The Effect Of Vascular Endothelial Growth Factor 165 On KCNQ1/KCNE1 Current In Human Embryonic Kidney 293 Cells And Its Mechanism

Backgrounds and Objectives:Vascular endothelial growth factor 165 has a strong role in promoting angiogenesis in vivo,because of this characteristic,people regard it as a potential therapeutic drug for ischemic diseases such as myocardial infarction.Many studies have shown that vascular endothelial growth factor 165 can protect myocardial cells by reducing infarct size,promote stem cell migration to ischemic sites,prevent ventricular remodeling,and resist apoptosis of myocardial cells.However,whether this cytoprotection is related to the direct effect of vascular endothelial growth factor165 on ion channels in cardiomyocytes has not been confirmed.Slowly activated delayed rectifier potassium channels(I_ks)are widely distributed in mammalian cardiomyocytes and play an important role in action potential repolarization.It has been demonstrated that I_ks is a complex composed of?subunits and?subunits encoded,respectively,by the KCNQ1 and KCNE1 genes.In this study,cells transfection and patch clamp techniques were used to analyze the effects of vascular endothelial growth factor 165 on KCNQ1/KCNE1 current in heterologous expression system and explore its possible mechanism.Materials and Methods:Human embryonic kidney 293 cells were randomly divided into control group,experimental group A and experimental group B.The control group cells were cultured normally without treatment.Group A cells were transfected with plasmids containing KDR,KCNQ1 and KCNE1 genes,while group B cells were transfected with plasmids containing KDR and KCNQ1 genes.About 48 hours after the exogenous gene transfection,the PCR technique was used to verify the success of the transfection..Human embryonic kidney 293 cells in group A and group B were treated with recombinant human vascular endothelial growth factor 165 at the same concentration.The current was recorded by whole cell patch clamp technique before and after treatment.Results:1.The results of PCR indicated that the target genes were successfully transfected into human embryonic kidney 293 cells in both group A and group B.2.Cells in group A were treated with VEGF 165,At-20,0,+20,+40 and+60mV,the normalized activated currents?0.078+0.060,0.243+0.090,0.465+0.080,0.750+0.060,1.000+0.000?before treatment were significantly higher than those after treatment?0.045+0.040,0.159+0.060,0.349+0.050,0.568+0.090,0.729+0.100?,the difference was statistically significant;The normalized tail currents?0.068+0.030,0.252+0.080,0.526+0.070,0.766+0.060,1.000+0.000?before treatment were significantly higher than those after treatment?0.054+0.030,0.167+0.090,0.409+0.050,0.607+0.060,0.803+0.030?,the difference was statistically significant.3.Cells in group B were treated with VEGF 165,At-20,0,+20,+40 and+60mV,there were no significant difference in the normalized activated currents before and after treatment,there were no significant difference in the normalized tail currents before and after treatment.Conclusion:At the concentration of 100 ng/mL,VEGF 165 could directly inhibit the current of KCNQ1/KCNE1 in human embryonic kidney 293 cell.VEGF 165 could not directly inhibit KCNQ1 current in human embryonic kidney 293 cells,suggesting that the inhibition of KCNQ1/KCNE1 current by VEGF 165 requires?subunit?KCNE1?.