Rho/ROCK Signal Cascade Mediates Asymmetric Dimethylarginine-Induced Vascular Smooth Muscle Cells Migration and Phenotype Change
Figure 1
ADMA upregulates RhoA activity. (a) Confluent VSMCs (starved for 48 h in FCS-free DMEM without L-arginine) were treated with ADMA at different concentrations (0-30 μM) for 30 min. (b) VSMCs were pretreated with L-arginine (1 mM) for 2 h and then incubated with ADMA or SDMA at the concentration of 10 μM for 30 min. (c) VSMCs were transfected with the reconstitute human DDAH2 overexpression plasmid vector (pAV-CMV-GFP-DDAH2) or the blank vector pAV-CMV-GFP for 48 h and then incubated with ADMA at the concentration of 10 μM for 30 min. VSMCs were lysed with an ice-cold cell lysis buffer. Cell lysates were incubated with GST-fused Rho-binding domain of rhotekin which were bound to glutathione-Sepharose beads for 1 h, and bound proteins were immunoblotted with anti- RhoA antibody. The graphs represent the ratio of pull-down RhoA to total RhoA of every group to VSMCs without ADMA treatment. Data shown are the mean ± SEM from three independent experiments, ; .
