Satoko Hori

A pericyte-derived angiopoietin-1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro.

Although tight‐junctions (TJs) at the blood–brain barrier (BBB) are important to prevent non‐specific entry of compounds into the CNS, molecular mechanisms regulating TJ maintenance remain still unclear. The purpose of this study was therefore to identify molecules, which regulate occludin expression, derived from astrocytes and pericytes that ensheathe brain microvessels by using conditionally immortalized adult rat brain capillary endothelial (TR‐BBB13), type II astrocyte (TR‐AST4) and brain pericyte (TR‐PCT1) cell lines. Transfilter co‐culture with TR‐AST4 cells, and exposure to conditioned medium of TR‐AST4 cells (AST‐CM) or TR‐PCT1 cells (PCT‐CM) increased occludin mRNA in TR‐BBB13 cells. PCT‐CM‐induced occludin up‐regulation was significantly inhibited by an angiopoietin‐1‐neutralizing antibody, whereas the up‐regulation by AST‐CM was not. Immunoprecipitation and western blot analyses confirmed that multimeric angiopoietin‐1 is secreted from TR‐PCT1 cells, and induces occludin mRNA, acting through tyrosine phosphorylation of Tie‐2 in TR‐BBB13 cells. A fractionated AST‐CM study revealed that factors in the molecular weight range of 30–100 kDa led to occludin induction. Conversely, occludin mRNA was reduced by transforming growth factor β1, the mRNA of which was up‐regulated in TR‐AST4 cells following hypoxic treatment. In conclusion, in vitro BBB model studies revealed that the pericyte‐derived multimeric angiopoietin‐1/Tie‐2 pathway induces occludin expression.

New approaches to in vitro models of blood-brain barrier drug transport.

The pharmaceutical industry has been searching for an in vitro blood-brain barrier (BBB) model that preserves in vivo transporter functions in CNS drug discovery and development. The application of conditionally immortalized cell lines derived from transgenic animals harboring temperature-sensitive SV40 large T-antigen gene, is a rational and promising approach to such a workable in vitro BBB model. The established brain capillary endothelial cell lines retain the in vivo transport rate of several compounds and various forms of gene expression. Furthermore, this new approach has enabled the development of stable and reproducible co-culture models with a pericyte cell line and/or an astrocyte cell line.

Functional expression of rat ABCG2 on the luminal side of brain capillaries and its enhancement by astrocyte-derived soluble factor(s)

The purpose of the present study was to clarify the expression, transport properties and regulation of ATP‐binding cassette G2 (ABCG2) transporter at the rat blood–brain barrier (BBB). The rat homologue of ABCG2 (rABCG2) was cloned from rat brain capillary fraction. In rABCG2‐transfected HEK293 cells, rABCG2 was detected as a glycoprotein complex bridged by disulfide bonds, possibly a homodimer. The protein transported mitoxantrone and BODIPY‐prazosin. In rat brain capillary fraction, rABCG2 protein was also detected as a glycosylated and disulfide‐linked complex. Immunohistochemical analysis revealed that rABCG2 was localized mainly on the luminal side of rat brain capillaries, suggesting that rABCG2 is involved in brain‐to‐blood efflux transport. For the regulation study, conditionally immortalized rat brain capillary endothelial (TR‐BBB13), astrocyte (TR‐AST4) and pericyte (TR‐PCT1) cell lines were used as an in vitro BBB model. Following treatment of TR‐BBB13 cells with conditioned medium of TR‐AST4 cells, the Ko143 (an ABCG2‐specific inhibitor)‐sensitive transport activity and rABCG2 mRNA level were significantly increased, whereas conditioned medium of TR‐PCT1 cells had no effect. These results suggest that rat brain capillaries express functional rABCG2 protein and that the transport activity of the protein is up‐regulated by astrocyte‐derived soluble factor(s) concomitantly with the induction of rABCG2 mRNA.

A functional in vitro model of rat blood-brain barrier for molecular analysis of efflux transporters

Physiological studies of the blood-brain barrier (BBB) are often performed in rats. We describe the functional characterization of a reproducible in vitro model of the rat BBB and its validation for investigating mechanisms involved in BBB regulation. Puromycin-purified primary cultures of brain endothelial cells, co-cultured with astrocytes in the presence of hydrocortisone (HC) and cAMP, presented low sucrose permeability (< or =0.1 x 10(-3) cm/min) and high transendothelial electrical resistance (> or =270 Omega cm(2)). Expression of specific BBB markers and their transcripts was detected by immunostaining and RT-PCR, respectively: tight junction proteins (claudin-3 and -5, ZO-1 and occludin) and transporters (P-gp, Bcrp and Oatp-2). RT-PCR experiments demonstrated a role of treatment by astrocytes, HC and cAMP in regulation of the transcript level of tight junction proteins (claudin-5 and ZO-1) as well as transporters (Mdr1a, Mrp3, Mrp4, Bcrp, Glut-1), while transcript level of Mdr1b was significantly decreased. The functionality of efflux pumps (P-gp, Mrps and Bcrp) was demonstrated in the presence of specific inhibitors (PSC833, MK571 or Ko143, respectively) by (i) assessing the uptake of the common substrates rhodamine 123 and daunorubicin and (ii) evaluating apical to basolateral and basolateral to apical polarized transport of daunorubicin. In addition, a good correlation (R=0.94) was obtained between the permeability coefficients of a series of compounds of various lipophilicity and their corresponding in vivo rodent blood-brain transfer coefficients. Taken together, our results provide compelling evidence that puromycin-purified rat brain endothelial cells constitute a reliable model of the rat BBB for physiological and pharmacological characterization of BBB transporters.

Distinct spatio-temporal expression of ABCA and ABCG transporters in the developing and adult mouse brain

Using in situ hybridization for the mouse brain, we analyzed developmental changes in gene expression for the ATP‐binding cassette (ABC) transporter subfamilies ABCA1–4 and 7, and ABCG1, 2, 4, 5 and 8. In the embryonic brains, ABCA1 and A7 were highly expressed in the ventricular (or germinal) zone, whereas ABCA2, A3 and G4 were enriched in the mantle (or differentiating) zone. At the postnatal stages, ABCA1 was detected in both the gray and white matter and in the choroid plexus. On the other hand, ABCA2, A3 and A7 were distributed in the gray matter. In addition, marked up‐regulation of ABCA2 occurred in the white matter at 14 days‐of‐age when various myelin protein genes are known to be up‐regulated. In marked contrast, ABCA4 was selective to the choroid plexus throughout development. ABCG1 was expressed in both the gray and white matters, whereas ABCG4 was confined to the gray matter. ABCG2 was diffusely and weakly detected throughout the brain at all stages examined. Immunohistochemistry of ABCG2 showed its preferential expression on the luminal membrane of brain capillaries. Expression signals for ABCG5 and G8 were barely detected at any stages. The distinct spatio‐temporal expressions of individual ABCA and G transporters may reflect their distinct cellular expressions in the developing and adult brains, presumably, to regulate and maintain lipid homeostasis in the brain.

Co-administration of proton pump inhibitors delays elimination of plasma methotrexate in high-dose methotrexate therapy

AIM To assess whether or not co-administration of proton pump inhibitors (PPIs) is a risk factor for delayed elimination of plasma methotrexate (MTX) in high-dose MTX (HDMTX) therapy for malignant diseases. METHODS To assess the effects of PPI co-administration on elimination of plasma MTX, we examined plasma MTX concentration data on 171 cycles of HDMTX therapy performed in 74 patients. We performed multiple logistic regression analysis to evaluate PPI co-administration as a risk factor. Inhibitory potencies of omeprazole, lansoprazole, rabeprazole and pantoprazole on MTX transport via breast cancer resistance protein (BCRP, ABCG2) were also investigated in an in vitro study using membrane vesicles expressing human BCRP. RESULTS We identified co-administration of PPIs as a risk factor for delayed elimination (odds ratio 2.65, 95% confidence interval 1.03, 6.82) as well as renal and liver dysfunction. All four PPIs inhibited BCRP-mediated transport of MTX, with half-maximal inhibitory concentrations of 5.5-17.6 microM--considerably higher than the unbound plasma concentrations of the PPIs. CONCLUSIONS Our results support previous findings suggesting that PPI co-administration is associated with delayed elimination of plasma MTX in patients with HDMTX therapy. This drug interaction, however, cannot be explained solely by the inhibitory effects of PPIs on BCRP-mediated MTX transport.

ATP-Binding Cassette Transporter G2 Mediates the Efflux of Phototoxins on the Luminal Membrane of Retinal Capillary Endothelial Cells

PurposeThe purpose of this study was to clarify the localization and function of the ATP-binding cassette transporter G2 (ABCG2; BCRP/MXR/ABCP) in retinal capillary endothelial cells, which form the inner blood–retinal barrier, as an efflux transport system.MethodsThe expression was determined by reverse transcriptase polymerase chain reaction and Western blotting. The localization was identified by immunostaining. The transport function of ABCG2 was measured by flow cytometry.ResultsWestern blotting indicated that ABCG2 was expressed as a glycosylated disulfide-linked complex in the mouse retina and in peripheral tissues, including liver, kidney, and small intestine. Double immunolabeling of ABCG2 and glucose transporter 1 suggested that ABCG2 was localized on the luminal membrane of mouse retinal capillary endothelial cells. ABCG2 mRNA and protein were found to be expressed in a conditionally immortalized rat retinal capillary endothelial cell line, TR-iBRB, and rat retina. Treatment with Ko143, an ABCG2 inhibitor, restored the accumulation of pheophorbide a and protoporphyrin IX in TR-iBRB cells.ConclusionABCG2 is expressed on the luminal membrane of retinal capillary endothelial cells, where ABCG2 acts as the efflux transporter for photosensitive toxins such as pheophorbide a and protoporphyrin IX. ABCG2 could play an important role at the inner blood–retinal barrier in restricting the distribution of phototoxins and xenobiotics in retinal tissue.

ATP-binding cassette transporter A1 (ABCA1) deficiency does not attenuate the brain-to-blood efflux transport of human amyloid-β peptide (1-40) at the blood-brain barrier

ATP-binding cassette transporter A1 (ABCA1) mediates apolipoprotein-dependent cholesterol release from cellular membranes. Recent studies using ABCA1 knockout mice have demonstrated that ABCA1 affects amyloid-beta peptide (A beta) levels in the brain and the production of senile plaque. Cerebral A beta(1-40) was eliminated from the brain to the circulating blood via the blood-brain barrier (BBB), which expresses ABCA1. Therefore, in the present study, we examined whether ABCA1 affects the brain-to-blood efflux transport of human A beta(1-40)(hA beta(1-40)) at the BBB. The apparent uptake of [125I]hA beta(1-40) into ABCA1-expressing HEK293 cells was not significantly different from that into parental HEK293 cells. In addition, the apparent uptake was not significantly affected even in the presence of apolipoprotein A-I as a cholesterol release acceptor. Moreover, [125I]hA beta(1-40) elimination from mouse brain across the BBB was not significantly different between ABCA1-deficient and wild-type mice 60 min after its administration into the cerebrum. These results suggest that ABCA1 does not directly transport hA beta(1-40) and a deficiency of ABCA1 does not attenuate the brain-to-blood efflux transport of hA beta(1-40) across the BBB.

Expression of nuclear receptor mRNA and liver X receptor-mediated regulation of ABC transporter A1 at rat blood-brain barrier.

The aim of the present study was to investigate the expression of nuclear receptor mRNA and regulation of the expression of ATP-binding cassette (ABC) transporters by nuclear receptor agonists in rat brain capillary endothelial cells, which form the blood-brain barrier, by using rat brain capillary fraction from 8-week-old rats and a conditionally immortalized brain capillary endothelial cell line (TR-BBB13). RT-PCR analysis revealed that liver X receptor alpha and beta, retinoid X receptor alpha and beta and peroxisome proliferator-activating receptor alpha and beta mRNAs were expressed in the rat brain capillary endothelial cells and TR-BBB cells. In contrast, pregnane X receptor, farnesoid X receptor and constitutive androstane receptor were not detected. Furthermore, treatment with a liver X receptor agonist increased the ABCA1 mRNA level in TR-BBB13 cells, while ABCG2 mRNA expression was not affected. Treatment with a rat pregnane X receptor agonist did not affect the ABCB1 mRNA level in TR-BBB13 cells. These results demonstrate that the rat blood-brain barrier has an expressional regulation mechanism via sterol-related nuclear receptor, and indicate that the blood-brain barrier in 8-week-old rats lacks ABCB1 regulation via pregnane X receptor.

24S-hydroxycholesterol induces cholesterol release from choroid plexus epithelial cells in an apical- and apoE isoform-dependent manner concomitantly with the induction of ABCA1 and ABCG1 expression

The release of cholesterol from choroid plexus epithelial cells (CPE) plays an important role in cholesterol homeostasis in the CSF. The purpose of this study was to clarify the molecules involved in cholesterol release in CPE and the regulation mechanisms of the cholesterol release by the liver X receptor (LXR) using a conditionally immortalized CPE line (TR‐CSFB3). The mRNA expression of LXRα, LXRβ and their target genes, ATP‐binding cassette transporter (ABC)A1, ABCG1, ABCG4 and ABCG5, were detected in rat choroid plexus. ABCA1 and ABCG1 protein were detected in the plasma membrane of TR‐CSFB3 cells. Following treatment with 24S‐hydroxycholesterol, an endogenous LXR ligand, the expression of ABCA1 and ABCG1 were induced in TR‐CSFB3 cells. Moreover, apolipoprotein (apo)AI‐ and high‐density lipoprotein (HDL)‐mediated cholesterol release to the apical side of TR‐CSFB3 cells was facilitated by this treatment, whereas that to the basal side was not affected. Following 24S‐hydroxycholesterol treatment, apoE3‐dependent cholesterol release from TR‐CSFB3 cells was enhanced more than the apoE4‐dependent release. These results suggest that LXR activation facilitates cholesterol release into the CSF from CPE through the functional induction of ABCA1 and ABCG1. The difference between apoE3 and apoE4 suggests that the cholesterol release from CPE is related to the development of neurodegenerative diseases.