Advances in the cell biology of transport via the inner blood-retinal barrier: establishment of cell lines and transport functions

The retinal capillary endothelial cells are connected to each other by tight junctions that play a key role in permeability as the inner blood-retinal barrier (inner BRB). Thus, understanding the inner BRB transport mechanism is an important step towards drug targeting of the retina. Nevertheless, inner BRB transport studies have been very limited in number since it is not easy to use the retinal capillaries, which are very small in size, for in vitro transport studies. Conditionally immortalized rat retinal capillary endothelial cells (TR-iBRB), pericytes (TR-rPCT) and Müller cell lines (TR-MUL) have been established from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. These cell lines possess respective cell type markers and maintain certain in vivo functions. Using a combination of newly developed cell lines and in vivo studies, we have elucidated the mechanism whereby vitamin C, L-cystine, and creatine are supplied to the retina. TR-iBRB cells are also able to identify transporters and apply to study regulation of transporters under pathophysiological conditions. Furthermore, these cell lines permit the investigation of cell-to-cell interactions and the expression of inner BRB-specific genes between TR-iBRB and other cell lines.     

Involvement of reduced folate carrier 1 in the inner blood-retinal barrier transport of methyltetrahydrofolate

The purpose of this study was to elucidate the mechanism of methyltetrahydrofolate (MTF) transport at the inner blood-retinal barrier (inner BRB). The characteristics and function of MTF transport at the inner BRB were examined using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as an in vitro model of the inner BRB. The [3H]MTF uptake by TR-iBRB2 cells increased with lowering extracellular pH and was Na+- and Cl--independent. The [3H]MTF uptake was concentration-dependent with a K(m) of 5.1 microM. This process was inhibited by reduced folate carrier 1 (RFC1) substrates, such as methotrexate and formyltetrahydrofolate, in a concentration-dependent manner with an IC50 of 8.7 and 2.8 microM, respectively, suggesting that RFC1 mediates MTF uptake in TR-iBRB2 cells. Although both RFC1 and proton-coupled folate transporter (PCFT) mRNA, which are pH-sensitive folate transporters, are expressed in TR-iBRB2 cells and isolated rat retinal vascular endothelial cells, the expression level of RFC1 mRNA was 83- and 49-fold greater than that of PCFT, respectively. Taken together, the above findings are consistent with the involvement of RFC1 in the inner BRB transport of MTF.     

Conditionally immortalized cell lines as a new in vitro model for the study of barrier functions

Conditionally immortalized brain and retinal capillary endothelial and choroid plexus epithelial cell lines were established from a transgenic rat (Tg rat) and mouse (Tg mouse) harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen. These cell lines exhibit temperature-sensitive cell growth due to the expression of ts SV 40 large T-antigen. Mouse brain (TM-BBB) and rat brain (TR-BBB) and rat retinal (TR-iBRB) capillary endothelial cell lines appear to have a spindle-fiber shaped morphology and exhibit the typical endothelial markers, such as von Willebrand factor and acetylated low-density lipoprotein uptake. These cell lines express in vivo influx and efflux transporters, such as P-glycoprotein (P-gp) and GLUT1, which is capable of 3-O-methyl-D-glucose transport. TM-BBB cells are able to undergo efflux transport of cyclosporin A, which is a substrate for P-gp transport activity. They may also express oatp2 and exhibit dehydroepiandrosterone sulfate and digoxin uptake activity. TR-BBB cells express the mRNA of multidrug resistance associated protein 1 (MRP1) and a large neutral amino acid transporter, which consists of LAT1 and 4F2hc. TR-iBRB cells exhibit pH-dependent L-lactic acid transport activity and express the mRNA of monocarboxylate transporter (MCT) 1 and 2. The choroid plexus epithelial cell line (TR-CSFB) has polygonal cell morphology, expresses the typical choroid plexus epithelial cell marker, transthyretin, and has Na+, K+-ATPase located on the apical side. TR-CSFB cells also exhibit amino acid transport activity which has been observed in vivo. These barrier cell lines established from the Tg rat and Tg mouse have in vivo transport functions and are good in vitro models for drug transport to the brain and retina and as a screen for drugs which might be capable of delivery to the brain and retina.     

Inner Blood-Retinal Barrier Transporters: Role of Retinal Drug Delivery

The inner blood-retinal barrier (inner BRB) forms complex tight junctions of retinal capillary endothelial cells to prevent the free diffusion of substances between the circulating blood and the neural retina. Thus, understanding of the inner BRB transport mechanisms could provide a basis for the development of strategies for drug delivery to the retina. Recent progress in inner BRB research has revealed that retinal endothelial cells express a variety of unique transporters which play a role in the influx transport of essential molecules and the efflux transport of xenobiotics. In this review we focus on the transport mechanism at the inner BRB in relation to its importance in influencing the inner BRB permeability of drugs.     

Vascular EndotheliumSelective Gene Induction by Tie2 Promoter/Enhancer in the Brain and Retina of a Transgenic Rat

Purpose To produce a transgenic rat harboring the Tie2 promoter/enhancer linked green fluorescence protein (GFP) gene and investigate the blood-brain barrier (BBB)- and inner blood-retinal barrier (iBRB)-selective GFP expression in the brain and retina.Methods Transgenic rats were produced by the microinjection of mouse Tie2/GFP gene into fertilized oocytes of Wistar rats. The expression of GFP was observed in tissue slices by confocal microscopy.Results One of the obtained transgenic lines showed intense GFP expression in vascular endothelial cells throughout the brain. After double immunostaining with glucose transporter 1, the GFP was found to be localized in the cytoplasmic compartment of brain capillary endothelial cells. In contrast, no fluorescence was observed in neural cells. In the retina of transgenic rats, intense GFP expression was detected in retinal capillary endothelial cells. No fluorescence was detected in other cells. In kidney and liver, GFP expression was detected in vascular endothelial cells, whereas the expressed region was limited.Conclusions Mouse Tie2 promoter/enhancer has the ability to induce gene expression selectively in vascular endothelial cells in the brain and retina of transgenic rats.     

Evaluation of amino acid-mustard transport as L-type amino acid transporter 1 (LAT1)-mediated alkylating agents

The L-type amino acid transporter 1 (LAT1, SLC7A5) is an Na+-independent neutral amino acid transporter the expression of which is located in retinal endothelial cells. Due to its broad substrate selectivity, LAT1 has been proposed to mediate the transport of amino acid-related drugs across the blood-tissue barriers. Here, we have investigated the transport screening of amino acid-mustards using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) which expresses LAT1. We synthesized 5 amino acid-mustards: tyrosine-mustard, phenylglycine-mustard, alanine-mustard, ornithine-mustard, and lysine-mustard. LAT1-mediated [3H]L-phenylalanine (Phe) uptake by TR-iBRB2 cells was inhibited in a competitive manner by tyrosine-mustard and phenylglycine-mustard as well as melphalan (phenylalanine-mustard). Phenylglycine-mustard was able to induce the efflux of [3H]Phe preloaded into the TR-iBRB2 cells expressing LAT1 through the obligatory exchange mechanism, although tyrosine-mustard, alanine-mustard, ornithine-mustard, lysine-mustard, and melphalan did not induce any significant efflux. These findings suggest that phenylglycine-mustard is a better substrate for LAT1 than melphalan and other amino acid-mustards.     

Involvement of a Novel Organic Cation Transporter in Verapamil Transport Across the Inner Blood-Retinal Barrier

Purpose

To clarify the transport and inhibition characteristics involved in verapamil transport across the inner blood-retinal barrier (inner BRB).

Methods

The transport of [³H]verapamil across the inner BRB was investigated using retinal uptake index and integration plot analyses in rats. The detailed transport characteristics were studied using TR-iBRB2 cells, a conditionally immortalized rat retinal capillary endothelial cell line that is an in vitro model of the inner BRB.

Results

The apparent influx permeability clearance of [³H]verapamil was 614 μL/(min·g retina), which is 4.7-fold greater than that of brain. The retinal uptake of [³H]verapamil was slightly increased by 3 mM verapamil and 10 mM qunidine and inhibited by 40 mM pyrilamine, supporting the carrier-mediated efflux and influx transport of verapamil across the inner BRB. TR-iBRB2 cells exhibited a concentration-dependent uptake of [³H]verapamil with a K _m of 61.9 μM, and the uptake was inhibited by several cations, such as pyrilamine, exhibiting a different profile from the identified transporters. These transport properties suggest that verapamil transport at the inner BRB takes place via a novel organic cation transporter.

Conclusions

Our findings suggest that a novel organic cation transporter is involved in verapamil transport from the blood to the retina across the inner BRB.     

Inner blood-retinal barrier mediates l-isomer-predominant transport of serine

D-serine, a coagonist for N-methyl-D-aspartate-type glutamate receptors, which mediate visual signal transmission, is thought to be generated from L-serine via serine racemase in the retina. However, the source of L-serine and D-serine in the retina are yet to be determined. The purpose of the present study was to investigate the characteristics of the blood-to-retina transport of serine at the inner blood-retinal barrier (BRB). In vivo study revealed the blood-to-retina transport of [(3) H]L-serine with an influx clearance of 49.9 μL/(min·g retina), which is greater than that of [(3) H]D-serine. This was consistent with the L-isomer-predominant uptake of serine by conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), an in vitro inner BRB model. [(3) H]L-Serine and [(3) H]D-serine uptake by TR-iBRB2 cells took place in an Na(+)-dependent and a concentration-dependent manner with Michaelis constant values of 97.5 μM and 9.63 mM, respectively. The uptake process of [(3) H]L-serine and [(3) H]D-serine was significantly inhibited by system ASC (alanine-serine-cysteine) substrates. Polymerase chain reaction analysis and immunocytochemistry revealed the expression of ASC transporters ASCT1 and ASCT2 in TR-iBRB2 cells. These results suggest that the system ASC at the inner BRB is a potent pathway for supplying serine in the form of the L-isomer from the circulating blood to the retina.     

Hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells

We have reported previously that taurine transporter (TauT) mediates γ-aminobutyric acid (GABA) as a substrate in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). This study investigates how TauT-mediated GABA transport is regulated in TR-iBRB2 cells under hypertonic conditions. [3H]GABA uptake by TR-iBRB2 cells exposed to 12 h- to 24 h-hypertonic culture medium was significantly greater than that of isotonic culture medium. [3H]GABA uptake by TR-iBRB2 cells was Na(+)-, Cl(-)-, and concentration-dependent with a Michaelis-Menten (K(m)) constant of 3.5 mM under isotonic conditions and K(m) of 0.324 and 5.48 mM under hypertonic conditions. Under hypertonic conditions, [3H]GABA uptake by TR-iBRB2 cells was more potently inhibited by substrates of TauT, such as taurine and β-alanine, than those of GABA transporters such as GABA, nipecotic acid, and betaine. These results suggest that an unknown high-affinity GABA transport process and TauT-mediated GABA transport are enhanced under hypertonic conditions. In conclusion, hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.     

Effects of Bisphosphonates on Glucose Transport in a Conditionally Immortalized Rat Retinal Capillary Endothelial Cell Line (TR-iBRB Cells)

The objective of the present study was to elucidate the effect of bisphosphonates, anti-osteoporosis agents, on glucose uptake in retinal capillary endothelial cells under normal and high glucose conditions. The change of glucose uptake by pre-treatment of bisphosphonates at the inner blood-retinal barrier (iBRB) was determined by measuring cellular uptake of [³H]3-O-methyl glucose (3-OMG) using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB cells) under normal and high glucose conditions. [³H]3-OMG uptake was inhibited by simultaneous treatment of unlabeled D-glucose and 3-OMG as well as glucose transport inhibitor, cytochalasin B. On the other hand, simultaneous treatment of alendronate or pamidronate had no significant inhibitory effect on [³H]3-OMG uptake by TR-iBRB cells. Under high glucose condition of TR-iBRB cells, [³H]3-OMG uptake was increased at 48 h. However, [³H]3-OMG uptake was decreased significantly by pre-treatment of alendronate or pamidronate compared with the values for normal and high glucose conditions. Moreover, geranylgeraniol (GGOH), a mevalonate pathway intermediate, increased the uptake of [³H]3-OMG reduced by bisphosphonates pre-treatment. But, pre-treatment of histamine did not show significant inhibition of [³H]3-OMG uptake. The glucose uptake may be down regulated by inhibiting the mevalonate pathway with pre-treatment of bisphosphonates in TR-iBRB cells at high glucose condition.     

ATP-binding cassette transporters in human heart failure

Adenosine triphosphate-binding cassette (ABC) transporters are involved in energy-dependent transport of substrates across biological membranes. We hypothesized that their expression is altered during human heart failure, suggesting a pathophysiologic basis. Messenger ribonucleic acid quantification of all known ABC transporters revealed multiple alterations in ABC transporter expression in failing human hearts (New York Heart Association classification III–IV) compared to nonfailing controls. These include a loss of ABCC7 chloride channels and an increased expression of the K_ATP channel regulatory subunits ABCC8. Moreover, ABCG2, an efflux pump for xenobiotics/drugs, was expressed at much higher levels in failing hearts compared to nonfailing control hearts. ABCG2 was found in cardiac capillary endothelial cells and cardiomyocytes. Experiments in cells stably transfected with human ABCG2 revealed that the peroxisome proliferator-activated receptor-γ agonist rosiglitazone was transported by ABCG2 but also inhibited the export of the prototypical ABCG2 substrate pheophorbide A (IC₅₀ 25 μM). These results suggest that altered ABC transporter expression in failing hearts might contribute to impaired channel conductance or might affect the cardiac disposition of drugs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. B.P. and T.F.S. contributed equally.     

Blood-to-retina transport of riboflavin via RFVTs at the inner blood-retinal barrier

Riboflavin (vitamin B₂) supply to the retina across the inner blood-retinal barrier (BRB) was investigated. In rats, the apparent influx permeability clearance of [³H]riboflavin (62.8 μL/(min·g retina)) was much higher than that of a non-permeable paracellular marker, suggesting the facilitative influx transport of riboflavin across the BRB. The retinal uptake index (RUI) of [³H]riboflavin was 59.0%, and significantly reduced by flavin adenine dinucleotide (FAD), but not by l-ascorbic acid, suggesting the substrate specificity of riboflavin transport. TR-iBRB2 cells, an in vitro model of the inner BRB, showed a temperature- and concentration-dependent [³H]riboflavin uptake with a K_m of 113 nM, suggesting that the influx transport of riboflavin across the inner BRB involves a carrier-mediated process. [³H]Riboflavin uptake by TR-iBRB2 cells was slightly altered by Na⁺- and Cl⁻-free buffers, suggesting that riboflavin transport at the inner BRB is preferentially Na⁺- and Cl⁻-independent. [³H]Riboflavin uptake by TR-iBRB2 cells was significantly reduced by riboflavin analogues while the uptake remained unchanged by other vitamins. The function and inhibition profile suggested the involvement of riboflavin transporters (SLC52A/RFVT) in riboflavin transport at the inner BRB, and this is supported by expression and knockdown analysis of rRFVT2 (Slc52a2) and rRFVT3 (Slc52a3) in TR-iBRB2 cells.     

Application of membrane permeability evaluated in in vitro analyses to estimate blood-retinal barrier permeability

The relationship between the in vitro membrane permeability and systemic blood-retinal barrier (BRB) permeability of drugs was investigated. To determine membrane permeability trend lines in this relationship, the apparent permeability (P(app)) and initial uptake rate (V) of 23 compounds were evaluated in a parallel artificial membrane permeability assay and the uptake study with a rat retinal endothelial cell line (TR-iBRB2 cells) for comparison with their retinal uptake index (RUI). The RUI values of compounds undergoing passive diffusion across the BRB were correlated with a log of the P(app) [RUI = 7.93 × 10 × exp (0.994 × log P(app)), r(2) = 0.660] and a log of the V [RUI = 26.5 × exp (1.55 × log V), r(2) = 0.581]. The RUI values of compounds undergoing carrier-mediated transport across the BRB were correlated with a log of the V [RUI = 26.5 × exp (0.887 × log V), r(2) = 0.559]. These results showed that the membrane permeability trend lines derived from the RUI and V values reflect the transport of drugs at the BRB, suggesting that an in vitro analysis-based estimation of the BRB permeability can be obtained using TR-iBRB2 cells and membrane permeability trend lines.     

Evaluation of an Immortalized Retinal Endothelial Cell Line as an In Vitro Model for Drug Transport Studies Across the Blood-Retinal Barrier

Purpose. To evaluate the growth and barrier properties of an immortalized rat retinal endothelial cell line (TR-iBRB) maintained on permeable membrane for drug transport studies. Methods. TR-iBRB cells were grown on permeable membrane filters. The effect of coating material on cell growth was investigated. Transport of [¹⁴C]-3-O-methyl-D-glucose (3-OMG), AGN 194716, AGN 195127, AGN 197075, acebutolol, alprenolol, atenolol, brimonidine, carbamazepine epoxide (CBZ-E), metoprolol, nadolol, rhodamine 123, and sotalol was measured across the cultured cell layer to determine the apparent permeability coefficients (P_app). Rhodamine 123 uptake into these cells in the presence of these test compounds was evaluated. Western blot was performed to detect the efflux transporter P-glycoprotein (P-gp). Bidirectional transport in MDR1-MDCK cell monolayers overexpressing the human P-gp was measured for AGN 197075. Results. TR-iBRB cells form confluent cell layers when grown on fibronectin-coated membrane and exhibit characteristic spindle-shaped morphology. A good correlation between P_app and cLogD (pH 7.4) of the compounds tested was observed, except for 3-OMG, AGN 197075, and rhodamine 123, which are substrates of carrier-mediated transport systems such as P-gp and a glucose transporter (GLUT1). When grown on permeable membrane, TR-iBRB cells expressed functional P-gp and GLUT1. Conclusions. TR-iBRB cells, when grown on permeable membrane, provide a useful tool for predicting permeability across the BRB. The usefulness of this model for high-throughput screening and rank ordering of drug candidates intended for the back of the eye in treatment of ocular diseases needs further characterization upon correlation with in vivo data.     

Blood-to-Retina Transport of Imperatorin Involves the Carrier-Mediated Transporter System at the Inner Blood-Retinal Barrier

This study investigated the mechanism of transporting imperatorin across the inner blood-retinal barrier (iBRB). The carotid artery single injection method was used to calculate the retinal uptake index (RUI) of [³H]imperatorin in vivo, whereas the retinal capillary endothelial cell lines were used for the in vitro uptake and mRNA expression assays. RUI value of [³H]imperatorin was greater than that of the reference compound ([¹⁴C]n-butanol). [³H]Imperatorin significantly reduced the RUI in the presence of neuroprotective organic cationic drugs at 10 mM. However, tetraethylammonium and p-aminohippuric acid showed no significant effects. [³H]Imperatorin uptake by TR-iBRB2 cells was time-, pH-, energy-, and concentration-dependent with a K_m value of 679 ± 130 μM. In addition, the uptake study showed insensitivity to sodium and membrane potential. Various organic cations including pyrilamine, nicotine, and clonidine significantly reduced the uptake of [³H]imperatorin, whereas organic anions and monocarboxylic acids did not. Furthermore, the mRNA expression level dropped markedly with rOCTN1, rOCTN2, rPMAT, and rMATE1 small interfering RNAs in the transfection study. Moreover, [³H]imperatorin uptake remained neutral with small interfering RNA transfections. Our results indicate that imperatorin transport across the iBRB involves carrier-mediated transporter system.     

Propranolol Transport Across the Inner Blood–Retinal Barrier: Potential Involvement of a Novel Organic Cation Transporter

The influx transport of propranolol across the inner blood–retinal barrier (BRB) was investigated. In the in vivo analysis of carotid artery single-injection method, [³H]propranolol uptake by the retina was greater than that of an internal reference compound, and was reduced by several organic cations. In the in vitro uptake study, TR-iBRB2 cells, an in vitro model of the inner BRB, showed a time-, concentration-, pH- and temperature-dependent [³H]propranolol uptake, suggesting the involvement of a carrier-mediated transport process in the influx of propranolol across the inner BRB. In the inhibition study, various organic cations, including drugs and candidates for the treatment of the retinal diseases, inhibited the [³H]propranolol uptake by TR-iBRB2 cells with no significant effects by the substrates and inhibitors of well-characterized organic cation transporters, suggesting that the influx transport of propranolol is performed by a novel transporter at the inner BRB. An analysis of the relationship between the inhibitory effect and the lipophilicity of inhibitors suggests a lipophilicity-dependent inhibitory effect of amines on the [³H]propranolol uptake by TR-iBRB2 cells. These results showed that influx transport of propranolol across the inner BRB is performed by a carrier-mediated transport process, suggesting the involvement of a novel organic cation transporter.     

Strategies for therapy of retinal diseases using systemic drug delivery: relevance of transporters at the blood-retinal barrier

INTRODUCTION: There is an increasing need for managing rapidly progressing retinal diseases because of the potential loss of vision. Although systemic drug administration is one possible route for treating retinal diseases, retinal transfer of therapeutic drugs from the circulating blood is strictly regulated by the blood-retinal barrier (BRB). AREAS COVERED: This review discusses the constraints and challenges of drug delivery to the retina. In addition, this article discusses the properties of drugs and the conditions of the BRB that affect drug permeability. The reader will gain insights into the strategies for developing therapeutic drugs that are able to cross the BRB for treating retinal diseases. Further, the reader will gain insights into the role of BRB physiology including barrier functions, and the effect of influx and efflux transporters on retinal drug delivery. EXPERT OPINION: When designing and selecting optimal drug candidates, it's important to consider the fact that they should be recognized by influx transporters and that efflux transporters at the BRB should be avoided. Although lipophilic cationic drugs are known to be transported to the brain across the blood-brain barrier, verapamil transport to the retina is substantially higher than to the brain. Therefore, lipophilic cationic drugs do have a great ability to increase influx transport across the BRB.     

Experimental models of growth factor-mediated angiogenesis and blood-retinal barrier breakdown

Following chronic ischemia, vascular endothelial growth factor (VEGF) is induced primarily in the ganglion cell layer of the retina. This often results in neovascularization (NV) that originates from the vascular bed closest to the ganglion cell layer. To study the effects of VEGF, independent lines of transgenic mice that express VEGF in the lens and in the retina have been generated. Expression in the lens results in excessive proliferation and accumulation of angioblasts and endothelial cells in proximity to the lens. However, VEGF expression is not sufficient to direct blood vessel organization or maturation in the prenatal mouse. Abnormal vessels do form on the retinal surface, but not until the second postnatal week. In transgenic mice expressing VEGF in the photoreceptors, NV originates from the deep capillary bed--the vascular bed closest to the photoreceptors. NV is accompanied by localized blood-retinal barrier breakdown. NV is also induced in PDGF-B transgenic mice. PDGF-B expression in the lens occurs prenatally and, during this time, mainly affects the perilenticular vessels. Postnatally, transgenic mice expressing PDGF-B in the lens or photoreceptors show a similar phenotype. In both models, a highly vascularized cell mass containing endothelial cells, pericytes, and glia forms in the superficial retina, and the formation of the deep capillary bed is inhibited. The phenotype suggests that an additional factor is necessary for the maturation and penetration of vascular endothelial cells into the retina to form the deep capillary bed.