| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2003: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
Actions of N^ε-(carboxymethyl)lysine (CML), one of advanced glycation end products (AGEs) were investigated on neovascularization of cultured choroidal explant of aged and streptozotocin (STZ)-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, the number of microvessel-like structures, an index of neovascularization, was increased from these explants. Budded and developed cells from the choroidal explant had a feature of fibroblasts, which had attenuated long cytoplasmic processes, long ellipsoid nuclei and numerous membrane-bound polymorphic vesicles. The cells which were isolated from fibrin bed by collagenase were both CD34^+ and CD34^-, suggesting that the cells are vascular endothelial progenitor cells rather than typical vascular endothelial cells. CML increased proliferation of CD34^+ cells and number of vessel-like structures of cul
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tured choroidal explants. Cultured choroidal explants of aged and STZ-diabetic rats increased the number of structures in age and diabetic duration-dependent manners, respectively. The activities of neovascularization were suppressed by anti-CML antibody, suggesting that CML is accumulated in cultured choroidal explants of aged and STZ-diabetic rats. CML increased the release of vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-alpha and platelet-derived growth factor (PDGF) from cultured choroidal explant during 4^<th> to 6^<th> day in culture. The releasing effect of CML was in the order VEGF > TNF-alpha > PDGF-B. The release of PDGF-B and TNF-alpha, and VEGF from aged choroidal explant was started from 0 to 2^<nd> day in culture, and from 2^<nd> to 4^<th> day in culture, respectively. The time course of release of VEGF was parallel to that of growth of vessel-like structure in the cultured aged choroid. Antibodies against TNF-alpha and VEGF suppressed the activity of aged rat, suggesting that the released VEGF facilitated neovascularization in the cultured aged choroid. The released TNF-alpha may increase the neovascularization through the release of VEGF. Nifedipine, a blocker of voltage-dependent L type Ca^<2+> channel decreased both diabetic stage- and TNF-alpha-increased growth of microvessels. The inhibitory pattern of nifedipine to the action of TNF-alpha was in a non-competitive manner. (3-Eudesmol isolated from Atractylodes lancea rhizome inhibited angiogenesis through the blockade of extracellular signal-regulated kinase (ERK) 1/2 activity induced by VEGF. These results indicate that Nifedipine and β-eudesmol are specific tools for understanding the mechanism of CML. Less
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