晚期糖基化终产物引起的RhoA和ROCK在人皮肤微血管内皮细胞的分布变化

目的观察在晚期糖基化终产物修饰的人血清白蛋白作用下,小G蛋白RhoA及其激活的激酶ROCK在人微血管内皮细胞中分布的变化。方法培养人皮肤微血管内皮细胞株,分别以糖基化修饰的人血清白蛋白(AGE-HSA)处理不同的浓度和时间,用免疫荧光染色法、激光共聚焦显微镜观察RhoA和磷酸化ROCK在细胞中的分布变化;并用活性型RhoA L63或失活型的RhoA N19转染细胞后再给予AGE-HSA刺激,观察磷酸化ROCK细胞内分布的变化。结果 AGE-HSA刺激可以导致RhoA在胞浆中分布增多,其变化随着AGE-HSA作用时间的延长和剂量的增加更加明显。RhoA的下游激酶被活化的磷酸化ROCK在人微血管内皮细胞中的分布与RhoA类似;用失活型RhoAN19先处理细胞后,AGE-HSA介导的ROCK分布变化被抑制。结论晚期糖基化终产物刺激引起小G蛋白RhoA在细胞内分布变化,并导致其下游激酶ROCK磷酸化和定位变化。

Advanced Glycation End Products Induced the Redistribution of RhoA and Phosphorylated ROCK in Human Dermal Microvascular Endothelial Cells

Aim To observe the changes of distribution of RhoA and phosphorylated RhoA kinase(ROCK) induced by advanced glycation end products(AGE) in human dermal microvascular endothelial cells(HMVEC-1). Methods Cultured HMVEC-1 were treated with AGE-modified human serum albumin(AGE-HSA).The distribution of RhoA and phospho-ROCK were detected by immunohistochemistry using anti-RhoA and anti-phospho-ROCK antibodies and observed under confocal microscope. Results The data revealed that RhoA located around the nucleus in quiescent HMVEC-1.The stimulation of AGE-HSA evoked an increased distribution of RhoA in cytoplasma,even an accumulation at the cellular edge.The administration of AGE-HSA also triggered a significant increase of phospho-ROCK localization in HMVEC-1 cytoplasma.The transfection of dominant negative RhoA N19 attenuated this AGE-induced phosphor-ROCK spreading while constitutive active RhoA L63 mimiced the AGE-induced response in phospho-ROCK distribution. Conclusion AGE stimulation causes the redistribution of RhoA and results in the phosphorylation of ROCK in human microvascular endothelial cells.