Department of Pathophysiology, Key Laboratory for Shock and Microcirculation Research, Southern Medical University, Guangzhou, P R China.
Cardiovasc Diabetol. 2012 Jan 17;11:7. doi: 10.1186/1475-2840-11-7.
The role of advanced glycation end products (AGEs) in the development of diabetes, especially diabetic complications, has been emphasized in many reports. Accumulation of AGEs in the vasculature triggers a series of morphological and functional changes in endothelial cells (ECs) and induces an increase of endothelial permeability. This study was to investigate the involvement of RhoA/ROCK-dependent moesin phosphorylation in endothelial abnormalities induced by AGEs.
Using human dermal microvascular endothelial cells (HMVECs), the effects of human serum albumin modified-AGEs (AGE-HSA) on the endothelium were assessed by measuring monolayer permeability and staining of F-actin in HMVECs. Activations of RhoA and ROCK were determined by a luminescence-based assay and immunoblotting. Transfection of recombinant adenovirus that was dominant negative for RhoA (RhoA N19) was done to down-regulate RhoA expression, while adenovirus with constitutively activated RhoA (RhoA L63) was transfected to cause overexpression of RhoA in HMVECs. H-1152 was employed to specifically block activation of ROCK. Co-immunoprecipitation was used to further confirm the interaction of ROCK and its downstream target moesin. To identify AGE/ROCK-induced phosphorylation site in moesin, two mutants pcDNA3/HA-moesinT(558A) and pcDNA3/HA-moesinT(558D) were applied in endothelial cells.
The results showed that AGE-HSA increased the permeability of HMVEC monolayer and triggered the formation of F-actin-positive stress fibers. AGE-HSA enhanced RhoA activity as well as phosphorylation of ROCK in a time- and dose-dependent manner. Down-regulation of RhoA expression with RhoA N19 transfection abolished these AGE-induced changes, while transfection of RhoA L63 reproduced the AGE-evoked changes. H-1152 attenuated the AGE-induced alteration in monolayer permeability and cytoskeleton. The results also confirmed the AGE-induced direct interaction of ROCK and moesin. Thr558 was further identified as the phosphorylating site of moesin in AGE-evoked endothelial responses.
These results confirm the involvement of RhoA/ROCK pathway and subsequent moesin Thr558 phosphorylation in AGE-mediated endothelial dysfunction.
高级糖基化终产物（AGEs）在糖尿病，特别是糖尿病并发症的发展中的作用，在许多报告中都有强调。AGEs 在血管中的积累会触发内皮细胞（ECs）的一系列形态和功能变化，并诱导内皮通透性增加。本研究旨在探讨 RhoA/ROCK 依赖性 moesin 磷酸化在 AGE 诱导的内皮异常中的作用。
使用人真皮微血管内皮细胞（HMVECs），通过测量 HMVECs 单层通透性和 F-肌动蛋白染色来评估人血清白蛋白修饰的 AGE（AGE-HSA）对内皮的影响。通过发光测定法和免疫印迹法测定 RhoA 和 ROCK 的激活。转染显性失活的 RhoA 重组腺病毒（RhoA N19）以下调 RhoA 表达，而转染组成型激活的 RhoA 重组腺病毒（RhoA L63）以使 RhoA 在 HMVECs 中过表达。使用 H-1152 特异性阻断 ROCK 的激活。共免疫沉淀进一步证实了 ROCK 与其下游靶标 moesin 的相互作用。为了鉴定 moesin 中的 AGE/ROCK 诱导的磷酸化位点，应用了两个突变体 pcDNA3/HA-moesinT(558A) 和 pcDNA3/HA-moesinT(558D)。
结果表明，AGE-HSA 增加了 HMVEC 单层的通透性，并引发了 F-肌动蛋白阳性应力纤维的形成。AGE-HSA 以时间和剂量依赖的方式增强了 RhoA 活性和 ROCK 的磷酸化。用 RhoA N19 转染下调 RhoA 表达消除了这些 AGE 诱导的变化，而转染 RhoA L63 则再现了 AGE 引起的变化。H-1152 减弱了 AGE 诱导的单层通透性和细胞骨架改变。结果还证实了 AGE 诱导的 ROCK 和 moesin 的直接相互作用。Thr558 进一步被确定为 AGE 诱导的内皮反应中 moesin 的磷酸化位点。
这些结果证实了 RhoA/ROCK 途径及其随后的 moesin Thr558 磷酸化在 AGE 介导的内皮功能障碍中的作用。
