Effects Of Rho/ROCK Signal Pathway On AGE-induced Morphological And Functional Changes In HMVECs

Objective:This study is to determine the effects of RhoA/Rho kinase(RhoA/ROCK) phosphorylation on morphological and functional changes induced by advanced glycation end products(AGEs) in endothelial cells.Methods:AGEs were prepared by incubating human serum albumin(HSA) in PBS with D-glucose for 8 weeks in a sterile environment at 37℃.The dialyzed end products were expressed as AGE-HSA in this paper.Using immortalized human dermal microvascular endothelial cells(HMVECs),the effects of AGE-HSA on endothelial morphological and functional changes were detected by staining F-actin with rhodamine-phalloidin and measuring monolayer permeability with tracer protein TRITC-albumin.The phosphorylation of RhoA and ROCK were determined by western blot with primary antibodies of RhoA or ROCK respectively.HMVECs were respectively incubated with AGE-HSA in different concentrations and timing.In some other cases,HMVECs were pretreated with ROCK inhibitor H-1152 or Y-27632.The recombinant adenovirus of dominant negative RhoA N19 was used to down-regulate the expression of RhoA,while a constitutively active RhoA L63 was used to activate RhoA in HMVECs.Results:The distribution of F-actin was significantly altered by AGE-HSA treatment in time- and dose-dependent patterns with the formation of F-actin stress fibers and the impairment of cortical F-actin ring.AGE-HSA administration also increased the permeability of endothelial monolayer.Inhibition of ROCK activation by pre-applicating ROCK inhibitor H-1152 or Y-27632 significantly attenuated those responses.Down-regulation of RhoA by transfecting recombinant adenovirus of dominant negative RhoA N19 abolished this AGE-induced F-actin reorganization and monolayer hyperpermeability.The transfection of recombinant adenovirus of constitutively active RhoA L63 mimicked the AGE-evoked formation of stress fiber and hyperpermeability in ECs.The phosphorylations of RhoA and ROCK were remarkably increased by AGE-HSA treatment in time-and concentratin-dependent manners,while Rho and ROCK protein levels were not affected.The phosphorylation of ROCK was blocked both by ROCK inhibitor H-1152 or Y-27632.The phosphorylation of RhoA was also restrained by surpression of RhoA expression with dominant negative RhoA N19.The constitutively active RhoA L63 alone increased the phosphorylation of RhoA.To explore the mechanism of RhoA/ROCK pathway activation in AGE-induced morphological and functional alterations in HMVECs,the phosphorylation of p38 MAPK were compared with or without ROCK inhibitor H-1152 or Y-27632.Interestingly,while inhibition of ROCK decreased the AGE-triggered p38 MAPK phosphorylation,inhibition of p38 activation with SB203580 also attenuated AGE-HSA-induced phorphorylations of RhoA and ROCK.Conclusions:The results obtained in this study confirm the involvement of Rho/ ROCK pathway in AGE-HSA-stimulated endothelial responses.The data also suggest that the phosphorylation of Rho/ROCK plays an important role in AGE-HSA -induced morphological and functional alterations in HMVECs.The results indicate that there would be a cross-talk between Rho/ROCK and p38 MAPK pathways in these AGE-HSA -stimulated cellular responses.While numerous evidences showed that Rho/ROCK regulates p38 MAPK activation,our data show that p38 might inversively regulate ROCK activity as well.