| Part I The regulation and mechanism research of autophagy on endothelial-to-mesenchymal transition induced by hypoxia in human cardiac microvascular endothelial cellsObjective1.To explore the effects of hypoxia on endothelial-to-mesenchymal transition(EndMT)and autophagy in human cardiac microvascular endothelial cells(HCMECs).2.To investigate the effects and precise mechanisms of autophagy on hypoxia-induced EndMT of HCMECs.Methods1.To explore EndMT of HCMECs under hypoxia conditions.Cells were divided into two groups including,(1)Control group: HCMECs were cultured under normoxia conditions.(2)Hypoxia group: HCMECs were cultured under hypoxia conditions(1%O2)for 3 days,Then perform next experiments: ?Observing the morphological changes under the light microscope.?The relative changes in CD31,E-cadherin,?SMA,FSP-1 and Snail mRNA were analysised by RT-PCR.?The expression of CD31,E-cadherin,?SMA,FSP-1 and Snail protein were examined by Western blot.? Double immunofluorescence staining of CD31/?SMA and CD31/FSP-1 was used to detected the cells undergoing EndMT.2.To explore autophagy of HCMECs under hypoxia conditions.?The dynamic changes of LC3 protein expression in HCMECs within 3 days under hypoxia condition,The protein expression of LC3 was examined in HCMECs at 0,2,4,6,1224,48,72 h by Western blot.?Cells were divided into four groups including,(1)Control group: HCMECs were cultured under normoxia conditions;(2)chloroquine(CQ)group: HCMECs were treated with CQ(20 ?M)for8 hours under normoxia conditions.(3)Hypoxia group: HCMECs were cultured under hypoxia conditions for 6 hours;(4)Hypoxia+CQ group: HCMECs were pretreatedwith CQ(20 ?M)for 2 hours and cultured under hypoxia conditions for another 6hours.The protein expression of LC3 was examined by Western blot in each group.?The dynamic changes of GFP-LC3 expression in HCMECs within 3 days under hypoxia condition.Observing GFP-LC3 punctate formation in HCMECs at 0,2,6,12,24,36,48,72 h under hypoxia hypoxia by confocal microscope.3.To investigate the effects and precise mechanisms of autophagy on hypoxia-induced EndMT of HCMECs.Cells were divided into five groups including,(1)Control group: HCMECs were cultured under normoxia conditions;(2)Hypoxia group: HCMECs were cultured under hypoxia conditions for 3 days;(3)Hypoxia+rapamycin(rap)group: HCMECs were pretreated with rapamycin(100 nM)for 2 hours and cultured under hypoxia conditions for 3 days;(4)Hypoxia+3-methyladenine(3-MA)group: HCMECs were pretreated with 3-MA(5mM)for 2 hours and cultured under hypoxia conditions(1% O2)for 3 days;(5)Hypoxia+CQ group: HCMECs were pretreated with CQ(20 ?M)for 2 hours and cultured under hypoxia conditions for 3 days.Then perform next experiments :?Double immunofluorescence staining of CD31/?SMA was used to detected the cells undergoing EndMT.The colocalization of LC3 and Snail in HCMECs of each group by confocal microscope.?The relative changes in CD31?E-Cadherin??SMA?FSP-1 ? Snail ? TGF?2 ? Smad2 and Smad3 were analysised by RT-PCR.?The expressions of CD31?E-cadherin??SMA?FSP-1?Snail?LC3?beclin1?TGF?2?Smad2?p-Smad2?Smad3 and p-Smad3 protein were examined by Western blot.Results1.Hypoxia triggered EndMT of HCMECs:?ECs changed gradually from a cobblestone phenotype to an elongated spindle-shaped morphology.?Compared with the control groups,cells acquired mesenchymal makers(?-SMA,FSP1 and Snail)and lost endothelial makers(CD31and E-cadherin)(P<0.05).?RT-PCR analysis showed that hypoxia decreased the expression of CD31 and E-cadherin mRNAs but increased ?-SMA,FSP1 and Snail mRNA levels(P<0.05).?Immunofluorescence results further showed that CD31+?-SMA/FSP1+ cells or CD31-?-SMA/FSP1+ cells were found in the hypoxia groups,and CD31+?-SMA/FSP1-cells were found in the normoxia group(P<0.05).2.Autophagic flux of endothelial cells was increased under hypoxia:?Western blot showed that the ratio of LC3 II/I increased gradually at 4 h,reaching maximum at 12 h to 24 h,and subsequently declined(P<0.05).?LC3turnover assay showed that accumulation of LC3-II increased by the treatment with CQ under normoxia conditions,but the difference in LC3-II levels in the presence and absence of CQ was larger under hypoxia than normoxia conditions(P<0.01).?GFP-LC3 puncta formation assay showed that the number of punctate GFP-LC3 increased gradually at 4 h,reaching maximum at 12 h to 24 h,and subsequently declined(P<0.05).3.The effects and precise mechanisms of autophagy on hypoxia-induced EndMT of HCMECs:?Immunofluorescence results showed that rapamycin decreased the number of CD31+?-SMA+ cells and CD31-?-SMA+ cells under hypoxia conditions,3-MA and CQ markedly increased the number of CD31-?-SMA+cells under hypoxia conditions(P<0.05).A large amount of orange-colored dots were visualized in the merged images under hypoxia alone or with CQ,which was confirmed by the colocalization of LC3 and Snail in ECs(P<0.05).?RT-PCR analysis showed that rapamycin partly inhibited the loss of CD31 and E-cadherin mRNA and the increase of ?-SMA,FSP-1mRNA induced by hypoxia(P<0.05).3-MA or CQ markedly downregulated the expression of CD31 and E-cadherin proteins and significantly upregulated the expression of mesenchymal makers and transcription factor Snail under hypoxia condition(P<0.05).TGF-?2,smad2,p-smad2,smad3,p-smad3 and snail mRNA did not change under hypoxia condition with or without rapamycin,3-MA or CQ.?Western blot showed that rapamycin partly inhibited the loss of CD31 and E-cadherin proteins and the increase of ?-SMA,FSP1 and Snail proteins induced by hypoxia,corresponding with the increased ratio of LC3 II/I and beclin1 expression.3-MA or CQ suppressed LC3 and beclin1 expression under hypoxia conditions(P<0.05),meawhile,markedly downregulated the expression of CD31 and E-cadherin proteins and significantly upregulated the expression of mesenchymal makers and transcription factor Snail(P<0.05).TGF-?2,smad2,p-smad2,smad3 and p-smad3 protein did not change under hypoxia condition with or withoutrapamycin,3-MA or CQ.Conclusions:1.EndMT and autophagy were induced simultaneously by hypoxia in ECs.2.The molecular mechanism of EndMT induced by hypoxia should be hypoxia-induced autocrine TGF-b 2 elevation,which leads to Snail upregulation triggering EndMT3.Autophagy serves as cytoprotective mechanism against EndMT of HCMECs by degrading Snail under hypoxia condition.Part II The regulation and mechanism of autophagy on endothelial-to-mensenchymal transition and cardiac fibrosis after acute myocardial infarctionObjective:To detect the feasibility of intervening autophagy meliorates cardiac fibrosis via inhibiting endothelial-to-mesenchymal transition after acute myocardial infarction(AMI).Methods:To investigate the effects and precise mechanisms of autophagy on cardiac fibrosis post-myocardial infarction(MI).Male SD rats(body weight around 200 g,age about 10 weeks)were divided rando mly into 4 groups:(1)Sham group.(2)AMI group,ligating the anterior descending coronary artery(LAD)in SD rats.(3)AMI +rap(2 mg/kg/day),ligating the anterior descending coronary arteryin SD rats.Rapamycin was injected intraperitoneally immediately and given once a day until 14 days after AMI.(4)AMI + 3-MA(15 mg/kg/day),ligating the anterior descending coronary artery in SD rats.3-MA was injected intraperitoneally immediately and given once a day until 14 days after AMI.Then perform next experiments:?echocardiography was performed to assess LVEF?LVFS?LVIDd and LVIDs at 14 days after AMI.?Masson's trichrome was performed to assess cardiac firosis and collagen volume fraction(CVF)was analysised in each group at 14 days after AMI.?The expressions of CD31,?SMA,COL1,LC3 and belin1 in the infarct border zone were examined by Western blot at 7 days after AMI.?Double immunofluorescence staining of CD31/?SMA was used to detecte the cells undergoing EndMT in the infarct border zone at 7 days after AMI.?Double immunofluorescence staining of CD31/LC3 to detecte the changes of LC3 in endothelial cells the infarction border zone in each group at 7 days after AMI.Results:The effects and precise mechanisms of autophagy on cardiac fibrosis post-MI:?Echocardiography showed that at 14 days after AMI,compared with the shamgroup,LAD ligation significantly decreased EF and LVFS while increased LVIDd,LVIDs and the fibrotic area in rats(P<0.05).Compared with the AMI group,rapamycin treatment increased EF and FS,decreased LVIDd and LVIDs(P<0.05).3-MA treatment showed no obvious effect on the cardiac function compared with the AMI group.?Masson's trichromes showd that compared with the AMI group,rapamycin treatment attenuated cardiac fibrosis(P<0.05),3-MA treatment worsen the cardiac fibrosis(P<0.05).?Western blot in the infarct border zone showed that?SMA and COL1 were upregulated in the AMI group compared with the sham group(P<0.05).Rapamycin treatment inhibited their upregulation but 3-MA treatment exacerbated their upregulation after 7 days of AMI(P<0.05).In contrast,CD31 was downregulated in AMI group compared with that in the sham group(P<0.05).Rapamycin treatment partially recovered CD31 expression but 3-MA treatment markedly decreased it(P<0.05).?Immunofluorescence results showed that CD31+?SMA+cells increased in cardiac tissue sections at 7 days after AMI compared with the sham group(P<0.05).Rapamycin treatment after LAD ligation decreased these double positive cells,compared with the AMI group,while 3-MA treatment after LAD ligation accelerated the emergence of these cells(P<0.05).CD31 and LC3 double immunofluorescence staining showed that there was no increase of LC3 expression in ECs in the infarction zone compared with that in the sham group.Rapamycin treatment upregulated LC3 expression of ECs and 3-MA treatment downregulated it compared with the AMI group(P<0.05).Conclusions:EndMT contributes to cardiac fibrosis post-MI.Enhanced autophagy inhibits EndMT after rapamycin treatment in AMI rats,attenuates cardiac fibrosis and dysfunction. |
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