Roles of AGE for mechanism of angiogenesis

观察晚期糖基化终末产物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血管生成的影响。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，外植体长出了轮辐状结构。血管内皮细胞标志物CD34抗体呈阳性反应。这些轮辐状结构具有不同大小的空间，与血管管腔兼容。显微镜下计数结构数目，作为血管生成的指标。1)CML可增加培养的正常幼鼠脉络膜外植体的辐状结构，其作用可被抗CML抗体(6D12)所抑制。2)培养的老年大鼠脉络膜外植体增加了年龄依赖性…的结构数量抗CML抗体可明显抑制老年大鼠的免疫功能。3)CML可促进脉络膜外植体释放血管内皮生长因子(VEGF)和肿瘤坏死因子(TNF)-α。抗血管内皮生长因子和肿瘤坏死因子-α的抗体可抑制老龄大鼠的活动。4)上述结果提示，随着年龄的增长，CML在脉络膜外植体中积聚，并通过释放血管内皮生长因子和肿瘤坏死因子-α而增加血管生成。5)晚期糖尿病大鼠的脉络膜外植体较早期糖尿病大鼠的脉络膜外植体增加的结构数量更多。抗CML抗体可完全抑制早期和晚期糖尿病大鼠的活动。6)抗血管内皮生长因子、肿瘤坏死因子-α和血小板衍生生长因子-B抗体以及抗CML抗体均可抑制糖尿病大鼠的活动增强。7)晚期糖尿病大鼠血清降低糖尿病脉络膜活性，早期糖尿病大鼠血清升高糖尿病脉络膜活性。8)电压依赖性L钙通道阻断剂硝苯地平可降低糖尿病大鼠的糖尿病分期，减少肿瘤坏死因子-α所致的结构数目增加。粉防己的主要成分粉防己碱、VDLC阻滞剂及其电压依赖性T型钙通道(VDTC)也可降低糖尿病脉络膜的活性。9)这些结果表明CML在糖尿病大鼠的脉络膜外植体中积聚，并通过释放VEGF、TNF-α和PDGF-B促进脉络膜血管生成。CML和TNF-α的作用可能与脉络膜毛细血管内VDLC和/或VDTC的活性有关。较少

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 … More 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