Bovine Brain Endothelial Cells Research Articles

Critical role of Babesia bovis spherical body protein 3 in ridge formation on infected red blood cells.

Babesia bovis, an apicomplexan intraerythrocytic protozoan parasite, causes serious economic loss to cattle industries around the world. Infection with this parasite leads to accumulation of infected red blood cells (iRBCs) in the brain microvasculature that results in severe clinical complications known as cerebral babesiosis. Throughout its growth within iRBCs, the parasite exports various proteins to the iRBCs that lead to the formation of protrusions known as "ridges" on the surface of iRBCs, which serve as sites for cytoadhesion to endothelial cells. Spherical body proteins (SBPs; proteins secreted from spherical bodies, which are organelles specific to Piroplasmida) are exported into iRBCs, and four proteins (SBP1-4) have been reported to date. In this study, we elucidated the function of SBP3 using an inducible gene knockdown (KD) system. Localization of SBP3 was assessed by immunofluorescence assay, and only partial colocalization was detected between SBP3 and SBP4 inside the iRBCs. In contrast, colocalization was observed with VESA-1, which is a major parasite ligand responsible for the cytoadhesion. Immunoelectron microscopy confirmed localization of SBP3 at the ridges. SBP3 KD was performed using the glmS system, and effective KD was confirmed by Western blotting, immunofluorescence assay, and RNA-seq analysis. The SBP3 KD parasites showed severe growth defect suggesting its importance for parasite survival in the iRBCs. VESA-1 on the surface of iRBCs was scarcely detected in SBP3 KD parasites, whereas SBP4 was still detected in the iRBCs. Moreover, abolition of ridges on the iRBCs and reduction of iRBCs cytoadhesion to the bovine brain endothelial cells were observed in SBP3 KD parasites. Immunoprecipitation followed by mass spectrometry analysis detected the host Band 3 multiprotein complex, suggesting an association of SBP3 with iRBC cytoskeleton proteins. Taken together, this study revealed the vital role of SBP3 in ridge formation and its significance in the pathogenesis of cerebral babesiosis.

Hypoxia increases the proliferation of brain capillary endothelial cells via upregulation of TMEM16A Ca2+-activated Cl− channels

The blood–brain barrier (BBB) is mainly formed by brain capillary endothelial cells (BCECs) and is exposed to hypoxic environments under pathological conditions. The effects of hypoxia on the expression and activity of Ca2+-activated Cl− (ClCa) channels, TMEM16A, were examined in bovine brain endothelial t-BBEC117 cells and mouse BCECs. The expression of TMEM16A was upregulated by hypoxia. Whole-cell ClCa currents increased under hypoxia. Hypoxia also increased cell proliferation and trans-endothelial permeability, which were attenuated by ClCa channel blockers or TMEM16A siRNA. These findings are useful for elucidating the pathological role of TMEM16A ClCa channels in the BBB during cerebral ischemia.

Brain endothelial cells metabolize glutamate via glutamate dehydrogenase to replenish TCA-intermediates and produce ATP under hypoglycemic conditions.

The endothelial cells of the blood-brain barrier participate in the regulation of glutamate concentrations in the brain interstitial fluid by taking up brain glutamate. However, endothelial glutamate metabolism has not been characterized, nor is its role in brain glutamate homeostasis and endothelial energy production known. The aim of this study was to investigate endothelial glutamate dehydrogenase (GDH) expression and glutamate metabolism and probe its functional significance. The primary brain endothelial cells were isolated from bovine and mouse brains, and human brain endothelial cells were derived from induced pluripotent stem cells. GDH expression on the protein level and GDH function were investigated in the model systems using western blotting, confocal microscopy, 13 C-glutamate metabolism, and Seahorse assay. In this study, it was shown that GDH was expressed in murine and bovine brain capillaries and in cultured primary mouse and bovine brain endothelial cells as well as in human-induced pluripotent stem cell-derived endothelial cells. The endothelial GDH expression was confirmed in brain capillaries from mice carrying a central nervous system-specific GDH knockout. Endothelial cells from all tested species metabolized 13 C-glutamate to α-ketoglutarate, which subsequently entered the tricarboxylic acid (TCA)-cycle. Brain endothelial cells maintained mitochondrial oxygen consumption rates, when supplied with glutamate alone, whereas glutamate supplied in addition to glucose did not lead to additional oxygen consumption. In conclusion, brain endothelial cells directly take up and metabolize glutamate and utilize the resulting α-ketoglutarate in the tricarboxylic acid cycle to ultimately yield ATP if glucose is unavailable.

Procoagulant activity of bovine neutrophils incubated with conditioned media or extracellular vesicles from Histophilus somni stimulated bovine brain endothelial cells

Histophilus somni is a Gram negative coccobacillus that causes respiratory, reproductive and central nervous system disease in cattle. The hallmark of H. somni infection is diffuse vasculitis and intravascular thrombosis that can lead to an acute central nervous system disease known as thrombotic meningoencephalitis (TME). Because neutrophils are major players in the pathophysiology of septic meningitis, we sought to determine their role in H. somni-induced fibrin clot formation in vitro. Bovine brain endothelial cells (TBBE cells) were exposed to H. somni cells at a 1:25 ratio, respectively. Conditioned media (CM) were collected after a 6 h incubation at 37 °C with 5% CO2, and then incubated with bovine peripheral blood polymorphonuclear neutrophils (PMNs). Following incubation, fibrin clot formation and tissue factor activity were assessed by a re-calcified plasma clotting assay. We found greater tissue factor activity in cell lysates and CM from H. somni-stimulated TBBE cells than unstimulated control TBBE cells. In addition, PMNs exposed to CM or extracellular vesicles from H. somni-stimulated TBBE cells expressed von Willenbrand factor, exhibited increased fibrin clot formation, and displayed greater tissue factor activity than PMNs exposed to CM or extracellular vesicles from unstimulated control TBBE cells. These results suggest that bovine PMNs might acquire extracellular vesicles from endothelial cells that leads to thrombus formation in bovine brain microvasculature and contribute to the process that characterizes TME.

Up-regulated expression of spherical body protein 2 truncated copy 11 in Babesia bovis is associated with reduced cytoadhesion to vascular endothelial cells

The factors involved in gain or loss of virulence in Babesia bovis are unknown. Spherical body protein 2 truncated copy 11 (sbp2t11) transcripts in B. bovis were recently reported to be a marker of attenuation for B. bovis strains. Increased cytoadhesion of B. bovis-infected red blood cells (iRBC) to vascular endothelial cells is associated with severe disease outcomes and an indicator of parasite virulence. Here, we created a stable B. bovis transfected line over-expressing sbp2t11 to determine whether up-regulation of sbp2t11 is associated with changes in cytoadhesion. This line was designated sbp2t11up and five B. bovis clonal lines were derived from the sbp2t11up line by limiting dilution for characterisation. We compared the ability of iRBCs from the sbp2t11up line and its five derivative clonal lines to adhere to bovine brain endothelial cells, using an in vitro cytoadhesion assay. The same lines were selected for in vitro cytoadhesion and the levels of sbp2t11 transcripts in each selected line were quantified. Our results demonstrate that up-regulation of sbp2t11 is accompanied by a statistically significant reduction in cytoadhesion. Confirmed up-regulation of sbp2t11 in B. bovis concomitant with the reduction of iRBC in vitro cytoadhesion to bovine brain endothelial cell is consistent with our previous finding that up-regulation of sbp2t11 is an attenuation marker in B. bovis and suggests the involvement of sbp2t11 transcription in B. bovis virulence.

Peptide-functionalized and high drug loaded novel nanoparticles as dual-targeting drug delivery system for modulated and controlled release of paclitaxel to brain glioma

A dual-targeting drug delivery system for paclitaxel (PTX) was developed by functionalizing novel polyester-based nanoparticles (NPs) with peptides possessing special affinity for low-density lipoprotein receptor (LDLR), overcoming the limitations of the current chemotherapeutics, to transport drug from blood to brain, and then target glioma cells. Employing novel biodegradable block co-polymers (P and 2P), PTX loaded and peptide-functionalized nanoparticles were prepared by a modified nano-co-precipitation method, carried out in one step only without emulsifier, allowing to obtain spherical nanometric (<200 nm), monodisperse (PDI ∼ 0.1), Poly (Ethylene Glycol) (PEG)-coated and high PTX loaded NPs with a slow and controlled release rate for a prolonged period of time. Peptide functionalization, confirmed by fluorimetric assay and HPLC amino acids analysis, enhanced the cellular uptake of functionalized-PTX-NPs by human primary glioblastoma cell line (U-87 MG) and Bovine Brain Endothelial Cells (BBMVECs), compared with non-functionalized-PTX-NPs. To confirm dual-targeting effect, transendothelial transport experiments in an in vitro BBB model and in vitro anti-tumoral activity against U-87 MG revealed that peptide-functionalized-PTX-NPs significantly increased the transport ratio of PTX across the BBB along with an improved anti-proliferative efficiency. Pharmacokinetics and biodistribution studies in rats, carried out by in vivo experiments with 125I radiolabelled dual-targeting PTX-NPs, confirmed the stealthy behavior of NPs and indicated slightly lower levels of penetration into brain tissue in comparison with peptides known to be able to cross the BBB. These promising results suggested that the dual-targeting drug delivery system might have great potential for glioma therapy in clinical applications.

The insulin receptor is expressed and functional in cultured blood-brain barrier endothelial cells but does not mediate insulin entry from blood to brain.

Insulin and its receptor are known to be present and functional in the brain. Insulin cerebrospinal fluid concentrations have been shown to correlate with plasma levels of insulin in a nonlinear fashion, indicative of a saturable transport pathway from the blood to the brain interstitial fluid. The aim of the present study was to investigate whether insulin was transported across brain endothelial cells in vitro via an insulin receptor-dependent pathway. The study showed that the insulin receptor was expressed at both the mRNA and protein levels in bovine brain endothelial cells. Luminally applied radiolabeled insulin showed insulin receptor-mediated binding to the endothelial cells. This caused a dose-dependent increase in Akt-phosphorylation, which was inhibited by coapplication of an insulin receptor inhibitor, s961, demonstrating activation of insulin receptor signaling pathways. Transport of insulin across the blood-brain barrier in vitro was low and comparable to that of a similarly sized paracellular marker. Furthermore, insulin transport was not inhibited by coapplication of an excess of unlabeled insulin or an insulin receptor inhibitor. The insulin transport and uptake studies were repeated in mouse brain endothelial cells demonstrating similar results. Although it cannot be ruled out that culture-induced changes in the cell model could have impaired a potential insulin transport mechanism, these in vitro data indicate that peripheral insulin must reach the brain parenchyma through alternative pathways rather than crossing the blood-brain barrier via receptor mediated transcytosis.

VEGF receptor-1 involvement in pericyte loss induced byEscherichia coliin anin vitromodel of blood brain barrier

The key aspect of neonatal meningitis is related to the ability of pathogens to invade the blood-brain barrier (BBB) and to penetrate the central nervous system. In the present study we show that, in an in vitro model of BBB, on the basis of co-culturing primary bovine brain endothelial cells (BBEC) and primary bovine retinal pericytes (BRPC), Escherichia coli infection determines changes of transendothelial electrical resistance (TEER) and permeability (Pe) to sodium fluorescein. In the co-culture model, within BBEC, bacteria are able to stimulate cytosolic and Ca(2+)-independent phospholipase A2 (cPLA2 and iPLA2 ) enzyme activities. In supernatants of E. coli-stimulated co-cultures, an increase in prostaglandins (PGE2) and VEGF production in comparison with untreated co-cultures were found. Incubation with E. coli in presence of AACOCF3 or BEL caused a decrease of PGE2 and VEGF release. SEM and TEM images of BBEC and BRPC showed E. coli adhesion to BBEC and BRPC but only in BBEC the invasion occurs. VEGFR-1 but not VEGFR-2 blockade by the specific antibody reduced E. coli invasion in BBEC. In our model of BBB infection, a significant loss of BRPC was observed. Following VEGFR-1, but not VEGFR-2 blockade, or in presence of AACOCF3 or BEL, elevated TEER values, reduced permeability and BRPC loss were found. These data suggest that VEGFR-1 negatively regulates BRPC survival and its blockade protects the barrier integrity. PGs and VEGF could exert a biological effect on BBB, probably by BRPC coverage ablation, thus increasing BBB permeability. Our results show the role played by the BBEC as well as BRPC during a bacterial attack on BBB. A better understanding of the mechanisms by which E. coli enter the nervous system and how bacteria alter the communication between endothelial cells and pericytes may provide exciting new insight for clinical intervention.

PECAM-1 is involved in neutrophil transmigration across Histophilus somni treated bovine brain endothelial cells

Histophilus somni ( H. somni) is a gram-negative bacterial pathogen that causes respiratory, reproductive, and central nervous system disease in cattle. The hallmark of systemic H. somni infection is diffused vasculitis that can lead to an acute central nervous system disease known as thrombotic meningoencephalitis (TME). Because platelet endothelial cell adhesion molecule-1 (PECAM-1) and endothelial nitric oxide synthase (eNOS) play fundamental roles in maintaining homeostasis in blood vessels, we sought to determine if PECAM-1 and eNOS expression play a role in events related to the pathogenesis of TME. Our findings demonstrate that neutrophil transmigration across H. somni-treated TBBEC (SV-40 transformed bovine brain endothelial cell line) was reduced by treatment with anti-PECAM-1 antibodies. Confocal microscopy indicated that H. somni treatment leads to redistribution of PECAM-1 and eNOS on the surface of TBBEC. These findings suggest that PECAM-1 and eNOS may play a role in the early pathogenesis of TME.

Lipopolysaccharide-induced apoptosis in transformed bovine brain endothelial cells and human dermal microvessel endothelial cells: the role of JNK.

Stimulation of transformed bovine brain endothelial cells (TBBEC) with LPS leads to apoptosis while human microvessel endothelial cells (HMEC) need the presence of cycloheximide (CHX) with LPS to induce apoptosis. To investigate the molecular mechanism of LPS-induced apoptosis in HMEC or TBBEC, we analyzed the involvement of MAPK and PI3K in TBBEC and HMEC. LPS-induced apoptosis in TBBEC was hallmarked by the activation of caspase 3, caspase 6, and caspase 8 after the stimulation of LPS, followed by poly(ADP-ribose) polymerase cleavage and lactate dehydrogenase release. We also observed DNA cleavage determined by TUNEL staining in TBBEC treated with LPS. Herbimycin A, a tyrosine kinase inhibitor, and SP600125, a JNK inhibitor, suppressed the activation of caspases and lactate dehydrogenase release. Moreover, a PI3K inhibitor (LY294002) suppressed activation of caspases and combined treatment with both SP600125 and LY294002 completely inhibited the activation of caspases. These results suggest that the JNK signaling pathway through the tyrosine kinase and PI3K pathways is involved in the induction of apoptosis in LPS-treated TBBEC. On the other hand, we observed sustained JNK activation in HMEC treated with LPS and CHX, and neither ERK1/2 nor AKT were activated. The addition of SP600125 suppressed phosphorylation of JNK and the activation of caspase 3 in HMEC treated with LPS and CHX. These results suggest that JNK plays an important role in the induction of apoptosis in endothelial cells.

Viable “Haemophilus somnus” Induces Myosin Light-Chain Kinase-Dependent Decrease in Brain Endothelial Cell Monolayer Resistance

"Haemophilus somnus" causes thrombotic meningoencephalitis in cattle. Our laboratory has previously reported that H. somnus has the ability to adhere to, but not invade, bovine brain endothelial cells (BBEC) in vitro. The goal of this study was to determine if H. somnus alters brain endothelial cell monolayer integrity in vitro, in a manner that would be expected to contribute to inflammation of the central nervous system (CNS). Monolayer integrity was monitored by measuring transendothelial electrical resistance (TEER) and albumin flux. BBEC incubated with H. somnus underwent rapid cytoskeletal rearrangement, significant increases in albumin flux, and reductions in TEER. Decreased monolayer TEER was preceded by phosphorylation of the myosin regulatory light chain and was partially dependent on tumor necrosis factor alpha and myosin light-chain kinase but not interleukin-1beta. Neither heat-killed H. somnus, formalin-fixed H. somnus, nor purified lipooligosaccharide altered monolayer integrity within a 2-h incubation period, whereas conditioned medium from H. somnus-treated BBEC caused a modest reduction in TEER. The data from this study support the hypothesis that viable H. somnus alters integrity of the blood-brain barrier by promoting contraction of BBEC and increasing paracellular permeability of the CNS vasculature.

Characteristics of the ATP-Induced Ca2+-Entry Pathway in the t-BBEC 117 Cell Line Derived From Bovine Brain Endothelial Cells

ATP-receptor (P2Y) stimulation induced sustained Ca2+-entry, which was essential for the enhanced cell-proliferation in t-BBEC117, an immortalized cell-line derived from bovine brain endothelial cells. Application of Ca2+ following store-depletion with thapsigargin in Ca2+-free solution induced Ca2+-entry through store-operated channels (SOCs). Ca2+-entry induced by ATP or 1-oleoyl-2-acetyl-sn-glycerol (OAG) together with Ca2+ was significantly larger than that by Ca2+ alone, suggesting the involvement of receptor-operated channels (ROCs) in the Ca2+-entry. Results obtained using pharmacological tools suggest that the contribution of Ca2+ sources to ATP-induced [Ca2+]i rise in t-BBEC117 is estimated as approximately 2:1:2 for Ca2+-release and Ca2+-entry though SOCs and ROCs, respectively.

Novel Functions of Small Conductance Ca2+-activated K+ Channel in Enhanced Cell Proliferation by ATP in Brain Endothelial Cells

Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB), which is essential for maintaining homeostasis of the brain. Net cellular turnover, which results from the balance between cell death and proliferation, is important in maintaining BBB homeostasis. Here we report a novel mechanism that underlies ATP-induced cell proliferation in t-BBEC 117, a cell line derived from bovine brain endothelial cells. Application of 0.1-30 mum ATP to t-BBEC 117 concentration-dependently increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in two phases: an initial transient phase and a later and smaller sustained one. These two phases of [Ca(2+)](i) rise were mainly due to Ca(2+) release and sustained Ca(2+) influx, respectively. The pretreatment with apamin, a selective blocker of small conductance Ca(2+)-activated K(+) channels (SK), significantly reduced both the [Ca(2+)](i) increase and K(+) current induced by ATP. Transcripts corresponding to P2Yx, SK2, and transient receptor potential channels were detected in t-BBEC 117. Knock down of SK2 protein, which was the predominant Ca(2+)-activated K(+) channel expressed in t-BBEC 117, by siRNA significantly reduced both the sustained phase of the [Ca(2+)](i) rise and the K(+) current induced by ATP. Cell proliferation was increased significantly by the presence of the stable ATP analogue ATPgammaS. This effect was blunted by UCL1684, a synthesized SK blocker. In conclusion, in brain endothelial cells ATP-induced [Ca(2+)](i) rise activates SK2 current, and the subsequent membrane hyperpolarization enhances Ca(2+) entry presumably through transient receptor potential channels. This positive feedback mechanism can account for the augmented cell proliferation by ATP.

Evaluation of Cattle Inoculated with Babesia bovis Clones Adhesive In Vitro to Bovine Brain Endothelial Cells

A comparative assessment of the virulence of Babesia bovis clones that adhere or not to bovine brain endothelial cells was done using two clones of B. bovis: (1) a clone phenotypically characterized as virulent (2F8) and (2) a clone of reduced virulence (RAD). Of these subpopulations, we selected those that had adhesive characteristics (a) or nonadhesive characteristics (na) in cultured endothelial cells. Twenty Holstein cattle, 12 months of age or older, were used in this study, and these cattle were randomly assigned to five groups of four animals each. The clones and their respective subpopulations were inoculated via intramuscular injection at a 0.5 x 10(7) infected erythrocyte dosage. Group A was inoculated with aRAD, group B with naRAD, group C with a2F8, group D with na2F8, and group E remained as a control. All inoculated animals showed a decrease in the packed cell volume (PCV), with group D showing the largest decrease (39.53%) and longest time (7 days) with rectal temperature above 39.5 degrees C. Babesia was observed in stained blood smears from only six cattle. While the four parasite subpopulations were pathogenic, significant differences were not noted among them, despite that the subpopulations considered to be virulent caused the greatest reduction in PCV per individual.

Roles of Cellular Activation and Sulfated Glycans inHaemophilus somnusAdherence to Bovine Brain Microvascular Endothelial Cells

Haemophilus somnus can cause a devastating fibrinopurulent meningitis with thrombotic vasculitis and encephalitis in cattle. The mechanisms used by H. somnus to migrate from the bloodstream into the central nervous system (CNS) are unknown. In this study, we demonstrate that H. somnus adheres to, but does not invade, bovine brain endothelial cells (BBEC) in vitro. The number of adherent H. somnus was significantly increased by prior activation of the BBEC with tumor necrosis factor alpha (TNF-alpha). Addition of exogenous glycosaminoglycans significantly reduced H. somnus adherence to resting and TNF-alpha-activated BBEC. Heparinase digestion of the endothelial cell's glycocalyx or sodium chlorate inhibition of endothelial cell sulfated glycan synthesis significantly reduced the number of adherent H. somnus. In contrast, addition of hyaluronic acid, a nonsulfated glycosaminoglycan, had no inhibitory effect. These findings suggest a critical role for both cellular activation and sulfated glycosaminoglycans in adherence of H. somnus to BBEC. Using heparin-labeled agarose beads, we demonstrated a high-molecular-weight heparin-binding protein expressed by H. somnus. Heparin was also shown to bind H. somnus in a 4 degrees C binding assay. These data suggest that heparin-binding proteins on H. somnus could serve as initial adhesins to sulfated proteoglycans on the endothelial cell surface, thus contributing to the ability of H. somnus to infect the bovine CNS.

In vitro models for the blood–brain barrier

The aim of the present study was to identify a model for the blood–brain barrier based on the use of a continuous cell line, and to investigate the specificity of this model. A set of test compounds, reflecting different transport mechanisms and different degrees of permeability, as well as different physiochemical properties was selected. In vivo data for transport across the blood–brain barrier of this set of test compounds was generated as part of the study using two different in vivo models. A computational prediction model was also developed, based on 74 proprietary Pharmacia compounds, previously tested in one of the in vivo models. Molsurf descriptors were calculated and 21 descriptors were correlated with log(Brain conc./Plasma conc.) using partial least squares projection to latent structures (PLS). However, the correlation between predicted and measured values was found to be rather low and differed between one and two log units for several of the compounds. The test compounds were analyzed in vitro using primary bovine and human brain endothelial cells co-cultured with astrocytes, and also using two different immortalized brain endothelial cell lines, one originating from rat and one from mouse. Cell models using cells not derived from the blood–brain barrier, ECV/C6, MDCK and Caco-2 cell lines, were also used. No linear correlation between in vivo and in vitro permeability was found for any of the in vitro models when all compounds were included in the analysis. The highest r 2 values were seen in the bovine brain endothelial cells ( r 2 = 0.43) and MDCKwt ( r 2 = 0.46) cell models. Higher correlations were seen when only passively transported compounds were included in the analysis, bovine brain endothelial cells ( r 2 = 0.74), MDCKwt ( r 2 = 0.65) and Caco-2 ( r 2 = 0.86). By plotting in vivo P app values against log D pH 7.4 it was possible to classify compounds into four different classes: (1) compounds crossing the blood–brain barrier by passive diffusion, (2) compounds crossing the blood–brain barrier by blood-flow limited passive diffusion, (3) compounds crossing the blood–brain barrier by carrier mediated influx, and (4) compounds being actively excreted from the brain by active efflux. P app and P e values obtained using the different in vitro models were also plotted against log D pH 7.4 and compared to the plot obtained when in vivo P app values were used. Several of the in vitro models could distinguish between passively distributed compounds and efflux substrates. Of the cell lines included in the present study, the MDCKmdr-1 cell line gave the best separation of passively and effluxed compounds. Ratios between AUC in brain and AUC in blood were also calculated for six of the compounds and compared to ratios between P e or P app for transport in the apical to basolateral and basolateral to apical direction. Again the MDCKmdr-1 cell line gave the best correlation with only one compound (AZT) giving large discrepancy between in vitro and in vivo data. None of the in vitro models could identify compounds known to be substrates for carrier mediated influxed as such, and the results indicate that a tighter in vitro blood–brain barrier model probably is needed in order to facilitate studies on carrier mediated influx. The findings presented also indicate that identification of “batteries” of in vitro tests are likely to be necessary in order to improve in vitro–in vivo correlations and to make it possible to perform acceptable predictions of in vivo brain distributions from in vitro data.

Release of prostaglandin E-2 in bovine brain endothelial cells after exposure to three unique forms of the antifungal drug amphotericin-B: role of COX-2 in amphotericin-B induced fever

Common formulations of amphotericin-B include a deoxycholate colloidal suspension (d-Amph), an amphotericin-B lipid complex (Ablc), and a liposomal product (l-Amph). The clinical incidence of infusion related fever is highest with d-Amph, intermediate with Ablc, and lowest with l-Amph. In the present study, we measured the activation of cyclooxygenase-2 (COX-2) and subsequent release of prostaglandin E-2 (PgE-2) from brain microvessel endothelium treated with these three formulations of amphotericin-B. Primary cultured bovine brain microvessel endothelial cells (BBMEC) were exposed to d-Amph, Ablc and l-Amph at concentrations that can be achieved in the plasma of patients receiving the drug. Media samples from the cells were collected and analyzed for PgE-2. Release of PgE-2 from BBMEC monolayers treated with l-Amph was similar to cells receiving culture media alone. In contrast, Ablc and d-Amph caused significantly greater release of PgE-2 from BBMEC monolayers compared to controls receiving culture media alone. PgE-2 release after d-Amph treatment was similar in magnitude to that observed with bacterial lipopolysaccharide (LPS). Western blot analysis indicated significant induction of COX-2 expression in BBMEC following LPS, Ablc or d-Amph treatment. Furthermore, PgE-2 release following exposure of BBMEC monolayers to either LPS or the various amphotericin-B formulations was reduced by the addition of the selective COX-2 inhibitor, NS-398. These studies indicate that amphotericin-B induces COX-2 expression in brain microvessel endothelium resulting in release of fever producing PgE-2. The magnitude of PgE-2 release from BBMEC following exposure to various amphotericin-B formulations mirrors the clinical observations regarding amphotericin-B induced fever and serves as initial support for the clinical use of COX-2 inhibitors to reduce amphotericin-B fever.

Glucose Transporter Asymmetries in the Bovine Blood-Brain Barrier

The transport of glucose across the mammalian blood-brain barrier is mediated by the GLUT1 glucose transporter, which is concentrated in the endothelial cells of the cerebral microvessels. Several studies supported an asymmetric distribution of GLUT1 protein between the luminal and abluminal membranes (1:4) with a significant proportion of intracellular transporters. In this study we investigated the activity and concentration of GLUT1 in isolated luminal and abluminal membrane fractions of bovine brain endothelial cells. Glucose transport activity and glucose transporter concentration, as determined by cytochalasin B binding, were 2-fold greater in the luminal than in the abluminal membranes. In contrast, Western blot analysis using a rabbit polyclonal antibody raised against the C-terminal 20 amino acids of GLUT1 indicated a 1:5 luminal:abluminal distribution. Western blot analysis with antibodies raised against either the intracellular loop of GLUT1 or the purified erythrocyte protein exhibited luminal:abluminal ratios of 1:1. A similar ratio was observed when the luminal and abluminal fractions were exposed to the 2-N-4[(3)H](1-azi-2,2,2,-trifluoroethyl)benzoxyl-1,3-bis-(d-mannos-4-yloxyl)-2-propylamine ([(3)H]ATB-BMPA) photoaffinity label. These observations suggest that either an additional glucose transporter isoform is present in the luminal membrane of the bovine blood-brain barrier or the C-terminal epitope of GLUT1 is "masked" in the luminal membrane but not in the abluminal membranes.

Molecular cloning of two bovine aquaporin-4 cDNA isoforms and their expression in brain endothelial cells

Two cDNA isoforms of bovine aquaporin-4 (bAQP4-A and bAQP4-B) were newly isolated. Sequence analysis of both cDNAs revealed open reading frames of 972 (bAQP4-A) and 906 nucleotides (bAQP4-B) with deduced proteins of 323 (bAQP4-A) and 301 amino acid residues (bAQP4-B). Partial 5′-genomic sequence analysis showed that the 5′-noncoding sequences specific to bAQP4-A and -B transcripts were contained in distinct exons, exon 0 for bAQP4-A and new exon X for bAQP4-B. RNase protection assay demonstrated the definite expression of both isoforms in bovine brain. The deduced amino acid sequence of bAQP4-A was highly homologous to the human (97%), rat (95%), and mouse (93%) AQP4. Reverse transcription-PCR detected the expression of AQP4 mRNAs in bovine brain endothelial cells as well as in a variety of bovine organs such as brain, lung, spleen, and kidney. Northern blot analysis indicated that a 6.0 kb message is predominantly expressed in bovine brain and lung.

Dexamethasone Regulation of P‐Glycoprotein Activity in an Immortalized Rat Brain Endothelial Cell Line, GPNT

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from the blood to the extracellular fluid environment of the brain. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain capillary endothelial cells, thus forming a functional barrier to lipid-soluble drugs, notably, antitumor agents. It is of interest to develop an in vitro BBB model that stably expresses P-gp to investigate the mechanisms of regulation in expression and activity. The rat brain endothelial cell line, GPNT, was derived from a previously characterized rat brain endothelial cell line. A strong expression of P-gp was found in GPNT monocultures, whereas the multidrug resistance-associated pump Mrp1 was not expressed. The transendothelial permeability coefficient of the P-gp substrate vincristine across GPNT monolayers was close to the permeability coefficient of bovine brain endothelial cells cocultured with astrocytes, a previously documented in vitro BBB model. Furthermore, the P-gp blocker cyclosporin A induced a large increase in apical to basal permeability of vincristine. Thus, P-gp is highly functional in GPNT cells. A 1-h treatment of GPNT cells with dexamethasone resulted in decreased uptake of vincristine without any increase in P-gp expression. This effect could be mimicked by protein kinase C (PKC) activation and prevented by PKC inhibition, strongly suggesting that activation of P-gp function may involve a PKC-dependent pathway. These results document the GPNT cell line as a valuable in vitro model for studying drug transport and P-gp function at the BBB and suggest that activation of P-gp activity at the BBB might be considered in chemotherapeutic treatment of cancer patients.