Ken-ichi Hosoya

A pericyte-derived angiopoietin-1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro.

Although tight‐junctions (TJs) at the blood–brain barrier (BBB) are important to prevent non‐specific entry of compounds into the CNS, molecular mechanisms regulating TJ maintenance remain still unclear. The purpose of this study was therefore to identify molecules, which regulate occludin expression, derived from astrocytes and pericytes that ensheathe brain microvessels by using conditionally immortalized adult rat brain capillary endothelial (TR‐BBB13), type II astrocyte (TR‐AST4) and brain pericyte (TR‐PCT1) cell lines. Transfilter co‐culture with TR‐AST4 cells, and exposure to conditioned medium of TR‐AST4 cells (AST‐CM) or TR‐PCT1 cells (PCT‐CM) increased occludin mRNA in TR‐BBB13 cells. PCT‐CM‐induced occludin up‐regulation was significantly inhibited by an angiopoietin‐1‐neutralizing antibody, whereas the up‐regulation by AST‐CM was not. Immunoprecipitation and western blot analyses confirmed that multimeric angiopoietin‐1 is secreted from TR‐PCT1 cells, and induces occludin mRNA, acting through tyrosine phosphorylation of Tie‐2 in TR‐BBB13 cells. A fractionated AST‐CM study revealed that factors in the molecular weight range of 30–100 kDa led to occludin induction. Conversely, occludin mRNA was reduced by transforming growth factor β1, the mRNA of which was up‐regulated in TR‐AST4 cells following hypoxic treatment. In conclusion, in vitro BBB model studies revealed that the pericyte‐derived multimeric angiopoietin‐1/Tie‐2 pathway induces occludin expression.

New approaches to in vitro models of blood-brain barrier drug transport.

The pharmaceutical industry has been searching for an in vitro blood-brain barrier (BBB) model that preserves in vivo transporter functions in CNS drug discovery and development. The application of conditionally immortalized cell lines derived from transgenic animals harboring temperature-sensitive SV40 large T-antigen gene, is a rational and promising approach to such a workable in vitro BBB model. The established brain capillary endothelial cell lines retain the in vivo transport rate of several compounds and various forms of gene expression. Furthermore, this new approach has enabled the development of stable and reproducible co-culture models with a pericyte cell line and/or an astrocyte cell line.

Role of blood-brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate, a uremic toxin: Its involvement in neurotransmitter metabolite clearance from the brain

Renal impairment is associated with CNS dysfunctions and the accumulation of uremic toxins, such as indoxyl sulfate, in blood. To evaluate the relevance of indoxyl sulfate to CNS dysfunctions, we investigated the brain‐to‐blood transport of indoxyl sulfate at the blood–brain barrier (BBB) using the Brain Efflux Index method. [3H]Indoxyl sulfate undergoes efflux transport with an efflux transport rate of 1.08 × 10−2/min, and the process is saturable with a Km of 298 µm. This process is inhibited by para‐aminohippuric acid, probenecid, benzylpenicillin, cimetidine and uremic toxinins, such as hippuric acidand 3‐carboxy‐4‐methyl‐5‐propyl‐2‐furanpropanoic acid. RT–PCR revealed that an OAT3 mRNA is expressed in conditionally immortalized rat brain capillary endothelial cell lines and rat brain capillary fraction. Xenopus oocytes expressing OAT3 were found to exhibit [3H]indoxyl sulfate uptake, which was significantly inhibited by neurotransmitter metabolites, such as homovanillic acid and 3‐methoxy‐4‐hydroxymandelic acid, and by acyclovir, cefazolin, baclofen, 6‐mercaptopurine, benzoic acid, and ketoprofen. These results suggest that OAT3 mediates the brain‐to‐blood transport of indoxyl sulfate, and is also involved in the efflux transport of neurotransmitter metabolites and drugs. Therefore, inhibition of the brain‐to‐blood transport involving OAT3 would occur in uremia and lead to the accumulation of neurotransmitter metabolites and drugs in the brain.

Thioredoxin interacting protein (TXNIP) induces inflammation through chromatin modification in retinal capillary endothelial cells under diabetic conditions

Chronic hyperglycemia and activation of receptor for advanced glycation end products (RAGE) are known risk factors for microvascular disease development in diabetic retinopathy. Thioredoxin‐interacting protein (TXNIP), an endogenous inhibitor of antioxidant thioredoxin (TRX), plays a causative role in diabetes and its vascular complications. Herein we investigate whether HG and RAGE induce inflammation in rat retinal endothelial cells (EC) under diabetic conditions in culture through TXNIP activation and whether epigenetic mechanisms play a role in inflammatory gene expression. We show that RAGE activation by its ligand S100B or HG treatment of retinal EC induces the expression of TXNIP and inflammatory genes such as Cox2, VEGF‐A, and ICAM1. TXNIP silencing by siRNA impedes RAGE and HG effects while stable over‐expression of a cDNA for human TXNIP in EC elevates inflammation. p38 MAPK‐NF‐κB signaling pathway and histone H3 lysine (K) nine modifications are involved in TXNIP‐induced inflammation. Chromatin immunoprecipitation (ChIP) assays reveal that TXNIP over‐expression in EC abolishes H3K9 tri‐methylation, a marker for gene inactivation, and increases H3K9 acetylation, an indicator of gene induction, at proximal Cox2 promoter bearing the NF‐κB‐binding site. These findings have important implications toward understanding the molecular mechanisms of ocular inflammation and endothelial dysfunction in diabetic retinopathy. J. Cell. Physiol. 221: 262–272, 2009.

The blood-brain barrier creatine transporter is a major pathway for supplying creatine to the brain.

Although creatine plays a pivotal role in the storage of phosphate-bound energy in the brain, the source of cerebral creatine is still unclear. The authors examined the contribution made by the creatine transporter (CRT) at the blood–brain barrier in supplying creatine to the brain from blood. An in vivo intravenous administration study suggested that creatine is continuously transported from the blood to the brain against the creatine concentration gradient that exists between brain and blood. Conditionally immortalized mouse brain capillary endothelial cells (TM-BBB) exhibited creatine uptake, which is Na+ and Cl− dependent and inhibited by CRT inhibitors, such as β-guanidinopropionate and guanidinoacetate. Northern blot and immunoblot analyses demonstrated that CRT is expressed in TM-BBB cells and isolated mouse brain microvessels. Moreover, high expression of CRT was observed in the mouse brain capillaries by confocal immunofluorescent microscopy. These results suggest that CRT plays an important role in supplying creatine to the brain via the blood–brain barrier.

Rat Organic Anion Transporter 3 (rOAT3) is Responsible for Brain-to-Blood Efflux of Homovanillic Acid at the Abluminal Membrane of Brain Capillary Endothelial Cells

The mechanism that removes homovanillic acid (HVA), an end metabolite of dopamine, from the brain is still poorly understood. The purpose of this study is to identify and characterize the brain-to-blood HVA efflux transporter at the rat blood–brain barrier (BBB). Using the Brain Efflux Index method, the apparent in vivo efflux rate constant of [3H]HVA from the brain, keff, was determined to be 1.69 × 10–2 minute–1. This elimination was significantly inhibited by para-aminohippuric acid (PAH), benzylpenicillin, indoxyl sulfate, and cimetidine, suggesting the involvement of rat organic anion transporter 3 (rOAT3). rOAT3-expressing oocytes exhibited [3H]HVA uptake (Km = 274 μmol/L), which was inhibited by several organic anions, such as PAH, indoxyl sulfate, octanoic acid, and metabolites of monoamine neurotransmitters. Neurotransmitters themselves did not affect the uptake. Furthermore, immunohistochemical analysis suggested that rOAT3 is localized at the abluminal membrane of brain capillary endothelial cells. These results provide the first evidence that rOAT3 is expressed at the abluminal membrane of the rat BBB and is involved in the brain-to-blood transport of HVA. This HVA efflux transport system is likely to play an important role in controlling the level of HVA in the CNS.

Blood—Brain Barrier Is Involved in the Efflux Transport of a Neuroactive Steroid, Dehydroepiandrosterone Sulfate, via Organic Anion Transporting Polypeptide 2

Abstract: We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood—brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [3H]DHEAS evaluated by the brain efflux index method was 2.68 × 10‐2 min‐1. DHEAS efflux transport was a saturable process with a Michaelis constant (Km) of 32.6 μM. Significant amounts of [3H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [3H]DHEAS was significantly inhibited by common rat organic anion‐transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone‐3‐sulfate. Moreover, the apparent efflux clearance of [3H]DHEAS across the BBB (118 μl/min‐g of brain) was 10.4‐fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 μl/min‐g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM‐BBB4), [3H]DHEAS uptake by TM‐BBB4 cells exhibited a concentration dependence with a Km of 34.4 μM and was significantly inhibited by the oatp2‐specific substrate digoxin. Conversely, [3H]digoxin uptake by TM‐BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [3H]DHEAS at 30 min was significantly increased under ATP‐depleted conditions, suggesting that an energy‐dependent efflux process may also be involved in TM‐BBB4. RT‐PCR and sequence analysis suggest that an oatp2 is expressed in TM‐BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT‐PCR suggest that there is oatp2‐mediated DHEAS transport at the BBB.

Screening of cationic compounds as an absorption enhancer for nasal drug delivery

Several cationic compounds were screened as potential nasal absorption enhancers to increase intranasal absorption of a model drug, fluorescein isothiocyanate labeled dextran (MW 4.4 kDa, FD-4), without nasal membrane damage in rats. Their effects were compared with those of classical enhancers. Various cationic compounds (poly-L-arginines with different molecular weights (MW 8.9, 45.5 and 92.0 kDa, poly-L-Arg (10), (50) and (100), respectively), L-arginine (L-Arg), L-lysine (L-Lys), and cetylpyridinium chloride (CPCL) were evaluated. Of the cationic compounds, poly-L-Arg and CPCL greatly enhanced the intranasal absorption of FD-4, as did chitosan, a cationic polysaccharide which has been reported to show a great effect on the transnasal delivery of peptide and protein drugs. The enhancing intensity by poly-L-Arg was dependent on its molecular weight. Rank order of the enhancing ratio, calculated from the AUC ratio for the enhancer treatment against the untreatment, was 0.5% poly-L-Arg (100) congruent with0.5% sodium dodecylsulfate congruent with0.5% CPCL?0.5% poly-L-Arg (50)?0.5% sodium deoxycholate congruent with0.5% sodium taurodihydrofusidate?0.5% polyoxyethylene-9-lauryl ether congruent with0.5% lysophosphatidylcholine?0.5% chitosan congruent with0.5% poly-L-Arg (10)>/=10% L-Arg congruent with10% L-Lys?0.5% sodium glycocholate congruent with0.5% sodium taurocholate congruent with0.5% EDTA. Only the poly-L-Args represented almost the same degree of hemolysis of cationic compounds compared with pH 7.0 phosphate buffered saline in the rat erythrocyte lysis experiment. The enhancing ratio by classical enhancers correlated with leaching of protein, phospholipids and LDH from isolated rabbit nasal mucosa. CPCL also fell on the regression lines between the enhancing ratio and their degree of leaching from classical enhancers. In contrast, the enhancing intensities by poly-L-Arg (10), (50) and (100) were greatly shifted from the regression line: the amount of leaching was markedly low in spite of a great enhancement of FD-4 absorption. These findings suggest that of the assessed enhancers only the poly-L-Args enhance the transnasal delivery of high molecular substances without severe damage to the nasal mucosal membrane. Poly-L-Arg is therefore a promising candidate having a good balance between enhancing activity and safety for nasal peptide and protein delivery.

Brain Insulin Impairs Amyloid-β(1-40) Clearance from the Brain

Cerebral amyloid-β peptide (Aβ) clearance plays a key role in determining the brain level of Aβ; however, its mechanism remains unclear. In this study, we investigated cerebral Aβ clearance across the blood-brain barrier (BBB) by using the Brain Efflux Index method. [125I]Aβ(1-40) was eliminated from rat brain to circulating blood with a half-life of 48.8 min and a half-saturation concentration of 8.15 nm. The Aβ(1-40) elimination rate was reduced by 30.5% in 23-month-old rats compared with 7-week-old rats. The intact form of Aβ(1-40) was detected in plasma after intracerebral administration, indicating the occurrence of efflux transport of intact Aβ(1-40). The Aβ(1-40) elimination rate was significantly inhibited by coadministration of 100 μg/ml insulin and 1 mm thiorphan by 44.6 and 34.0%, respectively. The level of intact [125I]Aβ(1-40) in the brain was increased by coadministration of insulin. Among insulin-degrading enzyme inhibitors, bacitracin inhibited the elimination rate, whereas N-ethylmaleimide and metal chelators had no effect. Receptor-associated protein, fucoidan, 3-bromo-5-t-butyl-4-hydroxy-benzylidenemalonitrile, anti-IGF-I receptor antibody, and l-tyrosine did not affect the Aβ(1-40) elimination rate, suggesting that the relevant receptors or transporters are not likely to be involved in the clearance. In conclusion, the present study has demonstrated the involvement of a proteolytic degradation process and an insulin-sensitive process in cerebral Aβ(1-40) clearance in the rat.

MCT1-mediated transport of L-lactic acid at the inner blood-retinal barrier: a possible route for delivery of monocarboxylic acid drugs to the retina.

AbstractPurpose. The aim of this study was to characterize L-lactic acid transport using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as a model of in vitro inner blood-retinal barrier (iBRB) to obtain a better understanding of the transport mechanism at the iBRB. Methods. TR-iBRB2 cells were cultured at 33°C, and L-lactic acid uptake was monitored by measuring [14C]L-lactic acid at 37°C. The expression and mRNA level of monocarboxylate transporter (MCT)1 and MCT2 were determined by reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR with specific primers, respectively. Results. The [14C]L-lactic acid uptake by TR-iBRB2 cells increased up to a pH of 5.0 and was inhibited in the presence of 10 mM L-lactic acid. The [14C]L-lactic acid uptake at pH 6.0 was both temperature- and concentration-dependent with a Michaelis-Menten constant of 1.7 mM and a maximum uptake rate of 15 nmol/(30 s · mg of protein). This process was reduced by carbonylcyanide p-trifluoromethoxy- phenylhydrazone (protonophore), α-cyano-4-hydroxycinnamate, and p-chloromercuribenzenesulfonate (typical inhibitors for H+-coupled monocarboxylic acid transport), suggesting that L-lactic acid uptake by TR-iBRB2 cells is a carrier-mediated transport process coupled with an H+ gradient. [14C]L-Lactic acid uptake was markedly inhibited by monocarboxylic acids but not dicarboxylic acids and amino acids. Moreover, salicylic and valproic acids competitively inhibited this process with an inhibition constant of 4.7 mM and 5.4 mM, respectively. Although MCT1 and MCT2 mRNA were found to be expressed in TR-iBRB2 cells, MCT1 mRNA was found to be present at a concentration 33-fold greater than that of MCT2 mRNA using quantitative real-time PCR. [14C]L-Lactic acid was significantly reduced by 5-(N,N-hexamethylene)-amiloride at pH 7.4 and Na+/H+ exchanger 1 mRNA was expressed in TR-iBRB2 cells. Conclusion. L-Lactic acid transport at the iBRB is an H+-coupled and carrier-mediated mechanism via MCT1 that is competitively inhibited by monocarboxylate drugs.