrier.

Paeonol has neuroprotective function, which could be useful for improving central nervous system disorder. The purpose of this study was to characterize the functional mechanism involved in brain transport of paeonol through blood-brain barrier (BBB). Brain transport of paeonol was characterized by internal carotid artery perfusion (ICAP), carotid artery single injection technique (brain uptake index, BUI) and intravenous (IV) injection technique in vivo. The transport mechanism of paeonol was examined using conditionally immortalized rat brain capillary endothelial cell line (TR-BBB) as an in vitro model of BBB. Brain volume of distribution (VD) of [3H]paeonol in rat brain was about 6-fold higher than that of [14C]sucrose, the vascular space marker of BBB. The uptake of [3H]paeonol was concentration-dependent. Brain volume of distribution of paeonol and BUI as in vivo and inhibition of analog as in vitro studies presented significant reduction effect in the presence of unlabeled lipophilic compounds such as paeonol, imperatorin, diphenhydramine, pyrilamine, tramadol and ALC during the uptake of [3H]paeonol. In addition, the uptake significantly decreased and increased at the acidic and alkaline pH in both extracellular and intracellular study, respectively. In the presence of metabolic inhibitor, the uptake reduced significantly but not affected by sodium free or membrane potential disruption. Similarly, paeonol uptake was not affected on OCTN2 or rPMAT siRNA transfection BBB cells. Interestingly. Paeonol is actively transported from the blood to brain across the BBB by a carrier mediated transporter system.

Phosphatase Actin Regulator-1 (PHACTR-1) Knockdown Suppresses Cell Proliferation and Migration and Promotes Cell Apoptosis in the bEnd.3 Mouse Brain Capillary Endothelial Cell Line.

BackgroundThe phosphatase actin regulator-1 (PHACTR-1) gene on chromosome 6 encodes an actin and protein phosphatase 1 (PP1) binding protein, Phactr-1, which is highly expressed in brain tissues. Phactr-1 expression is involved in physiological and pathological cerebral microvascular events. This study aimed to investigate the role of expression of Phactr-1 in a mouse brain capillary endothelial cell line, bEnd.3, by knockdown the PHACTR-1 gene.Material/MethodsThree bEnd.3 cell groups were studied, CON (normal control cells), NC (control scramble transfected cells), and KD (cells with PHACTR-1 gene knockdown). The PHACTR-1 gene was knocked down using transfection with small hairpin RNA (shRNA). In the three cell groups cell proliferation, migration, and apoptosis were studied by MTT and colony formation assays, transwell and scratch assays, and flow cytometry. The related cell pathways of associated with Phactr-1 knockdown were studied by Western blot.ResultsPhactr-1 knockdown suppressed bEnd.3 cell proliferation and migration, promoted cell apoptosis, and downregulated the expressions of migration-associated proteins, including matrix metalloproteinase (MMP)-2 and MMP-9 and upregulated apoptosis-associated proteins, including Bax, Bcl-2, cleaved caspase-3, and caspase-3.ConclusionsPhactr-1 was shown to have a role in the inhibition of endothelial cell proliferation and migration, promoted cell apoptosis, and regulated matrix metalloproteinases and apoptosis-associated proteins. These findings indicate that the expression of the Phactr-1 should be studied further in the cerebral microvasculature, both in vitro and in vivo, regarding its potential as a diagnostic and therapeutic target for cerebral microvascular disease.

The influence of receptor activity-modifying protein-1 overexpression on angiogenesis in mouse brain capillary endothelial cells.

Receptor activity-modifying protein-1 (RAMP1) is highly expressed in the heart and vasculature, indicating that it might be related to the vascular system. However, the effects of RAMP1 on angiogenesis and the intrinsic mechanisms underlying this process remain unclear. Here, we verified that RAMP1 is a critical regulator of angiogenesis in a mouse brain capillary endothelial cell line (bEnd.3). We first constructed a RAMP1 overexpression lentiviral vector system and stably transfected bEnd.3 cells. We further showed that RAMP1 overexpression could lead to bEnd.3 migration and capillary tube formation in Matrigel without exogenous calcitonin gene-related peptide (CGRP) treatment. At the same time, RAMP1 overexpression had little effect on proliferation. More importantly, vascular endothelial growth factor (VEGF) and CGRP expression levels were not significantly higher in RAMP1-overexpressing cells than in control cells (P > 0.05), indicating that RAMP1 did not function through upregulating VEGF or CGRP expression in bEnd.3 cells. Strikingly, RAMP1 transfection increased adrenomedullin 2 (AM2) expression levels ( P < 0.05). Taken together, these data contribute to a better understanding of the molecular mechanisms of RAMP1 in angiogenesis.

Interferon-stimulated gene 60 (ISG60) constitutes a negative feedback loop in the downstream of TLR3 signaling in hCMEC/D3 cells

Brain capillary endothelial cells are the component of blood brain barrier, and the first line of defense against viruses invading into brain. We demonstrate that treatment of hCMEC/D3 cells, a human brain capillary endothelial cell line, with a Toll-like receptor 3 (TLR3) agonist polyinosinic-polycytidylic acid (poly IC) induces the expression of interferon (IFN)-stimulated gene 60 (ISG60), and this reaction was mediated by IFN-β. Knockdown of ISG60 increased the poly IC-induced expression of IFN-β and an IFN-β-inducible chemokine CXCL10. This indicates that ISG60 constitutes a negative feedback loop in the downstream of TLR3/IFN-β. ISG60 in brain capillary endothelial cells may contribute to prevent excess immune reactions associated with viral infections.

L-Citrulline restores nitric oxide level and cellular uptake at the brain capillary endothelial cell line (TR-BBB cells) with glutamate cytotoxicity

ObjectiveGlutamate excitotoxicity provokes neuronal cell damage and death, leading to collapse of the blood-brain barrier (BBB). Recently, it has been reported that l-citrulline, a neutral amino acid and a major precursor of l-arginine in the nitric oxide (NO) cycle, can prevent both neuronal cell death and cerebrovascular cell loss in brain ischemia. Therefore, the objective of this study was to investigate the effect of l-citrulline on glutamate cytotoxicity in the BBB using the conditionally immortalized rat brain capillary endothelial cell line (TR-BBB cells) as an in vitro model of the BBB. MethodsCell viability was determined using MTT assay. Cellular uptake of [14C] l-citrulline and expression levels of rat large neutral amino acid transporter 1 (rLAT1), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) at mRNA level were performed using quantitative real-time polymerase chain reaction (PCR) analysis. NO production from TR-BBB cells was measured using Griess reagents. All experiments were performed after treatment of TR-BBB cells with glutamate alone or co-treatment with l-citrulline, l-arginine, and/or taurine for 24 h. Resultsl-Citrulline treatment increased cell viability, [14C] l-citrulline uptake, and the mRNA levels of LAT1 and eNOS in TR-BBB cells treated with glutamate. However, iNOS mRNA expression was inhibited by l-citrulline. NO production and transcript level of iNOS were markedly increased by glutamate treatment alone. However, co-treatment with l-citrulline, taurine, or both l-citrulline and taurine decreased NO levels and mRNA levels of iNOS in TR-BBB cells treated with glutamate. In co-treatment of TR-BBB cells with l-arginine, a NO donor, and glutamate, NO levels were increased and expression levels of iNOS mRNA were similar compared to those in cells treated with glutamate alone. Conclusionl-Citrulline can restore NO level and its cellular uptake in TR-BBB cells with glutamate cytotoxicity. Supplying l-citrulline at the BBB may provide neuroprotective effect to improve cerebrovascular dysfunction such as a brain ischemia.

Quantitative Targeted Absolute Proteomics (QTAP)-based Pharmacoproteomics: The Importance of International Collaboration.

Proteins such as membrane transporters, enzymes, receptors and channels play key roles in drug absorption, distribution, metabolism, and elimination, and also influence efficacy and the likelihood of adverse reactions. Therefore, if we can quantify the activities of these molecules, it may be possible to predict the behavior of candidate drugs in humans in disease states; such methodology would be extremely helpful for efficient drug development. We have developed an in silico method to select appropriate peptides within amino acid sequences in order to quantify targeted proteins by LC-MS/MS in selected reaction monitoring (SRM) mode. We have applied this method for the quantification of functional proteins in order to validate various in vitro and in vivo models. We found fairly good correlation between protein amounts and the enzymatic activities of microsomal cytochrome P450 (CYP) isoforms and uridine 5'-diphospho-glucuronosyltransferase (UGT) in human liver, as well as between protein amounts and the transport activities of multiple transporters in human lung cells. These results suggest that protein quantification can be useful in predicting activity. We have applied this approach to evaluate the usefulness and limitations of an immortalized human brain capillary endothelial cell line (D3 cells) and a P-glycoprotein humanized (hMDR1) mouse model by comparing the amounts of functional proteins in the models with those in isolated capillaries from human brain. In order to obtain sufficient human tissue specimens for further studies leading to clinical applications, we believe that international collaboration will be crucial.

Characteristics of L-citrulline transport through blood-brain barrier in the brain capillary endothelial cell line (TR-BBB cells)

BackgroundL-Citrulline is a neutral amino acid and a major precursor of L-arginine in the nitric oxide (NO) cycle. Recently it has been reported that L-citrulline prevents neuronal cell death and protects cerebrovascular injury, therefore, L-citrulline may have a neuroprotective effect to improve cerebrovascular dysfunction. Therefore, we aimed to clarify the brain transport mechanism of L-citrulline through blood-brain barrier (BBB) using the conditionally immortalized rat brain capillary endothelial cell line (TR-BBB cells), as an in vitro model of the BBB.MethodsThe uptake study of [14C] L-citrulline, quantitative real-time polymerase chain reaction (PCR) analysis, and rLAT1, system b0,+, and CAT1 small interfering RNA study were performed in TR-BBB cells.ResultsThe uptake of [14C] L-citrulline was a time-dependent, but ion-independent manner in TR-BBB cells. The transport process involved two saturable components with a Michaelis–Menten constant of 30.9 ± 1.0 μM (Km1) and 1.69 ± 0.43 mM (Km2). The uptake of [14C] L-citrulline in TR-BBB cells was significantly inhibited by neutral and cationic amino acids, but not by anionic amino acids. In addition, [14C]L-citrulline uptake in the cells was markedly inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), which is the inhibitor of the large neutral amino acid transporter 1 (LAT1), B0, B0,+ and harmaline, the inhibitor of system b0,+. Gabapentin and L-dopa as the substrates of LAT1 competitively inhibited the uptake of [14C] L-citrulline. IC50 values for L-dopa, gabapentin, L-phenylalanine and L-arginine were 501 μM, 223 μM, 68.9 μM and 33.4 mM, respectively. The expression of mRNA for LAT1 was predominantly increased 187-fold in comparison with that of system b0,+ in TR-BBB cells. In the studies of LAT1, system b0,+ and CAT1 knockdown via siRNA transfection into TR-BBB cells, the transcript level of LAT1 and [14C] L-citrulline uptake by LAT1 siRNA were significantly reduced compared with those by control siRNA in TR-BBB cells.ConclusionsOur results suggest that transport of L-citrulline is mainly mediated by LAT1 in TR-BBB cells. Delivery strategy for LAT1-mediated transport and supply of L-citrulline to the brain may serve as therapeutic approaches to improve its neuroprotective effect in patients with cerebrovascular disease.

Poly-(amidoamine) dendrimers with a precisely core positioned sulforhodamine B molecule for comparative biological tracing and profiling

We report on a simple robust procedure for synthesis of generation-4 poly-(amidoamine) (PAMAM) dendrimers with a precisely core positioned single sulforhodamine B molecule. The labelled dendrimers exhibited high fluorescent quantum yields where the absorbance and fluorescence spectrum of the fluorophore was not affected by pH and temperature. Since the stoichiometry of the fluorophore to the dendrimer is 1:1, we were able to directly compare uptake kinetics, the mode of uptake, trafficking and safety of dendrimers of different end-terminal functionality (carboxylated vs. pyrrolidonated) by two phenotypically different human endothelial cell types (the human brain capillary endothelial cell line hCMEC/D3 and human umbilical vein endothelial cells), and without interference of the fluorophore in uptake processes. The results demonstrate comparable uptake kinetics and a predominantly clathrin-mediated endocytotic mechanism, irrespective of dendrimer end-terminal functionality, where the majority of dendrimers are directed to the endo-lysosomal compartments in both cell types. A minor fraction of dendrimers, however, localize to endoplasmic reticulum and the Golgi apparatus, presumably through the recycling endosomes. In contrast to amino-terminated PAMAM dendrimers, we confirm safety of carboxylic acid- and pyrrolidone-terminated PAMAM dendrimers through determination of cell membrane integrity and comprehensive respiratory profiling (measurements of mitochondrial oxidative phosphorylation and determination of its coupling efficiency). Our dendrimer core-labelling approach could provide a new conceptual basis for improved understanding of dendrimer performance within biological settings.

Neofiscalin A and fiscalin C are potential novel indole alkaloid alternatives for the treatment of multidrug-resistant Gram-positive bacterial infections.

Ten indole alkaloids were obtained from the marine sponge-associated fungus Neosartorya siamensis KUFA 0017. We studied the antimicrobial properties of these and of three other compounds previously isolated from the soil fungus N. siamensis KUFC 6349. Only neofiscalin A showed antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE); with a minimum inhibitory concentration (MIC) of 8 μg mL(-1) against both strains. Another compound, fiscalin C, presented synergistic activity against MRSA when combined with oxacillin, although alone showed no antibacterial effect. Moreover, neofiscalin A, when present at sub-MICs, hampered the ability of both MRSA and VRE strains to form a biofilm. Additionally, the biofilm inhibitory concentration values of neofiscalin A against the MRSA and VRE isolates were 96 and 80 μg mL(-1), respectively. At a concentration of 200 μg mL(-1), neofiscalin A was able to reduce the metabolic activity of the biofilms by ∼50%. One important fact is that our results also showed that neofiscalin A had no cytotoxicity against a human brain capillary endothelial cell line.

Cytotoxicity Induced by Extracts of Pisolithus tinctorius Spores on Human Cancer and Normal Cell Lines—Evaluation of the Anticancer Potential

Fungi have been considered a potential source of natural anticancer drugs. However, studies on these organisms have mainly focused on compounds present in the sporocarp and mycelium. The aim of this study was to assess the anticancer potential of fungal spores using a bioassay-guided fractionation with cancer and normal cell lines. Crude extracts from spores of the basidiomycetous fungus Pisolithus tinctorius were prepared using five solvents/solvent mixtures in order to select the most effective crude extraction procedure. A dichloromethane/methanol (DCM/MeOH) mixture was found to produce the highest extraction yield, and this extract was fractionated into 11 fractions. Crude extracts and fractions were assayed for cytotoxicity in the human osteocarcinoma cell line MG63, the human breast carcinoma cell line T47D, the human colon adenocarcinoma cell line RKO, and the normal human brain capillary endothelial cell line hCMEC/D3. Cytotoxicity was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay. The results showed a reduction in cancer cell viability of approximately 95% with 4 of 11 fractions without a significant reduction in viability of hCMEC/D3 cells. Data demonstrated that spores of P. tinctorius might serve as an interesting source of compounds with potential anticancer properties.

Regulation of store-operated Ca2+ entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells

Store-operated Ca2+ entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca2+ influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells.

Marked Differences in the Effect of Antiepileptic and Cytostatic Drugs on the Functionality of P-Glycoprotein in Human and Rat Brain Capillary Endothelial Cell Lines

The expression of P-glycoprotein (Pgp) is increased in brain capillary endothelial cells (BCECs) of patients with pharmacoresistant epilepsy. This may restrict the penetration of antiepileptic drugs (AEDs) into the brain. However, the mechanisms underlying increased Pgp expression in epilepsy patients are not known. One possibility is that AEDs induce the expression and functionality of Pgp in BCECs. Several older AEDs that induce human cytochrome P450 enzymes also induce Pgp in hepatocytes and enterocytes, but whether this extends to Pgp at the human BBB and to newer AEDs is not known. This prompted us to study the effects of various old and new AEDs on Pgp functionality in the human BCEC line, hCMEC/D3, using the rhodamine 123 (Rho123) efflux assay. For comparison, experiments were performed in two rat BCEC lines, RBE4 and GPNT, and primary cultures of rat and pig BCECs. Furthermore, known Pgp inducers, such as dexamethasone and several cytostatic drugs, were included in our experiments. Under control conditions, GPNT cells exhibited the highest and RBE4 the lowest Pgp expression and Rho123 efflux, while intermediate values were determined in hCMEC/D3. Known Pgp inducers increased Rho123 efflux in all cell lines, but marked inter-cell line differences in effect size were observed. Of the various AEDs examined, only carbamazepine (100 μM) moderately increased Pgp functionality in hCMEC/D3, while valproate (300 μM) inhibited Pgp. These data do not indicate that treatment with AEDs causes a clinically relevant induction in Pgp functionality in BCECs that form the BBB.