RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular response

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-moesinT558A and pcDNA3/HA-moesinT558D 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.Advanced glycation end products (AGEs) are a heterogeneous group of complex compounds that are formed irreversibly in serum and tissues via a chain of non-enzymatic chemical reactions [1]. The role of AGEs in the development of diabetes, especially diabetic complications, ha