Rho/ROCK Signal Cascade Mediates Asymmetric Dimethylarginine-Induced Vascular Smooth Muscle Cells Migration and Phenotype Change
Figure 2
ADMA induces VSMCs migration and proliferation via RhoA/ROCK cell signaling transduction way. (a) The affinity precipitation of RhoA with GST-rhotekin fusion protein. The graphs represent the ratio of pull-down RhoA to total RhoA of every group to VSMCs without ADMA treatment. (b) Phosphorylation level of the MYTP-1 in VSMCs. Confluent VSMCs were pretreated with Y27632 (10 μM) or not and then incubated with ADMA at the concentration of 10 μM for 30 min. (c) The migration ability of VSMCs was assessed by the confluent area of VSMCs in a wound-healing assay. Confluent VSMCs were pretreated with Y27632 or not (10 μM, 2 h) and then incubated with ADMA at the concentration of 10 μM for 24 h. The upper, middle, and bottom images represent the beginning point (no incubation with ADMA), end point (incubation with ADMA for 24 h), and merged images, respectively. The red shadow represents the change in covered area. (e) The migration activity was expressed as the X-folds increase of the change in covered area by comparing to VSMCs without ADMA treatment. (d) The proliferation ability of VSMCs was assessed by the DNA synthesis rate of VSMCs in a BrdU incorporation assay. After incubation with mouse anti-BrdU antibody and Isotype-matched fluorescein isothiocyanate- (FITC-) conjugated anti-rat IgG1 secondary antibody, DAPI was employed to detect nuclei. The upper, middle, and bottom images represent the BrdU positive VSMCs, nucleolus of all VSMCs in an observation field, and merged images, respectively. (f) The proliferation activity was expressed as the X-folds increase of the synthesis rate by comparing to VSMCs without ADMA treatment. Data shown are the mean ± SEM from three independent experiments, ; .
