| OBJECTIVE:To investigate the effect of Angiotensin II Type 1 Receptor Associated Protein(ATRAP)on the apoptosis of cultured vascular smooth muscle cells(VSMC)in vitro and its related mechanism.To investigate the effect of adenovirus over-expression of ATRAP on intimal hyperplasia Caused by Carotid Artery Balloon Injury in rat.Thereby to enrich the mechanism of ATRAP inhibiting carotid intimal hyperplasia and provide a new therapy direction for the treatment of carotid restenosis.Method:In vitro experiments:1 acquisition,culture and identification of VSMCVSMC was purchased from Guangzhou Jennio Biotechnology Co.,Ltd.,taken from the thoracic aorta tissue of rat and obtained by enzyme digestion method.The cells were cultured in high glucose DMEM supplemented with 10%fetal bovine serum(FBS).Cell growth status and confluency were observed under a leica inverted microscope,and the VSMC phenotype was identified by a-SM-actin staining.2 The effect of ATRAP overexpression on VSMC apoptosis was detected by TUNEL assay2.1 Construction of ATRAP overexpression adenovirus and control adenovirusATRAP overexpression vector was constructed by Hanbio Biotech(Shanghai)Co.,Ltd.and the control adenovirus was also provided.2.2 The effect of ATRAP over-expression on VSMC apoptosisVSMCs were cultured to 5 to 7 passages and the confluency reached 90%.After trypsinization,the cells were inoculated into 96-well plates(1×105 cells/mL)and transfected with Adv.ATRAP or Adv.GFP.After 24 hours,the cells were replaced by hign glucose DMEM medium supplemented with 0.1%FBS for 24 hours for synchronization,Ang ?(100 nM)was added and continued to culture for 24 hours.End deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay(TUNEL assay)was used to determine the rate of apoptotic cells under the leica live cell workstation.2.3 RNA extraction and real-time fluorescence quantitative PCR to determine the mRNA expression changes of apoptosis-related genesVSMC seeded in 6-well plates and other processes was treated as described above.Total RNA was extracted from the VSMC using the RNA isoplus kit,reverse transcribed into cDNA according to the PrimeScript TM RT Master Mix reverse transcription kit instructions,and real-time fluorescence quantitative PCR was performed on Applied Biosystems QuantStudio(?)6 Flex to detect changes in the mRNA expression of the apoptotic relevant genes.2.4 Western blot detection of ATRAP and apoptosis-related protein expressionThe total protein of VSMC was extracted by RIPA and PI(20:1)mixture,and the expression of ATRAP and apoptosis related proteins(caspase3,caspase8,caspase9,bcl-2 and bax)were detected by western blot.3 Flow cytometry was used to determine the effect of ATRAP on VSMC apoptosis3.1 Construction of ATRAP siRNA and Negative ControlATRAP siRNA was synthesized by Genepharma ShangHai Co.,Ltd.and a negative control was also provided.3.2 Identification of Interference Efficiency of Interfering Sequences on ATRAP siRNAVSMCs were seeded on 6-well plates and transfected with Lipofectamine TM RNAiMAX Transfection Reagent(Invitrogen TM)to the medium when cell confluency reached 70-90%.After 48 hours treated,then total RNA was extracted by Trizol method and the interference efficiency of each group of interference sequences on ATRAP was detected by fluorescence quantitative PCR.3.3 Detection of protein changes of apoptosis-related genes by westen blot methodThe cells were treated as described above and the total cellular protein was extracted.The changes of ATRAP,cleaved-cas8,cleaved-cas-3,bcl-2 and bax were measured by western blotting.GAPDH was used as internal control.4 Mechanism investigation of over-expression of ATRAP on VSMC apoptosis4.1 The effect of ATRAP on Angiotensin-induced Akt phosphorylationVSMCs were inoculated into 6-well plates and transfected with Adv.ATRAP or Adv.GFP at the confluency of 50-70%.After 24 hours,the culture media was replaced with high glucose DMEM medium supplemented with 0.1%FBS for 24 hours.Phosphorylated protein induced by Ang ?(100 nM)was extracted at several time points(0 min,30 min,45 min,60 min,2 h,4 h),and the changes of p-Akt were detected by western blot.4.2 Effect of PTEN Inhibitors on the Inhibition of ATRAP on p-akt Phosphorylation induced by Ang ?After transfection with adenovirus as described above,PTEN inhibitor-bpv(200 nM)was added 4 h prior to Ang ? treatment and then incubated with Ang ?(100 nM)for 0 min,30 min,45 min,60 min,2 h and 4 h.The phosphorylated proteins were extracted and the changes of p-Akt were detected by western blot.4.3 The effect of PTEN inhibitor on VSMC apoptosis caused by over-expression of ATRAPTUNEL was used to determine the effect of PTEN inhibitor on VSMC apoptosis caused by ATRAP overexpression.The changes of mRNA and protein levels of apoptosis-related proteins were detected by real-time fluorescence quantitative PCR and western blot respectively.In vivo experiments5 The effect of adenovirus-transfected ATRAP on intimal hyperplasia induced by carotid artery balloon injury in rats5.1 Construction of rat carotid artery balloon injury model and adenovirus local transfectionLeft common carotid artery balloon injury in rats was caused by 2F fogarty thrombectomy catheter,and locally incubated by ATRAP over-expression adenovirus or control virus for 30 min and virus usage were 2*10 ^ 9 pfu.5.2 Effect of adenovirus transfected ATRAP on intimal hyperplasia induced by carotid artery balloon injury in ratsThe rats were sacrificed at different time points(3 days,7 days,14 days)and the left common carotid artery was divided into two parts.The RNA extracted from some specimens was used to detect the mRNA changes of ATRAP and apoptosis related proteins,and the other specimens were fixed with paraformaldehyde and carotid artery cross-sectional vascular elastography-Verhoeff-Van Gieson(VVG)staining was performed after fixation.5.3 Effect of adenovirus transfected ATRAP on VSMC apoptosis in balloon injuried carotid artery in ratsCell apoptosis in balloon-injured carotid artery in rats was determined by Tunel/DAPI immunofluorescence double staining.Result:1 VSMC acquisition,culture and identificationVSMCs showed adherent growth;a single cell was elongated fusiform;cell growed densely and arranged in parallel lines;the typical "peak-valley" growth under inverted fluorescence microscopy.After staining with a-SM-actin,VSMCs showed green fluorescence under inverted fluorescence microscopy.2 Adenovirus transfection of overexpression ATRAP promotes apoptosis of cultured rat VSMCs2.1 Cell processingAdenovirus of overexpression of ATRAP or control adenovirus were transfected to cultured VSMC for 24 hours,replaced with high glucose DMEM medium supplymentary with 0.1%FBS.Ang ? was added into media after 24 hours and continued to culture for 24 hours.TUNEL staining showed that over-expression of ATARP promoted VSMC apoptosis in vitro,and the effect had no significant correlation with Ang ? stimulation.The apoptosis rate of VSMC in Adv.ATRAP transfected group was significantly higher than that in Adv.GFP group in both with Ang ? stimulation(11.50 ± 1.66%vs.1.13 ± 0.27%,P<0.05)and without Ang? stimulation(13.17 ± 0.48%vs 1.20 ± 0.17%,P<0.05).2.2 Detection of ATRAP and Apoptosis-related Genes mRNA Expression by Fluorescent quantitative PCR methodThe results showed that ATRAP overexpression caused a significant upregulation in the mRNA expression of pro-apoptotic gene-bax and a downregulation in the mRNA expression of apoptosis suppressor gene-bcl-2.2.3 Western blot detection of ATRAP and apoptosis-related protein expressionWestern blot results showed that ATRAP and GFP did not form a fusion protein,cleaved-caspase 8,cleaved-caspase 3,bax protein expression were increased,bcl-2 protein expression was decreased and cleaved-caspase-9 expression did not change significantly,indicating that ATRAP overexpression induced pro-apoptotic protein expression increased and promoted VSMC apoptosis mainly through the death receptor pathway,that is exogenous pathway.3 Effect of ATRAP interference on VSMC Apoptosis3.1 Identification of interference effects of interference sequence on ATRAP by Fluorescence quantitative PCRThe results shows that ATRAP interference efficiency in 325 interference sequence group is higher than other groups.3.2 westen blot detection of apoptosis-related protein changesThe results of western blot showed that the expression of cleaved-caspase 8,cleaved-caspase 3 and bax was decreased and the expression of bcl-2 was increased after ATRAP interference,thus ATRAP silence could inhibit the apoptosis of VSMC.4 Mechanism of overexpression of ATRAP in VSMC apoptosis4.1 ATRAP inhibits Angiotensin ?-induced Akt PhosphorylationCompared with Adv.GFP group,the expression of p-Akt protein in Adv.ATRAP group was significantly decreased(P<0.05)at multiple time points-30 min,45 min,60 min,2 h,4 h,which suggested that overexpression of ATRAP inhibited Akt phosphorylation induced by Ang ? stimulation.4.2 PTEN inhibitors could reverse ATRAP Angiotensin ?-induced Akt phosphorylation inhibitionThe expression of p-Akt in Adv.ATRAP + bpv group was significantly higher than that in Adv.ATRAP group(P<0.05)at multiple time points-30 min,45 min,60 min,2 h,4 h.In VSMC,PTEN inhibitor reversed the inhibition of Akt phosphorylation induced by Ang II stimulation caused by ATRAP overexpression.4.3 PTEN inhibitors can reverse the promotion of VSMC apoptosis caused by ATRAP overexpressionCompared with Adv.ATRAP group,the apoptosis of VSMC in Adv.ATRAP +bpv group was significantly lower than that in Ang ? stimulation group(1.81 ±0.19%vs 11.50 ± 1.66%,P<0.05)and non-Ang II stimulation(1.45 ± 0.15%vs 13.17 ± 0.48%P<0.05);mRNA and protein expression also supported PTEN inhibitor reversed the apoptosis in VSMCs.In vivo experiments5 Adenovirus-transfected ATRAP inhibits intimal hyperplasia induced by carotid balloon injury in rats3 days after balloon injury and adenovirus incubation,there was no significant difference in the intima hyperplasia of left common carotid artery between Adv.ATRAP transfection group and Adv.GFP transfection group.However,7 days and 14 days after balloon injury and adenovirus incubation,the intima area and the ratio of intima/media(I/M)in Adv.ATRAP group were significantly lower than that in Adv.GFP group(P<0.05).6 Adenovirus-transfected ATRAP promotes VSMC apoptosis in the carotid artery of ratsTUNEL/DAPI immunofluorescence double staining results showed that the cell apoptosis in the intima of injuried left common carotid artery showed no significant difference between Adv.ATRAP transfected group and Adv.GFP transfected group 3 days after balloon injury and adenovirus incubation.The apoptotic rate of Adv.ATRAP group was significantly higher than that of Adv.GFP group 7 days and 14 days after balloon injury and adenovirus incubation(P<0.05).Conclusion:1 Adenovirus overexpression of ATRAP promotes apoptosis in cultured rat VSMCs 2 ATRAP overexpression inhibits Akt phosphorylation induced by Ang ±stimulation3 PTEN inhibitor significantly inhibited ATRAP-induced apoptosis in rat VSMCs4 PTEN inhibitors can reverse the inhibition of Akt phosphorylation induced by Ang ? stimulation caused by ATRAP overexpression5 RNA interference of ATRAP inhibits VSMC apoptosis6 Overexpression of ATRAP significantly inhibited neointimal formation after carotid artery balloon injury in rats7 Overexpression of ATRAP promotes cell apoptosis in the carotid artery in rats... |
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