The inner blood-retinal barrier in diabetes

The inner blood-retinal barrier is highly complex, dependent on a normally functioning retinal vascular endothelium and possibly adjacent perivascular cells. There are undoubtedly many different grades of barrier failure, and in the early stages an inbuilt compensatory mechanism may prevent significant fluid accumulation and delay associated structural and functional changes. In diabetes, early barrier failure may be a function of an increase in the rate of endothelial cell death and turnover, probably exaggerated by the early and widespread loss of the supporting cells and dilatation of the small blood vessels.     

Breakdown of the outer blood-retinal barrier in experimental commotio retinae

An animal model has been developed in this study to produce commotio retinae (Berlin's edema) by means of standardized, non-penetrating B.B. pistol injury to the cornea. Parameters have been established to ensure uniform blunt injury, enabling study of the retina and blood-retinal barrier by light- and electron microscopy, including use of horseradish peroxidase as a vascular tracer. Retinal whitening and swelling were found in the peripapillary and central regions, as occurs in the human. The earliest damage involved breakage of the connecting cilia of rods and cones, with rapid disorganization of the outer segments. Later changes included swelling of photoreceptor inner segments and breakdown of the outer blood-retinal barrier at the level of the retinal pigment epithelium. The outer blood-retinal barrier was re-established between 7 and 14 days, and at the longest survival (56 days), incompletely regenerated outer segments were present. This model for commotio retinae provides a new approach for the study of outer-segment regeneration in rods and cones after mechanical injury, as well as mechanisms that underlie re-establishment of the blood-retinal barrier following non-penetrating trauma to the eye.     

Model experiments of the outer blood-retinal barrier in vitro]

Because of the difficulty in conducting experiments on the outer blood-retinal barrier in vivo, we developed an in vitro model. Bovine retinal pigment epithelial cells were grown on semipermeable membranes, enabling separate manipulation of the apical and basal medium. As a parameter of barrier function, we measured the transepithelial resistance (TER). Barrier function was also tested with fluorescein. The transepithelial resistance increased under optimal culture conditions, in confluent cultures, by 200 omega and there was no fluorescein leakage. After exposure to trypsin in Ca/Mg-less medium or EDTA or after application of argon laser, we were able to induce a breakdown of the TER and fluorescein leakage. This happened immediately after laser exposure, 1 min after EDTA, and 4 min after trypsin application. We observed no morphological differences after breakdown of the barrier function on the intercellular connections compared to normal confluent cultures following EDTA or trypsin exposure. In all experiments there was a recovery of barrier function after returning the cells to control conditions. These first results demonstrate that our in vitro model is a sensitive method for investigating barrier function in retinal pigment epithelium in cell culture.     

In vivo imaging of breakdown of the inner and outer blood-retinal barriers.

Real-time contrast-enhanced magnetic resonance imaging (MRI) was used to distinguish between experimentally induced breakdown of the vascular (inner) and retinal pigment epithelial (RPE; outer) blood-retinal barrier (BRB) in vivo. Pigmented rabbits were treated with intravenous sodium iodate 30 mg/kg, (a specific RPE cell poison), intravitreal N-ethylcarboxamidoadenosine (NECA) 10(-3) mol/l (which specifically disrupts the vascular BRB), or retinal diode laser photocoagulation. Coronal T1-weighted proton images were acquired in a timed sequence after intravenous injection of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA). Images were analyzed to localize leakage of Gd-DTPA and determine the permeability surface area product normalized per unit area (PS). The pattern of enhancement observed in eyes treated with sodium iodate differed clearly from that in eyes treated with NECA. PS' values were significantly higher in eyes treated with sodium iodate than with NECA. Simultaneous leakage from the outer and inner BRB in eyes treated with dense retinal laser photocoagulation could be localized and quantitated independently.     

Influence of vital dyes on the function of the outer blood-retinal barrier in vitro

BACKGROUND: The use of vital dyes in "chromovitrectomy" allows the easier removal of less recognizable structures like epiretinal membranes (EM) or the inner limiting membrane (ILM). In recent years numerous studies demonstrated the use of indocyanine green (ICG), trypan blue (TB) and patent blue (PB) for this indication. Reports of the possible risk of these dyes, i. e. especially ICG, in respect of reduced visual acuity results, possible visual field defects or alterations of the retinal pigment epithelium (RPE) resulted in limitations in their application. MATERIAL AND METHODS: Human RPE cells and choroidal endothelial cells were cultured in monolayers on semipermeable membranes representing an in vitro model of the outer blood-retinal barrier. By measurement of the transepithelial electrical resistance (TER) the stable barrier function was determined. Two different models representing an air-filled and a fluid-filled eye were tested on the one hand by addition of the dye to the culture medium and, on the other, by direct application on the cell monolayer. In these two models ICG (5 mg/ml, 0.5 mg/ml, 0.125 mg/ml), TB (1.5 mg/ml, 0.15 mg/ml) and PB (2.4 mg/ml, 0.24 mg/ml) were applied for three minutes and the influence on the barrier function was determined. RPE cell growth was also tested in these two models after the application of ICG, TB and PB. Finally, monolayers of RPE cells were evaluated by transmission electron microscopy (TEM). RESULTS: After application of TB, PB and the lowest concentration of ICG of 0.125 mg/ml, the TER remained stable in both models. In contrast, ICG in a concentration of 5 mg/ml and 0.5 mg/ml caused a significant TER decrease in the model of the air-filled eye, whereas no influence on the function of the outer blood-retinal barrier was noted in the model of the fluid-filled eye. RPE cell growth rates were not influenced by the addition of the vital dyes, with the exception of ICG in a concentration of 5 mg/ml in the model of the air-filled eye, resulting in a temporary reduction of the cell count. In good correspondence to these results also in TEM intercellular blisters were noted after application of 5 mg/mL ICG for 3 minutes in the model of the air-filled eye. However, damage to the RPE cells themselves was not obvious. No pathological changes in the TEM were noted after application of TB and PB. CONCLUSIONS: The use of PB and TB at the posterior eye segment seems to be safe concerning damage to the PRE and its barrier function. In contrast, ICG in higher concentrations and with longer application times may cause a toxic effect on RPE morphology and function.     

Enzymatic digestion increases permeability of the outer blood-retinal barrier for high-molecular-weight substances

Background: The purpose of the study was to investigate whether lysosomal enzymes can participate in damaging the outer blood-retinal barrier and to examine the role of glycosaminoglycans in maintaining the barrier function for high-molecular-weight substances. Methods: The ciliary artery was cannulated in freshly enucleated pig eyes. Perfusion was performed with buffer (controls), with heparinase (substrate: heparan sulfate), or with lysosomal enzymes freshly prepared from pig retinal pigment epithelium at 36° C, followed by perfusion with the tracer native ferritin (NF) or the marker cationized ferritin (CF). The eyes were examined by electron microscopy. Results: In controls treated with buffer alone, NF was found in high concentration in the lumina of the choroidal capillaries; however, little NF was found in Bruch's membrane (BsM). The tracer did not penetrate to any extent beyond BsM. In eyes digested with heparinase or lysosomal enzymes, significantly higher numbers of tracer molecules were found in BsM. Furthermore, NF penetrated BsM and was apparent in the subretinal space and also inside retinal pigment epithelial cells, probably due to pinocytosis. Conclusions: The results indicate that heparan sulfate proteoglycan is important for the maintenance of the outer blood-retinal barrier and that lysosomal proteases may participate in damaging this barrier, causing increased permeability to high-molecular-weight substances.Presented in part at the Association for Research in Vision and Ophthalmology Annual Meeting, Sarasota, Florida, 28 April 1991     

Repair of outer blood-retinal barrier after severe ocular blunt trauma in rabbits

Retinal contusion is a leading cause of visual loss in ocular blunt trauma. However, its pathogenesis remains controversial. We established a rabbit model of severe retinal contusion with energy of about 2.87 J. Typical retinal edema and sometimes subretinal hemorrhage reproducibly occurred at the posterior pole after injury. These subsided 1 week later with depigmentation in the lesion. Histopathological examination revealed severe damage of the outer layer of retina, for example, disruption of retinal pigment epithelium (RPE) and photoreceptors. Electroretinography showed a decrease in the b wave by 38–47% in amplitudes (P<0.01) during the first 3 days and then returned, although not to normal level. To investigate the damage and repair of blood-retinal barrier (BRB), 5 ml 2% lanthanum solution (La) was injected via the common carotid artery 1–2 min before enucleation. La diffused in the interphotoreceptor space through the damaged junctions of RPE 1 h-3 days after injury. La also reached the nuclei level of photoreceptors up to 14 days after injury. Although a glial scar with scattered RPE cells attached to Bruch's membrane in the severely damage area, no La diffusion was found in the retina 4 weeks after trauma. These results showed incomplete repair of outer BRB after severe blunt trauma.     

Mitochondrial expression and activity of P-glycoprotein under oxidative stress in outer blood-retinal barrier

AIM: To investigate the role of oxidative stress in regulating the functional expression of P-glycoprotein (P-gp) in mitochondria of D407 cells. METHODS: D407 cells were exposed to different ranges of concentrations of H2O2. The mitochondrial location of P-gp in the cells subjected to oxidative stress was detected by confocal analysis. Expression of P-gp in isolated mitochondria was assessed by Western blot. The pump activity of P-gp was evaluated by performing the efflux study on isolated mitochondria with Rhodamine 123 (Rho-123) alone and in the presence of P-gp inhibitor (Tariquidar) using flow cytometry analysis. The cells were pretreated with 10 mmol/L N-acetylcysteine (NAC) for 30min before exposing to H2O2, and analyzed the mitochondrial extracts by Western blot and flow cytometry. RESULTS: P-gp was co-localized in the mitochondria by confocal laser scanning microscopy, and it was also detected in the mitochondria of D407 cells using Western blot. Exposure to increasing concentrations of H2O2 led to gradually increased expression and location of P-gp in the mitochondria of cells. Rho-123 efflux assay showed higher uptake of Rho-123 on isolated mitochondria in the presence of Tariquidar both in normal and oxidative stress state. H2O2 up-regulated P-gp in D407 cells, which could be reversed by NAC treatment. CONCLUSION: H2O2 could up-regulate the functional expression of P-gp in mitochondria of D407 cells, while antioxidants might suppress oxidative-stress-induced over-expression of functional P-gp. It is indicative that limiting the mitochondrial P-gp transport in retinal pigment epithelium cells would be to improve the effect of mitochondria-targeted antioxidant therapy in age-related macular degeneration-like retinopathy.     

Involvement of moesin phosphorylation in ischemia/reperfusion induced inner blood-retinal barrier dysfunction

AIM: To investigate the role of moesin and its underlying signal transduction in retinal vascular damage induced by retinal ischemia-reperfusion(RIR) insult.METHODS: C57 BL/6 mice were subjected to continued ischemia for 45 min, followed by blood reperfusion. The expression and phosphorylation of moesin in retinal vessels were detected by immunohistochemistry and Western blotting. The inner blood-retinal barrier was evaluated using FITCdextran leakage assay on whole-mount retina. Further studies were conducted to explore the effects of p38 mitogen-activated protein kinase(MAPK) pathway on the involvement of moesin in RIR-evoked retinal vascular hyperpermeability response. RESULTS: It revealed that RIR induced moesin phosphorylation in a time-dependent manner after reperfusion. The phosphorylation of moesin was alleviated by inhibitions of p38 MAPK, while this treatment also ameliorated the dysfunction of inner blood-retinal barrier. CONCLUSION: The results suggest that moesin is involved in RIR-evoked retinal vascular endothelial dysfunction and the phosphorylation of moesin is triggered via p38 MAPK activation.     

L-type amino acid transporter 1-mediated L-leucine transport at the inner blood-retinal barrier

PURPOSE: L-type amino acid transporters (LATs) prefer branched-chain and aromatic amino acids, including neurotransmitter precursors. The objective of this study was to clarify the expression and function of LAT at the inner blood-retinal barrier (BRB). METHODS: [3H]L-Leucine transport at the inner BRB was characterized by using in vivo integration plot analysis and a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2). The expression of the LAT1 was demonstrated by quantitative real-time RT-PCR, immunoblot, and immunohistochemical analyses. RESULTS: The apparent influx permeability clearance of [3H]L-leucine in the rat retina was found to be 203 microL/(min.g retina), supporting a carrier-mediated influx transport of L-leucine at the BRB. [3H]L-Leucine uptake by TR-iBRB2 cells was an Na+-independent and concentration-dependent process with a Km of 14.1 microM. This process was more potently cis inhibited by substrates of LAT1, D-leucine, D-phenylalanine, and D-methionine, than those of LAT2, L-alanine, and L-glutamine. [3H]L-Leucine efflux from TR-iBRB2 cells was trans-stimulated by substrates of LAT1. The expression of LAT1 mRNA was 100- and 15-fold greater than that of LAT2 in TR-iBRB2 and magnetically isolated rat retinal vascular endothelial cells, respectively. The expression of LAT1 protein was observed in TR-iBRB2 and primary cultured human retinal endothelial cells and immunostaining of LAT1 was observed along the rat retinal capillaries. CONCLUSIONS: LAT1 is expressed at the inner BRB and mediates blood-to-retina L-leucine transport. This transport system plays a key role in maintaining large neutral amino acids as well as neurotransmitters in the neural retina.     

胚胎发育中神经视网膜对血视网膜外屏障结构形成的作用

目的 研究胚胎发育中神经视网膜对血视网膜外屏障结构形成的作用。 方法 将孵化7、10、14 d的鸡胚眼各150、120和90只，分批分别剥离视网膜神经上皮层（RNE）和色素上皮层（RPE）。RNE用来制备不同条件的培养液（7drcS F3、10drcSF3和14drcSF3），将7 d和14 d的RPE细胞分别培养到12 mmTranswell滤膜上,其顶房分别用SF3或上述条件培养液，底房均用SF2培养液。形成单层RPE细胞后，测定此上皮层的电阻(TER)。最后固定细胞，用细胞免疫组织化学方法和透射电子显微镜检测RPE细胞间紧密连接形成的情况。 结果 不同培养液(SF3/S F2, 7drcSF3/SF2,10drcSF3/SF2, 14drcSF3/SF2)培养7d和14d鸡胚眼的RPE细胞TER之间的差异有非常显著性的意义(P＜0.01)。电子显微镜结果显示神经视网膜条件培养液可以使RPE细胞产生细胞极性，有效地促使紧密连接条带形成连续的网状结构。 结论 神经视网膜对血视网膜外屏障结构的形成起积极的促进作用。 （中华眼底病杂志，2004，20：237-240）     

补肾活血中药方血清对离体状态下血-视网膜内屏障模型的保护作用

目的　探讨补肾活血中药方血清对离体状态下血-视网膜内屏障（iBRB）模型的保护作用。方法　取新生7 d SD大鼠用于取材及细胞原代培养。将大鼠视网膜微血管内皮细胞（RMEC）和视网膜Müller胶质细胞（RMGC）分离及传代培养,利用Transwell小室将RMEC与RMGC共培养以构建正常条件下的体外iBRB模型,制备补肾活血中药方血清,并分别标记为中药含药血清和空白血清。本实验分8组。选取构建成功的体外iBRB细胞共培养模型,按不同培养条件进行分组。其中正常对照组用含体积分数20%空白血清的DMEM液培养;中药干预正常组用含体积分数20%中药含药血清的DMEM液培养;低糖基化终末产物（AGEs)组用含体积分数20%空白血清的DMEM液及终浓度50 mg·L^-1AGEs培养;中药干预低AGEs组用含体积分数20%中药含药血清的DMEM液及终浓度50 mg·L^-1AGEs培养;高AGEs组用含体积分数20%空白血清的DMEM液及终浓度100 mg·L^-1AGEs培养;中药干预高AGEs组用含体积分数20%中药含药血清的DMEM液及终浓度100 mg·L^-1AGEs培养;缺氧组用含体积分数20%空白血清的DMEM液及终浓度1.0 mmol·L^-1连二亚硫酸钠培养;中药干预缺氧组用含体积分数20%中药含药血清的DMEM液及终浓度1.0 mmol·L^-1连二亚硫酸钠培养。利用细胞电阻仪检测各组RMEC层的跨内皮细胞电阻（TEER）,采用免疫荧光双标法检测各组Occludin、ZO-1蛋白在RMEC层的表达。结果　在AGEs或缺氧条件下,低AGEs组、高AGEs组及缺氧组RMEC层TEER在细胞培养24 h、48 h和72 h后均低于正常对照组,差异均有统计学意义（均为P<0.05）。在细胞培养24 h后,中药干预正常组与正常对照组RMEC层TEER相比,差异无统计学意义（P>0.05）,在细胞培养48 h后中药干预正常组RMEC层TEER高于正常对照组,差异有统计学意义（P<0.05）,在细胞培养72 h后中药干预正常组RMEC层TEER低于正常对照组,差异有统计学意义（P<0.05）;中药干预低AGEs组、中药干预高AGEs组、中药干预缺氧组RMEC层TEER在细胞培养24 h、48 h和72 h后均高于同一时段相对应的非中药干预组,差异均有统计学意义（均为P<0.05）。低AGEs组、高AGEs组及缺氧组Occludin、ZO-1蛋白在RMEC层的阳性表达在细胞培养24 h、48 h和72 h后均弱于正常对照组。在细胞培养24 h、48 h和72 h后,中药干预正常组、中药干预低AGEs组、中药干预高AGEs组、中药干预缺氧组Occludin、ZO-1蛋白在RMEC层的阳性表达均强于同一时段相对应的非中药干预组。结论　AGEs及缺氧均可导致体外iBRB模型RMEC层TEER降低及Occludin、ZO-1蛋白表达减弱,补肾活血中药方血清能有效提高Occludin 和ZO-1蛋白的表达,抑制iBRB模型通透性增加,从而起到对体外iBRB模型的保护作用。     

Effect of photodynamic therapy on the function of the outer blood-retinal barrier in an in vitro model

Background Photodynamic therapy (PDT) is a well established clinical treatment for age-related macular degeneration (AMD), and comprises intravenous injection of verteporfin and subsequent application of a non-thermal laser beam to the area of AMD to induce selective vascular occlusion. Since there is evidence that PDT may cause outer blood-retinal barrier (BRB) breakdown and possibly RPE cell alteration, we investigated the effect of PDT on the BRB function of the RPE in an in vitro model.Methods Twenty-one monolayers of human RPE cells were cultured on semipermeable membranes until a stable barrier function was achieved as determined by transepithelial electrical resistance (TER) and sodium fluorescein permeability. To test the effect of PDT on the outer BRB function, non-thermal laser (692 nm), verteporfin or a combination of both were applied. TER assessment prior to and after PDT was utilized to identify changes in barrier function of the RPE in this in vitro model. Finally, monolayers of RPE cells were evaluated by transmission electron microscopy (TEM).Results No significant TER decrease was observed after application of non-thermal laser alone or after administration of verteporfin in therapeutic concentrations, but combination of these modalities resulted in significantly decreased TER within 4 h. Except for intercellular blisters, no damage to the RPE was evident in TEM. Verteporfin added at concentrations higher than therapeutic doses (2 mg/ml) resulted in an immediate decrease in TER and damage to the RPE cells.Conclusion The combination of a therapeutic concentration of verteporfin and application of non-thermal laser resulted in a morphologically and functionally detectable breakdown of the outer BRB function of the RPE without any damage to the RPE cells themselves in vitro. However, increasing the concentration of verteporfin can result in RPE cell damage.     

Detection of destruction of anionic sites in the outer blood-retinal barrier and damage caused by iron]

We studied the alteration of the anionic charge in the outer blood-retinal barrier in ocular siderosis by electron microscopy using the cationic probe, polyethyleneimine (PEI). The eyes of four days after injection of iron powder and control rabbit eyes demonstrated numerous PEI-positive sites at the basement membrane of the retinal pigment epithelium, collagen fiber of Bruch's membrane, and the basement membrane of the choriocapillaris. Seven days after injection of iron powder, no change was observed in the retina by light microscopy, but PEI positive sites at the basement membrane of the retinal pigment epithelium were decreased. At 14 days, it was seen that the outer layer of the retina disappeared and was replaced by pigment containing macrophages and Müller cells. Berlin blue reaction became positive in some of those macrophages and retinal pigment epithelia. PEI positive sites decreased. At 28 days, the retina was completely disorganized, and became thin, while the retinal pigment epithelium proliferated and became double layered. Berlin blue reaction was strongly positive in pigment-laden macrophages and retinal pigment epithelium. PEI-positive sites became scarce. As a result of these methods, anionic sites decreased, despite negative iron reaction of 7 days after injection of iron powder. It was shown that in ocular siderosis at an early stage, the barrier function of anionic sites between choroid and retina decreased before histological change.     

Outward permeability of the blood-retinal barrier

To characterize quantitatively the active transport mechanism of the blood-retinal barrier (BRB), we estimated the inward (Pin) and outward permeability (P_out) of the BRB in monkey eyes using vitreous fluorophotometry. P_in values for fluorescein (F) and fluorescein glucuronide (FG) were simulated by computer in separate experiments following measurements of intraocular fluorescence at 1 h following the intravenous injection of F or FG. The estimated mean P_in values for F and FG were 4.7 ± 1.6 and 5.9 ± 1.7 × 10^–6 cm/min, respectively, in ten eyes of five monkeys. At 1 month thereafter, F was injected into the right vitreous cavity and the same amount of FG was injected into the left vitreous cavity. The intraocular kinetics of fluorescence were measured at 6–24 h postinjection. As estimated by the simulation model, the mean P_out values were 7.7 ± 2.6 and 1.7 ± 0.9 × 10^–4 cm/min for F and FG, respectively. The P_out/P_in ratio was 160±78 for F and 26±9 for FG. The intraperitoneal injection of probenecid resulted in a significant decrease in the P_out value for F (P < 0.005) but had no significant effect on that for FG, suggesting that F is excreted from the retina via an active transport mechanism; as characterized by the P_out/P_in ratio, the actual magnitude of the latter is far greater than that previously reported.This research was supported in part by a 1988 Grant-in-Aid for the Encouragement of Young Scientists (63771366, to A.Y.) from the Ministry of Education, Science and Culture, Japan Offprint requests to: A. Yoshida     

Molecular basis of the inner blood-retinal barrier and its breakdown in diabetic macular edema and other pathological conditions

Breakdown of the inner endothelial blood-retinal barrier (BRB), as occurs in diabetic retinopathy, age-related macular degeneration, retinal vein occlusions, uveitis and other chronic retinal diseases, results in vasogenic edema and neural tissue damage, causing loss of vision. The central mechanism of altered BRB function is a change in the permeability characteristics of retinal endothelial cells caused by elevated levels of growth factors, cytokines, advanced glycation end products, inflammation, hyperglycemia and loss of pericytes. Subsequently, paracellular but also transcellular transport across the retinal vascular wall increases via opening of endothelial intercellular junctions and qualitative and quantitative changes in endothelial caveolar transcellular transport, respectively. Functional changes in pericytes and astrocytes, as well as structural changes in the composition of the endothelial glycocalyx and the basal lamina around BRB endothelium further facilitate BRB leakage. As Starling's rules apply, active transcellular transport of plasma proteins by the BRB endothelial cells causing increased interstitial osmotic pressure is probably the main factor in the formation of macular edema. The understanding of the complex cellular and molecular processes involved in BRB leakage has grown rapidly in recent years. Although appropriate animal models for human conditions like diabetic macular edema are lacking, these insights have provided tools for rational design of drugs aimed at restoring the BRB as well as for design of effective transport of drugs across the BRB, to treat the chronic retinal diseases such as diabetic macular edema that affect the quality-of-life of millions of patients.     

Blood-to-retina transport of biotin via Na-dependent multivitamin transporter (SMVT) at the inner blood-retinal barrier

The purpose of this study was to elucidate the mechanisms of biotin transport across the inner blood-retinal barrier (inner BRB). [³H]Biotin transport in the retina across the inner BRB was examined using an in vivo integration plot and retinal uptake index analyses in rats. The transport mechanism was characterized using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells) as an in vitro inner BRB model. The apparent influx permeability clearance (K_in) per gram retina of [³H]biotin was found to be 5.55 μL/(min g retina). The K_in of [³H]biotin was 8.9-fold greater than that of [³H]D-mannitol, a non-permeable paracellular marker. [³H]Biotin uptake by the retina was found to be significantly inhibited by biotin and pantothenic acid, supporting carrier-mediated influx transport of biotin at the inner BRB. [³H]Biotin uptake by TR-iBRB2 cells was Na⁺-, temperature-, and concentration-dependent with a K_m of 146 μM. These forms of transport were significantly inhibited by Na⁺-dependent multivitamin transporter (SMVT) substrates such as biotin, pantothenic acid, lipoic acid, and desthiobiotin. These transport properties are consistent with those of biotin transport by SMVT. SMVT mRNA was expressed in TR-iBRB2 cells and isolated rat retinal vascular endothelial cells. Our findings suggest that SMVT is involved in the transport of biotin from the circulating blood to the retina across the inner BRB.