H-1152 was employed to specifically block activation of ROCK

H-1152 was employed to specifically block activation of ROCK. was dominant unfavorable for RhoA (RhoA N19) was carried out 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 Flavopiridol HCl conversation 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. Results The results showed that AGE-HSA increased the permeability of HMVEC monolayer and brought on 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 conversation of ROCK and moesin. Thr558 was further identified as the phosphorylating site of moesin in AGE-evoked endothelial responses. Conclusion These results confirm the involvement of RhoA/ROCK pathway and subsequent moesin Rabbit polyclonal to annexinA5 Thr558 phosphorylation in AGE-mediated endothelial dysfunction. Keywords: advanced glycation end products (AGEs), vascular permeability, Flavopiridol HCl RhoA/ROCK pathway, moesin Background Advanced glycation end products (AGEs) are a heterogeneous group of complex compounds that are created irreversibly in serum and tissues via a chain of nonenzymatic chemical reactions [1]. The role of AGEs in the development of diabetes, especially diabetic complications, has been emphasized in many reports [2,3] and the level of AGEs is usually correlated with the severity of diabetic complications [4-7]. Direct effects of AGEs include formation of extracellular cross-links that may trap numerous unrelated macromolecules. Furthermore, AGEs can bind to numerous receptors, such as the receptor for AGE (RAGE) or AGE receptors 1-3 (AGER1-3), leading to complex effects on cellular function via complicated transduction pathways [8-10]. The major AGE receptor (RAGE) enhances inflammation, while Flavopiridol HCl AGER1 promotes the removal of AGEs and blocks inflammation [11]. The vascular endothelium is the front-line organ for vascular injury and a common target of various risk factors, with dysfunction of the microvascular endothelial barrier playing a critical role in the pathogenesis of insulin resistance and diabetes [12]. 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 [13]. It has been reported that AGEs cause significant disorganizations of the F-actin cytoskeleton, disruption of tight junctions and adherens junctions in cultured human umbilical venous endothelial cells (HUVECs), and increase the permeability of EC monolayers [14,15]. The Rho family of small GTPase proteins control a wide variety of cellular processes. RhoA is one of the Flavopiridol HCl best-known users of this family and the Rho kinases (ROCK) are the first and the best-characterized RhoA effectors. By modulating the organization of the actin cytoskeleton, RhoA/ROCK signaling regulates a wide range of cellular functions, such as contraction, motility, proliferation, and apoptosis. It has been shown that ROCK-dependent re-arrangement of the actin cytoskeleton and changes of cell contractility are involved in the regulation of endothelial permeability [16-19]. Our previous studies have also suggested the involvement of ROCK in Flavopiridol HCl AGE-induced endothelial responses. Inhibition of ROCK with Y-27632 was reported to reduce the AGE-evoked formation of actin stress fibers and the weakening of adherens junction [15] in HUVECs. Y-27632 also abolished the AGE-induced increase of ROCK phosphorylation [20]. Using anti-RAGE antibody, we and Hirose et al. have exhibited in HMVECs and HUVECs, respectively, that activation of RhoA depends on the binding of AGEs to RAGE [14,20]. Since ROCK does.