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The expression and functional characterization of ABCG2 in brain endothelial cells and vessels¹

Cerebrovascular Research Group, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada

²Correspondence: Institute for Biological Sciences, NRC, 1200 Montreal Road Campus, Bldg. M-54, Ottawa, Ontario, Canada K1A 0R6. E-mail: danica.stanimirovic{at}nrc-cnrc.gc.ca

SPECIFIC AIMS

The blood-brain barrier (BBB) is a dynamic endothelial barrier characterized by the presence of specific transporters and efflux proteins that control trafficking of essential nutrients and determine brain bioavailability of drugs. The goal was to identify a subset of ATP binding cassette (ABC) transporters expressed in human BBB endothelium using a combination of bioinformatics, molecular, and functional approaches (Fig. 1 ). The specific aim was to characterize the expression and functional role of ABCG2 in brain endothelium in vitro and in vivo.

View larger version (44K): [in this window] [in a new window]   Figure 1. Approaches used in this study to identify and characterize ABC transporters expressed in human BBB. 1. Bioinformatics approach was used to identify EST sequences expressed in human brain and human brain endothelial cells. 2. Functional validation of the most abundantly expressed clone, identical to ABCG2, was performed in vitro and in vivo as outlined in the schematic.

PRINCIPAL FINDINGS

1. Human brain endothelial cells in vitro and human brain vessels in vivo express ABCG2 gene and protein
EST database searches conducted using a conserved ATP binding cassette domain identified 15 ABC transporter sequences expressed in human brain cDNA libraries (Fig. 1 ). These EST clones were purchased from American Type Culture Collection (ATCC); specific primers were designed for each clone and their expression was determined in human cerebromicrovascular endothelial cells (HCEC) using RT-PCR and Northern blot analyses. One EST clone (dbEST id: 991288; EST name: 56968; GenBank accession #AA349979; ATCC #150857) highly expressed in HCEC was sequenced and analyzed. Sequencing analysis showed that this EST clone carries an insert of 1689 bp with a coding region of 1626 bp that encodes 542 amino acids, including the ATP binding domain, but lacks the 5' end sequence of the gene. A 2.2 kb cDNA fragment was subsequently recovered from a lambda ZAP II cDNA library constructed from primary HCEC and found to contain a full-length coding region of 1965 bp that encodes a 655 amino acid polypeptide (GenBank accession #AY289766) identical to ABCG2/BCRP. The expression of ABCG2 was then confirmed (Fig. 1) in multiple isolations of primary HCEC (Fig. 2 A, C) and fetal human astrocytes (FHA) (Fig. 2B, C ) using RT-PCR and Western blot analyses. Quantitative expression analyses of ABCG2 and two other ABC transporters, ABCB1 (MDR-1) and ABCC1 (MRP-1), were conducted using real-time Q-PCR. In whole-brain homogenates, ABCG2 and ABCC1 were expressed at similar and relatively higher levels than ABCB1. Cultured HCEC and FHA displayed an abundant ABCC1 expression (5- to 10-fold higher than that of ABCB1), consistent with previous reports of ABCC1 up-regulation in cultured cells. ABCG2 expression levels were similar to those of ABCB1 in HCEC and three- to fourfold lower in FHA. Immunohistochemistry on frozen-sectioned human brain tissues using a monoclonal anti-human ABCG2 antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) demonstrated a strong, predominantly vascular staining (Fig. 2D, E ) that colocalized with an endothelial marker, factor VIII-related antigen (data not shown). Collectively, the results indicate that ABCG2 is strongly expressed in endothelial cells of the BBB in vitro and in vivo.

View larger version (45K): [in this window] [in a new window]   Figure 2. Expression of ABCG2 in cultured human cerebromicrovascular endothelial cells (HCEC), fetal human astrocytes (FHA), and frozen human brain tissue sections. ABCG2 mRNA expression was determined in multiple isolations of HCEC (A) and FHA (B) using RT-PCR. ABCG2 protein expression (C) was determined in HCEC (lanes 1–4) and FHA (lane 5) using Western blot and in sections of human brain tissues (D; E-negative control) by immunohistochemistry (monoclonal mouse anti-human ABCG2 antibody from Santa Cruz Biotech).

2. ABCG2 is involved in directional efflux of drug substrates in an in vitro BBB model
Functional activity of ABCG2 was investigated by assessing substrate drug uptake and directional transport across an in vitro BBB model (Fig. 1) . Functional characterization of ABCG2 was performed in cerebromicrovascular endothelial cells (CEC) transfected with ABCG2. Since the stable overexpression of ABCG2 gene could not be achieved in primary human CEC, rat CEC immortalized by a stable transfection of SV-40 large T antigen (iRCEC) were used. Northern blot analysis using a [³²P]-labeled human ABCG2 (hABCG2) cDNA probe (EST56968/ ATCC #150857/GenBank accession #AA349979) showed no hybridization with iRCEC. Full-length cDNA for hABCG2 was cloned into a pcDNA3.1 expression vector and stably transfected into iRCEC; clones expressing hABCG2 were selected in the presence of Geneticin (Invitrogen, San Diego, CA, USA) and the expression of hABCG2 was confirmed by RT-PCR. hABCG2-expressing iRCEC accumulated 37%, 33%, and 55% less mitoxantrone, rhodamine 123 (R123), and fluorescein, respectively, than either nontransfected or empty vector-transfected iRCEC.

An in vitro BBB model was used to assess polarization of drug transport across the hABCG2-expressing iRCEC monolayer. Luminal to abluminal (top-to-bottom) transport of mitoxantrone (Fig. 3 A), fluorescein (Fig. 3B ), and R123 (Fig. 3C ) was significantly reduced across hABCG2-transfected iRCEC compared with empty vector-transfected iRCEC cells. In contrast, abluminal to luminal (bottom-to-top) transport of all three drugs was increased across hABCG2-transfected iRCEC monolayers compared with empty vector-transfected iRCEC cells (Fig. 3) . Therefore, hABCG2 expressed in iRCEC demonstrates a functional polarization and increases extrusion of drug substrates from the brain compartment of the in vitro BBB model.

View larger version (30K): [in this window] [in a new window]   Figure 3. Mitoxantrone (A), fluorescein (B), and rhodamine-123 (R123) (C) transport across an in vitro BBB model. The in vitro BBB model consists of immortalized rat cerebromicrovascular endothelial cells (iRCEC) grown as a monolayer on a porous membrane in the tissue culture insert. Media conditioned by primary rat astrocytes are applied to the bottom compartment. Mitoxantrone (20 µM), fluorescein (75 µM), or R123 (15 µM) were added to either the top (left panels) or bottom compartment (right panels) of the triplicate BBB assemblies and drug concentrations were determined in the opposite compartment by fluorescence detection. Each time point represents the mean ± SD for 3 replicate membranes. Collagen-coated empty membranes; iRCEC monolayer; iRCEC clone stably overexpressing hABCG2. *Significant difference (ANOVA, P<0.01) from iRCEC monolayer.

3. ABCG2 expression is regulated in human brain vessels and parenchyma in vivo in pathological conditions
To investigate the expression and regulation of ABCG2 in vivo, laser capture microdissection (LCM) microscopy coupled to real-time PCR, was used. The purpose of these studies was to compare the expression of ABCG2 in brain vessels and parenchyma of nonmalignant brain tissue with that of highly malignant and vascularized brain glial tumor, glioblastoma multiforme (GBM, WHO grade IV). GBM is known to exhibit resistance to multiple chemotherapeutics. We hypothesized that multiple drug resistance phenotype of GBM is the result of either an increased expression of ABC transporters in tumor vessels or in parenchymal tumor cells. Small (<120 µm) brain vessels in fresh-frozen sections of nonmalignant human brain lesions and surgically removed-and histopathologically classified GBMs were stained with the fluorescently-labeled lectin, Ulex Europeus agglutinin I (UEA-I). LCM of vessels and perivascular parenchyma was performed using a Pixcell II Laser Capture Microscope (Arcturus, Mountain View, CA, USA). Approximately 50–60 vessels were captured from each section; vessels from three or four sections were pooled and RNA was extracted (Absolutely RNA Microprep Kit, Stratagene, San Diego, CA, USA) yielding 0.05–0.1 µg. The expression of ABCG2, ABCB1, and ABCC1 in these samples was quantified using RT-PCR and real-time Q-PCR. ABCG2 expression was significantly up-regulated in vessels (4- to 20-fold) and parenchyma (2- to 4-fold) of GBMs compared to nonmalignant brain lesions. ABCC1 was down-regulated (3-fold) in vessels of GBMs compared to control vessels and strongly up-regulated (5- to 10-fold) in GBM parenchyma. ABCB1 expression was not detectable in any of the LCM-captured samples, most likely due to low RNA yield; real-time Q-PCR analyses showed that ABCB1 expression was substantially lower than that of ABCG2 and ABCC1 in whole-brain homogenates. The data suggest that drug resistance of GBM is likely due to combined changes in the expression of specific drug transporters in the tumor vessels and parenchyma.

CONCLUSIONS AND SIGNIFICANCE

A potential impact of BBB efflux pumps on brain bioavailability of drugs can be assessed only by monitoring the expression and functional activity of a full complement of transporters/efflux pumps expressed in a given physiological or disease state. This manuscript contributes the following discoveries:

1. The development of a new approach that combines bioinformatics, molecular, and functional studies to examine simultaneous changes in the expression of multiple ABC transporters in vessels and tissues in various disease states.

2. The first comprehensive analysis of ABCG2 expression in human brain endothelial cells in vitro and human brain vessels in vivo.

3. The demonstration of ABCG2 functional polarization and the ability to efflux drugs from the brain compartment of the in vitro BBB model. These findings implicate ABCG2 transporter in the multiple drug resistance phenotype of the BBB.

4. The study shows that ABCG2 is up-regulated in vessels of glioblastomas, likely contributing to drug resistance of these highly malignant brain tumors. Therefore, ABCG2 may be a new target to modulate vascular barrier in glioblastomas and improve efficacy of chemotherapy.

Drug delivery to the brain remains one of the most perplexing challenges facing the pharmaceutical industry and medical community. The identification of expression and functionality of ABC transporters in BBB, including ABCG2, will be important in understanding biodistribution and pharmacokinetics of brain-targeted drugs. General principles of the approaches described here are applicable to many other fields including cancer chemotherapy, drug development, and vascular diseases in general.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1131fje; doi: 10.1096/fj.02-1131fje

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