| Part One Lovastatin Inhibits Gap Junctional Communication in Cultured aortic smooth muscles cellsPURPOSE:To explore the effects of lovastatin and pravastatin on migration and gap-junctional intercellular communication(GJIC) of aortic smooth muscle cells,these results provide novel evidence for the key role of GJIC in migration of aortic smooth muscle cells and the possible mechamsm in anti-migration effect of statins.METHODS:1,Cell culture:smooth muscle cells were cultured as routine method.2,The cells quiesced for 72 h were suspended in DMEM with 0.4%FBS and added to the upper chamber of Transwell system.The lower chamber was filled with 2mL DMEM with 0.4%FBS with or without lovastatin.These were used to determine the migration of the cells.3,The cells were cultured to DMEM with 0.4%FBS with or without lovastatin or pravastatin at concentration of 0.08,0.4,2,or 10umol/L for 48 h,then subjected to FRAP analysis to determine the GJIC.Results:1,The migration of the cultured rabbit aortic smooth muscle cells were detected by Transwell system.Cells that had migrated below the membrane were counted in 20 high-power microscope fields.Cell migration was dose dependently inhibited with lovastatin.Compared with control(110±26),the number of migrated SMCs was significantly reduced to 72±24(P＜0.05),62±18(P＜0.01) and 58±19(P＜0.01) at the concentration of 0.4 umol/L,2 umol/L and 10 umol/L,per filed.2,The functions of GJIC in the cultured rat aortic smooth muscle cells were detected by FRAP techniques.The fluorescence intensity was gradually recovered at different times after bleaching.The mean fluorescence recovery rate of the control group(5 minutes) was 36.11%±10.53%compared with 24.38%±4.84%expressed in those treated with 10 uM lovastatin.Smaller concentrations of 2 uM,0.4 uM and 0.08 uM were also tested.It was found that gap junction communication was inhibited in a dose-dependent manner.Conclusions:1,Cell migration was dose dependently inhibited with lovastatin.2,Up to now,little work has been done on the effects of statins on gap junction intercellular communication(GJIC).Therefore,in the present study,we report the first functional study on the effects of statins on GJIC.On the basis of the results of this experiment,FRAP techniques could accurately reveal the inhibited effect of statins on the GJIC of rat aortic smooth muscle cells.These findings may explain the mechanisms of statins inhibiting SMCs migration partly. Part Two Effects of statins on gap-junctional intercellular communication and migration of aortic smooth muscle cells in vitroPURPOSE:To explore the effects of lovastatin on migration and gap-junctional intercellular communication(GJIC) of aortic smooth muscle cells,and compare the effects of lipophilic lovastatin and hydrophilic pravastatin,these results provide vital evidence for the key role of GJIC in migration of aortic smooth muscle cells and the possible mechamsm in anti-migration effect of statins.METHODS:1,Cell culture:smooth muscle cells were dissociated by collagenase and elastase digestion and cultured.2,The cells quiesced for 72 h were suspended in DMEM with 0.4%FCS and added to the upper chamber of Transwell(Costar) system.The lower chamber was filled with 2mL DMEM with 0.4%FBS with or without lovastatin or pravastatin.These were used to determine the migration of the cells.3,The cells were cultured to DMEM with 0.4%FBS with or without lovastatin at concentration of 0.08,0.4,2,or 10umol/L for 48 h,then subjected to FRAP analysis to determine the GJIC.Results:1,We found that both lovastatin and pravastatin could significantly inhibit SMC migration(43±7 and 81±15 cells in presence of 10 uM lovastatin and pravastatin,per field) compared to the control in which cells were cultured with simple medium alone (120±27 cells per field)(P＜0.05). 2,Both lovastatin and pravastatin inhibited gap junctional intercellular communication.Lovastatin 10uM and pravastatin 10 uM attenuated fluorescence recovery rates by 17.83%±6.49%and 29.54%±8.13%,respectively,as measured by FRAP(P＜0.01).The mean fluorescence recovery rate of the control group was 53.4%±13.84%;3,Compared with hydrophilic pravastatin,lipophilic lovastatin more significantly down-regulated GJIC and migration of rabbit aortic SMCs at the concentration of 2 umol/L and 10 umol/L(P＜0.05).Conclusions:1,Cell migration and GJIC were dose dependently inhibited with lovastatin or pravastatin.2,Compared with hydrophilic pravastatin,lipophilic lovastatin more significantly down-regulated GJIC and migration of rabbit aortic SMCs.3,The inhibited effects of statins on the GJIC may explain the mechanisms of statins inhibiting SMCs migration partly. |
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