Roles of AGE for neovascularization and proliferation of fibroblast-like cells in cultured rat choroidal explains

研究糖基化终末产物N‘ε-(羧甲基)赖氨酸对老年和链脲佐菌素糖尿病大鼠脉络膜外植体新生血管的作用。这些大鼠的脉络膜毛细血管外植体在含有胎牛血清、ε-氨基己酸和抗生素的Dulbecco‘s改良Eagle’s培养液的纤维蛋白凝胶中培养时，这些外植体的微血管样结构的数量增加，这是新生血管的指标。从脉络膜外植体中萌发和发育的细胞具有成纤维细胞的特征，其胞质突起变薄，核长椭圆形，并有大量的膜结合的多形性小泡。胶原酶从纤维蛋白床分离的细胞均为CD34^+和CD34^-，提示该细胞为血管内皮祖细胞，而不是典型的血管内皮细胞。慢性粒细胞白血病促进CD_(34)~+细胞增殖及CuL-…血管样结构增多更多的脉络膜外植体破裂。培养的老年和STZ糖尿病大鼠的脉络膜外植体分别以年龄和糖尿病病程依赖的方式增加结构数量。抗CML抗体抑制新生血管的活性，提示CML在老年和STZ糖尿病大鼠的脉络膜外植体中积聚。CML可促进培养的脉络膜外植体释放血管内皮生长因子(VEGF)、肿瘤坏死因子(TNF)-α和血小板衍生生长因子(PDGF)。CML的释放作用依次为：血管内皮生长因子-GT、肿瘤坏死因子-α、血小板衍生生长因子-B。老年脉络膜外植体在培养0~2天和培养2~4天开始分泌PDGF-B、TNF-α和VEGF。在培养的老年脉络膜中，血管内皮生长因子的释放过程与脉络膜血管样结构的生长过程是平行的。抗肿瘤坏死因子-α和血管内皮生长因子抗体抑制老年大鼠的活性，提示释放的血管内皮生长因子促进了培养的老年脉络膜新生血管的形成。释放的肿瘤坏死因子-α可能通过释放血管内皮生长因子而促进新生血管的形成。电压依赖性L型钙通道阻断剂硝苯地平可抑制糖尿病期和肿瘤坏死因子-α诱导的微血管生长。硝苯地平对肿瘤坏死因子-α的抑制作用呈非竞争性。从苍术中分离得到的3-桉叶酚通过阻断血管内皮生长因子诱导的细胞外信号调节激酶(ERK)1/2活性而抑制血管生成。这些结果表明，硝苯地平和β-eudesol是了解慢性粒细胞白血病发病机制的特异性工具。较少

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 … More 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