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

本实验观察了糖基化终产物（AGEs）之一N^ε-（羧甲基）赖氨酸（CML）对体外培养的老年大鼠和链脲佐菌素（STZ）糖尿病大鼠脉络膜新生血管的作用。当这些大鼠的脉络膜毛细血管的外植体培养在纤维蛋白凝胶与Dulbecco的改良Eagle培养基与胎牛血清，ε-氨基己酸和抗生素，微血管样结构的数量，新血管形成的指数，从这些外植体增加。来自脉络膜外植体的芽生和发育细胞具有成纤维细胞的特征，具有减弱的长胞质突起、长椭圆形核和许多膜结合的多态性囊泡。用胶原酶从纤维蛋白床分离的细胞均为CD 34 ^+和CD 34 ^-，表明该细胞为血管内皮祖细胞而不是典型的血管内皮细胞。CML增加了CD 34 ^+细胞的增殖和细胞内血管样结构的数量， 关于我们 脉络膜外植体破裂。培养的脉络膜外植体的老年和STZ糖尿病大鼠的结构数量增加的年龄和糖尿病持续时间依赖的方式，分别。抗CML抗体抑制新生血管的活动，表明CML在培养的老年和STZ糖尿病大鼠脉络膜外植体中积累。在培养的第4 ~ 6天，CML增加了培养的脉络膜组织中血管内皮生长因子（VEGF）、肿瘤坏死因子（TNF）-α和血小板衍生生长因子（PDGF）的释放<th><th>。CML对VEGF、TNF-α、PDGF-B的释放作用顺序为VEGF &gt; TNF-α&gt; PDGF-B。PDGF-B、TNF-α和VEGF分别从培养0 ~ 2<nd>天和培养2 <nd>~ 4天<th>开始释放。在培养的老年脉络膜中，VEGF的释放与血管样结构生长的时间过程是平行的。抗TNF-α和VEGF抗体抑制老年大鼠脉络膜的活性，表明VEGF的释放促进了培养的老年脉络膜中的新生血管形成。释放的TNF-α可能通过VEGF的释放增加新血管形成。硝苯地平是一种电压依赖性L型Ca^&lt;2+&gt;通道的阻断剂，它能抑制糖尿病阶段和TNF-α增加的微血管生长。硝苯地平对TNF-α作用的抑制模式为非竞争性。（3-Eudesmol）通过阻断VEGF诱导的细胞外信号调节激酶（ERK）1/2活性抑制血管生成。这些结果表明硝苯地平和β-桉叶醇是了解CML机制的特异性工具。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