| ObjectiveADMA is a risk factor of cardiovascular disease. DDAH1is the major metabolic enzyme to ADMA, which have been considered as an important target for the treatment of cardiovascular disease.we had found the third humer DDAH1transcript variant, but only the first transcript variant is positive correlation with ADMA metabolism. This study seeks to clarify simvastatin effect on the expression of DDAH1mRNA and protein, and to explore the different reactivity of DDAH1transcript variants to simvastatin.Methods(1)Primary cultured human umbilical vein endothelial cells were respectively treatment with DMSO, ADMA3μM, simvastatin1μM and AD MA3μM+simvastatin1μM to detect cell migration.(2)Three concentrations of simvastatin (0.2mM,1mM,2mM), were used to treat primary cultured human umbilical vein endothelial cells. The mRNA expression levels of DDAH1transcripts and protein were respectively determined by real-time polymerase chain reaction and western-blot.(3)Human umbilical vein endothelial cells were respectively treatment with2.5μg/ml Actinomycin D(ActD2.5μg/ml), middle and high concentrations of simvastatin combine with2.5μg/ml Actinomycin D for oh,lh,2h,4h,8h and12h. The mRNA expression levels of DDAH1transcripts were determined by real-time polymerase chain reaction.Results(1) Compared with DMSO, ADMA3mM+DMSO (1.30±0.30vs1±0.00, P=0.012) induced cell migration, simvastatin1mM (0.79±0.22vs1±0.00, P=0.10) only show a tendency of inhibiting cell migration. Compared with ADMA3mM, ADMA3mM+simvastatin1mM (0.94±0.086vs1.30±0.30, P=0.011) inverted the increased cell migration by ADMA.(2) Compared with DMSO, simvastatin did not affect the mRNA expression of DDAH1-V1, but decreased DDAH1-V2and DDAH1-V3mRNA expression, middle and high concentration simvastatin inhibited DDAH1-V2(0.52±0.24vs0.89±0.23, P=0.044and0.38±0.19vs0.89±0.23, P=0.0093) and DDAH1-V3(0.59±0.19vs0.86±0.05, P=0.028and0.40±0.15vs0.86±0.05, P=0.0013) encoding mRNA expression in a concentration-dependent way.(3)Compared with DMSO, simvastatin increased DDAH1(1.35±0.16vs1±0.00,P=0.03;1.90±0.29vs1±0.00, P=0.00014and1.95±0.04vsl±0.00, P=0.0001) protein expression in a concentration-dependent way when the treatment time was12hour. Extend the treatment time to24hour, although simvastatin show a tendency of increasing DDAH1(2.11±0.55vs1±0.00,P=0.0097) protein expression, but only high concentrations of simvastatin was statistically significant.(4)Compared with DMSO, ActD2.5μg/ml show a tendency of increasing DDAH1-V1mRNA ACT(CTDDAH1transcripts-CTGAPDH) value in a treatment time of1to12hour, but only at the treatment time of1hour (5.63±0.64vs4.64±0.37, P=0.011) was statistically significant. Compared with ActD2.5μg/ml in the same treatment time, Sim1μM+ActD reduced DDAH1-V1mRNA ACT in a treatment time of1to4hour, but only at the treatment time of1hour (4.66±0.27vs5.63±0.64, P=0.0085) and2hour (3.53±0.60vs5.89±0.15, P=0.021) were statistically significant; Sim2μM+ActD reduced DDAH1-V1mRNA ACT in a treatment time of1to4hour, but only at the treatment time of1hour (4.36±0.17vs5.63±0.64,P=0.0040) statistically significant. (5)Compared with DMSO, ActD2.5μg/ml show a tendency of increasing DDAH1-V2mRNA△CT(CTDDAH1transcripts-CTGAPDH) value in a treatment time of8to12hour, but only at the treatment time of8hour (8.06±0.49vs7.21±0.15, P=0.012) was statistically significant. Compared with ActD2.5μ.g/ml, Sim1μM+ActD and Sim2μM+ActD did not alter DDAH1-V2mRNA ACT value.(6)Compared with DMSO, ActD2.5μg/ml show a tendency of increasing DDAH1-V3mRNA ACT(CTDDAH1transcripts-CTGAPDH) value in a treatment time of2to12hour, but the difference did not statistically significant. Compared with2.5μg/ml Actinomycin D, Sim1μM+ActD and Sim2μM+ActD did not alter DDAH1-V3mRNA ACT value.Conclusions(1) Our results demonstrated that ADMA was able to induce cell migration, while simvastatin reversed the effection of ADMA on cell migration.(2)Simvastatin was able to upregulate of DDAH1-V1protein expression and downregulate DDAH1-V2and DDAH1-V3mRNA expression, but did not alter DDAH1-V1mRNA expression.(3) Simvastatin upregulated the expression of DDAH1-V1protein may by increasing DDAH1-V1mRNA stability. |
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