Published papers

© 2017 Elsevier Ltd The placenta is an organ that secretes prostaglandin (PG) E2 into the fetal-placental circulation to regulate both vascular tone and remodeling of the fetal ductus arteriosus. Placental PGE2 synthesis might be mediated by microsomal PGE synthase-1 (mPGES-1), in addition to cyclooxygenase (COX) isoforms. Thus, the purpose of this study is to clarify the temporal and spatial expression patterns of mPGES-1, together with COX-1 and COX-2, in murine placenta. We found that mPGES-1 and COX-1 protein levels continuously increased in the placental labyrinth from gestational day (GD) 13.5 to GD19.5, becoming higher than in the decidua or the junctional zone by GD17.5. The PGE2 level at GD17.5 was also highest in the labyrinth. Immunofluorescence stainings for mPGES-1 and COX-1 in the labyrinth at GD17.5 overlapped and were located on the fetal side of the signals for connexin 26, which forms gap junctions between maternal-facing (SynT-I) and fetal-facing (SynT-II) syncytiotrophoblast layers, and on the maternal side of the signals for glucose transporter 1 on the basal plasma membrane of SynT-II. On the other hand, the signals for COX-2 did not overlap with those for mPGES-1. These results indicate that COX-1 and mPGES-1 are co-localized in murine placental SynT-II, facing the fetal-placental circulation. Therefore, SynT-II could contribute to placental synthesis of PGE2 for release into the fetal-placental circulation.

© 2020 The Author(s) Molecular Biology; Endocrinology

Five immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg mouse). These cell lines expressed active large Tantigen and grew well at 33°C with a doubling time of about 20 to 30 hours. TM-BBBs also grew at 37°C but not at 39°C. However, growth was restored when the temperature of the culture was lowered to 33°C. Although significant amounts of large T-antigen were shown to be present in the cell culture at 33°C, there was less of this complex at 37°C and 39°C. TMBBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated lowdensity lipoprotein uptake activity. The alkaline phosphatase and γ-glutamyltranspeptidase activity in TM-BBBs were -10% and 50% to 80% of brain capillary fraction of normal mice, respectively. DMannitol transport in the both apical-to-basal and basal-to-apical directions across the TM-BBB was 2-fold greater than for inulin. TM-BBBs were found to express GLUT-1 but not GLUT-3, and exhibited concentration-dependent 3-O-methyl-D-glucose (3-OMG) uptake activity with a Michaelis-Menten constant of 6.59 ± 1.16 mmol/1. Moreover, Pglycoprotein (P-gp) with a molecular weight of-170 kDa was expressed in all TM-BBBs. Both mdr la and mdr 1b mRNA were detected in TM-BBB4 using reverse transcription-polymerase chain reaction (RT- 7 PCR) analysis. [3H]-Cyclosporin A uptake by TMBBB was significantly increased in the presence of 100 μmol/1 verapamil and vincristine, suggesting that TM-BBB exhibits efflux transport activity via P-gp. In conclusion, conditional brain capillary endothelial cell lines were established from Tg mice. This cell line expresses endothelial markers and transporters at the BBB and is able to regulate cell growth, due to the amount of active large T-antigen in the cell, by changing the culture temperature.

Tadalafil, a phosphodiesterase 5 (PDE5) inhibitor, is a candidate therapeutic agent for fetal growth restriction and hypertensive disorders of pregnancy. In this study, we elucidated the fetal transfer of tadalafil in comparison with that of sildenafil, the first PDE5 inhibitor to be approved. We also examined the contributions of multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) to fetal transfer. Tadalafil or sildenafil was administered to wild-type, Mdr1a/b-double-knockout or Bcrp-knockout pregnant mice by continuous infusion from gestational day (GD) 14.5 to 17.5, and the fetal-to-maternal plasma concentration ratio of unbound drug (unbound F/M ratio) was evaluated at GD 17.5. The values of unbound F/M ratio of tadalafil and sildenafil in wild-type mice were 0.80 and 1.6, respectively. The unbound F/M ratio of tadalafil was increased to 1.1 and 1.7 in Mdr1a/b-knockout and Bcrp-knockout mice, respectively, while the corresponding values for sildenafil were equal to or less than that in wild-type mice, respectively. A transcellular transport study revealed that basal-to-apical transport of both tadalafil and sildenafil was significantly higher than transport in the opposite direction in MDCKII-BCRP cells. Our research reveals that tadalafil is a newly identified substrate of human and mouse BCRP, and it appears that the fetal transfer of tadalafil is, at least in part, attributed to the involvement of BCRP within the placental processes in mice. The transfer of sildenafil to the fetus was not significantly constrained by BCRP, even though sildenafil was indeed a substantial substrate for BCRP.

There is growing evidence that aromatherapy can improve patients' stress and refreshment levels. Also, an explanation to patients of the anticipated pharmacological effects of drugs may serve to improve compliance and to enhance subjective treatment benefit. Here, we aimed to examine the effectiveness of counseling as a means of improving the stress-reducing efficacy of bergamot oil and the concentration-improving effect of rosemary oil in 23 healthy young women. Efficacy was evaluated by using a self-reported questionnaire and multi-dimensional fatigue inventory-20 (MFI-20) as subjective indicators, together with near-infrared spectroscopy (NIRS)-evaluated blood flow in the inferior frontal cortex and amylase concentration in saliva as objective indicators. Counseling combined with bergamot aromatherapy significantly improved physical fatigue and mental fatigue and also improved sleepiness, concentration, and activity (p<0.01, p<0.05). Counseling combined with rosemary aromatherapy significantly improved motivation (p<0.05). Regional blood flow in the inferior frontal cortex was decreased totally. Especially partial right field of the prefrontal cortex area was decreased significantly by exposure to bergamot oil with counseling. By contrast, rosemary oil with counseling was decreased significantly in partial left of the prefrontal cortex area. Interestingly, we found that levels of salivary amylase (a sensitive biological marker of stress) were increased by counseling in subjects with low scores for resilience (capacity of individuals to resist stress). This finding may indicate that counseling was stressful to these subjects. Overall, our results indicate that counseling intervention increased the effectiveness of aromatherapy. Focused counseling taking account of behavioral differences between individuals may be a promising strategy to maximize patients' benefit.

There is growing evidence that aromatherapy can improve mood. Since pharmacists may be requested to counsel patients about aromatherapy, this study was designed to evaluate the influence of counseling on the effectiveness of aromatherapy in 14 healthy young women. Participants completed a self-reported questionnaire (motivation, feeling of drowsiness and general fatigue) and multi-dimensional fatigue inventory-20 (MFI-20) in a room filled with the fragrance of bergamot, with or without counseling. Further, as an objective measure of changes in stress, blood flow in the inferior frontal cortex was evaluated by means of near-infrared spectroscopy (NIRS). MFI-20 showed that aromatherapy with counseling significantly improved activity and motivation, as compared to aromatherapy only. The self-reported questionnaire showed no significant difference. Regional blood flow in the inferior frontal cortex was significantly decreased by exposure to aromatherapy with counseling, compared to aromatherapy alone (p<0.01, p<0.05). The value of the laterality index (LI) derived from NIRS measurements, which is considered to be a measure of stress, was decreased by aromatherapy with counseling, indicating that bergamot oil reduces stress. Overall, the results indicated that counseling intervention markedly increased the effectiveness of aromatherapy.

Copyright © 2020 The Author(s). Two types of systems are used in ex vivo human placental perfusion studies to predict fetal drug exposures, that is, closed systems with recirculation of the maternal and fetal buffer and open systems using a single-pass mode without recirculation. The in vivo fetal/maternal (F:M) ratio of metformin, a cationic drug that crosses the placenta, is consistent with that reported in an open system ex vivo but not with that in a closed system. In the present study, we aimed to develop a pharmacokinetic (PK) model of transplacental transfer of metformin to predict in vivo fetal exposure to metformin and to resolve the apparent inconsistency between open and closed ex vivo systems. The developed model shows that the difference between open and closed systems is due to the difference in the time required to achieve the steady state. The model-predicted F:M ratio (approx. 0.88) is consistent with reported in vivo values [mean (95% confidence interval): 1.10 (0.69-1.51)]. The model incorporates bidirectional transport via organic cation transporter 3 (OCT3) at the basal plasma membrane, and simulations indicate that the use of trimethoprim (an OCT3 inhibitor) to prevent microbial growth in the placenta ex vivo has a negligible effect on the overall maternal-to-fetal and fetal-to-maternal clearances. The model could successfully predict in vivo fetal exposure using ex vivo human placental perfusion data from both closed and open systems. This transplacental PK modeling approach is expected to be useful for evaluating human fetal exposures to other poorly permeable compounds, besides metformin. SIGNIFICANCE STATEMENT: We developed a pharmacokinetic model of transplacental transfer of metformin, used to treat gestational diabetes mellitus, in order to predict in vivo fetal exposure and resolve the discrepancy between reported findings in open and closed ex vivo perfusion systems. The discrepancy is due to a difference in the time required to reach the steady state. The model can predict in vivo fetal exposure using data from both closed and open systems.

Importance of the field: Delivery of drugs to the retina remains a major challenge which needs to be addressed urgently because retinal disorders are leading causes of visual impairment and significantly affect a patient's quality of life. Systemic drug administration is one possible route for treating retinal disorders; however, retinal transfer of drugs from the circulating blood is strictly regulated by two bloodocular barrier systems, the bloodaqueous barrier and the bloodretinal barrier. Areas covered in this review: This review summarizes the latest biological research regarding bloodocular barrier drug transporters. What the reader will gain: The bloodocular barrier sites and their respective roles in aqueous humor dynamics and retinal homeostasis are briefly presented. The potential impact of ATP-binding cassette (ABC) and solute carrier (SLC) drug transporters, such as ABCB, ABCC, ABCG, SLC7, SLC16, SLC19, SLCO/SLC21A, SLC22A and SLC29 transporters, on the permeability of drugs across the bloodocular barriers is then illustrated. Take home message: As more information becomes available regarding the bloodocular barrier transporters, we may be able to design simpler and more effective routes for drug delivery to the retina and, consequently, improve the treatment of retinal diseases. © 2010 Informa UK, Ltd.

© 2018, Japanese Association of Anatomists. The A-subclass of ATP-binding cassette (ABC) transporters is a highly conserved superfamily of potent lipid transporters. Although the ABCA1-like subgroup of ABCA1-4, and A7 have been shown to mediate the transport of endogenous lipids, the roles of the ABCA6-like subgroup transporters, which have been identified as a unique gene cluster on human chromosome 17q24 (ABCA5, A6, A8, A9, and A10) and mouse chromosome 11 (Abca5, a6, a8a, a8b, and a9), remains largely unknown. The purpose of the present study was to clarify the spatial and temporal expression profiles of Abca6-like subgroup transporters in embryonic and postnatal mouse brains by a combination of in situ hybridization and quantitative polymerase chain reaction (PCR) using magnetically isolated brain vascular endothelial cells. In embryonic brains, the transcripts of Abca5, a8a and a8b were detected predominantly in the mantle zone, where postmitotic neurons differentiate. At the postnatal stages, they were expressed in various nuclei and neuronal layers. Abca9 mRNA was detected diffusely in the embryonic and postnatal brains and sequential and/or strong spotted signals were detected in the leptomeninges on the brain surface. PCR detected expression of Abca8a and Abca9 mRNAs in isolated vascular endothelial cells. Expression signals for Abca6 mRNA were hardly observed at any stages examined. These distinct spatio-temporal expression patterns of Abca6-like subgroup transporters may reflect their functional significance and diversity to regulate lipid transport, particularly in neurons, leptomeningeal cells, and vascular endothelial cells.

The purpose of this research was to examine whether the fatigue-relieving efficacy of a revitalizing medicinal product would be increased by counseling (advice to subjects regarding its expected efficacy), using both subjective and objective indicators. Twenty participants took the revitalizing medicinal product (drinkable product: 50 mL, once) on separate occasions, once with and once without counseling, with a 1-week interval. They completed a self-reported questionnaire (covering motivation, feeling of drowsiness, and general fatigue) and multi-dimensional fatigue inventory-20 (MFI-20) as subjective indicators, and blood flow in the inferior frontal cortex was evaluated by means of near-infrared spectroscopy (NIRS) as an objective indicator. The self-reported questionnaire showed that the revitalizing drink with counseling significantly improved general fatigue, as compared to the drink alone. MFI-20 showed no significant difference. Regional blood flow in the inferior frontal cortex was significantly increased by administration of the revitalizing drink with counseling, compared to without counseling (P < 0.05). Overall, the results indicated that counseling intervention increased the effectiveness of the revitalizing medicinal product.

The purpose of this research was to noninvasively evaluate the influence of aromatherapy on work performance in 14 healthy young adults. Participants in a room filled with the fragrance of lavender completed a self-reported questionnaire and multi-dimensional fatigue inventory-20 (MFI-20). Further, as an objective measure, blood flow in the inferior frontal cortex was evaluated via near-infrared spectroscopy (NIRS) as a parameter of working memory capacity. Compared to the control stage (no aromatherapy), exposure to aromatherapy achieved a significant reduction in general and mental fatigue according to MFI-20. Self-reported questionnaires also indicated improvement, but the differences were not statistically significant. NIRS measurement during the task performance of an N-back program indicated that regional blood flow in the inferior frontal cortex was significantly increased through exposure to aromatherapy, compared to without. The prefrontal area in the brain is involved in working memory, attention concentration and judgment. These results suggest that lavender may improve both cognitive ability and mood. A larger study with more aroma oils seems warranted.

The purpose of this research was to clarify the effect of repeated administration of 2 kinds of energy drink,1 containing vitamins,taurine and caffeine (simple-type),and the other containing vitamins,taurine,caffeine and several botanical herb extracts (herb-type),on work performance using cognitively subjective and objective indicators. The simple-type and herb-type drinks were given to 14 healthy young adults in a consecutive double-blind,randomized,2-period cross-over clinical trial.Participants took each energy drink once a day for five days during separate periods.Motivation,feeling of drowsiness,and general fatigue were evaluated on a subjective scale using a self-reported questionnaire both before and after administration.Other subjective evaluations were those based on the fatigue-visual analog scale (Fatigue-VAS) and multi-dimensional fatigue inventory-20 (MFI-20).As an objective measure,work performance was examined using the Kreapelin mental work test. Compared to the simple-type drink,the herb-type drink achieved a significant reduction in drowsiness and significantly increased motivation based on the self-reported questionnaire results for the post-administration assessment.In the Kreapelin mental work test,the mean amount of work was significantly increased by both drinks.MFI-20 revealed significant decreases in 2 categories of physical fatigue and general fatigue due to the 2 drinks.The fatigue-VAS also indicated decreases but they were not statistically significant. Both types of energy drink appeared to be effective in terms of subjective and objective indicators.Though the herb-type drink was found to be superior to the simple-type drink regarding subjective measures,there was no significant difference between the 2 regarding the objective measure.

Digoxin is used as first-line therapy to treat fetal supraventricular tachycardia; however, because of the narrow therapeutic window, it is essential to estimate digoxin exposure in the fetus. The data from ex vivo human placental perfusion study are used to predict in vivo fetal exposure noninvasively, but the ex vivo fetal-to-maternal concentration (F:M) ratios observed in digoxin perfusion studies were much lower than those in vivo. In the present study, we developed a human transplacental pharmacokinetic model of digoxin using previously reported ex vivo human placental perfusion data. The model consists of maternal intervillous, fetal capillary, non-perfused tissue, and syncytiotrophoblast compartments, with multidrug resistance protein (MDR) 1 and influx transporter at the microvillous membrane (MVM) and influx and efflux transporters at the basal plasma membrane (BM). The model-predicted F:M ratio was 0.66, which is consistent with the mean in vivo value of 0.77 (95% confidence interval: 0.64-0.91). The time to achieve the steady state from the ex vivo perfusion study was estimated as 1,500 minutes, which is considerably longer than the reported ex vivo experimental durations, and this difference is considered to account for the inconsistency between ex vivo and in vivo F:M ratios. Reported digoxin concentrations in a drug-drug interaction study with MDR1 inhibitors quinidine and verapamil were consistent with the profiles simulated by our model incorporating inhibition of efflux transporter at the BM in addition to MVM. Our modeling and simulation approach should be a powerful tool to predict fetal exposure and DDIs in human placenta. SIGNIFICANCE STATEMENT We developed a human transplacental pharmacokinetic model of digoxin based on ex vivo human placental perfusion studies in order to resolve inconsistencies between reported ex vivo and in vivo fetal-to-maternal concentration ratios. The model successfully predicted the in vivo fetal exposure to digoxin and the drug-drug interactions of digoxin and P-glycoprotein/multidrug resistance protein 1 inhibitors in human placenta.

© 2021 Elsevier Inc. Inadequate trophoblast invasion and impaired trophoblast-induced vascular remodeling are features of preeclampsia. In this context, an angiogenesis-related microRNA, miR-126, is abnormally expressed in preeclampsia placentas, but its role in trophoblast development remains unclear. The purpose of this study was to investigate the roles of miR-126 in the proliferation, migration, and invasion processes of trophoblast cells using the human choriocarcinoma-derived JEG-3 cell line as a model. The mRNA expression profiling of JEG-3 cells with and without miR-126 overexpression, in combination with bioinformatics analysis, identified LIN28A as a putative target of miR-126. The results of real-time RT-PCR and luciferase assay were consistent with this idea. Overexpression of miR-126 in JEG-3 cells decreased the invasive ability of the cells without affecting proliferation or migration. The invasiveness of JEG-3 cells was significantly reduced to a similar extent by knockdown of LIN28A with siRNA and by miR-126-overexpression-induced downregulation of LIN28A, although the level of LIN28A protein was much lower in the siLIN28A-transfected cells. These results indicate that miR-126 suppresses JEG-3 cell invasion by targeting LIN28A, and suggest that miR-126-mediated downregulation of LIN28A might contribute to the onset/deterioration of preeclampsia.

Purpose: The retina is a neural tissue especially differentiated for vision and, thus, the inner blood-retinal barrier (inner BRB) specific molecules may play an essential role in maintaining neural functions in the retina. The purpose of the present study was to identify selectively expressed genes at the inner blood-retinal barrier compared with the blood-brain barrier (BBB). Methods: A comparison of expressed genes between conditionally immortalized rat retinal (TR-iBRB) cell lines and brain capillary endothelial (TR-BBB) cell lines was performed using mRNA differential display analysis and quantitative real time PCR analysis. The rat M-cadherin gene was cloned by performing 5' RACE, and its protein expression was detected by immunoblot analysis. Results: Eight clones were identified as highly expressed genes in TR-iBRB cells including GATA-binding protein-3 (GATA-3), cytosolic branched chain amino transferase (BCATc), and M-cadherin (cadherin-15). The rat M-cadherin gene was cloned from TR-iBRB cells, for the first time, and has >86% amino acid sequence identity to the previously cloned mammalian M-cadherins. Rat M-cadherin expression in TR-iBRB cells was much greater than that in TR-BBB cells as far as mRNA and protein levels were concerned. Conclusions: M-cadherin, GATA-3, and BCATc are highly expressed in TR-iBRB cells compared with TR-BBB cells and may indeed be involved in unique functions at the inner BRB. © 2004 Molecular Vision.

The purpose of this study is to clarify the subcellular localizations of multidrug resistance protein 1 (MDR1)/ABCB1 and breast cancer resistance protein (BCRP)/ABCG2 in the rodent placental SynT bilayer, i.e., a maternal-facing (SynT-I) layer and a fetal-facing (SynT-II) layer. In double immunofluorescence staining, the signals of MDR1 and BCRP appeared midway between the signals of glucose transporter 1 on the apical membrane of SynT-I and the basal plasma membrane of SynT-II, and mostly overlapped with signals of connexin 26, which forms gap junctions between SynT-I and SynT-II. In detail, median intensities (pixels) of the MDR1 and BCRP signals were significantly closer to the fetal circulation as compared to the location of connexin 26 signals. In double in situ hybridization studies, the signals of Mdr1b mRNA mostly overlapped with those of Syncytin-B, a SynT-II marker. In conclusion, MDR1 and BCRP are expressed on apical membranes of the rodent placental SynT-II layer.

Brain and retinal capillary endothelial cells (BCECs and RCECs, respectively) exhibit a barrier structure and function. Comparison of gene expression in these cells could clarify the selective function of each barrier. The purpose of this study was to identify the genes selecti vely expressed at the blood-brain barrier (BBB) and to clarify the function of the selective gene, androgen receptor (AR). Gene expression was compared by a differential display using conditionally immortalized rat BCECs and RCECs (TR-BBB and TR-iBRB, respectively). A total of 12 gene fragments were identified as the selective genes dominantly expressed in TR-BBB cells. The most selective fragment in TR-BBB cells had the highest homology with the 3′-UTR of human and mouse AR. Rat AR mRNA was detected in TR-BBB cells and the brain capillary rich fraction, but not in TR-iBRB cells. Expression of organic anion transporter 3 (OAT3) mRNA in TR-BBB cells was induced by treatment with dihydrotestosterone (DHT), an AR ligand, and this induction was suppressed by flutamide. Moreover, uptake of benzylpenicillin by TR-BBB cells was also induced by DHT treatment. In contrast, OAT3 mRNA expression in TR-iBRB cells was not affected by DHT treatment. The brain-to-blood efflux rate of benzylpenicillin was not affected by gender. These results suggest that AR is involved in the functional regulation of OAT3 at the BBB, but not at the inner blood-retinal barrier (iBRB), and this regulation is not affected by gender. The BBB function will be affected by the androgen levels in the brain and/or plasma via AR. © 2005 Wiley-Liss, Inc.

The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell lines derived from newly developed tsA58/EGFP transgenic rats harboring the temperature-sensitive simian virus 40 (SV40) large T-antigen and enhanced green fluorescent protein (EGFP). Endothelial cells were isolated from the transgenic rats by intraluminal enzymatic digestion. The cloned cell lines were named TR-LE (temperature-sensitive rat lymphatic endothelial cells from thoracic duct) and TR-BE (temperature-sensitive rat blood-vessel endothelial cells from inferior vena cava), respectively, and cultured on fibronectin-coated dishes in HuMedia-EG2 supplemented with 20% fetal bovine serum and Endothelial Mitogen at a permissive temperature, 33°C. A temperature shift to 37°C resulted in a decrease in proliferation with degradation of the large T-antigen and cleavage of poly (ADP-ribose) polymerase. TR-LE cells expressed lymphatic endothelial markers VEGFR-3 (vascular endothelial growth factor receptor), LYVE-1 (a lymphatic endothelial receptor), Prox-1 (a homeobox gene product), and podoplanin (a glomerular podocyte membrane mucoprotein), together with endothelial markers CD31, Tie-2, and VEGFR-2, whereas TR-BE cells expressed CD31, Tie-2, and VEGFR-2, but no lymphatic endothelial markers. Thus, these conditionally immortalized and EGFP-expressing lymphatic and vascular endothelial cell lines might represent an important tool for the study of endothelial cell functions in vitro. © 2006 Springer-Verlag.

The purpose of this study was to establish and characterize a retinal pericyte cell line from retinal capillaries of transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), and to apply this to the co-culture with a retinal capillary endothelial cell line. The conditionally immortalized rat retinal pericyte cell lines (TR-rPCTs), which express a temperature-sensitive large T-antigen, were obtained from two tsA58 Tg rats. These cell lines had a multicellular nodule morphology and reacted positively with von Kossa staining, a marker of calcification. TR-rPCTs cells expressed mRNA of pericyte markers such as rat intercellular adhesion molecule-1, platelet-derived growth factor-receptor β, angiopoietin-1, and osteopontin. Western blot analysis indicated that α-smooth muscle actin (α-SMA) was expressed in TR-rPCT3 and 4 cells. In contrast, α-SMA was induced by transforming growth factor-β1 and its enhancement was reduced by basic fibroblast growth factor in TR-rPCT1 and 2 cells. When TR-rPCT1 cells were cultured with a rat retinal endothelial cell line (TR-iBRB2) in a contact co-culture system, the number of TR-iBRB2 cells were significantly reduced in comparison with that of a single culture of TR-iBRB2 cells, suggesting that physical contact between pericytes and retinal endothelial cells is important for the growth of retinal endothelial cells. In conclusion, conditionally immortalized retinal pericyte cell lines were established from tsA58 Tg rats. These cell lines exhibited the properties of retinal pericytes and can be applied in co-culture systems with a retinal capillary endothelial cell line.

The purpose of the present study was to quantify transporter gene levels at the inner blood-retinal barrier (inner BRB) using a combination of magnetic isolation method for rat retinal vascular endothelial cells (RVEC) and real-time quantitative PCR analysis. The transcript levels of CD31, Tie-2, claudin-5, occludin, Jam-1, mdr1a, oatp2, and oatp14 in the RVEC fraction were more than 100-fold greater than those in the non-RVEC fraction, suggesting that these genes are predominantly expressed at the inner BRB. The transcript levels of GLUT1 and MCT1 in the RVEC fraction were the most abundant in the respective transporter family, suggesting that GLUT1 and MCT1 play a predominant role in D-glucose and monocarboxylate transport, respectively, at the inner BRB. In conclusion, application of magnetically isolated RVEC is able to determine transporter gene levels at the inner BRB thereby increasing our understanding of inner BRB functions at a molecular level.