Brain Microvessel Endothelial Cell Monolayers Research Articles

培養牛脳微小血管内皮細胞を用いたｉｎ ｖｉｔｒｏ ＢＢＢモデルにおけるｌｉｐｏｓｏｍｅ封入ｃｉｓｐｌａｔｉｎ透過性の基礎的検討

Liposomes have been proved to be effective as carriers of water-soluble drugs into the central nervous system, but the mechanisms by which water-soluble drugs encapsulated in liposomes permeate the blood-brain barrier(BBB) are not clear. In order to elucidate the mechanisms, the authors investigated the permeability of liposome encapsulated cisplatin [cis-diamminedichloroplatinum (II), CDDP] across in vitro BBB model compared with that of free CDDP. This in vitro BBB model is consisted of primarily cultured bovine brain microvessel endothelial cell (BMEC) monolayer onto the collagen-coated polycarbonate membrane which is put in side by side diffusion cells. This in vitro BBB model matches in vitro BBB in morphology and enzymatic activity, and has been used in many transport studies. The result showed that liposome encapsulated CDDP permeated across BMEC monolayers and the permeability was significantly attenuated by metabolic inhibitor, 2-deoxyglucose treatment (p<0.05 by Student's t-test). Our findings confirmed that CDDP encapsulated in liposomes could be transported through BBB by way of energy-dependent transcellular pathway.

Leucine Enkephalin Effects on Paracellular and Transcellular Permeation Pathways Across Brain Microvessel Endothelial Cell Monolayers

Leucine enkephalin (YGGFL) effects on markers for transcellular and paracellular permeation across the blood-brain barrier (BBB) were investigated in vitro with bovine brain microvessel endothelial cell (BMEC) monolayers in primary culture. Intact YGGFL, but not metabolites of YGGFL, stimulated BMEC uptake of lucifer yellow (LY), a marker for fluid-phase endocytosis, in a concentration-dependent manner. However, D-[Ala2]-leucine enkephalin (YAGFL), a YGGFL analogue that altered BMEC monolayer permeability, had no effect on LY uptake. In part, these results suggested that YGGFL's effects on fluid-phase uptake might not relate directly to enhanced BMEC transcellular permeability in the presence of the peptide. The measurement of the fluorescence anisotropy of membrane-bound diphenyl-hexatriene probes did not show substantial peptide-induced changes in membrane lipid packing order (i.e., membrane fluidity) and indicated a limited role for membrane perturbations in YGGFL-induced changes in BMEC monolayer permeability. Conversely, the apparent permeability coefficients showed size-dependent YGGL-induced alterations for passage of membrane-impermeant substances across BMEC monolayers. The apparent permeability coefficients of low-molecular-weight (low-mol-wt) molecules (mannitol, sucrose, and fluorescein) were increased on exposure to YGGFL. The apparent permeability coefficients for high-mol-wt molecules, FITC dextran conjugates (4, 20, and 71.6 Kd), were not affected by exposure to YGGFL. Transmission electron micrographs of lanthanum (Stoke's radius, 10 A) exclusion from BMEC intercellular junctions supported these observations. Collectively, results from this study suggest that YGGFL enhanced BMEC permeability either by altering paracellular openings or through formation of a small pore in the monolayers to allow preferential penetration of low-mol-wt or small molecular size (< 10 A) substances.

Changes in brain microvessel endothelial cell monolayer permeability induced by adrenergic drugs

Brain microvessel endothelial cell monolayers have been shown to be a suitable blood-brain barrier in vitro system to study adrenergic regulation of permeability. We tested adrenergic drugs on bovine brain microvessel endothelial cell monolayer permeability to a biomembrane impermeant molecule, sodium fluorescein. Endonenous catecholamines noradrenaline and adrenaline were tested as well as the α-adrenoceptor agonist phenylephrine, the β-adrenoceptor agonist clenbuterol and the α-adrenoceptor antagonist prazosin. Results showed an α-adrenoceptor mediated increase and a β-adrenoceptor mediated decrease in monolayer permeability. Both α- and β-adrenoceptor mediated changes in permeability were abolished by inhibiting fluid-phase pinocytosis, either by vincristine or by avoiding bovine brain microvessel endothelial cell's energy utilization. The reverse transport (i.e., from brain to blood side) was also influenced by adrenergic drugs; α- or β-adrenoceptor stimulation induced a permeability-reducing effect. We conclude that α-adrenoceptor stimulation increases bovine brain microvessel endothelial cell monolayer permeability and that β-adrenoceptor stimulation has the opposite effect. Reverse transport results obtained with β-adrenoceptor stimulation seem controversial and deserve further study. These results also support in vivo findings that demonstrated adrenergic influences on blood-brain barrier permeability.

Uptake of surfactant-coated poly(methyl methacrylate)-nanoparticles by bovine brain microvessel endothelial cell monolayers

The objective of this study was to determine the influence of coating of radiolabeled poly(methylmethacrylate) nanoparticles (PMMA nanoparticles) with surfactants on their uptake by bovine microvessel endothelial cell cultures (BMEC cultures). For this purpose, BMEC cultures were grown in 24-well culture plates, where they formed confluent monolayers 10–12 days after seeding. The nanoparticle suspensions were then incubated with the cell cultures at 37°C, and the radioactivity within the cell cultures and the supernatant was measured after 30 min, 2 h and 6 h. The rates of uptake of the coated nanoparticles and of a control group of uncoated nanoparticles were compared.

Leucine enkephalin effects on brain microvessel endothelial cell monolayer permeability.

Leucine enkephalin effects on brain microvessel endothelial cell monolayer permeability.

Hydrogen bonding potential as a determinant of the in vitro and in situ blood-brain barrier permeability of peptides.

With the exception of various central nervous system (CNS)-required nutrients for which specific, saturable transport systems exist, the passage of most water-soluble solutes through the blood-brain barrier (BBB) is believed to depend largely on the lipid solubility of the solutes. Most peptides, therefore, do not enter the CNS because of their hydrophilic character. Recently, utilizing homologous series of model peptides and Caco-2 cell monolayers as a model of the intestinal mucosa, it was concluded that the principal determinant of peptide transport across the intestinal cellular membrane is the energy required to desolvate the polar amide bonds in the peptide (P. S. Burton et al., adv. Drug Deliv. Rev. 7:365, 1991). To determine whether this correlation can be extended to the BBB, the permeabilities of the same peptides were determined using an in vitro as well as an in situ BBB model. The peptides, blocked on the N- and C-terminal ends, consisted of D-phenylalanine (F) residues: AcFNH2, AcF2NH2, AcF3NH2, AcF2(NMeF)NH2, AcF(NMeF)2NH2, Ac(NMeF)3NH2, and Ac(NMeF)3NHMe. A good correlation among the permeabilities of these model peptides across the bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the BBB, and their permeabilities across the BBB in situ was observed (r = 0.928, P < 0.05). The permeabilities of these peptides did not correlate with the octanol-buffer partition coefficients of the peptides (r = 0.389 in vitro and r = 0.155 in situ; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Leucine-enkephalin metabolism in brain microvessel endothelial cells

The metabolism of leucine-enkephalin was investigated in primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. Leucine-enkephalin was hydrolyzed slowly with less than 40% of the peptide metabolized following a 5-h incubation with intact BMEC monolayers at 37°C. Following separation and extraction of BMEC enzymes into cytosolic and membrane-bound fractions, leucine-enkephalin was observed to be labile in the presence of both cytosolic and membrane-associated enzymes. A much greater concentration of enkephalin-hydrolyzing enzyme was associated with the cytosolic fraction. Resulting metabolite profiles for leucine-enkephalin appeared to be the result of interactions of the peptide with primarily aminopeptidases and angiotensin-converting enzyme. Our results suggested that extensive metabolism of leucine-enkephalin soley by BMECs in the cerebrovasculature would require internalization by the cells and presentation to cytosolic enzymes.

Biotin uptake and transport across bovine brain microvessel endothelial cell monolayers.

Primary cultures of bovine brain microvessel endothelial cells (BMECs) were used to characterize blood-brain barrier (BBB) uptake and transport of biotin. Both the uptake and the transcellular transport of either radiolabeled or fluorescein-conjugated biotin by confluent monolayers of BMECs were measured. Biotin uptake (Km = 123 microM) and bidirectional transport across BMEC monolayers was a saturable process and could be competed for by unlabeled biotin, biocytin, and biotinmethyl ester. Pantothenic and nonanoic acid were found not to be effective competitors for either biotin uptake or transport. The metabolic inhibitor, 2-deoxyglucose, had only small effects on the saturable apical-to-basolateral transport and apical uptake of biotin by BMECs. In contrast, basolateral-to-apical transport of biotin was substantially attenuated by 2-deoxyglucose pretreatment. Results supported the existence of specific and saturable uptake and efflux carrier systems for biotin in BMEC monolayers. The function of these systems was dependent to some degree on the metabolic status of the BMECs. Our findings confirm the existence of a biotin uptake system at the BBB in vivo and provide the first indication of an efflux system for biotin in BMECs.

The effect of protein binding on ivermectin uptake by bovine brain microvessel endothelial cells.

The effect of albumin binding on ivermectin uptake and transfer across the endothelial component of the blood-brain barrier (BBB) was determined with an in vitro model comprised of bovine brain microvessel endothelial cell (BMEC) monolayers. Cellular uptake of ivermectin was limited in the absence of albumin and 90% inhibited in the presence of 10% albumin. Cell membrane association of ivermectin, as followed by fluorescent probe labelling, was observed only at high (micromolar) concentrations of the drug. Membrane association was about 75% inhibited in the presence of albumin. Similarly, transfer across BMEC monolayers was restricted, equivalent to that of BBB impermeant markers. Unlike the uptake studies, however, albumin had little effect on the transfer of ivermectin across BMEC monolayers. These results support recent in vivo findings on the distribution of ivermectin into the brain and suggest that ivermectin has only a limited affinity for the endothelial component of the normal BBB.

Angiotensin Peptide Regulation of Bovine Brain Microvessel Endothelial Cell Monolayer Permeability

The passage of fluid-phase endocytosis markers--Lucifer yellow and a fluorescein isothiocyanate-conjugated dextran--across confluent primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers was characterized in the presence of angiotensin II (Ang II) peptides. Exposure to nanomolar concentrations of saralasin, an Ang II agonist, attenuated the passage of the fluorophores across the monolayers by 50-75%. In contrast, exposure to sarathrin, an Ang II antagonist, had no effect on passage of the fluorophores across the BMEC monolayers. Sarathrin, however, did inhibit the saralasin-induced reduction in transendothelial monolayer permeability and suggested specific Ang II binding site mediation. Other experiments performed suggest that saralasin-induced changes in BMEC monolayer permeability can be blocked by pretreatment with inhibitors of prostaglandin synthesis and that substantial saralasin-induced changes in BMEC monolayer permeability occurred only following exposure on the apical side of the endothelial cells. Results were consistent with previous studies demonstrating the existence of specific Ang II binding sites on BMECs and Ang II regulation of fluid-phase endocytosis. In addition, these studies support the role of angiotensin peptides in modulating permeability properties of the blood-brain barrier.

Some characteristics of specific angiotensin II binding sites on bovine brain microvessel endothelial cell monolayers

Angiotensin II (Ang II) binding sites were characterized in primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. Binding of [ 3H]Ang II to BMECs was time dependent and saturable. Scatchard plot analysis of dose-dependent [ 3H]Ang II binding revealed a single population of binding sites (K d=3.1 nM, B max=52 fmoles/mg protein). Sarathrin, an Ang II antagonist, and saralasin, a partial agonist, inhibited [ 3H]Ang II binding to BMEC monolayers, whereas two unrelated peptides, bradykinin and arginine-vasopressin, had no effect on the specific binding of [ 3H]Ang II. At 37°C, [ 3H]Ang II was internalized in BMECs and this uptake appeared to be saturable. Nanomolar concentrations of Ang II and saralasin stimulated [ 3H]thymidine uptake in serum-free starved BMEC monolayers, corresponding to an increase in DNA synthesis. On the other hand, sarathrin had no effect on [ 3H]thymidine uptake. The affinity of the single population of Ang II of binding sites was consistent with the concentration range of Ang II actions demonstrated in several cell types including BMECs. The Ang II-mediated actions on DNA synthesis suggest that this peptide-hormone may possess growth regulating properties in BMECs through either surface or internal site interactions. Collective findings support the complex nature of Ang II in regulating vascular and nonvascular cell growth and permeability characteristics.

Adsorptive endocytosis and membrane recycling by cultured primary bovine brain microvessel endothelial cell monolayers

The dynamics of membrane recycling were examined in primary cultures of brain microvessel endothelial cells (BMECs). Because the BMEC surface was dominated by galactosylated glycoconjugates, ricin agglutinin (RCAI) was used as a tracer to follow the endocytosis and recycling of RCAI binding sites. These binding sites accounted for 75% of the iodinatable or most externally disposed plasma membrane proteins. Because greater than 90% of the RCAI that had bound to BMECs was removed by a brief, nontoxic treatment with galactose, the amounts and kinetics for internalization and efflux of [125I]RCAI were measured. Both endocytosis and efflux were energy dependent. By using pseudo-first-order kinetics, the t1/2 values for RCAI binding, internalization and efflux were 5, 18 and 13-14 min, respectively. By comparing efflux with and without galactose present, we found that 60% of the RCAI binding sites that had been internalized were returned to the cell surface and reinternalized. Quantifying the distribution of gold-RCAI following internalization showed kinetics consistent with that obtained using radiolabeled RCAI. Both horseradish peroxidase (HRP) and gold-conjugated RCAI that had bound BMEC at 4 degrees C became localized within more caveolae within 2.5 min of warming to 37 degrees C to permit endocytosis. With time, RCAI appeared within endosomes and tubules and vesicles of which some were located in the trans-Golgi network (TGN). The distribution of HRP-RCAI contrasted with that of free HRP, which was not routed to the TGN. The absence of RCAI conjugates in association with the basolateral membrane domain suggested the presence of functional tight junctions and maintenance of polarity throughout the duration of these experiments. These results showed that membrane recycling was more extensive and much slower than fluid-phase endocytosis in cultured BMECs. Moreover, we found that endocytosis of membrane by BMECs in culture was similar to that reported for brain endothelium in vivo in that a fraction of the cell surface membrane was routed to the TGN.

Fluid-phase endocytosis by primary cultures of bovine brain microvessel endothelial cell monolayers

Blood-brain barrier (BBB) fluid-phase endocytosis was examined in primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. By fluorescence spectroscopy, Lucifer yellow (LY, a fluorescent, soluble molecule used as a marker for pinocytosis) accumulation by BMEC was observed to be linear over a concentration range of 0.05 to 1.0 mg/ml. Time-dependent uptake of LY exhibited curvilinear kinetics composed of an initially rapid uptake rate of 1338 ng of LY/mg protein per hour at 0.5 mg/ml LY. Within 20 min, the rate of LY accumulation slowed to a steady-state rate of 23 ng of LY/mg protein per hour. Accumulation of LY was inhibited in the presence of metabolic inhibitors, potassium cyanide or 2-deoxyglucose, and was decreased, but not completely inhibited, at 4°. Pulse-chase experiments revealed that efflux of LY was very rapid with at least 80% of the accumulated LY being lost within 2 min and was not sensitive to low temperature. Only 3–5% of the LY initially accumulated by BMEC remained cell-associated after a 30-min chase. The calculated turnover of the endocytic compartment's total volume (per hour) is 8- to 20-fold less than values for fibroblasts and macrophages, respectively. We have interpreted these data to suggest that the efflux of most of the LY involves loss from a rapidly recycled compartment of finite volume, possibly caveolae, that had sequestered marker during accumulation and suggest that these results are consistent with the present understanding of BBB pinocytosis in vivo.

In-vitro characterization of blood-brain barrier permeability to delta sleep-inducing peptide.

The diffusion of delta sleep-inducing peptide (DSIP) across the blood-brain barrier (BBB) has been investigated with an in-vitro model comprised of primary cultures of brain microvessel endothelial cell (BMEC) monolayers. The BMEC monolayers were mounted in a side-by-side diffusion apparatus and the transendothelial flux of DSIP analysed by HPLC with UV detection at 280 nm. The transendothelial flux of the peptide was linear with time and increasing concentrations of DSIP (non-saturable), but was not altered by reduced temperature. The apparent permeability coefficient for DSIP penetration of BMEC monolayers was in a range similar to water-soluble substances (e.g. fluorescein, fluorescein isothiocyanate dextrans) that penetrate the blood-brain barrier to a limited degree based on molecular weight. DSIP flux across the BMEC monolayers was also found to be bidirectional, insensitive to metabolic inhibitors, and not altered by high concentrations of tryptophan. Little degradation (apparent t1/2 about 10 h) of DSIP to major metabolites, tryptophan (trp) and des-trp DSIP, occurred over the time of the diffusion experiments. The results of these studies support and confirm observations in-vivo indicating that intact DSIP crosses the BBB by simple transmembrane diffusion.

Metabolism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by bovine brain microvessel endothelial cells

Incubation of bovine brain microvessel endothelial cell monolayers with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine led to the monoamine oxidase (MAO)-mediated formation of the oxidative metabolites 1-methyl-4-phenyl-2,3-dihydropyridine and 1-methyl-4-phenylpyridine (MPP +). The flux of the low nanomolar concentrations of [ 3H]MPP + across endothelial cell monolayers appeared to be restricted, probably due to the oxidation by MAO. [ 3H]MPP + did not significantly cross endothelial cell monolayers.

Demonstration of Acid Hydrolase Activity in Primary Cultures of Bovine Brain Microvessel Endothelium

The existence of lysosomes and acid hydrolase activity was demonstrated in an in vitro blood-brain barrier (BBB) model comprising primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. BMEC lysosomes were observed by the uptake of acridine orange and fluorophore-labeled acetylated low-density lipoprotein by fluorescence microscopy. Cytochemical localization of the acid hydrolase, sulfatase, and acid phosphatase (AcP) activities with light microscopy also revealed hydrolase-positive vacuoles or lysosomes that varied in number from cell to cell. BMEC monolayers were fractionated and biochemical assays of the sulfatase, AcP, and beta-galactosidase were performed. Significant activities of the acid hydrolases were found to be associated with lysosome and microsome fractions (69-77%). The majority of beta-galactosidase (approximately 48%) and total sulfatase (approximately 58%) activity was associated with the lysosome fraction of the BMECs. In contrast, approximately 52% of AcP activity was associated with the microsome fraction of the cells. The results of this study are consistent with the demonstration in vivo of acid hydrolases as potential factors in the endocytic pathway for transport of proteins through the BBB and as contributors to the BBB's enzymatic barrier function.

Differential adhesion of metastatic rat mammary carcinoma cells to organ-derived microvessel endothelial cells and subendothelial matrix.

The in vitro adhesion rates of rat 13762NF mammary adenocarcinoma cell clones of different spontaneous metastatic potentials to cloned microvessel endothelial cell monolayers and their subendothelial extracellular matrix were investigated. In this system, high rates of adhesion of the cloned tumor cell lines to syngeneic target (lung) organ-derived subendothelial matrix correlated with spontaneous metastatic potential, whereas adhesion to the lung microvessel endothelial cell apical surfaces occurred at lower rates and was not highly significantly different among the tumor cell lines. Adhesion rates to bovine aortic large vessel, and human brain and human meningeal microvessel endothelial cell monolayers were, in general, lower than those found with syngeneic lung microvessel endothelial cells, and did not correlate with spontaneous metastatic potential. Growth of endothelial cells in fetal bovine serum or platelet-poor horse serum did not affect the results, suggesting that in this system metastasis-associated organ-adhesive specificity is determined at the level of the subendothelial matrix.

The application of bovine brain microvessel endothelial-cell monolayers grown onto polycarbonate membranes in vitro to estimate the potential permeability of solutes through the blood-brain barrier.

Previously our laboratory (Rim et al., Int. J. Pharm. 32:79-84. 1986) described an in vitro blood-brain barrier (BBB) model consisting of cultured bovine brain microvessel endothelial cells (BMECs) grown onto regenerated cellulose acetate membranes. However, the utility of this in vitro BBB model system was limited because the regenerated cellulose acetate membrane and not the monolayer of bovine BMECs was rate limiting for the permeability of very lipophilic compounds. Therefore, in this study we have evaluated polycarbonate membranes as supports for growing bovine BMECs and for conducting in vitro drug permeability studies. Bovine BMECs were cultured on collagen-coated polycarbonate membranes (13-mm diameter, 12-microns pore size) which were then mounted into side-by-side diffusion cells for transport studies. The permeabilities of a series of solutes of varying lipophilicity (progesterone, estrone, testosterone, haloperidol, propranolol, antipyrine, caffeine, urea, acyclovir, ganciclovir, ribavirin, and glycerol) were determined and an excellent correlation (r = 0.97) was established between the permeability coefficients of the solutes and their log partition coefficients (PC)/(MW)1/2. These results suggest that bovine BMECs cultured onto polycarbonate membranes can be used as an in vitro model system for estimating the potential permeability of a solute through the BBB in vivo.

Aluminum effects on brain microvessel endothelial cell monolayer permeability

The permeability of primary cultures of brain microvessel endothelial cell (BMEC) monolayers has been shown to be sensitive to micromolar concentrations of aluminum. Aluminum increased BMEC permeability to a biomembrane impermeant molecule, fluorescein sodium, in a concentration-dependent manner with maximal effects observed after a 5 min preincubation. Aluminum attenuated BMEC pinocytosis as quantitated by the uptake of Lucifer yellow, a fluorescent marker for fluid-phase pinocytosis. Following labeling BMEC membranes with either diphenylhexatriene or trimethylammonium diphenylhexatriene, fluorescence anisotropy measurements showed that aluminum has no effect on membrane lipid order neither at the core of BMEC membranes nor near the surface of the BMEC membrane. The absence of aluminum-induced increases in BMEC pinocytosis, the absence of aluminum effects on BMEC lipid order, and the rapid onset of concetration-dependent increases in BMEC monolayer permeability suggests the potential site of aluminum-induced alterations of BBB permeability may originate at the luminal BMEC surface. Results confirm and support in vivo findings of aluminum-induced increases in blood-brain barrier (BBB) permeability to peptides and non-peptides. We conclude that primary cultures of BMEC monolayers provide an appropriate in vitro model for molecular/cellular level elucidation of mechanisms modulating BBB permeability to blood-borne factors.

Bovine brain microvessel endothelial cell monolayers as a model system for the blood-brain barrier.

Investigation of blood-brain barrier permeability and metabolic processes, and their regulation by endogenous or exogenous factors, will be important for development of efficient and selective delivery of therapeutic agents to the central nervous system. Primary cultures of brain microvessel endothelial cells offer a potentially powerful tool for studying at the cellular level the biochemical mechanisms regulating BBB function. Using this in vitro model, our studies are directed at characterization of the BBB processes that might be exploited as new schemes for drug delivery to the central nervous system.