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| 1.
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Iba, Tomohiro ; Naito, Hisamichi ; Shimizu, Shota ; Rahmawati, Fitriana Nur ; Wakabayashi, Taku ; Takakura, Nobuyuki ; 内藤, 尚道 ; 高倉, 伸幸
概要:
Background: During sprouting angiogenesis, stalk cells, localized behind tip cells, generate endothelial cells (ECs) for
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the elongation of new vessels. We hypothesized that stalk cells may have endothelial progenitor cell properties because of their highly proliferative ability. We conducted Hoechst dye DNA staining in ECs of preexisting blood vessels from hind limb muscle and found that endothelial-side population (E-SP) cells, which efflux Hoechst rapidly with abundant ABC transporters, show highly producing ability of ECs. We previously showed the existence of E-SP cells in hind limb muscle, retina, and liver, but not in other tissues such as adipose tissue, skin, and placenta. Methods: We investigated the existence of E-SP cells and analyzed their proliferative ability among CD31+CD45- ECs from adipose tissue, skin, and placenta of adult mice. We also analyzed the neovascular formation of E-SP cells from adipose tissue in vivo. Results: We detected E-SP cells in all tissues examined. However, by in vitro colony formation analysis on OP9 cells, we found that E-SP cells from adipose tissue and skin, but not from placenta, have highly proliferative ability. Moreover, E-SP cells from adipose tissue could contribute to the neovascular formation in hind limb ischemia model. Conclusion: The adipose tissue and skin are available sources to obtain endothelial stem cells for conducting therapeutic angiogenesis in regenerative medicine. © 2019 The Author(s).
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| 2.
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Wakabayashi, Taku ; Naito, Hisamichi ; Takara, Kazuhiro ; Kidoya, Hiroyasu ; Sakimoto, Susumu ; Oshima, Yusuke ; Nishida , Kohji ; Takakura, Nobuyuki ; 内藤, 尚道 ; 高倉, 伸幸
概要:
Purpose. The neovascular form of age-related macular degeneration (AMD) is characterized by the growth of abnormal new b
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lood vessels from the choroid, termed choroidal neovascularization (CNV). The origin of the new vessels in CNV, however, has not been elucidated fully to our knowledge. The purpose of this study is to identify vascular endothelial side population (SP) cells in the preexisting choroidal vessels, and investigate their potential role in AMD. Methods. We made single cell suspensions of freshly isolated mouse choroidal, retinal, and brain tissue by enzymatic digestion. Vascular endothelial SP cells were isolated using flow cytometry based on the ability to efflux the DNA-binding dye, Hoechst 33342, via ATP-binding cassette (ABC) transporters. Results. In the choroid, 2.8% of CD31+CD45- vascular endothelial cells (ECs) showed a typical SP staining pattern. They were not bone marrow-derived and possessed high colony-forming capacity in vitro. They proliferated during laser-induced CNV in vivo. In contrast, stereotypic SP staining pattern was not observed in retinal and brain ECs. Retinal and brain EC-SP cells included increased SP populations with less colony-forming capacity within the SP compartment, because they contained cells with and without proliferative potential. The latter still could efflux the dye due to high levels of ABC transporters, such as ABCB1a, ABCC4, and ABCC6. Conclusions. The EC-SP cells in the choroid may represent vessel-residing endothelial stem/progenitor cells contributing mainly to angiogenesis, and may be useful for augmenting vascular regeneration or for developing new antiangiogenic therapy in AMD. © 2013 The Association for Research in Vision and Ophthalmology, Inc.
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| 3.
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Aung, Khin Thuzar ; Yoshioka, Kazuaki ; Aki, Sho ; Ishimaru, Kazuhiro ; Takuwa, Noriko ; Takuwa, Yoh ; 吉岡, 和晃 ; 安藝, 翔 ; 多久和, 典子 ; 多久和, 陽
概要:
金沢大学医薬保健研究域医学系<br />Pinocytosis is an important fundamental cellular process that is used by the cell to transport fluid
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and solutes. Phosphoinositide 3-kinases (PI3Ks) regulate a diverse array of dynamic membrane events. However, it is not well-understood which PI3K isoforms are involved in specific mechanisms of pinocytosis. We performed knockdown studies of endogenous PI3K isoforms and clathrin heavy chain (CHC) mediated by small interfering RNA (siRNA). The results demonstrated that the class II PI3K PI3K-C2α and PI3K-C2β, but not the class I or III PI3K, were required for pinocytosis, based on an evaluation of fluorescein-5-isothiocyanate (FITC)–dextran uptake in endothelial cells. Pinocytosis was partially dependent on both clathrin and dynamin, and both PI3K-C2α and PI3K-C2β were required for clathrin-mediated—but not clathrin-non-mediated—FITC-dextran uptake at the step leading up to its delivery to early endosomes. Both PI3K-C2α and PI3K-C2β were co-localized with clathrin-coated pits and vesicles. However, PI3K-C2β, but not PI3K-C2α, was highly co-localized with actin filament-associated clathrin-coated structures and required for actin filament formation at the clathrin-coated structures. These results indicate that PI3K-C2α and PI3K-C2β play differential, indispensable roles in clathrin-mediated pinocytosis. © 2018, The Physiological Society of Japan and Springer Japan KK, part of Springer Nature.<br />Embargo Period 12 months
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| 4.
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Takuwa, Yoh ; Okamoto, Yasuo ; Yoshioka, Kazuaki ; Takuwa, Noriko
概要:
金沢大学医薬保健研究域医学系<br />The plasma lysophospholipid mediator sphingosine-1-phosphate (S1P) is produced exclusively by sphing
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osine kinase (SPHK) 1 and SPHK2 in vivo, and plays diverse biological and pathophysiological roles by acting largely through three members of the G protein-coupled S1P receptors, S1P1, S1P2 and S1P3. S1P1 expressed on endothelial cells mediates embryonic vascular maturation and maintains vascular integrity by contributing to eNOS activation, inhibiting vascular permeability and inducing endothelial cell chemotaxis via Gi-coupled mechanisms. By contrast, S1P2, is expressed in high levels on vascular smooth muscle cells (VSMCs) and certain types of tumor cells, inhibiting Rac and cell migration via a G12/13-and Rho-dependent mechanism. In rat neointimal VSMCs, S1P1 is upregulated to mediate local production of platelet-derived growth factor, which is a key player in vascular remodeling. S1P3 expressed on endothelial cells also mediates chemotaxis toward S1P and vasorelaxation via NO production in certain vascular bed, playing protective roles for vascular integrity. S1P3 expressed on VSMCs and cardiac sinoatrial node cells mediates vasopressor and negative chronotropic effect, respectively. In addition, S1P3, together with S1P2 and SPHK1, is suggested to play a protective role against acute myocardial ischemia. However, our recent work indicates that overexpressed SPHK1 is involved in cardiomyocyte degeneration and fibrosis in vivo, in part through S1P activation of the S1P3 signaling. We also demonstrated that exogenously administered S1P accelerates neovascularization and blood flow recovery in ischemic limbs, suggesting its usefulness for angiogenic therapy. These results provide evidence for S1P receptor subtype-specific pharmacological intervention as a novel therapeutic approach to cardiovascular diseases and cancer. © 2008 Elsevier B.V. All rights reserved.
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Echigo, Takeshi ; Hasegawa, Minoru ; Shimada, Yuka ; Takehara, Kazuhiko ; Sato, Shinichi
概要:
Background: Fractalkine (FKN) induces activation and adhesion of leukocytes expressing its receptor, CX3CR1. FKN is released from the cell surface through proteolytic cleavage as soluble FKN (sFKN). Objective: We sought to assess FKN
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and CX3CR1 expression in the skin, serum sFKN levels, and CX3CR1 expression on blood leukocytes in patients with atopic dermatitis (AD). Methods: FKN and CX3CR1 expression in the skin was examined immunohistochemically. mRNA expression of FKN, thymus and activation-regulated chemokine, and macrophage-derived chemokine in the skin was assessed by means of real-time RT-PCR. Serum sFKN levels were assessed by using ELISA. Blood leukocytes were stained for CX3CR1 by means of flow cytometric analysis. Results: FKN was strongly expressed on endothelial cells in skin lesions of patients with AD and psoriasis but not in normal skin. FKN mRNA levels in AD lesional skin increased to a similar extent to thymus and activation-regulated chemokine and macrophage-derived chemokine mRNA levels. CX3CR1-expressing cells in the affected skin of patients with AD or psoriasis increased compared with those in normal skin. Serum sFKN levels were increased in patients with AD but not in patients with psoriasis relative to levels in healthy control subjects. Serum sFKN levels were associated with the disease severity and decreased with the improvement of skin lesions in patients with AD. CX3CR1+ cell frequencies and CX3CR1 expression levels were decreased in CD8+ T cells, monocytes, and natural killer cells from patients with AD, but this was not observed in patients with psoriasis. Conclusions: These results suggest that through functions in both membrane-bound and soluble forms, FKN plays an important role in the trafficking of CX3CR1+ leukocytes during the inflammation caused by AD.
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| 6.
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Takuwa, Yoh ; Okamoto, Yasuo ; Yoshioka, Kazuaki ; Takuwa, Noriko
概要:
金沢大学医薬保健研究域医学系<br />The plasma lysophospholipid mediator sphingosine-1-phosphate (S1P) is produced exclusively by sphing
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osine kinase (SPHK) 1 and SPHK2 in vivo, and plays diverse biological and pathophysiological roles by acting largely through three members of the G protein-coupled S1P receptors, S1P1, S1P2 and S1P3. S1P1 expressed on endothelial cells mediates embryonic vascular maturation and maintains vascular integrity by contributing to eNOS activation, inhibiting vascular permeability and inducing endothelial cell chemotaxis via Gi-coupled mechanisms. By contrast, S1P2, is expressed in high levels on vascular smooth muscle cells (VSMCs) and certain types of tumor cells, inhibiting Rac and cell migration via a G12/13-and Rho-dependent mechanism. In rat neointimal VSMCs, S1P1 is upregulated to mediate local production of platelet-derived growth factor, which is a key player in vascular remodeling. S1P3 expressed on endothelial cells also mediates chemotaxis toward S1P and vasorelaxation via NO production in certain vascular bed, playing protective roles for vascular integrity. S1P3 expressed on VSMCs and cardiac sinoatrial node cells mediates vasopressor and negative chronotropic effect, respectively. In addition, S1P3, together with S1P2 and SPHK1, is suggested to play a protective role against acute myocardial ischemia. However, our recent work indicates that overexpressed SPHK1 is involved in cardiomyocyte degeneration and fibrosis in vivo, in part through S1P activation of the S1P3 signaling. We also demonstrated that exogenously administered S1P accelerates neovascularization and blood flow recovery in ischemic limbs, suggesting its usefulness for angiogenic therapy. These results provide evidence for S1P receptor subtype-specific pharmacological intervention as a novel therapeutic approach to cardiovascular diseases and cancer. © 2008 Elsevier B.V. All rights reserved.
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| 7.
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高安, 達典 ; Takayasu, Tatsunori
概要:
薬毒物の毒性は致死量などで示されているが,この量と細胞死における薬毒物の濃度は一定の関係があるかどうか,血管内皮細胞を用いて,薬毒物毒性評価の可能性を研究した。血管内皮細胞を用いた細胞死の濃度は上記の4物質で各々,5,5,5及び0.1mMで
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あった。その他57の薬毒物についても同細胞でスクリーニングした。それらの中で毒性が最も低かったものはメタノール2Mで最も高かったものは亜砒酸で0.01mMであった。一方ヒトの推定致死量を文献で調査したところ,メタンフェタミン,コカイン,フェノバルビタール及びパラコートで各々1.3-20,7-10,14-200及び14-40mg/kgであった。この値を比較のために単位をmg/Lと置き換えると濃度になり,各々の0.007-0.1,0.02-0.03,0.06-0.9,0.05-0.16mMと計算される。メタンフェタミン,コタイン,フェノバルビタールでは1/6から1/700の低濃度で個体では死亡していた。しかし,パラコートでは略同レベルの濃度であった。同様に他の40種の薬毒物で計算し,それらの値と血管内皮細胞の細胞死した濃度を比較したところ,それらの相関係数は0.310と低い相関であった。以上,物質の個体及び細胞への毒性は個々の物質により異なることが改めて示されたが,今回の結果からは薬毒物の毒性評価を推定するまでには至らなかった。今後さらに極々の因子の検索も含め詳細に調査することが必要と考えられた。<br />Toxicity of drugs and poisons shows as lethal dose in human. In this study, relationship between the lethal dose and dose of the cell death was examined using human endothelial cells(HUVEC). Death of HUVEC judged with morphological changes in culture fluids mixed with drugs and poisons. Doses of the cell death using methamphetamine(MA), cocaine, phenobarbital(PB), paraquat(PQ) showed 5,5,5 and 0.1 mM, respectively. The other 57 drugs and poisons were screened with the same method. The most poisonous substance was arsenite sodium (0.01mM) and the least poisonous one was methanol(2M). By the references, lethal doses of human(including presumption values) in MA, cocaine, PB and PQ were 1.3-20,7-10,14-200 and 14-40mg/kg, respectively. To compare the values between lethal dose and dose of cell death, the unit made mg/L of mg/kg. The concentrations of MA, cocaine, PB and PQ were 0.007-0.1,0.002-0.003,0.06-0.9 and 0.05-0.16mM, respectively. In MA, cocaine and PB lethal doses of human showed 1/6 to 1/700 comparing to cell death of HUVEC. In PQ, however, the dose of human was same level as cell death of HUVEC. In the same manner, 49 substances were plotted between lethal dose of human and the cell death of HUVEC. When the relation line was made, the correlation coefficient(r) was 0.310, indicating poor relationship. In conclusion, morphological changes of HUVEC showed poor relationship to lethal dose of human in total 61 drugs and poisons examined. Further examination needs to determine the relationship between lethal dose and endothelial cell death in human.<br />研究課題/領域番号:14570383, 研究期間(年度):2002-2003
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| 8.
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小泉, 晶一 ; Koizumi, Shoichi
概要:
ヘムオキシゲナーゼ1(HO-1)はヘムを一酸化炭素(CO)とビリルビンとフェリチン(Feより誘導される)に代謝する律則酵素である。われわれが世界で初めて発見した「ヒトHO-1欠損」症例(1999)は2歳頃からの全身性慢性炎症が増悪し,6歳で
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死亡した。本症例のこれまでの詳細な病態解析とin vitro実験的研究によって、HO-1の、生体へのストレス防御因子としての役割を解明し、下記の研究成果を得た。(1)患児の生検材料やリンパ球細胞株でHO-1の発現が認めらないことと、HO-1遺伝子の異常から本疾患が確立された。(2)HO-1欠損症の臨床特徴は、主として造血系単球と血管内皮細胞及び腎尿細管細胞の障害に起因する。(3)HO-1欠損症では、外因性酸化ストレスによって、全身性炎症と凝固線溶系の著しい亢進を惹起し、ストレスが長期に継続すると、免疫系や凝固線溶系が消耗、破壊される。(4)単球細胞表面抗原の異常、貪食能の低下が認められた。また、CD16high/CCR2-の細胞亜群にHO-1発現が強く、この細胞群の動態は細菌性感染症とウイルス性感染症で異なっていることから、単球のHO-1発現が各種感染症の病態表現に深くかかわっていることが示唆された。さらに、単球のHb/Hp/CD163コンプレックスがHO-1誘導と強く相関することがわかった。(5)各種腎疾患の生検材料、及びin vitro細胞株の検討から、HO-1は腎尿細管上皮がストレス防御に有効に働いていることが強く示唆された。さらに尿沈さ細胞中のHO-1濃度測定は、腎尿細管傷害を観察する非侵襲的な方法として有用であろうと思われた。(6)以上、HO-1欠損症の詳細な検討は、システミックな炎症発症における新しい病態機序の解明に役立ち、そして、新しい治療法の開発に繋がるかの知れない。<br />Heme oxygenase (HO) is the rate-limiting enzyme that adds an oxygen molecule to the porphyrin ring of heme, thereby catalyzing the oxidation of heme to biliverdin/ bilirubin, free iron, and carbon monoxide (CO). By producing these metabolites, HO plays a crucial role in humans as a defense factor against a variety of oxidative stresses. The first case of human HO - 1 deficiency was reported by Yachie, et. Al. in our laboratory in 1999. A series of clinical and laboratory investigation of the patient with HO-1 deficiency revealed that (1) The boy completely lacked HO-1, genetically having a two-base-pair deletion in exon 3 of the paternal allele of the gene and a deletion of exon 2 of the maternal allele with genomic exon-deletion (1,730bp) mediated by Alu-Alu, recombination. (2) Dysfunction of both monocytes and endothelial cells was remarkably demonstrated. (2) External continuous stresses triggered excessive systemic inflammatory reactions and marked abnormalities of the coagulation/ fibrinolysis system, resulting finally in exhaustion of immune and coagulation system. (3) Morphological abnormality of monocytes and significant reduction of their surface molecules resulted in a markedly impaired phagocytosis of monocytes. Furthermore, the selective expansion of a CD16+++, CCR2- subpopulation of monocytes that preferentially produced HO-1 mRNA was shown during the acute phase of infections including both bacterial and viral infections. HO-1 production by alveolar macrophages in childhood pulmonary hypertension correlated with overexpression of CD1G3 surface molecules was also demonstrated. (4) Progressive renal tubulointerstitial injury was remarkable in the patient with HO-1 deficiency. Enhanced production of HO-lin proximal tubular epithelial cells as compared with mesangial cells was shown in variety of renal biopsy specimen as well as in vitro experiment. Recently HO-1 production by urinary tract cells and evaluation of tubulointestinal injury of the kidney was investigated by MA analysis of urinary sediments. Detailed analysis of HO-I deficiency may offer a valuable tool to understand the pathogenesis of and to develop a novel therapeutic approach to systemic inflammatory illnesses.<br />研究課題/領域番号:17390298, 研究期間(年度):2005-2007<br />出典：「ヘムオキシゲナーゼ1欠損と全身性慢性炎症に対する防御機構の破綻」研究成果報告書 課題番号17390298 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）) 本文データは著者版報告書より作成
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