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