| Project/Area Number |
13672310
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
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| Research Institution | Hokuriku University |
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Principal Investigator |
KOBAYASHI Shinjiro Hokuriku University, Faculty of Pharmaceutical Sciences, Department of Pharmacology, Associate Professor, 薬学部, 助教授 (10186744)
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| Co-Investigator(Kenkyū-buntansha) |
HORIUCHI Seikoh Kumamoto University School of Medicine, Department of Biochemistry, Professor, 医学部, 教授 (10117377)
HAGINO Nobuyoshi The University of Texas Health Science Center at San Antonio, Department of Cellular and Structural Biology, Professor
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | Advanced Glycation End Product / N^ε-(carboxymethyl)lysine / Angiogenesis / Choroidal Capillary / Tumor Necrosis Factor-alpha / VEGF / Nifedipine / Tetrandrine / 腫瘍懐死因子-alpha / Advanced Glycation Endproduct / 網膜 / 糖尿病性網膜症 / VEGF / THF-α |
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| Research Abstract |
Actions of N^ε-(carboxymethyl)lysine (CML), one of advanced glycation end products (AGEs) were investigated on angiogenesis of cultured choroidal explant of aged and streptozotocin-diabetic rats. When explants of choroidal capillaries of these rats were cultured in fibrin gel with Dulbecco's modified Eagle's medium with fetal bovine serum, ε-amino caproic acid and antibiotics, spoke-like structures were sprouted from the explants. Cells organized spoke-like structures were positive with antibody against CD34, a marker of vascular endothelial cell (EC). These spoke-like structures had spaces of variable sizes that are compatible with vascular lumena. Number of structures were counted with a microscope and used as index of angiogenesis. 1) CML increased number of spoke-like structures of cultured choroidal explants of normal young rat and the action of CML was suppressed by anti-CML antibody (6D12). 2) Cultured choroidal explants of aged rats increased number of structures in an age-depe
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ndent manner and the activity of aged rat was suppressed by anti-CML antibody. 3) CML increased the release of vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-alpha from cultured choroidal explant. Antibodies against VEGF and TNF-alpha suppressed the activity of aged rat. 4) These results suggest that aging accumulates CML in choroidal explant and increases angiogenesis through the release of VEGF and TNF-alpha. 5) Choroidal explants of advanced stage-diabetic rat increased number of structures greater than those of early stage-diabetic rat did. The activities of early and advanced diabetic rats were completely suppressed by anti-CML antibody. 6) Antibodies against VEGF, TNF-alpha and platelet-derived growth factor (PDGF)-B as well as anti-CML antibody suppressed the enhanced activity of diabetic rat. 7) Serum of advanced diabetic rat decreased the activity whereas serum of early diabetic rat increased the activity of diabetic choroid. 8) Nifedipine, a blocker of voltage-dependent L type Ca^<2+> channel (VDLC) decreased both diabetic stage and TNF-alpha-increased number of structures. Tetrandrine of a main compound of Stephania Tetrandra radix, a blocker of VDLC and it voltage-dependent T type Ca^<2+> channel (VDTC) also decreased the activity of diabetic choroid. 9) These results demonstrate that CML is accumulated in the choroidal explants of diabetic rat and facilitates choroidal angiogenesis through the release of VEGF, TNF-alpha and PDGF-B. The actions of CML and TNF-alpha may be associated with the activity of VDLC and/or VDTC in choroidal capillary. Less
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