Research projects

妊婦における薬物治療を有効かつ安全に行うためには、定常状態ヒト胎児/母体血漿中薬物濃度比（F/M比）は必須だが、情報に乏しい。本研究では、出産後のヒト胎盤のex vivo灌流に、生理学的薬物動態モデルを用いたin silicoでのパラメータ推定を組み合わせ、従来のex vivoヒト胎盤灌流のみでは実測できなかった低膜透過性薬物のF/M比推定手法（ex vivo-PBPKハイブリッド解析）を確立することを目指す。本研究は、多くの薬物のヒトF/M比が不明な現状を改善し、妊婦と胎児でのアンメットメディカルニーズに応える意義を有している。

Although the prediction accuracy of the steady-state fetal/maternal plasma concentration ratio (F/M ratio) of some low-membrane-permeability drugs by human placental perfusion has been low, the present study demonstrated that the steady-state F/M ratio of can be estimated by combining the originally developed pharmacokinetic model. Moreover, we clarified that one of the reasons for the lower F/M ratio in rats compared to humans is the species difference in protein binding. This can be overcome by employing the unbound F/M ratio in rats. Furthermore, we observed that the specific expression of MATE1 in rat placenta and the rodent-specific localization of placental MDR1 contribute to the species differences in the fetal transfer of substrate drugs.

Syncytin-2, a molecule specifically expressed in the placenta, has membrane fusion capacities and contributes to the barrier formation through syncytialization. In this study, it is indicated that the uptake of JEG-3 cell-derived exosome to human hepatic HepG2 cells was significantly enhanced when the expression of syncytin-2 in JEG-3 cells was induced. The placenta-specific miRNA is detected from HepG2 cells exposed to JEG-3 cell-derived exosomes, implying that exosomal syncytin-2 is capable to mediate cell-to-cell communication through the exosome. However, the expression of MFSD2A, a receptor of syncytin-2, is found not to be constitutively expressed in the mouse liver. Therefore, it is necessary to further confirm whether syncytin-2-expressing exosome is preferably binds to hepatic cells. We also found that MFSD2A involves in the supply of docosahexaenoic acid to the fetus.

The purpose of the present study was to clarify the transporters mediating the fetal transfer of tryptophan and examine the importance of an adequate supply of tryptophan into the fetus. This study described the amount and localization of LAT1 protein, a transporter for large neutral amino acids including tryptophan, at the placental barrier. It is also observed that the tissue distribution of LAT1 substrate is suppressed by an increase of LAT1-substrate amino acids in the plasma. For serotonin, we have clarified OCTN1 protein accepts it as a substrate and is expressed at the placental barrier at term. However, the effect of tryptophan imbalance in the diet was negligible in the plasma concentration of amino acids.

The placental barrier regulates fetal transfer of maternal substances and thus maintains fetal environment. Therefore, it is important to elucidate functional regulations at the placental barrier. In this study, we clarified regulations of transporter expression at the placental barrier by (1) differentiation to the placental barrier, (2) gestational diabetes, and (3) caloric restriction. These results represent that the molecular expression at the placental barrier are regulated by placental differentiation and maternal nutrition environment in order to maintain fetal environment.

前年度において、温度感受性SV40T抗原遺伝子導入トランスジェニックラットから樹立した条件的不死化網膜および脳毛細血管内皮細胞株(TR-iBRB細胞およびTR-BBB細胞)をin vitroモデルとして用い、両細胞株の発現遺伝子をdifferential display法を用いて解析した。その結果、TR-BBB細胞と比較してTR-iBRB細胞に高発現する遺伝子を8遺伝子検出することができた。今年度は、TR-iBRB細胞高発現遺伝子の特性を詳細に解析することを目的とした。TR-iBRB細胞高発現遺伝子のうち、TR-BBB細胞との発現量の差が最も大きい遺伝子はマウスM-cadherinの3'末端非翻訳領域と高い相同性を示した。マウスおよびヒトM-cadherinの配列をもとにプライマーを設計し,ラットM-cadherinをクローニングした。ラットM-cadherinのアミノ酸配列はマウス,ヒトとそれぞれ97%および86%の相同性を示し、細胞外繰り返し構造および細胞外領域におけるCa^<2+>結合モチーフなどcadherinに特徴的な配列を有していることが推定された。リアルタイム定量PCRを用いた解析の結果、ラットM-cadherinのmRNA発現量比は,TR-iBRB細胞においてTR-BBB細胞の93倍であった。さらに、Western blot法を用いた解析の結果、タンパク質レベルでもM-cadherinがTR-iBRB細胞に高発現していることが示された。以上の結果から、内側血液網膜関門にはM-cadherinが高発現し、細胞接着において内側血液網膜関門は血液脳関門とは一部異なる機構を有している可能性が示された。

本研究は血液網膜関門(BRB)には多様な異物排出機構が存在し、薬物の網膜移行性や網膜内老廃物の除去に機能していることを証明することを目的とした。[^3H]estradiol 17-β glucuronide (E17βG)もしくは[^3H]dehydroepiandrosterone sulfate (DHEAS)と、bulk flowマーカーとして[^<14>C]D-mannitolを同時に硝子体内に投与し、microdialysisプローブを用いて投与化合物の硝子体内濃度推移を測定し、BRBを介した排出輸送を評価した。[^3H]E17βGおよび[^3H]DHEASの硝子体からの消失速度定数は[^<14>C]D-mannitolよりも約2倍高く、E17βGおよびDHEASはBRBを介して排出されていることが示唆された。[^3H]E17βGおよび[^3H]DHEASの消失は各々非標識E17βGとDHEASで自己阻害された。さらに、[^3H]E17βGおよび[^3H]DHEASの消失はsulfobromophthaleinおよびprobenecid存在下、およびorganic anion transporting polypeptide 2(oatp2)特異的な基質であるdigoxin存在下で有意に阻害された。磁気分離したラット網膜血管内皮細胞画分におけるmRNA発現量を解析した結果、oatp2および14の内側BRBにおける発現が示された。さらに、網膜血管内皮細胞画分を用いた解析および免疫組織化学解析の結果、内側BRBの網膜側膜にはorganic anion transporter (oat) 3の発現が示された。 以上の結果から、内側BRBにはoatp2、oatp14、およびoat3が発現し、さらに少なくともoatp2はBRBを介したE17βGおよびDHEAS排出輸送に関与していることを示唆することができた。

We have clarified the protein expression level of nutrient transporters at the brush-border membrane of the human placental barrier formed by single syncytiotrophoblast layer. We have also clarified the protein expression in rat placental barrier formed by double syncytiotrophoblast layers, for estimating placental permeability of transporter substrates across the rat placenta. The higher placental permeability of transporter substrates was observed, however, the estimated permeability was not highly consistent with the measured permeability. There have been thought that two syncytiotrophoblast layers in rodents act functionally as a single layer, but this appears not to be true since we found that MDR1 and BCRP transporters are localized in the second syncytiotrophoblast layer. We need to consider the presence of transporters in the second syncytiotrophoblast layer for estimating placental permeability.

We have clarified the developmental change in the placental transporters such as glucose transporter 1 (GLUT1) and monocarboxylate transporter 1 (MCT1), while the expressions of ABC transporters such as MDR1 and BCRP were unchanged during pregnancy. We also found a placenta-derived miRNA which may work for maturation of both placental and blood-brain barrier. These observations are helpful for examining the interorgan network in the fetoplacental circulation for establishing and managing barriers in the placenta and the brain.

We aimed to examine the fetal transfer mechanism of methyl donors, such as methionine, 5-methyltatrahydrofolate (MTHF), and betaine, across the placenta. LAT1, a transporter for methionine, was observed to be specifically localized at the brush-border membrane of rat placental syncytiotrophoblast layer I, suggesting the role of LAT1 in the fetal transfer of amino acids. Fetal transfer rate of 5-methyltatrahydrofolate (MTHF) and betaine appears to be low compared with methionine transported by LAT1, implying the importance of methionine in terms of placental permeability. SNAT2 mediates the transfer of betaine, and its functional expression in placental brush-border membrane was found to be detected in rat but not in human. This suggests the presence of the species difference in betaine transport. Methylated CpG level in the promoter region of the Abcg2 gene was observed to be very low.

網膜ペリサイトから分泌される因子が網膜毛細血管内皮細胞の増殖を制御しているという仮説から、網膜毛細血管内皮細胞株と網膜ペリサイト株の共培養解析を行った結果、網膜ペリサイト株培養後の培地の濃縮液を網膜毛細血管内皮細胞株に添加すると内皮細胞増殖を著しく抑制することを見いだした。本研究は、網膜ペリサイト細胞株が分泌する網膜毛細血管内皮細胞増殖抑制因子を同定することを目的とした。網膜ペリサイト株培養後の培地の濃縮液からタンパクを回収し、分子量で分画し、網膜毛細血管内皮細胞株の増殖を抑制するフラクションから2次元電気泳動でタンパクを分離した。分離したタンパクをMALDI-TOFMSで分析し、タンパクを同定した。同定したタンパクはtoropomyosin (TM)と高い相同性を示した。Rat TM3を特異的に認識するプライマーを作製してRT-PCR法でオープンリーディングフレームのクローニングを行った。さらに、RT-PCR解析からラット網膜、網膜ペリサイト株に発現が示された。増幅された網膜毛細血管周皮細胞株のPCR産物をDNAシークエンサー解析した結果、rat TM3、rat TM6と異なる配列を示した。得られた遺伝子配列をpGEX-6P-1 vectorに導入し、大腸菌を用いて大量培養した。大腸菌からタンパクを精製し、リコンビナントタンパクを作製した。リコンビナントタンパクはMALDI-TOFMSによってタンパク質量を測定し、さらにprotein sequencerを用いてN末端アミノ酸配列の解析を行い、同定したタンパクであることを確認した。網膜毛細血管内皮細胞株の増殖に対し、同定したタンパクは1μM以上の添加で顕著な細胞増殖抑制効果を示した。以上の結果から、網膜ペリサイトはTMと高い相同性を示すタンパクを分泌していること、およびこのタンパクは網膜毛細血管内皮細胞の増殖抑制効果を示すことが明らかとなった。

Vitamin C, which is an antioxidant in the body, plays a key role in protecting the retina from oxidative stress and is present in the retina at a high concentration compared with its presence in other organs. The purpose of study was to elucidate the mechanisms of vitamin C transport across the blood-retinal barrier (BRB) in vive and in vitro. [^<14>C]Dehydroascorbic acid (DHAA) and [^<14>C]ascorbic acid (AA) transport in the retina were examined using in vivo integration plot analysis in rats and the transport mechanism was characterized using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB) as an in vitro model of the inner BRB. The apparent influx permeability clearance (CL) of [^<14>C]DHAA of the retina was about 38-fold greater than that of [^<14>C]AA, whereas there was no major difference in the heart. HPLC analysis revealed that most of the vitamin C accumulates in AA form in the retina. These results suggest that vitamin C is mainly transported in DHAA form across the BRB and accumulates in AA form in the rat retina. [^<14>C]DHAA uptake by TR-iBRB cells took place in an Na^+-independent and concentration-dependent manner with a Michaelis constant of 93.4 microM. This process was inhibited by facilitative glucose transporter (GLUT)s' substrates and inhibitors. [^<14>C]DHAA uptake was inhibited by glucose in a concentration-dependent manner with a 50% inhibition concentration of 5.56 mM. These results suggest that GLUTs are involved in DHAA transport at the inner BRB. To quantify GLUTS' gene levels at the inner BRB using a combination of magnetic isolation method for rat retinal vascular endothelial cells and real-time quantitative PCR analysis, GLUT1 expression was the largest of three GLUTs, whereas GLUT3 was not detected at the inner BRB. The expression of GLUT4 was 100-fold less than that of GLUT1, suggesting that GLUT1 is responsible for DHAA transport at the inner BRB. When glucose concentration in the blood was high as 20 mM in streptozotocin-induced diabetic rats, CL of [^<14>C]DHAA of the retina was inhibited by 31%. In conclusion, vitamin C is predominantly transported as DHAA via GLUT1 at the inner BRB and accumulates as AA in the retina. Hyperglycemia reduces the supply of the vitamin C to the retina. The physiological role of GLUT1 at the inner BRB appears to involve the supply of vitamin C as well as glucose from the circulating blood to the retina to protect the retina against oxidative stress.

Placenta is an interface between mother and fetus and it has a role of nutrients transfer. The placenta has the function mitigating the osmotic stress exposed from maternal blood. Part of amino acid is transferred via amino acid transport system A from mother to fetus in the placenta. In this study, we clarified that expression of the system A molecule can be more quickly increased in response to the osmotic stress than other molecules and we investigated how the system A molecules is induced by hypertonic stress.

Organic anion transporter (OAT) 4 expressed at the basolateral membrane of the placental barrier appears to play a principal role in the uptake of 16α-OH DHEAS and contribute to the E3 synthesis during pregnancy. Moreover, the expression of OAT4

In formation process of a blood-tissue barrier, there is an interaction between specific tissue cells and bone marrow(BM)-derived endothelial progenitor cells(EPC). An assay system using the established cell lines from the specific tissue is desirable to obtain reproducible analysis. By using an established bone marrow-derived vascular endothelium precursor cell line, TR-BME, docetaxel and paclitaxel directly inhibited EPC-initiated vasculogenesis at low(non-cytotoxic) concentrations, causing suppression of tumor growth.