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Arie Reijerkerk,
M Alejandro Lopez-Ramirez,
Bert van Het Hof,
Joost A R Drexhage,
Wouter W Kamphuis,
Gijs Kooij,
Joost B Vos,
Tineke C T M van der Pouw Kraan,
Anton J van Zonneveld,
Anton J Horrevoets,
Alexandre Prat, Ignacio A Romero,
Helga E de Vries
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ABSTRACT: Blood-brain barrier (BBB) dysfunction is a major hallmark of many neurological diseases, including multiple sclerosis (MS). Using a genomics approach, we defined a microRNA signature that is diminished at the BBB of MS patients. In particular, miR-125a-5p is a key regulator of brain endothelial tightness and immune cell efflux. Our findings suggest that repair of a disturbed BBB through microRNAs may represent a novel avenue for effective treatment of MS.
Journal of Neuroscience 04/2013; 33(16):6857-6863. · 7.11 Impact Factor
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ABSTRACT: Since the first attempts in the 1970s to isolate cerebral microvessel endothelial cells (CECs) in order to model the blood--brain barrier (BBB) in vitro, the need for a human BBB model that closely mimics the in vivo phenotype and is reproducible and easy to grow, has been widely recognized by cerebrovascular researchers in both academia and industry. While primary human CECs would ideally be the model of choice, the paucity of available fresh human cerebral tissue makes wide-scale studies impractical. The brain microvascular endothelial cell line hCMEC/D3 represents one such model of the human BBB that can be easily grown and is amenable to cellular and molecular studies on pathological and drug transport mechanisms with relevance to the central nervous system (CNS). Indeed, since the development of this cell line in 2005 over 100 studies on different aspects of cerebral endothelial biology and pharmacology have been published. Here we review the suitability of this cell line as a human BBB model for pathogenic and drug transport studies and we critically consider its advantages and limitations.
Fluids and barriers of the CNS. 03/2013; 10(1):16.
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ABSTRACT: During neuroinflammation, cytokines such as TNF-α and IFN-γ secreted by activated leukocytes and/or CNS resident cells have been shown to alter the phenotype and function of brain endothelial cells (BECs) leading to blood-brain barrier breakdown. In this study, we show that the human BEC line hCMEC/D3 expresses the receptors for TNF-α, TNF receptor 1 and TNF receptor 2, and for IFN-γ. BEC activation with TNF-α alone or in combination with IFN-γ induced endothelial leakage of paracellular tracers. At high cytokine concentrations (10 and 100 ng/ml), this effect was associated with caspase-3/7 activation and apoptotic cell death as evidenced by annexin V staining and DNA fragmentation (TUNEL) assays. In addition, inhibition of JNK and protein kinase C activation at these doses partially prevented activation of caspase-3/7, although only JNK inhibition was partially able to prevent the increase in BEC paracellular permeability induced by cytokines. By contrast, lower cytokine concentrations (1 ng/ml) also led to effector caspase activation, increased paracellular flux, and redistribution of zonula occludens-1 and VE-cadherin but failed to induce apoptosis. Under these conditions, specific caspase-3 and caspase-9, but not caspase-8, inhibitors partially blocked cytokine-induced disruption of tight and adherens junctions and BEC paracellular permeability. Our results suggest that the concentration of cytokines in the CNS endothelial microenvironment determines the extent of caspase-mediated barrier permeability changes, which may be generalized as a result of apoptosis or more subtle as a result of alterations in the organization of junctional complex molecules.
The Journal of Immunology 08/2012; 189(6):3130-9. · 5.79 Impact Factor
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ABSTRACT: The use of gold nanoparticles (AuNPs) for diagnostic applications and for drug and gene-delivery is currently under intensive investigation. For such applications, biocompatibility and the absence of cytotoxicity of AuNPs is essential. Although generally considered as highly biocompatible, previous in vitro studies have shown that cytotoxicity of AuNPs in certain human epithelial cells was observed. In particular, the degree of purification of AuNPs (presence of sodium citrate residues on the particles) was shown to affect the proliferation and induce cytotoxicity in these cells. To expand these studies, we have examined if the effects are related to nanoparticle size (10, 11 nm, 25 nm), to the presence of sodium citrate on the particles' surface or they are due to a varying degree of internalization of the AuNPs. Since two cell types are present in the major barriers to the outside in the human body, we have also included endothelial cells from the vasculature and blood brain barrier.
Transmission electron microscopy demonstrates that the internalized gold nanoparticles are located within vesicles. Increased cytotoxicity was observed after exposure to AuNPs and was found to be concentration-dependent. In addition, cell viability and the proliferation of both endothelial cells decreased after exposure to gold nanoparticles, especially at high concentrations. Moreover, in contrast to the size of the particles (10 nm, 11 nm, 25 nm), the presence of sodium citrate on the nanoparticle surface appeared to enhance these effects. The effects on microvascular endothelial cells from blood vessels were slightly enhanced compared to the effects on brain-derived endothelial cells. A quantification of AuNPs within cells by ICP-AES showed that epithelial cells internalized a higher quantity of AuNPs compared to endothelial cells and that the quantity of uptake is not correlated with the amount of sodium citrate on the nanoparticles' surface.
In conclusion the higher amount of citrate on the particle surface resulted in a higher impairment of cell viability, but did not enhance or reduce the uptake behavior in endothelial or epithelial cells. In addition, epithelial and endothelial cells exhibited different uptake behaviors for citrate-stabilized gold nanoparticles, which might be related to different interactions occurring at the nanoparticle-cell-surface interface. The different uptake in epithelial cells might explain the higher reduction of proliferation of these cells after exposure to AuNPs treatment although more detailed investigations are necessary to determine subcellular events. Nevertheless an extrinsic effect of sodium-citrate stabilized particles could not be excluded. Thus, the amount of sodium citrate should be reduced to a level on which the stability of the particles and the safety for biomedical applications are guaranteed.
Particle and Fibre Toxicology 07/2012; 9:23. · 7.25 Impact Factor
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ABSTRACT: Alterations to blood-brain barrier (BBB) adhesion molecules and junctional integrity during neuroinflammation can promote central nervous system (CNS) pathology. The chemokine CCL2 is elevated during CNS inflammation and is associated with endothelial dysfunction. The effects of CCL2 on endothelial adherens junctions (AJs) have not been defined. We demonstrate that CCL2 transiently induces Src-dependent disruption of human brain microvascular endothelial AJ. β-Catenin is phosphorylated and traffics from the AJ to PECAM-1 (platelet endothelial cell adhesion molecule-1), where it is sequestered at the membrane. PECAM-1 is also tyrosine-phosphorylated, an event associated with recruitment of the phosphatase SHP-2 (Src homology 2 domain-containing protein phosphatase) to PECAM-1, β-catenin release from PECAM-1, and reassociation of β-catenin with the AJ. Surface localization of PECAM-1 is increased in response to CCL2. This may enable the endothelium to sustain CCL2-induced alterations in AJ and facilitate recruitment of leukocytes into the CNS. Our novel findings provide a mechanism for CCL2-mediated disruption of endothelial junctions that may contribute to BBB dysfunction and increased leukocyte recruitment in neuroinflammatory diseases.
Laboratory Investigation 05/2012; 92(8):1213-33. · 3.64 Impact Factor
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Willem Bintig,
Daniela Begandt,
Barbara Schlingmann,
Linda Gerhard,
Maria Pangalos,
Lutz Dreyer,
Natalija Hohnjec,
Pierre-Olivier Couraud, Ignacio A. Romero,
Babette B. Weksler,
Anaclet Ngezahayo
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ABSTRACT: The expression and physiology of purine receptors of the human blood–brain barrier endothelial cells were characterised by
application of molecular biological, gene-silencing and Ca2+-imaging techniques to hCMEC/D3 cells. Reverse transcription polymerase chain reaction showed the expression of the G-protein-coupled
receptors P2Y2-, P2Y6-, P2Y11- as well as the ionotropic P2X4-, P2X5- and P2X7-receptors. Fura-2 ratiometry revealed that adenosine triphosphate (ATP) or uridine triphosphate (UTP) mediated a change in
the intracellular Ca2+ concentration ([Ca2+]i) from 150 to 300 nM in single cells. The change in [Ca2+]i corresponded to a fourfold to fivefold increase in the fluorescence intensity of Fluo-4, which was used for high-throughput
experiments. Pharmacological dissection using different agonists [UTPγS, ATPγS, uridine diphosphate (UDP), adenosine diphosphate
(ADP), BzATP, αβ-meATP] and antagonist (MRS2578 or NF340) as well as inhibitors of intracellular mediators (U73122 and 2-APB)
showed a PLC-IP3 cascade-mediated Ca2+ release, indicating that the nucleotide-induced Ca2+ signal was mainly related to P2Y2, 6 and 11 receptors. The gene silencing of the P2Y2 receptor reduced the ATP- or UTP-induced Ca2+ signal and suppressed the Ca2+ signal mediated by P2Y6 and P2Y11 more specific agonists like UDP (P2Y6), BzATP (P2Y11) and ATPγS (P2Y11). This report identifies the P2Y2 receptor subtype as the main purine receptor involved in Ca2+ signalling of the hCMEC/D3 cells.
KeywordsP2 receptors–G-Protein–Neurovascular unit–Gene silencing–siRNA
Purinergic Signalling 04/2012; 8(1):71-80. · 3.16 Impact Factor
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ABSTRACT: The mechanisms that underpin the passage of lamotrigine at the blood-brain barrier to its site of action in the brain is poorly understood. Lamotrigine has been postulated to be delivered to its site of action in the brain favourably despite its physicochemical properties. The aim of this study was to investigate the transport of lamotrigine in an in-vitro model of the BBB. In this study, lamotrigine was found to have a distribution coefficient of 0 at pH 7.4 indicating that it was not highly lipophilic. Human brain endothelial cells (hCMEC/D3) were used to probe the interaction of lamotrigine with drug transporters. The uptake of lamotrigine into hCMEC/D3 cells was found to be an active process (K(m) = 62 ± 14 μM; V(max) = 385 ± 30 pmol/min/million cells). Furthermore, use of a panel of transporter inhibitors indicated that this active uptake was mediated by organic cation transporter 1 (OCT1). OCT1 mRNA and protein were shown to be expressed in hCMEC/D3 cells. KCL22 cells overexpressing OCT1 were then used to validate these findings. Lamotrigine was confirmed to be a substrate and inhibitor in OCT1-transfected KCL22 cells. A putative pharmacokinetic drug-drug interaction (DDI) between quetiapine and lamotrigine was recently reported in patients and we show here that quetiapine is a potent inhibitor of the OCT1-mediated transport of lamotrigine. This is the first time that a specific influx transporter has been shown to transport lamotrigine. The clinical implications of these findings with respect to the efficacy of lamotrigine and its potential for DDI require further investigation.
Biochemical pharmacology 03/2012; 83(6):805-14. · 4.25 Impact Factor
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ABSTRACT: The aim of this study was to characterize the hCMEC/D3 cell line, an in vitro model of the human Blood Brain Barrier (BBB) for the expression of brain endothelial specific claudins-3 and -12.
hCMEC/D3 cells express claudins-3 and -12. Claudin-3 is distinctly localized to the TJ whereas claudin -12 is observed in the perinuclear region and completely absent from TJs. We show that the expression of both proteins is lost in cell passage numbers where the BBB properties are no longer fully conserved. Expression and localization of claudin-3 is not modulated by simvastatin shown to improve barrier function in vitro and also recommended for routine hCMEC/D3 culture.
These results support conservation of claudin-3 and -12 expression in the hCMEC/D3 cell line and make claudin-3 a potential marker for BBB characteristics in vitro.
Fluids and barriers of the CNS. 02/2012; 9:6.
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ABSTRACT: In central nervous system (CNS)-directed gene therapy, efficient targeting of brain parenchyma through the vascular route is prevented by the endothelium and the epithelium of the blood-brain and the blood-cerebrospinal fluid barriers, respectively. In this study, we evaluated the feasibility of the combined genetic and chemical adenovirus capsid modification technology to enable transcellular delivery of targeted adenovirus (Ad) vectors across the blood-brain barrier (BBB) in vitro models. As a proof-of-principle ligand, maleimide-activated full-length human transferrin (hTf) was covalently attached to cysteine-modified Ad serotype 5 vectors either to its fiber or hexon protein. In transcytosis experiments, hTf-coupled vectors were shown to be redirected across the BBB models, the transcytosis activity of the vectors being dependent on the location of the capsid modification and the in vitro model used. The transduction efficiency of hTf-targeted vectors decreased significantly in confluent, polarized cells, indicating that the intracellular route of the vectors differed between unpolarized and polarized cells. After transcellular delivery the majority of the hTf-modified vectors remained intact and partly capable of gene transfer. Altogether, our results demonstrate that i) covalent attachment of a ligand to Ad capsid can mediate transcellular targeting across the cerebral endothelium in vitro, ii) the attachment site of the ligand influences its transcytosis efficiency and iii) combined genetic/chemical modification of Ad vector can be used as a versatile platform for the development of Ad vectors for transcellular targeting.
PLoS ONE 01/2012; 7(9):e45977. · 4.09 Impact Factor
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ABSTRACT: Human African trypanosomiasis (HAT) is a parasitic disease affecting sub-Saharan Africa. The parasites are able to traverse the blood-brain barrier (BBB), which marks stage 2 (S2) of the disease. Delivery of anti-parasitic drugs across the BBB is key to treating S2 effectively and the difficulty in achieving this goal is likely to be a reason why some drugs require highly intensive treatment regimes to be effective. This study aimed to investigate not only the drug transport mechanisms utilised by nifurtimox at the BBB, but also the impact of nifurtimox-eflornithine combination therapy (NECT) and other anti-HAT drug combination therapies (CTs) on radiolabelled-nifurtimox delivery in an in vitro model of drug accumulation and the human BBB, the hCMEC/D3 cell line. We found that nifurtimox appeared to use several membrane transporters, in particular breast-cancer resistance protein (BCRP), to exit the BBB cells. The addition of eflornithine caused no change in the accumulation of nifurtimox, nor did the addition of clinically relevant doses of the other anti-HAT drugs suramin, nifurtimox or melarsoprol, but a significant increase was observed with the addition of pentamidine. The results provide evidence that anti-HAT drugs are interacting with membrane transporters at the human BBB and suggest that combination with known transport inhibitors could potentially improve their efficacy.
Brain research 12/2011; 1436:111-21. · 2.46 Impact Factor
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Nicola F Fletcher,
Garrick K Wilson,
Jacinta Murray,
Ke Hu,
Andrew Lewis,
Gary M Reynolds,
Zania Stamataki,
Luke W Meredith,
Ian A Rowe,
Guangxiang Luo,
Miguel A Lopez-Ramirez,
Thomas F Baumert,
Babette Weksler,
Pierre-Olivier Couraud,
Kwang Sik Kim, Ignacio A Romero,
Catherine Jopling,
Susan Morgello,
Peter Balfe,
Jane A McKeating
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ABSTRACT: Hepatitis C virus (HCV) infection leads to progressive liver disease and is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. However, it is unclear whether such cognitive abnormalities are a function of systemic disease, impaired hepatic function, or virus infection of the CNS.
We measured levels of HCV RNA and expression of the viral entry receptor in brain tissue samples from 10 infected individuals (and 3 uninfected individuals, as controls) and human brain microvascular endothelial cells by using quantitative polymerase chain reaction and immunochemical and confocal imaging analyses. HCV pseudoparticles and cell culture-derived HCV were used to study the ability of endothelial cells to support viral entry and replication.
Using quantitative polymerase chain reaction, we detected HCV RNA in brain tissue of infected individuals at significantly lower levels than in liver samples. Brain microvascular endothelia and brain endothelial cells expressed all of the recognized HCV entry receptors. Two independently derived brain endothelial cell lines, hCMEC/D3 and HBMEC, supported HCV entry and replication. These processes were inhibited by antibodies against the entry factors CD81, scavenger receptor BI, and claudin-1; by interferon; and by reagents that inhibit NS3 protease and NS5B polymerase. HCV infection promotes endothelial permeability and cellular apoptosis.
Human brain endothelial cells express functional receptors that support HCV entry and replication. Virus infection of the CNS might lead to HCV-associated neuropathologies.
Gastroenterology 11/2011; 142(3):634-643.e6. · 11.68 Impact Factor
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Ganta V Chaitanya,
Walter E Cromer,
Shannon R Wells,
Merilyn H Jennings,
P Olivier Couraud, Ignacio A Romero,
Babette Weksler,
Anat Erdreich-Epstein,
J Michael Mathis,
Alireza Minagar,
J Steven Alexander
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ABSTRACT: The glio-vascular unit (G-unit) plays a prominent role in maintaining homeostasis of the blood-brain barrier (BBB) and disturbances in cells forming this unit may seriously dysregulate BBB. The direct and indirect effects of cytokines on cellular components of the BBB are not yet unclear. The present study compares the effects of cytokines and cytokine-treated astrocytes on brain endothelial barrier. 3-dimensional transwell co-cultures of brain endothelium and related-barrier forming cells with astrocytes were used to investigate gliovascular barrier responses to cytokines during pathological stresses. Gliovascular barrier was measured using trans-endothelial electrical resistance (TEER), a sensitive index of in vitro barrier integrity. We found that neither TNF-α, IL-1β or IFN-γ directly reduced barrier in human or mouse brain endothelial cells or ECV-304 barrier (independent of cell viability/metabolism), but found that astrocyte exposure to cytokines in co-culture significantly reduced endothelial (and ECV-304) barrier. These results indicate that the barrier established by human and mouse brain endothelial cells (and other cells) may respond positively to cytokines alone, but that during pathological conditions, cytokines dysregulate the barrier forming cells indirectly through astrocyte activation involving reorganization of junctions, matrix, focal adhesion or release of barrier modulating factors (e.g. oxidants, MMPs).
Journal of Neuroinflammation 11/2011; 8:162. · 3.83 Impact Factor
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Willem Bintig,
Daniela Begandt,
Barbara Schlingmann,
Linda Gerhard,
Maria Pangalos,
Lutz Dreyer,
Natalija Hohnjec,
Pierre-Olivier Couraud, Ignacio A Romero,
Babette B Weksler,
Anaclet Ngezahayo
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ABSTRACT: The expression and physiology of purine receptors of the human blood-brain barrier endothelial cells were characterised by application of molecular biological, gene-silencing and Ca(2+)-imaging techniques to hCMEC/D3 cells. Reverse transcription polymerase chain reaction showed the expression of the G-protein-coupled receptors P2Y(2)-, P2Y(6)-, P2Y(11)- as well as the ionotropic P2X(4)-, P2X(5)- and P2X(7)-receptors. Fura-2 ratiometry revealed that adenosine triphosphate (ATP) or uridine triphosphate (UTP) mediated a change in the intracellular Ca(2+) concentration ([Ca(2+)](i)) from 150 to 300 nM in single cells. The change in [Ca(2+)](i) corresponded to a fourfold to fivefold increase in the fluorescence intensity of Fluo-4, which was used for high-throughput experiments. Pharmacological dissection using different agonists [UTPγS, ATPγS, uridine diphosphate (UDP), adenosine diphosphate (ADP), BzATP, αβ-meATP] and antagonist (MRS2578 or NF340) as well as inhibitors of intracellular mediators (U73122 and 2-APB) showed a PLC-IP(3) cascade-mediated Ca(2+) release, indicating that the nucleotide-induced Ca(2+) signal was mainly related to P2Y(2, 6 and 11) receptors. The gene silencing of the P2Y(2) receptor reduced the ATP- or UTP-induced Ca(2+) signal and suppressed the Ca(2+) signal mediated by P2Y(6) and P2Y(11) more specific agonists like UDP (P2Y(6)), BzATP (P2Y(11)) and ATPγS (P2Y(11)). This report identifies the P2Y(2) receptor subtype as the main purine receptor involved in Ca(2+) signalling of the hCMEC/D3 cells.
Purinergic Signalling 09/2011; 8(1):71-80. · 3.16 Impact Factor
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ABSTRACT: In vitro models of the blood-brain barrier (B-BB) generally utilise murine or porcine brain endothelium and rat astrocytes which are commonly grown in foetal calf serum supplemented conditions which modulate cell growth rates. Consequently, results gained from these experimental models can be difficult to extrapolate to the human in vivo situation since they are not of human origin. The proposed in vitro Transwell model of the B-BB is a multi-culture human cell system. It requires reconstruction of the human derived B-BB components in vitro (cerebral microvascular endothelial cells, astrocytes, and brain vascular pericytes) in a three-dimensional (3D) configuration based on Transwell filters. Different cell permutations (mono-, co-, and tri-cultivation) were investigated to find the most effective model in terms of tight junction resistance of the human cerebral microvascular endothelial cells. The B-BB model permutations comprised of human astrocytes (CC-2565 and SC-1810), human brain vascular pericytes (HBVP), and human cerebral microvascular endothelial cells (hCMEC/D3), under human serum supplementation. The models were assessed by trans-endothelial electrical resistance (TEER) measurements using an epithelial voltohmmeter, to validate the tight junction formation between hCMEC/D3 cells. Mono-, co-, and tri-cultivation Transwell models constructed with human brain-derived cells under human serum supplementation demonstrated that co-cultivation of astrocytes with endothelial cells produced the most successful model, as determined by TEER. Pericytes on the other hand improved tight junction formation when co-cultured with endothelial cells but did not improve the model to such an extent when grown in tri-cultivation with astrocytes.
Journal of neuroscience methods 05/2011; 199(2):223-9. · 2.30 Impact Factor
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ABSTRACT: Breakdown of the inner blood-retinal barrier and the blood-brain barrier is associated with changes in tight and adherens junction-associated proteins that link vascular endothelial cells. This study aimed to test the hypothesis that transforming growth factor (TGF)-β1 increases the paracellular permeability of vascular endothelial monolayers through tyrosine phosphorylation of VE-cadherin and claudin-5. Bovine retinal and human brain capillary endothelial cells were grown as monolayers on coated polycarbonate membranes. Paracellular permeability was studied by measuring the equilibration of (14)C-inulin or fluorescence-labelled dextran. Changes in VE-cadherin and claudin-5 expression were studied by immunocytochemistry (ICC) and quantified by cell-based enzyme linked immunosorbent assays (ELISA). Tyrosine phosphorylation of VE-cadherin and claudin-5 was studied by ICC, immunoprecipitation and Western blotting. We found that exposure of endothelial cells to TGF-β1 caused a dose-dependent increase in paracellular permeability as reflected by increases in the equilibration of (14)C-inulin. This effect was enhanced by the tyrosine phosphatase inhibitor orthovanadate and attenuated by the tyrosine kinase inhibitor lavendustin A. ICC and cell-based ELISA revealed that TGF-β1 induced both dose- and time-dependent decreases in VE-cadherin and claudin-5 expression. Assessment of cell viability indicated that changes in these junction-associated proteins were not due to endothelial death or injury. ICC revealed that tyrosine phosphorylation of endothelial monolayers was greatly enhanced by TGF-β1 treatment, and immunoprecipitation of cell lysates showed increased tyrosine phosphorylation of VE-cadherin and claudin-5. Our results suggest that tyrosine phosphorylation of VE-cadherin and claudin-5 is involved in the increased paracellular permeability of central nervous system-derived vascular endothelium induced by TGF-β1.
European journal of cell biology 04/2011; 90(4):323-32. · 3.31 Impact Factor
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ABSTRACT: The blood-brain barrier (BBB) of the central nervous system (CNS) consists of a unique subset of endothelial cells that possess tight junctions which form a relatively impervious physical barrier to a large variety of blood components. Until recently, there have been no good in vitro models for studying the human BBB without the co-culture of feeder cells. The hCMEC/D3 cell line is the first stable, well-differentiated human brain endothelial cell line that grows independently in culture with characteristics that closely resemble those of resident human brain endothelial cells. As our previously published findings demonstrated the importance of adenosine receptor (AR) signaling for lymphocyte entry into the CNS, we wanted to determine if human brain endothelial cells possess the capacity to generate and respond to extracellular adenosine. Utilizing the hCMEC/D3 cell line, we determined that these cells express CD73, the cell surface enzyme that converts extracellular AMP to adenosine. When grown under normal conditions, these cells also express the A(1), A(2A), and A(2B) AR subtypes. Additionally, hCMEC/D3 cells are responsive to extracellular AR signaling, as cAMP levels increase following the addition of the broad spectrum AR agonist 5'-N-ethylcarboxamidoadenosine (NECA). Overall, these results indicate that human brain endothelial cells, and most likely the human BBB, have the capacity to synthesize and respond to extracellular adenosine.
Purinergic Signalling 02/2011; 7(2):265-73. · 3.16 Impact Factor
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ABSTRACT: The targeting potential of OX-26-decorated immunoliposomes was investigated, using the human brain endothelial cell line hCMEC/D3 as a model of the blood-brain barrier (BBB). Immuno-nanoliposomes were prepared by the biotin/streptavidin ligation strategy, and their uptake by hCMEC/D3 cells and permeability through cell monolayers was studied. In order to elucidate the mechanisms of uptake, pH-sensitive fluorescence signal of HPTS was used, while transport was measured using double labeled immunoliposomes (with aqueous and lipid membrane fluorescent tags). PEGylated and non-specific-IgG-decorated liposomes were studied under identical conditions, as controls. CHO-K1 cells (which do not overexpress the transferrin receptor) were studied in some cases for comparative purposes. Experimental results reveal that hCMEC/D3 cells are good models for in vitro screening of BBB-targeting nanoparticulate drug delivery systems. Uptake and transcytosis of immunoliposome-associated dyes by cell monolayers was substantially higher compared to those of control liposomes. HPTS-entrapping OX-26-immunoliposome uptake indicated lysosomal localization and receptor-mediated mechanism. The ratio of aqueous/lipid label transport is affected by pre-incubation with antibody, or use of high lipid doses, suggesting that vesicles are transported intact after lysosome saturation. Co-decoration with a second ligand slightly decreases OX-26-decorated vesicle uptake, but not transcytosis, proving that the biotin-streptavidin technique can be applied for the generation of dual-targeting nanoliposomes.
European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 02/2011; 77(2):265-74. · 3.15 Impact Factor
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ABSTRACT: A polarized layer of endothelial cells that comprises the blood-brain barrier (BBB) precludes access of systemically administered medicines to brain tissue. Consequently, there is a need for drug delivery vehicles that mediate transendothelial transport of such medicines. Endothelial cells use a variety of endocytotic pathways for the internalization of exogenous materials, including clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis. The different modes of endocytosis result in the delivery of endocytosed material to distinctive intracellular compartments and therewith correlated differential processing. To obtain insight into the properties of drug delivery vehicles that direct their intracellular processing in brain endothelial cells, we investigated the intracellular processing of fixed-size nanoparticles in an in vitro BBB model as a function of distinct nanoparticle surface modifications. Caveolar endocytosis, adsorptive-mediated endocytosis, and receptor-mediated endocytosis were promoted by the use of uncoated 500-nm particles, attachment of the cationic polymer polyethyleneimine (PEI), and attachment of prion proteins, respectively. We demonstrate that surface modifications of nanoparticles, including charge and protein ligands, affect their mode of internalization by brain endothelial cells and thereby their subcellular fate and transcytotic potential.
Molecular Therapy 11/2010; 19(2):318-25. · 6.87 Impact Factor
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ABSTRACT: Organ-specific vascular targeting, for example, to the blood-brain barrier, requires the identification of unique molecular addresses on a subset of endothelial cells. The present study describes a crucial step towards tapping the exquisite specificity of the peptide/HLA class I system for this goal. We utilized a novel T-cell receptor (TCR) mimic antibody of high affinity and specificity, which is restricted by HLA-A2 and has been generated to recognize a peptide epitope derived from p68 RNA helicase (YLLPAIVHI). The parent protein is highly expressed by brain endothelial cells. Flow cytometry and confocal imaging showed that the antibody binds to HLA-A2-positive human brain-derived endothelial cells, both immortalized hCMEC/D3 cells and primary cells. The TCR mimic antibody undergoes internalization into vesicles, where significant colocalization occurs with the early endosomal marker EEA-1, but barely with caveolin-1. To our knowledge internalization of neither MHC class I protein nor TCR mimics by brain endothelial cells has been previously observed. Knock down of p68 protein expression by siRNA reduced the presentation of YLLPAIVHI-peptide/HLA-A2 complexes on the cell membrane by half as measured by flow cytometry 48 h later. We also found that brain endothelial cells isolated from HLA-A2 transgenic mouse strains express the A2 transgene, and brain endothelial cells of one of these strains also present YLLPAIVHI-peptide/HLA-A2, making these mouse strains suitable models for studying TCR mimic antibodies in vivo. In conclusion, these data strongly support the notion that TCR mimic antibodies could be a new class of therapeutic targeting agents in a wide variety of diseases.
Journal of Cellular Physiology 11/2010; 225(3):664-72. · 3.87 Impact Factor
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ABSTRACT: Transport of drugs to the brain is limited by the blood-brain barrier. New, specific brain endothelium ligands can facilitate brain-specific delivery of drugs.
We used phage display in an in situ brain perfusion model to screen for new brain endothelium peptide ligands.
Two phage clones, displaying 15 amino acid-peptides (GLA and GYR) that were selected for brain binding in the mouse model, showed significant binding to human brain endothelium (hCMEC/D3), compared to a random control phage. This binding was not seen for other human endothelial cells (HUVEC). Binding to hCMEC/D3 cells was dose dependent. When phage GLA and GYR were individually perfused through the murine brain, their ability to bind to the brain was 6-fold (GLA) and 5-fold (GYR) higher than the control phage. When compared to lung perfusion, phage showed an 8.5-fold (GYR) and 48-fold (GLA) preference for brain over lung compared to the control.
These results indicate that two new peptide ligands have been identified that may be used for specific targeting of drugs to the blood-brain barrier.
Pharmaceutical Research 02/2010; 27(4):673-82. · 4.09 Impact Factor
