Elsevier

Microvascular Research

Volume 75, Issue 1, January 2008, Pages 68-72
Microvascular Research

Regular Article
Gene expression profiles of ATP-binding cassette transporter A and C subfamilies in mouse retinal vascular endothelial cells

https://doi.org/10.1016/j.mvr.2007.05.002Get rights and content

Abstract

The purpose of this study was to quantify gene expression levels of the ATP-binding cassette (ABC) transporter A and C subfamilies ABCA1–A9, and ABCC1–6/Mrp1–6, C10/Mrp7 in mouse retinal vascular endothelial cells (RVEC) using a combination of a magnetic isolation method for mouse RVEC and real-time quantitative PCR analysis. The transcript level of endothelial cell markers, such as CD31, Tie-2, claudin-5, occludin, ABCB1a/mdr1a, and ABCG2, were more than 20-fold higher than those in the non-RVEC fraction, suggesting that RVEC in the RVEC fraction are concentrated at least 20-fold compared with those of the non-RVEC fraction. In the ABCA1 to A9 families, the transcript level of ABCA3 and A9 in the RVEC fraction was 1.2- and 32-fold higher than that in the non-RVEC fraction. Although ABCA3 was expressed in both the RVEC and non-RVEC fractions, A9 is predominantly expressed in the RVEC fraction. In the ABCC1 to C6 and C10 families, the transcript level of ABCC3, C4, and C6 in the RVEC fraction was 27-, 251-, and 242-fold higher, respectively, than that in the non-RVEC fraction, suggesting that ABCC3, C4, and C6 are predominantly expressed in the RVEC. In conclusion, ABCA3, ABCA9, ABCC3, ABCC4, and ABCC6 mRNAs are predominantly expressed at the inner blood–retina barrier (inner BRB) and appear to play a major role in the efflux transport of their substrates at the inner BRB.

Introduction

Retinal vascular endothelial cells (RVEC) form the inner blood–retinal barrier (inner BRB) with complex tight junctions. RVEC regulates the exchange of a variety of compounds, including lipids and drugs, between the blood and the retina using transporter molecules (Hosoya and Tomi, 2005). Although understanding the molecular basis of the inner BRB transporters will provide us with important information about lipid and drug distribution in the retina and the effective drug delivery to the retina, only a limited number of transporters have been identified at the inner BRB.

ATP-binding cassette (ABC) transporters play a central role in the efflux transport of lipids, ions, drugs, and their metabolites. Although 49 ABC transporters have been identified on human chromosomes (Takahashi et al., 2005), only two ABC transporters, i.e., ABCB1a/mdr1a (Greenwood, 1992) and ABCG2 (Asashima et al., 2006) have been identified at the inner BRB. Since ABCB1a and ABCG2 cannot fully account for the mechanisms of lipid transport and drug permeation at the inner BRB, RVEC appears to express additional ABC transporters. ABCA and ABCC/multidrug resistance protein (MRP) transporter subfamilies have been reported to be involved in the efflux transport of lipids such as cholesterol, phospholipids, and a variety of lipophilic anions (Kaminski et al., 2006, Kruh and Belinsky, 2003) although the functional role of ABCA and ABCC transporters is only beginning to be revealed. Recently, ABCA1 and ABCC4/MRP4 transporters have been found in the brain vascular endothelial cells which form the blood–brain barrier (BBB) (Nies et al., 2004, Ohtsuki et al., 2004, Panzenboeck et al., 2002, Zhang et al., 2000, Zhang et al., 2004). Studies by Leggas et al. (2004) using Abcc4 knockout mice further revealed that ABCC4 functions as the efflux pump of drugs at the BBB. Therefore, it is conceivable that ABCA and ABCC transporters make a major contribution to retinal lipid distribution and drug permeation at the inner BRB. However, currently, there are no available data on the gene expression profiles of ABCA and C transporters at the in vivo inner BRB. It should be noted that this may be due to the difficulty in obtaining enough purified RVEC as RVEC represents a small portion of the weight of the entire retina and the retina itself is a very small tissue. Indeed, although it is easy to measure the comprehensive gene expression profiles in immortalized RVEC lines using RT–PCR analysis, there is no way of knowing whether those expression levels are changed by culture passages and conditions. To address this, we have applied the magnetic purification technique of mouse RVEC to quantify the gene expression levels of transporters at the in vivo inner BRB.

The purpose of this study was to quantify the gene expression levels of ABCA and ABCC transporters in mouse RVEC using a combination of a magnetic isolation method for mouse RVEC and quantitative real-time PCR analysis.

Section snippets

Animals

Male ddY mice (6 weeks) were purchased from SLC (Shizuoka, Japan). The investigations using mice described in this report conformed to the provisions of the Animal Care Committee, University of Toyama (#2006-4) and the ARVO Statement on the Use of Animals in Opthalmic and Vision Research.

Isolation of RVEC

Mouse RVEC isolation was performed using a modification of the procedure described by Tomi and Hosoya (2004). Rat anti-mouse CD-31 antibodies (BD Pharmingen, Franklin Lakes, NJ, USA) were incubated with

Results and discussion

In order to isolate RVEC from the mouse retinal homogenate, magnetic beads coated with anti-CD31 antibodies were used and the magnetically collected and non-collected cells were isolated as RVEC and non-RVEC fractions, respectively. Although we reported application of this method for rat RVEC isolation (Tomi and Hosoya, 2004), the transcript levels of endothelial markers in RVEC and non-RVEC fractions were analyzed to validate the mouse RVEC isolation due to the scale differences between rats

Acknowledgments

This study was supported, in part, by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and a grant for Research on Sensory and Communicative Disorders by the Ministry of Health, Labor, and Welfare, Japan.

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