Fasudil Hydrochloride Hydrate Suppresses VEGF Induced Pulmonary Artery Smooth Muscle Cell Proliferation

Objective:Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a critical role in the development of pulmonary artery hypertension, and inhibition of PASMC proliferation has been shown to be beneficial to patients with this disease. Recent studies indicate that Rho/ROCK is critically involved in the proliferation of smooth muscle cells. However, the signal transduction of Rho/ROCK and its downstream signaling are not fully understood. In the present study, we investigated the antiproliferation effect of fasudil hydrochloride hydrate, a Rho-kinase inhibitor, on rat PASMC proliferation, and its possible relation with Rho/ROCK.Methods:Adult clean Sprague Dawley (SD) rats were selected, peritoneal injection with pentobarbitol sodium, pulmonary artery was separated in sterile condition, endothelium was removered, tunica media was cut into tissue pieces of approximate lmm x lmm x lmm with eye scissor, tissue pieces then were inoculated into 50 ml culture bottle in a density of 1 piece/cm2, cultured in routine methods, cells were identified with immunohistochemistry method. Experiments were carried out using cells of 3-5 generation with healthy growth. Cell proliferation was tested with MTT method, cell cycle was tested with Flow CytoMeter, ROCK-1 mRNA was measured with RT-PCR method.Results:The results indicate that fasudil effectively inhibited vascular endothelial growth factor (VEGF)-induced PASMC proliferation, the inhibition extents depend on the concentrations of fasudil, as evaluated by MTT assay. Flow cytometry analysis showed that fasudil markedly blocked VEGF-induced cell-cycle progression by arresting the cells in the Go/G1 phase. After stimulated by 10ng/ml VEGF for 24 h, proportions of PASMCs in the S phase increased significantly from 17.8% to 26.3%, in the mean time, proportions of PASMCs in the G1 phase decreased significantly from 75.3% to 54%. Fasudil of different concentrations (10,30,50, 100μmol/L) made the proportions of PASMCs in the S phase decreased to 15.2%, 11.1%,12.7% and 10.9%, proportions of PASMCs blocked in the G0/G1 phase increased to 62.9%,72.4%,74.7%and 76.3%. Consistently, VEGF-induced ROCK-1 mRNA expression and MYPT-1 phosphorylation were markedly suppressed by fasudil, and the inhibition extents depend on the concentrations of fasudil.Conclusions:Fasudil effectively suppressed VEGF-induced PASMC proliferation and cell-cycle progression. Rho/ROCK is involved.